Nina Bhardwaj, M.D., Ph.D.

DCs and NK cells: critical effectors in the immune response to HIV-1
Altfeld, Marcus; Fadda, Lena; Frleta, Davor; Bhardwaj, Nina
2011 Mar;11(3):176-186, Nature reviews. Immunology
Dendritic cells (DCs) and natural killer (NK) cells have central roles in antiviral immunity by shaping the quality of the adaptive immune response to viruses and by mediating direct antiviral activity. HIV-1 infection is characterized by a severe dysregulation of the antiviral immune response that starts during early infection. This Review describes recent insights into how HIV-1 infection affects DC and NK cell function, and the roles of these innate immune cells in HIV-1 pathogenesis. The importance of understanding DC and NK cell crosstalk during HIV infection for the development of effective antiviral strategies is also discussed
— id: 134140, year: 2011, vol: 11, page: 176, stat: Journal Article,

TLR4 Engagement during TLR3-Induced Proinflammatory Signaling in Dendritic Cells Promotes IL-10-Mediated Suppression of Antitumor Immunity
Bogunovic, Dusan; Manches, Olivier; Godefroy, Emmanuelle; Yewdall, Alice; Gallois, Anne; Salazar, Andres M; Marie, Isabelle; Levy, David E; Bhardwaj, Nina
2011 Aug 15;71(16):5467-5476, Cancer research
Toll-like receptor (TLR) agonists are promising adjuvants for immune therapy of cancer, but their potential efficacy as single or combinatorial agents has yet to be fully evaluated. Here, we report that among all TLR agonists tested, dendritic cells (DC) stimulated with the TLR3 agonist polyI:C displayed the strongest activity in stimulating proinflammatory responses and the production of melanoma antigen-specific CD8(+) T cells. Simultaneous treatment with TLR7/8 agonists further improved these responses, but the inclusion of bacterial lipopolysaccharide (LPS), a TLR4 agonist, suppressed proinflammatory cytokine production. This inhibition was contingent upon rapid induction of the suppressive cytokine interleukin (IL)-10 by LPS, leading to dysregulated immune responses and it could be reversed by signal transducers and activators of transcription 3 knockdown, p38 blockade or antibodies to IL-10 and its receptor. Our findings show how certain TLR agonist combinations can enhance or limit DC responses associated with antitumor immunity, through their relative ability to induce IL-10 pathways that are immune suppressive. Cancer Res; 71(16); 5467-76. (c)2011 AACR
— id: 136637, year: 2011, vol: 71, page: 5467, stat: Journal Article,

miR-30b/30d Regulation of GalNAc Transferases Enhances Invasion and Immunosuppression during Metastasis
Gaziel-Sovran, Avital; Segura, Miguel F; Di Micco, Raffaella; Collins, Mary K; Hanniford, Douglas; Vega-Saenz de Miera, Eleazar; Rakus, John F; Dankert, John F; Shang, Shulian; Kerbel, Robert S; Bhardwaj, Nina; Shao, Yongzhao; Darvishian, Farbod; Zavadil, Jiri; Erlebacher, Adrian; Mahal, Lara K; Osman, Iman; Hernando, Eva
2011 Jul 12;20(1):104-118, Cancer cell
To metastasize, a tumor cell must acquire abilities such as the capacity to colonize new tissue and evade immune surveillance. Recent evidence suggests that microRNAs can promote the evolution of malignant behaviors by regulating multiple targets. We performed a microRNA analysis of human melanoma, a highly invasive cancer, and found that miR-30b/30d upregulation correlates with stage, metastatic potential, shorter time to recurrence, and reduced overall survival. Ectopic expression of miR-30b/30d promoted the metastatic behavior of melanoma cells by directly targeting the GalNAc transferase GALNT7, resulted in increased synthesis of the immunosuppressive cytokine IL-10, and reduced immune cell activation and recruitment. These data support a key role of miR-30b/30d and GalNAc transferases in metastasis, by simultaneously promoting cellular invasion and immunosuppression
— id: 135264, year: 2011, vol: 20, page: 104, stat: Journal Article,

Matrix Metalloproteinase-2 Conditions Human Dendritic Cells to Prime Inflammatory T(H)2 Cells via an IL-12- and OX40L-Dependent Pathway
Godefroy, Emmanuelle; Manches, Olivier; Dreno, Brigitte; Hochman, Tsivia; Rolnitzky, Linda; Labarriere, Nathalie; Guilloux, Yannick; Goldberg, Judith; Jotereau, Francine; Bhardwaj, Nina
2011 Mar 8;19(3):333-346, Cancer cell
Matrix metalloproteinase-2 (MMP-2) is a proteolytic enzyme degrading the extracellular matrix and overexpressed by many tumors. Here, we documented the presence of MMP-2-specific CD4(+) T cells in tumor-infiltrating lymphocytes (TILs) from melanoma patients. Strikingly, MMP-2-specific CD4(+) T cells displayed an inflammatory T(H)2 profile, i.e., mainly secreting TNF-alpha, IL-4, and IL-13 and expressing GATA-3. Furthermore, MMP-2-conditioned dendritic cells (DCs) primed naive CD4(+) T cells to differentiate into an inflammatory T(H)2 phenotype through OX40L expression and inhibition of IL-12p70 production. MMP-2 degrades the type I IFN receptor, thereby preventing STAT1 phosphorylation, which is necessary for IL-12p35 production. Active MMP-2, therefore, acts as an endogenous type 2 'conditioner' and may play a role in the observed prevalence of detrimental type 2 responses in melanoma
— id: 127238, year: 2011, vol: 19, page: 333, stat: Journal Article,

Oligonucleotide Motifs That Disappear during the Evolution of Influenza Virus in Humans Increase Alpha Interferon Secretion by Plasmacytoid Dendritic Cells
Jimenez-Baranda, Sonia; Greenbaum, Benjamin; Manches, Olivier; Handler, Jesse; Rabadan, Raul; Levine, Arnold; Bhardwaj, Nina
2011 Apr;85(8):3893-3904, Journal of virology
CpG motifs in an A/U context have been preferentially eliminated from classical H1N1 influenza virus genomes during virus evolution in humans. The hypothesis of the current work is that CpG motifs in a uracil context represent sequence patterns with the capacity to induce an immune response, and the avoidance of this immunostimulatory signal is the reason for the observed preferential decline. To analyze the immunogenicity of these domains, we used plasmacytoid dendritic cells (pDCs). pDCs express pattern recognition receptors, including Toll-like receptor 7 (TLR7), which recognizes guanosine- and uridine-rich viral single-stranded RNA (ssRNA), including influenza virus ssRNA. The signaling through TLR7 results in the induction of inflammatory cytokines and type I interferon (IFN-I), an essential process for the induction of specific adaptive immune responses and for mounting a robust antiviral response mediated by IFN-alpha. Secretion of IFN-alpha is also linked to the activation of other immune cells, potentially amplifying the effect of an initial IFN-alpha secretion. We therefore also examined the role of IFN-alpha-driven activation of NK cells as another source of selective pressure on the viral genome. We found direct evidence that CpG RNA motifs in a U-rich context control pDC activation and IFN-alpha-driven activation of NK cells, likely through TLR7. These data provide a potential explanation for the loss of CpG motifs from avian influenza viruses as they adapt to mammalian hosts. The selective decrease of CpG motifs surrounded by U/A may be a viral strategy to avoid immune recognition, a strategy likely shared by highly expressed human immune genes
— id: 128792, year: 2011, vol: 85, page: 3893, stat: Journal Article,

MAGE-A Inhibits Apoptosis in Proliferating Myeloma Cells through Repression of Bax and Maintenance of Survivin
Nardiello, Tricia; Jungbluth, Achim A; Mei, Anna; Diliberto, Maurizio; Huang, Xiangao; Dabrowski, Ania; Andrade, Valeria C C; Wasserstrum, Rebecca; Ely, Scott; Niesvizky, Ruben; Pearse, Roger; Coleman, Morton; Jayabalan, David S; Bhardwaj, Nina; Old, Lloyd J; Chen-Kiang, Selina; Cho, Hearn Jay
2011 Jul 1;17(13):4309-4319, Clinical cancer research
PURPOSE: The type I Melanoma Antigen GEnes (MAGEs) are commonly expressed in cancers, fueling speculation that they may be therapeutic targets with oncogenic potential. They form complexes with RING domain proteins that have E3 ubiquitin ligase activity and promote p53 degradation. MAGE-A3 was detected in tumor specimens from patients with multiple myeloma and its expression correlated with higher frequencies of Ki-67(+) malignant cells. In this report, we examine the mechanistic role of MAGE-A in promoting survival of proliferating multiple myeloma cells. EXPERIMENTAL DESIGN: The impact of MAGE-A3 expression on survival and proliferation in vivo was examined by immunohistochemical analysis in an independent set of tumor specimens segregated into two groups: newly diagnosed, untreated patients and patients who had relapsed after chemotherapy. The mechanisms of MAGE-A3 activity were investigated in vitro by silencing its expression by short hairpin RNA interference in myeloma cell lines and primary cells and assessing the resultant effects on proliferation and apoptosis. RESULTS: MAGE-A3 was detected in a significantly higher percentage of relapsed patients compared with newly diagnosed, establishing a novel correlation with progression of disease. Silencing of MAGE-A showed that it was dispensable for cell cycling, but was required for survival of proliferating myeloma cells. Loss of MAGE-A led to apoptosis mediated by p53-dependent activation of proapoptotic Bax expression and by reduction of survivin expression through both p53-dependent and -independent mechanisms. CONCLUSIONS: These data support a role for MAGE-A in the pathogenesis and progression of multiple myeloma by inhibiting apoptosis in proliferating myeloma cells through two novel mechanisms. Clin Cancer Res; 17(13); 4309-19. (c)2011 AACR
— id: 134910, year: 2011, vol: 17, page: 4309, stat: Journal Article,

Spatiotemporal trafficking of HIV in human plasmacytoid dendritic cells defines a persistently IFN-alpha-producing and partially matured phenotype
O'Brien, Meagan; Manches, Olivier; Sabado, Rachel Lubong; Baranda, Sonia Jimenez; Wang, Yaming; Marie, Isabelle; Rolnitzky, Linda; Markowitz, Martin; Margolis, David M; Levy, David; Bhardwaj, Nina
2011 Mar 1;121(3):1088-1101, Journal of clinical investigation
Plasmacytoid DCs (pDCs) are innate immune cells that are specialized to produce IFN-alpha and to activate adaptive immune responses. Although IFN-alpha inhibits HIV-1 replication in vitro, the production of IFN-alpha by HIV-activated pDCs in vivo may contribute more to HIV pathogenesis than to protection. We have now shown that HIV-stimulated human pDCs allow for persistent IFN-alpha production upon repeated stimulation, express low levels of maturation molecules, and stimulate weak T cell responses. Persistent IFN-alpha production by HIV-stimulated pDCs correlated with increased levels of IRF7 and was dependent upon the autocrine IFN-alpha/beta receptor feedback loop. Because it has been shown that early endosomal trafficking of TLR9 agonists causes strong activation of the IFN-alpha pathway but weak activation of the NF-kappaB pathway, we sought to investigate whether early endosomal trafficking of HIV, a TLR7 agonist, leads to the IFN-alpha-producing phenotype we observed. We demonstrated that HIV preferentially traffics to the early endosome in human pDCs and therefore skews pDCs toward a partially matured, persistently IFN-alpha-secreting phenotype
— id: 130297, year: 2011, vol: 121, page: 1088, stat: Journal Article,

Human immunodeficiency virus type 1 modified to package simian immunodeficiency virus vpx efficiently infects macrophages and dendritic cells
Sunseri, Nicole; O'Brien, Meagan; Bhardwaj, Nina; Landau, Nathaniel R
2011 Jul;85(13):6263-6274, Journal of virology
The lentiviral accessory protein Vpx is thought to facilitate the infection of macrophages and dendritic cells by counteracting an unidentified host restriction factor. Although human immunodeficiency virus type 1 (HIV-1) does not encode Vpx, the accessory protein can be provided to monocyte-derived macrophages (MDM) and monocyte-derived dendritic cells (MDDC) in virus-like particles, dramatically enhancing their susceptibility to HIV-1. Vpx and the related accessory protein Vpr are packaged into virions through a virus-specific interaction with the p6 carboxy-terminal domain of Gag. We localized the minimal Vpx packaging motif of simian immunodeficiency virus SIVmac(239) p6 to a 10-amino-acid motif and introduced this sequence into an infectious HIV-1 provirus. The chimeric virus packaged Vpx that was provided in trans and was substantially more infectious on MDDC and MDM than the wild-type virus. We further modified the virus by introducing the Vpx coding sequence in place of nef. The resulting virus produced Vpx and replicated efficiently in MDDC and MDM. The virus also induced a potent type I interferon response in MDDC. In a coculture system, the Vpx-containing HIV-1 was more efficiently transmitted from MDDC to T cells. These findings suggest that in vivo, Vpx may facilitate transmission of the virus from dendritic cells to T cells. In addition, the chimeric virus could be used to design dendritic cell vaccines that induce an enhanced innate immune response. This approach could also be useful in the design of lentiviral vectors that transduce these relatively resistant cells
— id: 134443, year: 2011, vol: 85, page: 6263, stat: Journal Article,

CTLA-4 blockade increases antigen-specific CD8(+) T cells in prevaccinated patients with melanoma: three cases
Yuan, Jianda; Ginsberg, Brian; Page, David; Li, Yanyun; Rasalan, Teresa; Gallardo, Humilidad F; Xu, Yinyan; Adams, Sylvia; Bhardwaj, Nina; Busam, Klaus; Old, Lloyd J; Allison, James P; Jungbluth, Achim; Wolchok, Jedd D
2011 Aug;60(8):1137-1146, Cancer immunology immunotherapy
BACKGROUND: Anti-cytotoxic T-lymphocyte antigen-4 (CTLA-4) antibodies, such as ipilimumab, have generated measurable immune responses to Melan-A, NY-ESO-1, and gp100 antigens in metastatic melanoma. Vaccination against such targets has potential for immunogenicity and may produce an effector-memory T-cell response. METHODS: To determine the effect of CTLA-4 blockade on antigen-specific responses following vaccination, in-depth immune monitoring was performed on three ipilimumab-treated patients prevaccinated with gp100 DNA (IMF-24), gp100(209-217) and tyrosinase peptides plus GM-CSF DNA (IMF-32), or NY-ESO-1 protein plus imiquimod (IMF-11); peripheral blood mononuclear cells were analyzed by tetramer and/or intracellular cytokine staining following 10-day culture with HLA-A*0201-restricted gp100(209-217) (ITDQVPFSV), tyrosinase(369-377) (YMDGTMSQV), or 20-mer NY-ESO-1 overlapping peptides, respectively. Tumors from IMF-32 were analyzed by immunohistochemistry to help elucidate mechanism(s) underlying tumor escape. RESULTS: Following vaccination, patients generated weak to no CD4(+) or CD8(+) T-cell response specific to the vaccine antigen but demonstrated increases in effector-memory (CCR7(lo)CD45RA(lo)) tetramer(+)CD8(+) T cells. After ipilimumab induction, patients experienced a robust, although sometimes transient, antigen-specific response for gp100 (IMF-32 and IMF-24) or NY-ESO-1 (IMF-11) and produced polyfunctional intracellular cytokines. Primary and metastatic tumors expressed tyrosinase but not gp100 or class I/II MHC molecules. CONCLUSION: Vaccination induced a measurable antigen-specific T-cell response that increased following CTLA-4 blockade, potentially 'boosting' the vaccine-primed response. Tumor escape may be related to antigen loss or lack of MHC expression necessary for immune activity. These results in a limited number of patients support the need for further research into combining vaccination with ipilimumab and provide insight into mechanisms underlying tumor escape
— id: 144815, year: 2011, vol: 60, page: 1137, stat: Journal Article,

Reply to Murali and Scolyer: Immune profile and mitotic index in advanced melanoma
Bogunovic, D; Bhardwaj, N
2010 MAR 30 ;107(13):E47-E47, Proceedings of the National Academy of Sciences of the United States of America
— id: 109066, year: 2010, vol: 107, page: E47, stat: Journal Article,

HIV-1 impairs in vitro priming of naive T cells and gives rise to contact-dependent suppressor T cells
Che, Karlhans F.; Sabado, Rachel L.; Shankar, Esaki M.; Tjomsland, Veronica; Messmer, Davorka; Bhardwaj, Nina; Lifson, Jeffrey D.; Larsson, Marie
2010 AUG ;40(8):2248-2258, European journal of immunology
Priming of T cells in lymphoid tissues of HIV-infected individuals occurs in the presence of HIV-1. DC in this milieu activate T cells and disseminate HIV-1 to newly activated T cells, the outcome of which may have serious implications in the development of optimal antiviral responses. We investigated the effects of HIV-1 on DC-naive T-cell interactions using an allogeneic in vitro system. Our data demonstrate a dramatic decrease in the primary expansion of naive T cells when cultured with HIV-1-exposed DC. CD4(+) and CD8(+) T cells showed enhanced expression of PD-1 and TRAIL, whereas CTLA-4 expression was observed on CD4(+) T cells. It is worth noting that T cells primed in the presence of HIV-1 suppressed priming of other naive T cells in a contact-dependent manner. We identified PD-1, CTLA-4, and TRAIL pathways as responsible for this suppresion, as blocking these negative molecules restored T-cell proliferation to a higher degree. In conclusion, the presence of HIV-1 during DC priming produced cells with inhibitory effects on T-cell activation and proliferation, i.e. suppressor T cells, a mechanism that could contribute to the enhancement of HIV-1 pathogenesis
— id: 112188, year: 2010, vol: 40, page: 2248, stat: Journal Article,

LXR promotes the maximal egress of monocyte-derived cells from mouse aortic plaques during atherosclerosis regression
Feig, Jonathan E; Pineda-Torra, Ines; Sanson, Marie; Bradley, Michelle N; Vengrenyuk, Yuliya; Bogunovic, Dusan; Gautier, Emmanuel L; Rubinstein, Daniel; Hong, Cynthia; Liu, Jianhua; Wu, Chaowei; van Rooijen, Nico; Bhardwaj, Nina; Garabedian, Michael; Tontonoz, Peter; Fisher, Edward A
2010 Dec 1;120(12):4415-4424, Journal of clinical investigation
We have previously shown that mouse atherosclerosis regression involves monocyte-derived (CD68+) cell emigration from plaques and is dependent on the chemokine receptor CCR7. Concurrent with regression, mRNA levels of the gene encoding LXRalpha are increased in plaque CD68+ cells, suggestive of a functional relationship between LXR and CCR7. To extend these results, atherosclerotic Apoe-/- mice sufficient or deficient in CCR7 were treated with an LXR agonist, resulting in a CCR7-dependent decrease in plaque CD68+ cells. To test the requirement for LXR for CCR7-dependent regression, we transplanted aortic arches from atherosclerotic Apoe-/- mice, or from Apoe-/- mice with BM deficiency of LXRalpha or LXRbeta, into WT recipients. Plaques from both LXRalpha and LXRbeta-deficient Apoe-/- mice exhibited impaired regression. In addition, the CD68+ cells displayed reduced emigration and CCR7 expression. Using an immature DC line, we found that LXR agonist treatment increased Ccr7 mRNA levels. This increase was blunted when LXRalpha and LXRbeta levels were reduced by siRNAs. Moreover, LXR agonist treatment of primary human immature DCs resulted in functionally significant upregulation of CCR7. We conclude that LXR is required for maximal effects on plaque CD68+ cell expression of CCR7 and monocyte-derived cell egress during atherosclerosis regression in mice
— id: 119227, year: 2010, vol: 120, page: 4415, stat: Journal Article,

A needle in the 'cancer vaccine' haystack
Gallois, Anne; Bhardwaj, Nina
2010 Aug;16(8):854-856, Nature medicine
— id: 111589, year: 2010, vol: 16, page: 854, stat: Journal Article,

Toll-like receptor agonists: are they good adjuvants?
Gnjatic, Sacha; Sawhney, Nikhil B; Bhardwaj, Nina
2010 Jul-Aug;16(4):382-391, Cancer journal
Therapeutic immunization leading to cancer regression remains a significant challenge. Successful immunization requires activation of adaptive immunity, including tumor specific CD4 T cells and CD8 T cells. Generally, the activation of T cells is compromised in patients with cancer because of immune suppression, loss of tumor antigen expression, and dysfunction of antigen-presenting cells. Antigen-presenting cells such as dendritic cells (DCs) are key for the induction of adaptive antitumor immune responses. Recently, attention has focused on novel adjuvants that enhance dendritic cell function and their ability to prime T cells. Agonists that target toll-like receptors are being used clinically either alone or in combination with tumor antigens and showing initial success both in terms of enhancing immune responses and eliciting antitumor activity. This review summarizes the application of these adjuvants to treat cancer and the potential for boosting responses in vivo
— id: 111592, year: 2010, vol: 16, page: 382, stat: Journal Article,

Cellular immune responses against CT7 (MAGE-C1) and humoral responses against other cancer-testis antigens in multiple myeloma patients
Lendvai, Nikoletta; Gnjatic, Sacha; Ritter, Erika; Mangone, Michael; Austin, Wayne; Reyner, Karina; Jayabalan, David; Niesvizky, Ruben; Jagannath, Sundar; Bhardwaj, Nina; Chen-Kiang, Selina; Old, Lloyd J; Cho, Hearn Jay
2010 ;10:4-4, Cancer imunity
The type I melanoma antigen gene (MAGE) proteins CT7 (MAGE-C1) and MAGE-A3 are commonly expressed in multiple myeloma (MM), and their expression correlates with increased plasma cell proliferation and poor clinical outcome. They belong to the cancer-testis antigen (CTAg) group of tumor-associated proteins, some of which elicit spontaneous immune responses in cancer patients. CT7 and MAGE-A3 are promising antigenic targets for therapeutic tumor vaccines in myeloma; therefore, it is critical to determine if they are immunogenic in MM patients. We analyzed cellular and humoral immune responses against CTAgs in patients with plasma cell dyscrasias: MM, monoclonal gammopathy of undetermined significance (MGUS), and Waldenstrom's macroglobulinemia (WM). Bone marrow lymphocytes from two of four untreated MM patients exhibited CT7-specific cellular immune responses as measured by an autologous cellular immunity assay, the first such immune response to CT7 to be reported in cancer patients. Sera from 24 patients were screened by ELISA for humoral immune responses to CTAgs. Two patients with MM demonstrated positive titers, one for MAGE-A1 and the other for SSX1. These data demonstrate that CTAgs, particularly CT7, are immunogenic in MM patients and merit further exploration as targets of immunological therapy in MM
— id: 106505, year: 2010, vol: 10, page: 4, stat: Journal Article,

"Corrigendum to ""Efficient in vitro expansion of JC virus-specific CD8+ T-cell responses by JCV peptide-stimulated dendritic cells from patients with progressive multifocal leukoencephalopathy"" [Virology 383 (2009) 173-177] (DOI:10.1016/j.virol.2008.10.046)"
Marzocchetti A.; Lima M.; Tompkins T.; Kavanagh D.G.; Gandhi R.T.; O'Neill D.W.; Bhardwaj N.; Koralnik I.J.
2010 ;396(2):349-350, Virology
— id: 106196, year: 2010, vol: 396, page: 349, stat: Journal Article,

Directing dendritic cell immunotherapy towards successful cancer treatment
Sabado, Rachel Lubong; Bhardwaj, Nina
2010 Jan;2(1):37-56, Immunotherapy
The use of dendritic cells (DCs) for tumor immunotherapy represents a powerful approach for harnessing the patient's own immune system to eliminate tumor cells. However, suboptimal conditions for generating potent immunostimulatory DCs, as well as the induction of tolerance and suppression mediated by the tumors and its microenvironment have contributed to limited success. Combining DC vaccines with new approaches that enhance immunogenicity and overcome the regulatory mechanisms underlying peripheral tolerance may be the key to achieving effective and durable anti-tumor immune responses that translate to better clinical outcomes
— id: 111350, year: 2010, vol: 2, page: 37, stat: Journal Article,

Evidence of dysregulation of dendritic cells in primary HIV infection
Sabado, Rachel Lubong; O'Brien, Meagan; Subedi, Abhignya; Qin, Li; Hu, Nan; Taylor, Elizabeth; Dibben, Oliver; Stacey, Andrea; Fellay, Jacques; Shianna, Kevin V; Siegal, Frederick; Shodell, Michael; Shah, Kokila; Larsson, Marie; Lifson, Jeffrey; Nadas, Arthur; Marmor, Michael; Hutt, Richard; Margolis, David; Garmon, Donald; Markowitz, Martin; Valentine, Fred; Borrow, Persephone; Bhardwaj, Nina
2010 Nov 11;116(19):3839-3852, Blood
Myeloid and plasmacytoid dendritic cells (DCs) are important mediators of both innate and adaptive immunity against pathogens such as HIV. During the course of HIV infection, blood DC numbers fall substantially. In the present study, we sought to determine how early in HIV infection the reduction occurs and whether the remaining DC subsets maintain functional capacity. We find that both myeloid DC and plasmacytoid DC levels decline very early during acute HIV in-fection. Despite the initial reduction in numbers, those DCs that remain in circulation retain their function and are able to stimulate allogeneic T-cell responses, and up-regulate maturation markers plus produce cytokines/chemokines in response to stimulation with TLR7/8 agonists. Notably, DCs from HIV-infected subjects produced significantly higher levels of cytokines/chemokines in response to stimulation with TLR7/8 agonists than DCs from uninfected controls. Further examination of gene expression profiles indicated in vivo activation, either directly or indirectly, of DCs during HIV infection. Taken together, our data demonstrate that despite the reduction in circulating DC numbers, those that remain in the blood display hyperfunctionality and implicates a possible role for DCs in promoting chronic immune activation
— id: 114507, year: 2010, vol: 116, page: 3839, stat: Journal Article,

CD8+ T cell priming by dendritic cell vaccines requires antigen transfer to endogenous antigen presenting cells
Yewdall, Alice W; Drutman, Scott B; Jinwala, Felecia; Bahjat, Keith S; Bhardwaj, Nina
2010 ;5(6):e11144-e11144, PLoS ONE
BACKGROUND: Immunotherapeutic strategies to stimulate anti-tumor immunity are promising approaches for cancer treatment. A major barrier to their success is the immunosuppressive microenvironment of tumors, which inhibits the functions of endogenous dendritic cells (DCs) that are necessary for the generation of anti-tumor CD8+ T cells. To overcome this problem, autologous DCs are generated ex vivo, loaded with tumor antigens, and activated in this non-suppressive environment before administration to patients. However, DC-based vaccines rarely induce tumor regression. METHODOLOGY/PRINCIPAL FINDINGS: We examined the fate and function of these DCs following their injection using murine models, in order to better understand their interaction with the host immune system. Contrary to previous assumptions, we show that DC vaccines have an insignificant role in directly priming CD8+ T cells, but instead function primarily as vehicles for transferring antigens to endogenous antigen presenting cells, which are responsible for the subsequent activation of T cells. CONCLUSIONS/SIGNIFICANCE: This reliance on endogenous immune cells may explain the limited success of current DC vaccines to treat cancer and offers new insight into how these therapies can be improved. Future approaches should focus on creating DC vaccines that are more effective at directly priming T cells, or abrogating the tumor induced suppression of endogenous DCs
— id: 110668, year: 2010, vol: 5, page: e11144, stat: Journal Article,

Hematopoietic progenitor kinase 1 is a negative regulator of dendritic cell activation
Alzabin, Saba; Bhardwaj, Nina; Kiefer, Friedemann; Sawasdikosol, Sansana; Burakoff, Steven
2009 May 15;182(10):6187-6194, Journal of immunology
Hematopoietic progenitor kinase 1 (HPK1) is a hematopoietic cell-restricted member of the Ste20 kinases that acts as a negative regulator of T cell functions through the AP-1, NFAT, and NFkappaB pathways. Using HPK1-deficient (HPK1(-/-)) mice, we report in this study a novel role for HPK1 in dendritic cells (DCs). Specifically, we observed that matured HPK1(-/-) bone marrow-derived DCs (BMDCs) are superior to their wild-type (WT) counterpart in stimulating T cell proliferation in vivo and in vitro. Several characteristics of HPK1(-/-) BMDCs may account for this enhanced activity: Matured HPK1(-/-) BMDCs express higher levels of costimulatory molecules CD80, CD86, and I-A(b) as well as produce more proinflammatory cytokines IL-12, IL-1beta, TNF-alpha, and IL-6 than their WT littermates. The role of HPK1 as a proapoptotic molecule was assessed post activation with LPS, and results indicated that HPK1(-/-) BMDCs are significantly resistant to LPS-induced apoptosis. Our results led us to investigate the role of HPK1(-/-) BMDCs in tumor immunotherapy. Using a s.c. murine model of Lewis Lung Carcinoma, we found that HPK1(-/-) BMDCs eliminate established s.c. Lewis Lung Carcinoma more efficiently than their WT counterpart. Our data reveal a novel role for HPK1 as a negative regulator of DC functions, identifying its potential as a molecular target for DC-based immunotherapy against cancers
— id: 98016, year: 2009, vol: 182, page: 6187, stat: Journal Article,

Immune regulation
Bhardwaj, Nina; O'Neill, David; Thomas Waldmann, Thomas
Essential clinical immunology Cambridge UK : Cambridge University Press, 2009,
— id: 5429, year: 2009, vol: , page: ?, stat: Chapter,

Vaccination with recombinant NY-ESO-1 protein elicits immunodominant HLA-DR52b-restricted CD4+ T cell responses with a conserved T cell receptor repertoire
Bioley, Gilles; Dousset, Christelle; Yeh, Alice; Dupont, Bo; Bhardwaj, Nina; Mears, Gregory; Old, Lloyd J; Ayyoub, Maha; Valmori, Danila
2009 Jul 1;15(13):4467-4474, Clinical cancer research
PURPOSE: ESO is a tumor-specific antigen with wide expression in human tumors of different histologic types and remarkable spontaneous immunogenicity. We have previously shown that specific T(H)1 and antibody responses can be elicited in patients with no detectable preexisting immune responses by vaccination with rESO administered with Montanide ISA-51 and CpG ODN 7909. The purpose of the present study was to characterize vaccine-induced ESO-specific CD4(+) T cell responses. EXPERIMENTAL DESIGN: We generated CD4(+) T cell clones from patient C2, who had the highest CD4(+) T cell response to the vaccine, and analyzed their fine specificity and HLA class II restriction to determine the recognized epitope. We then assessed the response to the identified epitope in all vaccinated patients expressing the corresponding HLA class II allele. RESULTS: We found that ESO-specific CD4(+) T cell clones from patient C2 recognize peptide ESO(119-143) (core region 123-137) presented by HLA-DR52b (HLA-DRB3*0202), a MHC class II allele expressed by about half of Caucasians. Importantly, following vaccination, all patients expressing DR52b developed significant responses to the identified epitope, accounting for, on average, half of the total CD4(+) T cell responses to the 119-143 immunodominant region. In addition, analysis of ESO-specific DR52b-restricted CD4(+) T cells at the clonal level revealed significant conservation of T cell receptor usage among different individuals. CONCLUSIONS: The identification of a DR52b-restricted epitope from ESO that is immunodominant in the context of vaccine-elicited immune responses is instrumental for the immunologic monitoring of vaccination trials targeting this important tumor antigen
— id: 133705, year: 2009, vol: 15, page: 4467, stat: Journal Article,

Vaccination with a recombinant protein encoding the tumor-specific antigen NY-ESO-1 elicits an A2/157-165-specific CTL repertoire structurally distinct and of reduced tumor reactivity than that elicited by spontaneous immune responses to NY-ESO-1-expressing Tumors
Bioley, Gilles; Guillaume, Philippe; Luescher, Immanuel; Bhardwaj, Nina; Mears, Gregory; Old, Lloyd; Valmori, Danila; Ayyoub, Maha
2009 Feb-Mar;32(2):161-168, Journal of immunotherapy (Hagerstown)
In a recent vaccination trial assessing the immunogenicity of an NY-ESO-1 (ESO) recombinant protein administered with Montanide and CpG, we have obtained evidence that this vaccine induces specific cytolytic T lymphocytes (CTL) in half of the patients. Most vaccine-induced CTLs were directed against epitopes located in the central part of the protein, between amino acids 81 and 110. This immunodominant region, however, is distinct from another ESO CTL region, 157-165, that is a frequent target of spontaneous CTL responses in A2+ patients bearing ESO tumors. In this study, we have investigated the CTL responses to ESO 157-165 in A2+ patients vaccinated with the recombinant protein. Our data indicate that after vaccination with the protein, CTL responses to ESO 157-165 are induced in some, but not all, A2+ patients. ESO 157-165-specific CTLs induced by vaccination with the ESO protein were functionally heterogeneous in terms of tumor recognition and often displayed decreased tumor reactivity as compared with ESO 157-165-specific CTLs isolated from patients with spontaneous immune responses to ESO. Remarkably, protein-induced CTLs used T-cell receptors similar to those previously isolated from patients vaccinated with synthetic ESO peptides (Vbeta4.1) and distinct from those used by highly tumor-reactive CTLs isolated from patients with spontaneous immune responses (Vbeta1.1, Vbeta8.1, and Vbeta13.1). Together, these results demonstrate that vaccination with the ESO protein elicits a repertoire of ESO 157-165-specific CTLs bearing T-cell receptors that are structurally distinct from those of CTLs found in spontaneous immune responses to the antigen and that are heterogeneous in terms of tumor reactivity, being often poorly tumor reactive
— id: 95133, year: 2009, vol: 32, page: 161, stat: Journal Article,

HLA class I - associated immunodominance affects CTL responsiveness to an ESO recombinant protein tumor antigen vaccine
Bioley, Gilles; Guillaume, Philippe; Luescher, Immanuel; Yeh, Alice; Dupont, Bo; Bhardwaj, Nina; Mears, Gregory; Old, Lloyd J; Valmori, Danila; Ayyoub, Maha
2009 Jan 1;15(1):299-306, Clinical cancer research
PURPOSE: Vaccination with full-length human tumor antigens aims at inducing or increasing antitumor immune responses, including CD8 CTL in cancer patients across the HLA barrier. We have recently reported that vaccination with a recombinant tumor-specific NY-ESO-1 (ESO) protein, administered with Montanide and CpG resulted in the induction of specific integrated antibody and CD4 T cell responses in all vaccinated patients examined, and significant CTL responses in half of them. Vaccine-induced CTL mostly recognized a single immunodominant region (ESO 81-110). The purpose of the present study was to identify genetic factor(s) distinguishing CTL responders from nonresponders. EXPERIMENTAL DESIGN: We determined the HLA class I alleles expressed by CTL responders and nonresponders using high-resolution molecular typing. Using short overlapping peptides spanning the ESO immunodominant CTL region and HLA class I/ESO peptide tetramers, we determined the epitopes recognized by the majority of vaccine-induced CTL. RESULTS: CTL induced by vaccination with ESO protein mostly recognized distinct but closely overlapping epitopes restricted by a few frequently expressed HLA-B35 and HLA-Cw3 alleles. All CTL responders expressed at least one of the identified alleles, whereas none of the nonresponders expressed them. CONCLUSIONS: Expression of HLA-B35 and HLA-Cw3 is associated with the induction of immunodominant CTL responses following vaccination with recombinant ESO protein. Because recombinant tumor-specific proteins are presently among the most promising candidate anticancer vaccines, our findings indicate that the monitoring of cancer vaccine trials should systematically include the assessment of HLA association with responsiveness
— id: 95135, year: 2009, vol: 15, page: 299, stat: Journal Article,

Immune profile and mitotic index of metastatic melanoma lesions enhance clinical staging in predicting patient survival
Bogunovic, Dusan; O'Neill, David W; Belitskaya-Levy, Ilana; Vacic, Vladimir; Yu, Yi-Lo; Adams, Sylvia; Darvishian, Farbod; Berman, Russell; Shapiro, Richard; Pavlick, Anna C; Lonardi, Stefano; Zavadil, Jiri; Osman, Iman; Bhardwaj, Nina
2009 Dec 1;106(48):20429-20434, Proceedings of the National Academy of Sciences of the United States of America
Although remission rates for metastatic melanoma are generally very poor, some patients can survive for prolonged periods following metastasis. We used gene expression profiling, mitotic index (MI), and quantification of tumor infiltrating leukocytes (TILs) and CD3+ cells in metastatic lesions to search for a molecular basis for this observation and to develop improved methods for predicting patient survival. We identified a group of 266 genes associated with postrecurrence survival. Genes positively associated with survival were predominantly immune response related (e.g., ICOS, CD3d, ZAP70, TRAT1, TARP, GZMK, LCK, CD2, CXCL13, CCL19, CCR7, VCAM1) while genes negatively associated with survival were cell proliferation related (e.g., PDE4D, CDK2, GREF1, NUSAP1, SPC24). Furthermore, any of the 4 parameters (prevalidated gene expression signature, TILs, CD3, and in particular MI) improved the ability of Tumor, Node, Metastasis (TNM) staging to predict postrecurrence survival; MI was the most significant contributor (HR = 2.13, P = 0.0008). An immune response gene expression signature and presence of TILs and CD3+ cells signify immune surveillance as a mechanism for prolonged survival in these patients and indicate improved patient subcategorization beyond current TNM staging
— id: 105312, year: 2009, vol: 106, page: 20429, stat: Journal Article,

Suppression of human dendritic cell function during acute HIV infection
Frleta, D; O'Brien, M; Haynes, B; Kessler, B; Bhardwaj, N
2009 DEC ;6(2-3):115-115, Retrovirology
— id: 105706, year: 2009, vol: 6, page: 115, stat: Journal Article,

A randomized therapeutic vaccine trial of canarypox-HIV-pulsed dendritic cells vs. canarypox-HIV alone in HIV-1-infected patients on antiretroviral therapy
Gandhi, Rajesh T; O'Neill, David; Bosch, Ronald J; Chan, Ellen S; Bucy, R Pat; Shopis, Janet; Baglyos, Lynn; Adams, Elizabeth; Fox, Lawrence; Purdue, Lynette; Marshak, Ann; Flynn, Theresa; Masih, Reena; Schock, Barbara; Mildvan, Donna; Schlesinger, Sarah J; Marovich, Mary A; Bhardwaj, Nina; Jacobson, Jeffrey M
2009 Oct 9;27(43):6088-6094, Vaccine
Targeting canarypox (CP)-HIV vaccine to dendritic cells (DCs) elicits anti-HIV-1 immune responses in vitro. We conducted a phase I/II clinical trial to evaluate whether adding DC to a CP-HIV vaccine improved virologic control during analytic treatment interruption (ATI) in HIV-1-infected subjects. Twenty-nine subjects on suppressive antiretroviral therapy were randomized to vaccination with autologous DCs infected with CP-HIV+keyhole limpet hemocyanin (KLH) (arm A, n=14) or CP-HIV+KLH alone (arm B, n=15). The mean viral load (VL) setpoint during ATI did not differ between subjects in arms A and B. A higher percentage of subjects in the DC group had a VL setpoint < 5,000 c/mL during ATI (4/13 or 31% in arm A compared with 0/13 in arm B, p=0.096), but virologic control was transient. Subjects in arm A had a greater increase in KLH lymphoproliferative response than subjects in arm B; however, summed ELISPOT responses to HIV-1 antigens did not differ by treatment arm. We conclude that a DC-CP-HIV vaccine is well-tolerated in HIV-1-infected patients, but does not lower VL setpoint during ATI compared with CP-HIV alone. New methods to enhance the immunogenicity and antiviral efficacy of DC-based vaccines for HIV-1 infection are needed
— id: 133722, year: 2009, vol: 27, page: 6088, stat: Journal Article,

Dendritic cells as targets for therapy in rheumatoid arthritis
Khan, Shaukat; Greenberg, Jeffrey D; Bhardwaj, Nina
2009 Oct;5(10):566-571, Nature reviews. Rheumatology
Dendritic cells (DCs) are central in inducing immunity and in mediating immune tolerance in their role as professional antigen-presenting cells. In the absence of DCs, a fatal autoimmunity develops in animal models. Although the role of DCs has been investigated extensively in the pathogenesis of rheumatoid arthritis (RA), it remains unclear whether DCs initiate autoimmunity in this disease. Nevertheless, evidence points towards a significant role for DCs in disease maintenance and progression. Current biologic therapies target cytokine products of antigen-presenting cells, such as tumor necrosis factor, interleukin-1 and interleukin-6. Emerging therapies for RA exploit the tolerogenic capacity of DCs. 'Tolerogenic' DCs can be generated from myeloid precursors ex vivo, loaded with antigen, and manipulated to suppress autoimmune responses in vivo, through the induction of activation-induced cell death, anergy, and/or regulatory T cells. Cells that are primed by DCs, such as B cells, type 1 and type 17 T helper cells, and that have been implicated in certain models of autoimmunity, are also being considered as additional targets for immune-based therapy. Studies to validate these approaches to ameliorate autoimmunity will be necessary before their application in the clinic
— id: 103156, year: 2009, vol: 5, page: 566, stat: Journal Article,

In vitro priming recapitulates in vivo HIV-1 specific T cell responses, revealing rapid loss of virus reactive CD4 T cells in acute HIV-1 infection
Lubong Sabado, Rachel; Kavanagh, Daniel G; Kaufmann, Daniel E; Fru, Karlhans; Babcock, Ethan; Rosenberg, Eric; Walker, Bruce; Lifson, Jeffrey; Bhardwaj, Nina; Larsson, Marie
2009 ;4(1):e4256-e4256, PLoS ONE
BACKGROUND: The requirements for priming of HIV-specific T cell responses initially seen in infected individuals remain to be defined. Activation of T cell responses in lymph nodes requires cell-cell contact between T cells and DCs, which can give concurrent activation of T cells and HIV transmission. METHODOLOGY: The study aim was to establish whether DCs pulsed with HIV-1 could prime HIV-specific T cell responses and to characterize these responses. Both infectious and aldrithiol-2 inactivated noninfectious HIV-1 were compared to establish efficiencies in priming and the type of responses elicited. FINDINGS: Our findings show that both infectious and inactivated HIV-1 pulsed DCs can prime HIV-specific responses from naive T cells. Responses included several CD4(+) and CD8(+) T cell epitopes shown to be recognized in vivo by acutely and chronically infected individuals and some CD4(+) T cell epitopes not identified previously. Follow up studies of acute and recent HIV infected samples revealed that these latter epitopes are among the earliest recognized in vivo, but the responses are lost rapidly, presumably through activation-induced general CD4(+) T cell depletion which renders the newly activated HIV-specific CD4(+) T cells prime targets for elimination. CONCLUSION: Our studies highlight the ability of DCs to efficiently prime naive T cells and induce a broad repertoire of HIV-specific responses and also provide valuable insights to the pathogenesis of HIV-1 infection in vivo
— id: 95134, year: 2009, vol: 4, page: e4256, stat: Journal Article,

Resolution of immune activation defines nonpathogenic SIV infection
Manches, O; Bhardwaj, N
2009 DEC ;119(12):3512-3515, Journal of clinical investigation
Natural nonhuman primate hosts of SIV do not succumb to AIDS despite significant viral replication, a phenomenon attributed to reduced levels of chronic and deleterious 'immune activation.' Two studies in this issue of the JCI, by Bosinger et al. and Jacquelin et al., now show that SIV induces vigorous immune activation and upregulation of IFN-stimulated genes in both natural and susceptible hosts, but strikingly, the responses resolve only in the former (see the related articles beginning on pages 3556 and 3544, respectively). Thus, natural hosts for SIV actively engage mechanisms to abort sustained immune activation and its associated harmful effects
— id: 105714, year: 2009, vol: 119, page: 3512, stat: Journal Article,

HIV-activated human plasmacytoid DCs induce Tregs through an indoleamine 2,3-dioxygenase-dependent mechanism
Manches, O; Munn, D; Fallahi, A; Lifson, J; Chaperot, L; Plumas, J; Bhardwaj, N
2009 DEC ;6(2-3):115-115, Retrovirology
— id: 105708, year: 2009, vol: 6, page: 115, stat: Journal Article,

Efficient in vitro expansion of JC virus-specific CD8(+) T-cell responses by JCV peptide-stimulated dendritic cells from patients with progressive multifocal leukoencephalopathy
Marzocchetti, Angela; Lima, Marco; Tompkins, Troy; Kavanagh, Daniel G; Gandhi, Rajesh T; O'Neill, David W; Bhardwaj, Nina; Koralnik, Igor J
2009 Jan 20;383(2):173-177, Virology
Progressive multifocal leukoencephalopathy (PML) is a demyelinating disease of the brain caused by JC virus (JCV) for which there is no cure. PML patients who have JCV-specific CD8(+) cytotoxic T lymphocytes (CTL) in their blood have a better clinical outcome. We compared JCV-specific CTL responses in vitro elicited either by JCV peptide-loaded dendritic cells (DC) or by direct peptide stimulation of lymphocytes from 20 HLA-A0201(+) healthy controls, HIV(+) and PML patients. JCV peptide-loaded DC elicited a stronger CTL expansion in 13/15 responders. DC can induce a potent JCV-specific CTL response in vitro, and may constitute a promising approach for PML immunotherapy
— id: 95136, year: 2009, vol: 383, page: 173, stat: Journal Article,

Dendritic cell function in HIV infection
Miller E.; Bhardwaj N.; O'Brien M.
2009 ;3(5):527-537, HIV Therapy
Dendritic cells (DCs) are antigen-presenting cells that serve as critical links between innate and adaptive immunity. Recent advances have revealed that DC interactions with HIV are pleiotropic. As direct antimicrobial effector cells, DCs may play a role in whether HIV infection is established upon mucosal exposure. As potent antigen-presenting cells, DCs probably modulate control of chronic HIV disease through the priming of adaptive immune responses. By contrast, DCs may contribute to HIV pathogenesis through the enhancement of T-cell infection, production of inflammatory cytokines that lead to chronic immune activation (and a proapoptotic state) and the formation of suppressive T-regulatory cells. In this article, recent progress in the field of HIV-DC interactions, including mucosal infection and HIV transmission to T cells, DC infection and activation, DC number and function in acute and chronic HIV infection, and DC immunoregulatory effects are discussed. copyright 2009 Future Medicine Ltd
— id: 108915, year: 2009, vol: 3, page: 527, stat: Journal Article,

Phosphorylated 4E-BP1 is associated with poor survival in melanoma
O'Reilly, Kathryn E; Warycha, Melanie; Davies, Michael A; Rodrik, Vanessa; Zhou, Xi K; Yee, Herman; Polsky, David; Pavlick, Anna C; Rosen, Neal; Bhardwaj, Nina; Mills, Gordon; Osman, Iman
2009 Apr 15;15(8):2872-2878, Clinical cancer research
PURPOSE: Both phosphatidylinositol 3-kinase/AKT and RAS/mitogen-activated protein kinase signal transduction pathways mediate 4E-BP1 phosphorylation, releasing 4E-BP1 from the mRNA cap and permitting translation initiation. Given the prevalence of PTEN and BRAF mutations in melanoma, we first examined translation initiation, as measured by phosphorylated 4E-BP1 (p-4E-BP1), in metastatic melanoma tissues and cell lines. We then tested the association between amounts of total and p-4E-BP1 and patient survival. EXPERIMENTAL DESIGN: Seven human metastatic melanoma cells lines and 72 metastatic melanoma patients with accessible metastatic tumor tissues and extended follow-up information were studied. Expression of 4E-BP1 transcript, total 4E-BP1 protein, and p-4E-BP1 was examined. The relationship between 4E-BP1 transcript and protein expression was assessed in a subset of patient tumors (n = 41). The association between total and p-4E-BP1 levels and survival was examined in the larger cohort of patients (n = 72). RESULTS: 4E-BP1 was hyperphosphorylated in 4 of 7 melanoma cell lines harboring both BRAF and PTEN mutations compared with untransformed melanocytes or RAS/RAF/PTEN wild-type melanoma cells. 4E-BP1 transcript correlated with 4E-BP1 total protein levels as measured by the semiquantitative reverse-phase protein array (P = 0.012). High levels of p-4E-BP1 were associated with worse overall and post-recurrence survival (P = 0.02 and 0.0003, respectively). CONCLUSION: Our data show that translation initiation is a common event in human metastatic melanoma and correlates with worse prognosis. Therefore, effective inhibition of the pathways responsible for 4E-BP1 phosphorylation should be considered to improve the treatment outcome of metastatic melanoma patients
— id: 99295, year: 2009, vol: 15, page: 2872, stat: Journal Article,

Aberrant miR-182 expression promotes melanoma metastasis by repressing FOXO3 and microphthalmia-associated transcription factor
Segura, Miguel F; Hanniford, Douglas; Menendez, Silvia; Reavie, Linsey; Zou, Xuanyi; Alvarez-Diaz, Silvia; Zakrzewski, Jan; Blochin, Elen; Rose, Amy; Bogunovic, Dusan; Polsky, David; Wei, Jianjun; Lee, Peng; Belitskaya-Levy, Ilana; Bhardwaj, Nina; Osman, Iman; Hernando, Eva
2009 Feb 10;106(6):1814-1819, Proceedings of the National Academy of Sciences of the United States of America
The highly aggressive character of melanoma makes it an excellent model for probing the mechanisms underlying metastasis, which remains one of the most difficult challenges in treating cancer. We find that miR-182, member of a miRNA cluster in a chromosomal locus (7q31-34) frequently amplified in melanoma, is commonly up-regulated in human melanoma cell lines and tissue samples; this up-regulation correlates with gene copy number in a subset of melanoma cell lines. Moreover, miR-182 ectopic expression stimulates migration of melanoma cells in vitro and their metastatic potential in vivo, whereas miR-182 down-regulation impedes invasion and triggers apoptosis. We further show that miR-182 over-expression promotes migration and survival by directly repressing microphthalmia-associated transcription factor-M and FOXO3, whereas enhanced expression of either microphthalmia-associated transcription factor-M or FOXO3 blocks miR-182's proinvasive effects. In human tissues, expression of miR-182 increases with progression from primary to metastatic melanoma and inversely correlates with FOXO3 and microphthalmia-associated transcription factor levels. Our data provide a mechanism for invasion and survival in melanoma that could prove applicable to metastasis of other cancers and suggest that miRNA silencing may be a worthwhile therapeutic strategy
— id: 92154, year: 2009, vol: 106, page: 1814, stat: Journal Article,

Developing a multidisciplinary prospective melanoma biospecimen repository to advance translational research
Wich, Lindsay G; Hamilton, Heather K; Shapiro, Richard L; Pavlick, Anna; Berman, Russell S; Polsky, David; Goldberg, Judith D; Hernando, Eva; Manga, Prashiela; Krogsgaard, Michelle; Kamino, Hideko; Darvishian, Farbod; Lee, Peng; Orlow, Seth J; Ostrer, Harry; Bhardwaj, Nina; Osman, Iman
2009 ;1(1):35-43, American Journal of Translational Research
Several challenges face the development and operation of a biospecimen bank linked to clinical information, a critical component of any effective translational research program. Melanoma adds particular complexity and difficulty to such an endeavor considering the unique characteristics of this malignancy. We describe here a review of biospecimen bank and our experience in establishing a multi-disciplinary, prospective, integrated clinicopathological-biospecimen database in melanoma. The Interdisciplinary Melanoma Cooperative Group (IMCG), a prospective clinicopathological and biospecimen database, was established at the New York University (NYU) Langone Medical Center. With patients' informed consent, biospecimens from within and outside NYU, clinicopathological data, and follow-up information are collected using developed protocols. Information pertaining to biospecimens is recorded in 35 fields, and clinicopathological information is recorded in 371 fields within 5 modules in a virtual network system. Investigators conducting research utilizing the IMCG biospecimen resource are blind to clinicopathological information, and molecular data generated using biospecimens are linked independently with clinicopathological data by biostatistics investigators. This translational research enterprise acts as a valuable resource to efficiently translate laboratory discoveries to the clinic
— id: 105566, year: 2009, vol: 1, page: 35, stat: Journal Article,

Lack of Functionally Active Melan-A(26-35)-Specific T Cells in the Blood of HLA-A2(+) Vitiligo Patients
Adams, Sylvia; Lowes, Michelle A; O'Neill, David W; Schachterle, Stephen; Romero, Pedro; Bhardwaj, Nina
2008 Aug;128(8):1977-1980, Journal of investigative dermatology
Vitiligo, a skin disorder characterized by the spontaneous destruction of melanocytes, is believed to be of autoimmune origin. We investigated the presence and functionality of CD8(+) T-cells specific for the melanocyte-associated antigens Melan-A, gp100, tyrosinase, and TRP-2 in the blood of HLA-A2(+) vitiligo patients. We enumerated antigen-specific CD8(+) T cells by major histocompatibility complex multimer staining directly ex vivo, as well as after 9 days of in vitro stimulation and assessed IFN-gamma secretion by enzyme-linked immunospot (Elispot) assay. Tyrosinase-, gp100-, or TRP-2-specific CD8(+) T cells could not be identified in the peripheral blood of individuals with vitiligo. Although Melan-A-specific T cells were detectable at levels comparable to Flu-MP-specific T cells by multimer staining, these lymphocytes did not express the skin-homing receptor cutaneous lymphocyte antigen, were phenotypically naive (CD45RA(+)), and were unresponsive in the IFN-gamma Elispot assay, suggesting that they are unlikely to be involved in the etiopathogenesis of vitiligo.Journal of Investigative Dermatology advance online publication, 13 March 2008; doi:10.1038/jid.2008.31
— id: 76847, year: 2008, vol: 128, page: 1977, stat: Journal Article,

Immunization of Malignant Melanoma Patients with Full-Length NY-ESO-1 Protein Using TLR7 Agonist Imiquimod as Vaccine Adjuvant
Adams, Sylvia; O'Neill, David W; Nonaka, Daisuke; Hardin, Elizabeth; Chiriboga, Luis; Siu, Kimberly; Cruz, Crystal M; Angiulli, Angelica; Angiulli, Francesca; Ritter, Erika; Holman, Rose Marie; Shapiro, Richard L; Berman, Russell S; Berner, Natalie; Shao, Yongzhao; Manches, Olivier; Pan, Linda; Venhaus, Ralph R; Hoffman, Eric W; Jungbluth, Achim; Gnjatic, Sacha; Old, Lloyd; Pavlick, Anna C; Bhardwaj, Nina
2008 Jul 1;181(1):776-784, Journal of immunology
T cell-mediated immunity to microbes and to cancer can be enhanced by the activation of dendritic cells (DCs) via TLRs. In this study, we evaluated the safety and feasibility of topical imiquimod, a TLR7 agonist, in a series of vaccinations against the cancer/testis Ag NY-ESO-1 in patients with malignant melanoma. Recombinant, full-length NY-ESO-1 protein was administered intradermally into imiquimod preconditioned sites followed by additional topical applications of imiquimod. The regimen was very well tolerated with only mild and transient local reactions and constitutional symptoms. Secondarily, we examined the systemic immune response induced by the imiquimod/NY-ESO-1 combination, and show that it elicited both humoral and cellular responses in a significant fraction of patients. Skin biopsies were assessed for imiquimod's in situ immunomodulatory effects. Compared with untreated skin, topical imiquimod induced dermal mononuclear cell infiltrates in all patients composed primarily of T cells, monocytes, macrophages, myeloid DCs, NK cells, and, to a lesser extent, plasmacytoid DCs. DC activation was evident. This study demonstrates the feasibility and excellent safety profile of a topically applied TLR7 agonist used as a vaccine adjuvant in cancer patients. Imiquimod's adjuvant effects require further evaluation and likely need optimization of parameters such as formulation, dose, and timing relative to Ag exposure for maximal immunogenicity
— id: 79260, year: 2008, vol: 181, page: 776, stat: Journal Article,

Gene expression profile for metastatic melanoma and patient survival
Bogunovic D; O'Neill D; Adams S; Wang J; Darvishian F; Pavlick AC; Shapiro RL; Zavadil J; Osman I; Bhardwaj N
2008 May 20;26(Suppl):?-? #9049, Proceedings (American Society of Clinical Oncology)
Background: Although remission rates for metastatic melanoma are generally very poor, some patients can survive for prolonged periods following metastasis. Here we use gene expression profiling of metastatic melanoma samples to search for a molecular basis for this observation. Methods: We analyzed gene expression profiles of 44 metastatic melanoma specimens collected from 38 patients who were followed clinically for a median of 20 months after surgery. RNA was processed using standard methods and hybridized to Affymetrix Human Genome HU133 Plus 2.0 arrays. Data were analyzed using GeneSpring and PathwayAssist software, normalizing using a PLIER algorithm and utilizing ANOVA statistical tests. Results: Unsupervised clustering yielded 4 distinct groups of subjects, 2 of which had large differences in overall survival. We then used supervised clustering to compare patients whose post-surgery survival was less then 1.5 years (11 subjects, 9.5 month median survival) to those who survived greater than 1.5 years (23 subjects, 26 month median survival, 17 alive at last follow-up). Only genes with changes in expression of > 2 with a p value of < 0.05 were accepted, resulting in a group of approximately 200 genes. Genes associated with immune response (TNFa, IRF1, Granzyme B, CD8a, CXCL11, IL27AR, GBP1, CXCL10, CCBP2, GBP2, CCR9 and IFIT4) or with cell proliferation (FGFR1, MET, MDM2, CDC25A, RFC2, SOS1, HOXA3, MCM4, MCM7, ORC5L, KIF23 and VAV3) were highly represented. Prolonged survival was associated with elevated expression of immune response genes and decreased expression of genes associated with cell proliferation. Conclusions: Gene expression profiling of metastatic melanoma samples identified a set of genes associated with patient survival, and suggests that immune surveillance is a mechanism for prolonged survival in these patients
— id: 106301, year: 2008, vol: 26, page: ?, stat: Journal Article,

Innate immune responses in primary HIV-1 infection
Borrow, Persephone; Bhardwaj, Nina
2008 Jan;3(1):36-44, Current Opinion in HIV & AIDS
PURPOSE OF REVIEW: Events occurring in acute HIV-1 infection are now recognized to be critical determinants of the subsequent disease course. Innate responses constitute the first line of defence against pathogens, and also play a key role in triggering the early adaptive response; as such, the innate responses activated in acute HIV-1 infection and their contribution to control of viral replication or disease pathogenesis are the focus of much current research. We review recent advances in this area. RECENT FINDINGS: Dendritic cell subsets can play pleiotropic roles in acute HIV-1 infection, with in-vitro studies illustrating that HIV-dendritic cell interactions may have outcomes as diverse as virion destruction, virus dissemination, T-cell triggering or subversion of dendritic cell functions. Natural killer cells can be activated in acute HIV-1 infection, and mounting evidence suggests that they contribute to determining the ensuing course of disease; however, much remains to be learned about how they mediate their effects. SUMMARY: The importance of innate responses as determinants of the outcome of HIV infection is increasingly evident, but more work is needed to understand how innate immunity can be harnessed to combat this infection
— id: 133576, year: 2008, vol: 3, page: 36, stat: Journal Article,

Microparticles and Apoptotic Cells Generated from Apoptotic T Cells Inhibit Human Dendritic Cell Cytokine Production
Frleta, D; Skoberne, M; Cutler, TS; Smith, NG; Borrow, P; Haynes, B; Bhardwaj, N
2008 OCT ;24(1):82-82, AIDS research & human retroviruses
— id: 91418, year: 2008, vol: 24, page: 82, stat: Journal Article,

Unique Spectrum of Pro- and Anti-Inflammatory Molecules Induced on DCs by Simultaneous Stimulation with TLR8 plus TLR3 or TLR4 Ligands
Kavanagh, DG; Bogunovic, D; Meier, A; Kaufmann, DE; Ryan, KP; Altfeld, M; Bhardwaj, N; Gandhi, RT
2008 OCT ;24(1):38-39, AIDS research & human retroviruses
— id: 91409, year: 2008, vol: 24, page: 38, stat: Journal Article,

HIV-activated human plasmacytoid DCs induce Tregs through an indoleamine 2,3-dioxygenase-dependent mechanism
Manches, Olivier; Munn, David; Fallahi, Anahita; Lifson, Jeffrey; Chaperot, Laurence; Plumas, Joel; Bhardwaj, Nina
2008 Oct;118(10):3431-3439, Journal of clinical investigation
Plasmacytoid DCs (pDCs) have been implicated as crucial cells in antiviral immune responses. On recognizing HIV, they become activated, secreting large amounts of IFN-alpha and inflammatory cytokines, thereby potentiating innate and adaptive antiviral immune responses. Here, we have shown that HIV-stimulated human pDCs can also induce the differentiation of naive CD4+ T cells into Tregs with suppressive function. This differentiation was independent of pDC production of IFN-alpha and primarily dependent on pDC expression of indoleamine 2,3-dioxygenase, which was induced through the TLR/MyD88 pathway, following binding of HIV to CD4 and triggering of TLR7 by HIV genomic RNA. Functionally, the Tregs induced by pDCs were shown to inhibit the maturation of bystander conventional DCs. This study therefore reveals what we believe to be a novel mechanism by which pDC may regulate and potentially limit anti-HIV immune responses
— id: 95137, year: 2008, vol: 118, page: 3431, stat: Journal Article,

The anergic state in sarcoidosis is associated with diminished dendritic cell function
Mathew, Sneha; Bauer, Kristy L; Fischoeder, Arne; Bhardwaj, Nina; Oliver, Stephen J
2008 Jul 1;181(1):746-755, Journal of immunology
Sarcoidosis is a chronic inflammatory disease of unknown cause, characterized by granuloma formation similar to tuberculosis, but without clear evidence of a microbial infection. Because sarcoidosis is linked with clinical anergy and other evidence of diminished cellular immunity, we hypothesized that decreased skin delayed-type hypersensitivity (DTH) responses to recall Ags in affected individuals would be associated with decreased function of their blood dendritic cells (DCs). Our study involved ex vivo isolation, phenotyping, and functional testing of myeloid DCs (mDCs), plasmacytoid DCs, and T lymphocytes from blood of normal healthy volunteers and sarcoidosis subjects with active, untreated pulmonary disease. We found mDC function in the allogeneic MLR directly corresponded to the magnitude of skin DTH reactions to recall Ags in both sarcoidosis subjects and normal volunteers. However, both of these outcomes were significantly decreased in the sarcoidosis group. Diminished mDC function occurred despite up-regulated costimulatory and maturation markers. Clinical relevance is suggested by the inverse relationship between both mDC allogeneic responses and skin DTH responses with clinical disease severity as measured by chest radiograms. Because granulomas form when cellular immunity fails to clear antigenic stimuli, attenuated mDC function in sarcoidosis may contribute to susceptibility and persistence of the chronic inflammation characteristic of this disease
— id: 80312, year: 2008, vol: 181, page: 746, stat: Journal Article,

Transfection of dendritic cells (DCs) with mRNA encoding IL-12p70 enhances anti-tumor immunity in melanoma patients
Minkis K; Kavanagh D; O'Neill D; Alter G; Sunderji S; Adams S; Walker B; Pavlick AC; Gandhi R; Bhardwaj N
2008 May 20;26(Suppl):?-? #22217, Proceedings (American Society of Clinical Oncology)
Background: DCs are widely used in experimental immune-based therapies for melanoma. Prior to injection, DCs are matured with a cocktail of recombinant cytokines and PGE-2, which preserves the ability of DCs to migrate to draining lymph nodes. However, PGE-2 also suppresses the ability of DCs to express IL-12p70. To overcome this paradox, we transfected mature DCs with synthetic mRNA encoding IL-12p70 and evaluated both their innate and adaptive immune functions in vitro. Methods: DCs from healthy donors and melanoma patients were cultured in vitro and transfected with IL-12p70 mRNA. The ability of these DCs to secrete bioactive IL-12p70 was tested by ELISA. Bioactivity of the secreted IL-12 was tested using NK cell assays and allogeneic MLRs. T cell assays were also performed to assess TH1 skewing towards melanoma antigens: T cells from healthy donors and melanoma patients were isolated and stimulated with autologous DCs previously transfected with MART-1 and IL-12p70 mRNA. The quantity and quality of the primed MART-1 specific T cells was determined. For comparison, melanoma tumor-infiltrating T cells (TILs) isolated from a resected tumor were used to examine TH phenotype. Results: IL-12p70-transfected DCs produced bioactive IL-12p70, and enhanced the response of NK cells to MHC class I-negative tumor targets. IL-12p70 transfected DC skewed TH responses away from TH2 in allogeneic MLRs. Cotransfection of DCs derived from the blood of healthy donors with IL-12p70 and MART-1 enhanced in vitro priming of melanoma- specific CD8 T cells, enhanced their capacity to secrete IFN? and inhibited the production of the TH2 cytokines IL-4 and IL-5, compared to MART-1 alone. In contrast, TILs from resected tumor exhibited a strong TH2 phenotype. T cell lines primed in vitro from the blood of melanoma patients by autologous DCs also showed strong TH2 skewing that was reversed by IL-12p70 transfection. Conclusions: Clinical DC preparations may be improved by IL-12p70 transfection, which enhances both innate and antigen-specific anti-tumor immune responses in vitro. This approach can be applied in vivo, thereby contributing to effective immune-based therapy against melanoma
— id: 79466, year: 2008, vol: 26, page: ?, stat: Journal Article,

Type 2 Bias of T cells expanded from the blood of melanoma patients switched to type 1 by IL-12p70 mRNA-transfected dendritic cells
Minkis, Kira; Kavanagh, Daniel G; Alter, Galit; Bogunovic, Dusan; O'Neill, David; Adams, Sylvia; Pavlick, Anna; Walker, Bruce D; Brockman, Mark A; Gandhi, Rajesh T; Bhardwaj, Nina
2008 Nov 15;68(22):9441-9450, Cancer research
Melanoma patients may exhibit a T(H)2-skewed cytokine profile within blood and tumor-infiltrating lymphocytes. Therapies that induce beneficial T(H)1-type tumor-specific immune responses, therefore, are highly desirable. Dendritic cells (DC) are widely used as immune adjuvants for cancer. Before their administration, DC are generally induced to mature with a cocktail of recombinant cytokines [interleukin (IL)-1beta, tumor necrosis factor alpha, and IL-6] and prostaglandin E(2) (PGE(2)), which is added to preserve the ability of DC to migrate to draining lymph nodes. However, PGE(2) suppresses the production of IL-12p70, a cytokine essential for differentiation of T(H)1 responses. In this study, human DC were transfected with IL-12p70 mRNA and tested for their ability to alter the T(H)2 type bias manifested by blood T cells of patients with melanoma. Transfected DC secreted high levels of bioactive IL-12p70, as indicated by their capacity to enhance natural killer cell activity, skew T(H)1 responses in allogeneic mixed lymphocyte reactions through reduction of IL-4 and IL-5, and prime CD8(+) T cells to the melanoma-associated antigen Melan A/MART-1. Furthermore, T-cell lines primed in vitro from the blood of melanoma patients showed strong type 2 skewing that was dramatically reversed by IL-12p70 transfection of autologous DC. Thus, IL-12p70 transfection of clinical DC preparations may enhance type 1 antitumor responses and may thereby contribute to effective immune-based therapy
— id: 90929, year: 2008, vol: 68, page: 9441, stat: Journal Article,

Quantitative effect of suboptimal codon usage on translational efficiency of mRNA encoding HIV-1 gag in intact T cells
Ngumbela, Kholiswa C; Ryan, Kieran P; Sivamurthy, Rohini; Brockman, Mark A; Gandhi, Rajesh T; Bhardwaj, Nina; Kavanagh, Daniel G
2008 ;3(6):e2356-e2356, PLoS ONE
BACKGROUND: The sequences of wild-isolate strains of Human Immunodeficiency Virus-1 (HIV-1) are characterized by low GC content and suboptimal codon usage. Codon optimization of DNA vectors can enhance protein expression both by enhancing translational efficiency, and by altering RNA stability and export. Although gag codon optimization is widely used in DNA vectors and experimental vaccines, the actual effect of altered codon usage on gag translational efficiency has not been quantified. METHODOLOGY AND PRINCIPAL FINDINGS: To quantify translational efficiency of gag mRNA in live T cells, we transfected Jurkat cells with increasing doses of capped, polyadenylated synthetic mRNA corresponding to wildtype or codon-optimized gag sequences, measured Gag production by quantitative ELISA and flow cytometry, and estimated the translational efficiency of each transcript as pg of Gag antigen produced per microg of input mRNA. We found that codon optimization yielded a small increase in gag translational efficiency (approximately 1.6 fold). In contrast when cells were transfected with DNA vectors requiring nuclear transcription and processing of gag mRNA, codon optimization resulted in a very large enhancement of Gag production. CONCLUSIONS: We conclude that suboptimal codon usage by HIV-1 results in only a slight loss of gag translational efficiency per se, with the vast majority of enhancement in protein expression from DNA vectors due to altered processing and export of nuclear RNA
— id: 95138, year: 2008, vol: 3, page: e2356, stat: Journal Article,

Blood Dendritic Cell Subsets in Early HIV Infection
Sabado, RL; O'Brien, M; Subedi, A; Taylor, E; Hutt, R; Haggerty, R; Marmor, M; Margolis, D; Valentine, F; Borrow, P; Bhardwaj, N
2008 OCT ;24(1):84-85, AIDS research & human retroviruses
— id: 91419, year: 2008, vol: 24, page: 84, stat: Journal Article,

KBMA Listeria monocytogenes is an effective vector for DC-mediated induction of antitumor immunity
Skoberne, Mojca; Yewdall, Alice; Bahjat, Keith S; Godefroy, Emmanuelle; Lauer, Peter; Lemmens, Edward; Liu, Weiqun; Luckett, Will; Leong, Meredith; Dubensky, Thomas W; Brockstedt, Dirk G; Bhardwaj, Nina
2008 Dec;118(12):3990-4001, Journal of clinical investigation
Vaccine strategies that utilize human DCs to enhance antitumor immunity have yet to realize their full potential. Approaches that optimally target a spectrum of antigens to DCs are urgently needed. Here we report the development of a platform for loading DCs with antigen. It is based on killed but metabolically active (KBMA) recombinant Listeria monocytogenes and facilitates both antigen delivery and maturation of human DCs. Highly attenuated KBMA L. monocytogenes were engineered to express an epitope of the melanoma-associated antigen MelanA/Mart-1 that is recognized by human CD8+ T cells when presented by the MHC class I molecule HLA-A*0201. The engineered KBMA L. monocytogenes induced human DC upregulation of costimulatory molecules and secretion of pro-Th1 cytokines and type I interferons, leading to effective priming of Mart-1-specific human CD8+ T cells and lysis of patient-derived melanoma cells. KBMA L. monocytogenes expressing full-length NY-ESO-1 protein, another melanoma-associated antigen, delivered the antigen for presentation by MHC class I and class II molecules independent of the MHC haplotype of the DC donor. A mouse therapeutic tumor model was used to show that KBMA L. monocytogenes efficiently targeted APCs in vivo to induce protective antitumor responses. Together, our data demonstrate that KBMA L. monocytogenes may be a powerful platform that can both deliver recombinant antigen to DCs for presentation and provide a potent DC-maturation stimulus, making it a potential cancer vaccine candidate
— id: 91460, year: 2008, vol: 118, page: 3990, stat: Journal Article,

Imiquimod -- a TLR 7 agonist as vaccine adjuvant
Adams S; O'Neill D; Pavlick A; Hardin E; Nonaka D; Chiriboga L; Siu K; Shapiro R; Berman R; Strober B; Cruz C; Angiulli A; Manchez O; Berner N; Mukhi V; Shao Y; Bhardwaj N
2007 ;:- #8545, Proceedings (American Society of Clinical Oncology)
— id: 73377, year: 2007, vol: , page: , stat: Journal Article,

Harnessing the immune system to treat cancer
Bhardwaj, Nina
2007 May;117(5):1130-1136, Journal of clinical investigation
A major challenge for the immune system is to recognize and eliminate cells undergoing carcinogenesis. Immune defense against tumors is complex. It can be mediated early by the innate immune system (i.e., phagocytes, NK cells, NKT cells, cytokines, and complement proteins) and later by the adaptive immune system (i.e., B cells and T cells). The eight articles in this Review series on tumor immunology discuss the mechanisms underlying immune surveillance of tumors, the regulation of carcinogenesis by immune inflammatory mediators, current approaches to controlling tumor growth through immunotherapy, and novel targets of immunotherapy.
— id: 72872, year: 2007, vol: 117, page: 1130, stat: Journal Article,

Biology of plasmacytoid dendritic cells and natural killer cells in HIV-1 infection
Lakshmanan, Viswanathan; Alter, Galit; Altfeld, Marcus; Bhardwaj, Nina
2007 May;2(3):189-200, Current Opinion in HIV & AIDS
PURPOSE: This review summarizes recent literature on the biology of dendritic cells and natural killer cells in HIV-1 infection and the importance of crosstalk between them in the development of strong antiviral immunity. RECENT FINDINGS: Type I interferons produced by dendritic cells in response to HIV-1 have been suggested to act as a double-edged sword, stemming HIV-1 replication on the one hand and causing T-cell loss on the other. Recent epidemiologic evidence demonstrates a strong association between the natural killer cell receptor KIR3DS1 (along with its presumed ligand HLA-B Bw4-80I) in the control of HIV-1 replication. SUMMARY: Dendritic and natural killer cells play a central role in the innate immune response to viral infections through both the direct elimination of infected cells and modulation of each other's function
— id: 133524, year: 2007, vol: 2, page: 189, stat: Journal Article,

Dendritic cells as a therapeutic vaccine against Progressive Multifocal Leukoencephalopathy
Marzocchetti, A; Lima, M; Tompkins, T; Kavanagh, DG; Ghandi, RT; Bhardwaj, N; Koralnik, IJ
2007 JAN ;13(1):101-102, Journal of neurovirology
— id: 75947, year: 2007, vol: 13, page: 101, stat: Journal Article,

Armed and ready: how effector T cells deploy in reactive lymph nodes to modulate immunity
O'Neill, David W; Bhardwaj, Nina
2007 Jul;8(7):679-681, Nature immunology
— id: 78744, year: 2007, vol: 8, page: 679, stat: Journal Article,

Exploiting dendritic cells for active immunotherapy of cancer and chronic infections
O'Neill, David W; Bhardwaj, Nina
2007 Jun;36(2):131-141, Molecular biotechnology
Dendritic cells (DCs) are important antigen-presenting cells (APCs) that can prime naive T cells and control adaptive immune responses with respect to magnitude, memory and self-tolerance. Understanding the biology of these cells is central to the development of new generation immunotherapies for cancer and chronic infections. This review presents a brief overview of DC biology and of the preparation and use of DC-based vaccines
— id: 74685, year: 2007, vol: 36, page: 131, stat: Journal Article,

Pathways utilized by dendritic cells for binding, uptake, processing and presentation of antigens derived from HIV-1
Sabado, Rachel L; Babcock, Ethan; Kavanagh, Daniel G; Tjomsland, Veronica; Walker, Bruce D; Lifson, Jeffrey D; Bhardwaj, Nina; Larsson, Marie
2007 Jul;37(7):1752-1763, European journal of immunology
The outcome following HIV infection depends on the nature and durability of the HIV-specific T cell response induced initially. The activation of protective T cell responses depends upon dendritic cells (DC), antigen-presenting cells which have the capacity to process and present viral antigens. DC pulsed with aldrithiol-2-inactivated HIV and delivered in vivo were reported to induce immune responses and promote virologic control in chronically HIV-1-infected subjects. To gain an understanding of this phenomenon, we characterized the steps involved in the presentation of antigens derived from aldrithiol-2-treated vs. infectious HIV-1 by DC. Antigen presentation, on both MHC class I and II, was independent of DC-specific ICAM-3-grabbing integrin, DEC-205 and macrophage mannose receptor, C-type lectins expressed by the DC. Inhibitor studies showed that presentation on MHC class I was dependent on viral fusion in a CD4/coreceptor-dependent manner, both at the cell surface and within endosomes, and access to the classical endosomal processing pathway. MHC class II presentation of HIV-associated antigens was dependent on active endocytosis, probably receptor-mediated, and subsequent degradation of virions in acidified endosomes in the DC. Our study brings forth new facts regarding the binding, uptake, and processing of chemically inactivated virions leading to efficient antigen presentation and should aid in the design of more effective HIV vaccines
— id: 78745, year: 2007, vol: 37, page: 1752, stat: Journal Article,

Vaccination with NY-ESO-1 protein and CpG in Montanide induces integrated antibody/Th1 responses and CD8 T cells through cross-priming
Valmori, Danila; Souleimanian, Naira E; Tosello, Valeria; Bhardwaj, Nina; Adams, Sylvia; O'Neill, David; Pavlick, Anna; Escalon, Juliet B; Cruz, Crystal M; Angiulli, Angelica; Angiulli, Francesca; Mears, Gregory; Vogel, Susan M; Pan, Linda; Jungbluth, Achim A; Hoffmann, Eric W; Venhaus, Ralph; Ritter, Gerd; Old, Lloyd J; Ayyoub, Maha
2007 May 22;104(21):8947-8952, Proceedings of the National Academy of Sciences of the United States of America
The use of recombinant tumor antigen proteins is a realistic approach for the development of generic cancer vaccines, but the potential of this type of vaccines to induce specific CD8(+) T cell responses, through in vivo cross-priming, has remained unclear. In this article, we report that repeated vaccination of cancer patients with recombinant NY-ESO-1 protein, Montanide ISA-51, and CpG ODN 7909, a potent stimulator of B cells and T helper type 1 (Th1)-type immunity, resulted in the early induction of specific integrated CD4(+) Th cells and antibody responses in most vaccinated patients, followed by the development of later CD8(+) T cell responses in a fraction of them. The correlation between antibody and T cell responses, together with the ability of vaccine-induced antibodies to promote in vitro cross-presentation of NY-ESO-1 by dendritic cells to vaccine-induced CD8(+) T cells, indicated that elicitation of NY-ESO-1-specific CD8(+) T cell responses by cross-priming in vivo was associated with the induction of adequate levels of specific antibodies. Together, our data provide clear evidence of in vivo cross-priming of specific cytotoxic T lymphocytes by a recombinant tumor antigen vaccine, underline the importance of specific antibody induction for the cross-priming to occur, and support the use of this type of formulation for the further development of efficient cancer vaccines
— id: 72446, year: 2007, vol: 104, page: 8947, stat: Journal Article,

Expression of the cancer/testis antigen NY-ESO-1 in primary and metastatic malignant melanoma (MM)--correlation with prognostic factors
Velazquez, Elsa F; Jungbluth, Achim A; Yancovitz, Molly; Gnjatic, Sacha; Adams, Sylvia; O'Neill, David; Zavilevich, Kira; Albukh, Tatyana; Christos, Paul; Mazumdar, Madhu; Pavlick, Anna; Polsky, David; Shapiro, Richard; Berman, Russell; Spira, Joanna; Busam, Klaus; Osman, Iman; Bhardwaj, Nina
2007 ;7:11-11, Cancer imunity
Cancer/testis (CT) antigens are potential targets for cancer immunotherapy, with NY-ESO-1 being among the most immunogenic. In several clinical trials in malignant melanoma (MM) patients, NY-ESO-1 protein/peptides showed clear evidence of inducing specific immunity. However, little is known about NY-ESO-1 expression in primary and metastatic MM and its relationship to disease progression. We analyzed NY-ESO-1 expression immunohistochemically in a series of primary and metastatic MMs and its relation to prognostic parameters and survival. We studied 61 primary and 63 metastatic MM specimens (from 61 and 56 patients, respectively). The prevalence of NY-ESO-1 expression was significantly higher in metastatic versus primary tumors [18/56 (32%) versus 8/61 (13%), P = 0.015]. There was a significant association between initial stage at presentation and NY-ESO-1 expression [stage I (3.45%), stage II (9.52%) and stage III (45.45%), P = 0.0014]. Primary MMs expressing NY-ESO-1 were significantly thicker than NY-ESO-1 negative cases (median thickness 4.7 mm versus 1.53 mm respectively, P = 0.03). No significant difference was seen in overall survival. In conclusion, NY-ESO-1 is more frequently expressed in metastatic than in primary MM and its expression is associated with thicker primary lesions and a higher frequency of metastatic disease, indicative of a worse prognosis. Our study suggests that patients with metastatic MM who express NY-ESO-1 may benefit from NY-ESO-1-based immunotherapy
— id: 73347, year: 2007, vol: 7, page: 11, stat: Journal Article,

Clinical relevance of neutral endopeptidase (NEP/CD10) in melanoma
Velazquez, Elsa F; Yancovitz, Molly; Pavlick, Anna; Berman, Russell; Shapiro, Richard; Bogunovic, Dusan; O'Neill, David; Yu, Yi-Lo; Spira, Joanna; Christos, Paul J; Zhou, Xi Kathy; Mazumdar, Madhu; Nanus, David M; Liebes, Leonard; Bhardwaj, Nina; Polsky, David; Osman, Iman
2007 ;5:2-2, Journal of translational medicine
BACKGROUND: Overexpression of Neutral Endopeptidase (NEP) has been reported in metastatic carcinomas, implicating NEP in tumor progression and suggesting a role for NEP inhibitors in its treatment. We investigated the role of NEP expression in the clinical progression of cutaneous melanoma. METHODS: We screened 7 melanoma cell lines for NEP protein expression. NEP-specific siRNA was transfected into the lines to examine the role of gene transcription in NEP expression. Immunohistochemistry was done for 93 specimens and correlated with clinicopathologic parameters. Thirty-seven metastatic melanoma specimens were examined for NEP transcript expression using Affymetrix GeneChips. In a subset of 25 specimens for which both transcript and protein expression was available, expression ratios were used to identify genes that co-express with NEP in GeneChip analysis. RESULTS: NEP was overexpressed in 4/7 human melanoma cell lines, and siRNA knock-down of NEP transcripts led to downregulation of its protein expression. NEP protein overexpression was significantly more common in metastatic versus primary tumors (P = 0.002). Twelve of 37 (32%) metastatic tumors had increased NEP transcript expression, and an association was observed between NEP transcript upregulation and protein overexpression (P < 0.0001). Thirty-eight genes were found to significantly co-express with NEP (p < 0.005). Thirty-three genes positively correlated with NEP, including genes involved in the MAP kinase pathway, antigen processing and presentation, apoptosis, and WNT signaling pathway, and 5 genes negatively correlated with NEP, including genes of focal adhesion and the notch signaling pathways. CONCLUSION: NEP overexpression, which seems to be largely driven by increased transcription, is rare in primary melanoma and occurs late in melanoma progression. Functional studies are needed to better understand the mechanisms of NEP regulation in melanoma
— id: 74679, year: 2007, vol: 5, page: 2, stat: Journal Article,

Differential expression of type I MAGE in new and relapsed multiple myelorna: Evidence for association with proliferation and progression of disease
Cho, HJ; Ely, S; Austin, WR; Niesvizkyl, R; Pearse, R; Coleman, M; Bhardwaj, N; DiLiberto, M; Chen-Kiang, S; Old, LJ; Jungbluth, AA
2006 NOV 16 ;108(11):970A-970A, Blood
— id: 71384, year: 2006, vol: 108, page: 970A, stat: Journal Article,

Expansion of HIV-specific CD4+ and CD8+ T cells by dendritic cells transfected with mRNA encoding cytoplasm- or lysosome-targeted Nef
Kavanagh, Daniel G; Kaufmann, Daniel E; Sunderji, Sherzana; Frahm, Nicole; Le Gall, Sylvie; Boczkowski, David; Rosenberg, Eric S; Stone, David R; Johnston, Mary N; Wagner, Bradford S; Zaman, Mohammad T; Brander, Christian; Gilboa, Eli; Walker, Bruce D; Bhardwaj, Nina
2006 Mar 1;107(5):1963-1969, Blood
Transfection with synthetic mRNA is a safe and efficient method of delivering antigens to dendritic cells for immunotherapy. Targeting antigens to the lysosome can sometimes enhance the CD4+ T-cell response. We transfected antigen-presenting cells (APCs) with mRNA encoding Gag-p24 and cytoplasmic, lysosomal, and secreted forms of Nef. Antigen-specific cytotoxic T cells were able to lyse the majority of transfected targets, indicating that transfection was efficient. Transfection of APCs with a Nef construct bearing lysosomal targeting signals produced rapid and prolonged antigen presentation to CD4+ and CD8+ T cells. Polyclonal CD4+ and CD8+ T-cell lines recognizing multiple distinct epitopes were expanded by coculture of transfected dendritic cells with peripheral blood mononuclear cells from viremic and aviremic HIV-infected subjects. Importantly, lysosome-targeted antigen drove a significantly greater expansion of Nef-specific CD4+ T cells than cytoplasmic antigen. The frequency of recognition of CD8 but not CD4 epitopes by mRNA-expanded T cells was inversely proportional to sequence entropy and was similar to ex vivo responses from a large chronic cohort. Thus human dendritic cells transfected with mRNA encoding lysosome-targeted HIV antigen can expand a broad, polyclonal repertoire of antiviral T cells, offering a promising approach to HIV immunotherapy
— id: 63119, year: 2006, vol: 107, page: 1963, stat: Journal Article,

Host immune responses against CT antigens in multiple myeloma patients
Lendvai, N; Gnjatic, S; Ritter, E; Chen, YT; Coughlin, C; Vonderheide, RH; Niesvizky, R; Bhardwaj, N; Chen-Kiang, S; Old, LJ; Cho, HJ
2006 NOV 16 ;108(11):996A-996A, Blood
— id: 71385, year: 2006, vol: 108, page: 996A, stat: Journal Article,

Altered dendritic cell function in sarcoidosis
Saladino, K; Mathew, S; Addrizzo-Harris, D; Bhardwaj, N; Oliver, SJ
2006 SEP ;54(9):S539-S539, Arthritis & rheumatism
— id: 70125, year: 2006, vol: 54, page: S539, stat: Journal Article,

The apoptotic-cell receptor CR3, but not alphavbeta5, is a regulator of human dendritic-cell immunostimulatory function
Skoberne, Mojca; Somersan, Selin; Almodovar, Wanda; Truong, Tuan; Petrova, Kseniya; Henson, Peter M; Bhardwaj, Nina
2006 Aug 1;108(3):947-955, Blood
Dendritic cells (DCs) that capture apoptotic cells (ACs) in the steady state mediate peripheral tolerance to self-antigens. ACs are recognized by an array of receptors on DCs, the redundancy of which is not completely defined. We made use of an AC surrogate system to address the individual roles of the alphavbeta5 and complement receptors (CRs) in the phagocytosis and induction of immunity. CR3 and CR4, while substantially less efficient than alphavbeta5 in internalizing ACs, initiate signals that render DCs tolerogenic. Responding T cells show impaired proliferation and IFNgamma production and subsequently die by apoptosis. While tolerogenic DCs are not induced via alphavbeta5, coligation of CR3 and alphavbeta5 maintains the DC's tolerogenic profile. This immunomodulatory role, however, is countered by a significant inflammatory stimulus such as bacterial infection. Overall, our data suggest that under steady-state conditions, signaling via CRs predominates to render DCs tolerogenic
— id: 68772, year: 2006, vol: 108, page: 947, stat: Journal Article,

Expression of cancer testis (CT) antigen NY-ESO-1 in primary and metastatic malignant melanoma (MM), correlation with prognostic factors and potential role in a melanoma vaccine
Velazquez, EF; Jungbluth, AA; Osman, I; Yancovitz, M; Adams, S; O'Neill, D; Zavilevich, K; Albukh, T; Pavlick, A; Polsky, D; Shapiro, R; Berman, R; Spira, J; Busam, K; Bhardwaj, N
2006 JAN ;19(4):89A-89A, Modern pathology
— id: 61434, year: 2006, vol: 19, page: 89A, stat: Journal Article,

Expression of cancer testis (CT) antigen NY-ESO-1 in primary and metastatic malignant melanoma (MM), correlation with prognostic factors and potential role in a melanoma vaccine
Velazquez, EF; Jungbluth, AA; Osman, I; Yancovitz, M; Adams, S; O'Neill, D; Zavilevich, K; Albukh, T; Pavlick, A; Polsky, D; Shapiro, R; Berman, R; Spira, J; Busam, K; Bhardwaj, N
2006 JAN ;86(4):89A-89A, Laboratory investigation
— id: 62615, year: 2006, vol: 86, page: 89A, stat: Journal Article,

Neutral endopeptidase (NEP) overexpression is associated with progression in malignant melanoma (MM) and is a potential target of treatment
Velazquez, EF; Yancovitz, M; Sorhaindo, L; Bogunovic, D; O'Neill, D; Shapiro, R; Pavlick, A; Berman, R; Bhardwaj, N; Spira, J; Christos, P; Nanus, D; Polsky, D; Osman, I
2006 JAN ;19(4):89A-89A, Modern pathology
— id: 61435, year: 2006, vol: 19, page: 89A, stat: Journal Article,

Neutral endopeptidase (NEP) overexpression is associated with progression in malignant melanoma (MM)and is a potential target of treatment
Velazquez, EF; Yancovitz, M; Sorhaindo, L; Bogunovic, D; O'Neill, D; Shapiro, R; Pavlick, A; Berman, R; Bhardwaj, N; Spira, J; Christos, P; Nanus, D; Polsky, D; Osman, I
2006 JAN ;86(4):89A-89A, Laboratory investigation
— id: 62616, year: 2006, vol: 86, page: 89A, stat: Journal Article,

Clinical relevance of neutral endopeptidase overexpression in melanoma
Yancovitz, M; Velazquez, E; Christos, P; Pavlick, A; Berman, R; Shapiro, R; Bhardwaj, N; Nanus, D; Polsky, D; Osman, I
2006 JUN 20 ;24(18):459S-459S, Journal of clinical oncology
— id: 69301, year: 2006, vol: 24, page: 459S, stat: Journal Article,

Spontaneous immune reponses to melanoma-associated antigens in melanoma, vitiligo and healthy controls
Adams S; Lowes M; Pavlick A; Schachterle S; O'Neill D; Bhardwaj N
2005 ;23:16S-16S abstact #9682, Proceedings (American Society of Clinical Oncology)
— id: 60240, year: 2005, vol: 23, page: 16S, stat: Journal Article,

Recent advances in dendritic cell biology
Adams, Sylvia; O'Neill, David W; Bhardwaj, Nina
2005 Mar;25(2):87-98, Journal of clinical immunology
Dendritic cells are professional antigen presenting cells that are central to the induction and regulation of immunity. This review discusses recent advances in the understanding of dendritic cell biology
— id: 55977, year: 2005, vol: 25, page: 87, stat: Journal Article,

Recent advances in dendritic cell biology
Adams, Sylvia; O'Neill, David W; Bhardwaj, Nina
2005 May;25(3):177-188, Journal of clinical immunology
Dendritic cells are professional antigen presenting cells that are central to the induction and regulation of immunity. This review discusses recent advances in the understanding of dendritic cell biology.
— id: 58180, year: 2005, vol: 25, page: 177, stat: Journal Article,

Endocytosis of HIV-1 activates plasmacytoid dendritic cells via Toll-like receptor-viral RNA interactions
Beignon, Anne-Sophie; McKenna, Kelli; Skoberne, Mojca; Manches, Olivier; DaSilva, Ida; Kavanagh, Daniel G; Larsson, Marie; Gorelick, Robert J; Lifson, Jeffrey D; Bhardwaj, Nina
2005 Nov;115(11):3265-3275, Journal of clinical investigation
HIV-1 directly activates human plasmacytoid DCs (pDCs) by upregulating the expression of costimulatory and MHC molecules and maturation markers, increasing T cell stimulatory activity, and inducing the production of type I interferons and TNF-alpha. A consequence of this activation is the bystander maturation of myeloid DCs and overall enhancement of antigen-presenting function. However, little is known about the mechanism(s) of pDC activation by HIV-1. Here we demonstrate by in vitro studies that IFN-alpha production by pDC in response to HIV-1 requires at least 2 interactions between the cell and virus. Initially, envelope-CD4 interactions mediate endocytosis of HIV-1, as demonstrated through the use of inhibitors of binding, fusion, endocytosis, and endosomal acidification. Subsequently, endosomally delivered viral nucleic acids, particularly RNA, stimulate pDCs through TLRs, as activation is reproduced with purified genomic RNA but not viral RNA packaging-deficient HIV-1 and blocked with different inhibitory TLR ligands. Finally, by using genetic complementation, we show that TLR7 is the likely primary target. Viral RNA rather than DNA in early retrotranscripts appears to be the active factor in HIV-1 that induces IFN-alpha secretion by pDCs. Since the decline in pDCs in chronic HIV-1 infection is associated with high viral loads and opportunistic infections, exploiting this natural adjuvant activity of HIV-1 RNA might be useful in the development of vaccines for the prevention of AIDS
— id: 62364, year: 2005, vol: 115, page: 3265, stat: Journal Article,

Dendritic cell chic
Bhardwaj N
2005 Jan;26(3):215-219, Springer seminars in immunopathology
— id: 55978, year: 2005, vol: 26, page: 215, stat: Journal Article,

CT7 (MAGE-C1)-specific cellular immune responses in the bone marrow microenvironment of multiple myeloma patients
Cho, HJ; Farol, L; Truong, JT; Austin, WR; Chen, YT; Bhardwaj, N; Niesvizky, R; Old, LJ; Chen-Kiang, S
2005 NOV 16 ;106(11):108A-108A, Blood
— id: 61460, year: 2005, vol: 106, page: 108A, stat: Journal Article,

Combining radiotherapy and immunotherapy: a revived partnership
Demaria, Sandra; Bhardwaj, Nina; McBride, William H; Formenti, Silvia C
2005 Nov 1;63(3):655-666, International journal of radiation oncology biology physics
Ionizing radiation therapy (RT) is an important local modality for the treatment of cancer. The current rationale for its use is based largely on the ability of RT to kill the cancer cells by a direct cytotoxic effect. Nevertheless, considerable evidence indicates that RT effects extend beyond the mere elimination of the more radiosensitive fraction of cancer cells present within a tumor at the time of radiation exposure. For instance, a large body of evidence is accumulating on the ability of RT to modify the tumor microenvironment and generate inflammation. This might have far-reaching consequences regarding the response of a patient to treatment, especially if radiation-induced tumor cell kill were to translate into the generation of effective antitumor immunity. Although much remains to be learned about how radiation can impact tumor immunogenicity, data from preclinical studies provide the proof of principle that different immunotherapeutic strategies can be combined with RT to enhance antitumor effects. Conversely, RT could be a useful tool to combine with immunotherapy. This article will briefly summarize what is known about the impact of RT on tumor immunity, including tumor-associated antigens, antigen-presenting cells, and effector mechanisms. In addition, the experimental evidence supporting the contention that RT can be used as a tool to induce antitumor immunity is discussed, and a new approach to radioimmunotherapy of cancer is proposed
— id: 62122, year: 2005, vol: 63, page: 655, stat: Journal Article,

The cancer-testis antigens CT7 (MAGE-C1) and MAGE-A3/6 are commonly expressed in multiple myeloma and correlate with plasma-cell proliferation
Jungbluth, Achim A; Ely, Scott; DiLiberto, Maurizio; Niesvizky, Ruben; Williamson, Barbara; Frosina, Denise; Chen, Yao-Tseng; Bhardwaj, Nina; Chen-Kiang, Selina; Old, Lloyd J; Cho, Hearn Jay
2005 Jul 1;106(1):167-174, Blood
Multiple myeloma is a malignancy of plasma cells. Vaccine immunotherapy is among the novel therapeutic strategies under investigation for this disease. To identify myeloma-associated antigens as potential targets for vaccine immunotherapy, we surveyed a comprehensive panel of bone marrow specimens from patients with monoclonal gammopathy of undetermined significance (MGUS) and multiple myeloma for expression of cancer-testis (CT) antigens. Immunohistochemistry (IHC) demonstrated that 82% of stage-III myeloma specimens expressed the CT antigen CT7 (also known as melanoma antigen C1 [MAGE-C1]) and 70% expressed MAGE-A3/6. Messenger RNA for CT7 and MAGE-A family members was detected in 87% and 100% of stage-III samples, respectively. CT7 protein expression increased with advanced stage of disease. Higher levels of CT7 and MAGE-A3/6 proteins also correlated with elevated plasma-cell proliferation. These results show that CT7 and MAGE-A3/6 are promising myeloma-associated antigens for application in vaccine immunotherapy. Furthermore, the common expression and correlation with proliferation suggest a possible pathogenic role for these proteins in myeloma
— id: 63121, year: 2005, vol: 106, page: 167, stat: Journal Article,

Transmission and accumulation of CTL escape variants drive negative associations between HIV polymorphisms and HLA
Leslie, Alasdair; Kavanagh, Daniel; Honeyborne, Isobella; Pfafferott, Katja; Edwards, Charles; Pillay, Tilly; Hilton, Louise; Thobakgale, Christina; Ramduth, Danni; Draenert, Rika; Le Gall, Sylvie; Luzzi, Graz; Edwards, Anne; Brander, Christian; Sewell, Andrew K; Moore, Sarah; Mullins, James; Moore, Corey; Mallal, Simon; Bhardwaj, Nina; Yusim, Karina; Phillips, Rodney; Klenerman, Paul; Korber, Bette; Kiepiela, Photini; Walker, Bruce; Goulder, Philip
2005 Mar 21;201(6):891-902, Journal of experimental medicine
Human immunodeficiency virus (HIV)-1 amino acid sequence polymorphisms associated with expression of specific human histocompatibility leukocyte antigen (HLA) class I alleles suggest sites of cytotoxic T lymphocyte (CTL)-mediated selection pressure and immune escape. The associations most frequently observed are between expression of an HLA class I molecule and variation from the consensus sequence. However, a substantial number of sites have been identified in which particular HLA class I allele expression is associated with preservation of the consensus sequence. The mechanism behind this is so far unexplained. The current studies, focusing on two examples of 'negatively associated' or apparently preserved epitopes, suggest an explanation for this phenomenon: negative associations can arise as a result of positive selection of an escape mutation, which is stable on transmission and therefore accumulates in the population to the point at which it defines the consensus sequence. Such negative associations may only be in evidence transiently, because the statistical power to detect them diminishes as the mutations accumulate. If an escape variant reaches fixation in the population, the epitope will be lost as a potential target to the immune system. These data help to explain how HIV is evolving at a population level. Understanding the direction of HIV evolution has important implications for vaccine development
— id: 63120, year: 2005, vol: 201, page: 891, stat: Journal Article,

Plasmacytoid dendritic cells: linking innate and adaptive immunity
McKenna, Kelli; Beignon, Anne-Sophie; Bhardwaj, Nina
2005 Jan;79(1):17-27, Journal of virology
— id: 47906, year: 2005, vol: 79, page: 17, stat: Journal Article,

Differentiation of peripheral blood monocytes into dendritic cells
O'Neill, David W; Bhardwaj, Nina
2005 Jul;Chapter 22:Unit 22F.4-Unit 22F.4, Current protocols in immunology
Dendritic cells (DCs) are potent antigen-presenting cells (APC) that are important in the initiation and control of cellular immune responses. Commonly used in T cell-stimulation experiments, DCs are typically 'matured' in vitro with microbial products or proinflammatory cytokines, and then loaded with antigens from any number of sources, including peptides, whole proteins, cell lysates, RNA, microbes, or killed tumor cells. This unit presents a simple and commonly used method for the generation of mature human dendritic cells--differentiating them from peripheral blood monocytes
— id: 78743, year: 2005, vol: Chapter 22, page: Unit 22F.4, stat: Journal Article,

Exploiting dendritic cells for active immunotherapy of cancer and chronic infection
O'Neill, David; Bhardwaj, Nina
2005 ;109:1-18, Methods in molecular medicine
Dendritic cells (DC) are important antigen-presenting cells (APC) that can prime naive T-cells and control lymphocyte-mediated adaptive immune responses with respect to magnitude, memory, and self-tolerance. Understanding the biology of these cells is central to the development of new generation immunotherapies for cancer and chronic infection. This chapter presents a brief overview of DC biology and the preparation and use of DC-based vaccines
— id: 48879, year: 2005, vol: 109, page: 1, stat: Journal Article,

Generation of autologous peptide- and protein-pulsed dendritic cells for patient-specific immunotherapy
O'Neill, David; Bhardwaj, Nina
2005 ;109:97-112, Methods in molecular medicine
This chapter presents a detailed protocol for the generation of mature, monocyte-derived dendritic cells (DC) that are loaded or 'pulsed' with tumor-associated peptide antigens for use as patient-specific immunotherapy. The protocol can easily be adapted for the treatment of patients with a variety of tumors or chronic viral infections by simply changing the peptide antigens used. The vaccine may use major histocompatibility complex (MHC) class I- or class II-restricted peptides to elicit stimulation of CD8+ cytotoxic T-lymphocytes (CTL) or CD4+ T-helper cells, respectively. It also provides an example of loading DC with a purified protein antigen-in this instance, keyhole limpet hemocyanin (KLH). KLH may be used to boost the immunogenicity of the vaccine and as a control to test for the induction of CD4+ T-helper-cell responses. Other antigenic proteins may be added or substituted. Once prepared, the DC are frozen in aliquots and samples are tested for identity, purity, viability, and sterility. After these 'release' criteria are met, vaccine aliquots can be thawed, drawn up into syringes, and injected back into the patient
— id: 48880, year: 2005, vol: 109, page: 97, stat: Journal Article,

Apoptotic cells at the crossroads of tolerance and immunity
Skoberne, M; Beignon, A S; Larsson, M; Bhardwaj, N
2005 ;289:259-292, Current topics in microbiology & immunology
Clearance of apoptotic cells by phagocytes can result in either anti-inflammatory and immunosuppressive effects or prostimulatory consequences through presentation of cell-associated antigens to T cells. The differences in outcome are due to the conditions under which apoptosis is induced, the type of phagocytic cell, the nature of the receptors involved in apoptotic cell capture, and the milieu in which phagocytosis of apoptotic cells takes place. Preferential ligation of specific receptors on professional antigen-presenting cells (dendritic cells) has been proposed to induce potentially tolerogenic signals. On the other hand, dendritic cells can efficiently process and present antigens from pathogen-infected apoptotic cells to T cells. In this review, we discuss how apoptotic cells manipulate immunity through interactions with dendritic cells
— id: 51103, year: 2005, vol: 289, page: 259, stat: Journal Article,

Maturation matters: importance of maturation for antitumor immunity of dendritic cell vaccines
Adams, Sylvia; O'Neill, David; Bhardwaj, Nina
2004 Sep 15;22(18):3834-3835, Journal of clinical oncology
— id: 63123, year: 2004, vol: 22, page: 3834, stat: Journal Article,

Killed but metabolically active recombinant Listeria monocytogenes as an antigen delivery and activation platform for human dendritic cell-based cancer immumotherapy
Brockstedt, DG; Skoberne, M; Yewdall, A; Bahjat, KS; Corash, L; Dubensky, TW; Bhardwaj, N
2004 NOV 16 ;104(11):939A-939A, Blood
— id: 49321, year: 2004, vol: 104, page: 939A, stat: Journal Article,

CT7 (MAGE-C1) is a widely expressed Cancer-Testis antigen in multiple myeloma
Cho, HJ; Jungbluth, AA; Williamson, B; Kolb, D; Ely, S; Cheng, YT; Bhardwaj, N; Coleman, M; Niesvizky, R; Old, L
2004 JUL 15 ;22(14):571S-571S, Journal of clinical oncology
— id: 48687, year: 2004, vol: 22, page: 571S, stat: Journal Article,

Lack of phenotypic and functional impairment in dendritic cells from chimpanzees chronically infected with hepatitis C virus
Larsson, Marie; Babcock, Ethan; Grakoui, Arash; Shoukry, Naglaa; Lauer, Georg; Rice, Charles; Walker, Christopher; Bhardwaj, Nina
2004 Jul;78(12):6151-6161, Journal of virology
Dendritic cells (DCs), which are potent antigen-presenting cells (APCs), are used as adjuvants for the treatment of cancer and infectious diseases in human and nonhuman primates, with documented clinical efficacy. The hepatitis C virus (HCV)-chimpanzee model is the best available model for testing the immunotherapeutic effects of DCs in the setting of a chronic infection, as chimpanzees develop a persistent infection resembling that seen in humans. However, several reports have suggested that DCs derived from chronically infected individuals or nonhuman primates are functionally compromised. As a prelude to clinical studies, we evaluated whether functionally mature DCs could be generated in chimpanzee plasma by good manufacturing practice using CD14(+) mononuclear precursors from chronically infected chimpanzees. DCs generated in a medium with HCV-negative plasma and treated with a defined cocktail of cytokines or a CD40 ligand trimer matured fully, as measured by the induction of CD83 expression and the upregulation of costimulatory molecules. Furthermore, the expression of CCR7 was induced, suggesting an acquisition of migration capacity. Mature DCs were capable of stimulating allogeneic T cells, antigen-specific memory CD4(+) T cells, and HCV-specific CD8(+)-T-cell clones. In all cases, there was no evidence of HCV infection in DCs. Furthermore, these DCs maintained their phenotype and APC function after cryopreservation. Finally, no discernible differences were noted between DCs derived from HCV-infected and uninfected chimpanzees. In summary, precursor cells from HCV-infected chimpanzees are fully capable of differentiating into functional, mature DCs, which can now be reproducibly prepared for investigations of their immunotherapeutic potential in the setting of chronic HCV infection
— id: 46174, year: 2004, vol: 78, page: 6151, stat: Journal Article,

DC-virus interplay: a double edged sword
Larsson, Marie; Beignon, Anne-Sophie; Bhardwaj, Nina
2004 Jun;16(3):147-161, Seminars in immunology
Myeloid and plasmacytoid dendritic cells, a family of professional antigen presenting cells, are crucial in generating and maintaining anti-viral immunity. Many viruses have evolved to avoid, subvert, and even counterattack them. In this article, we focus on the tuning of innate and adaptive responses induced by human dendritic cells, and on the inhibition of their functions by viruses of medical significance. A constant 'tug of war' goes on between dendritic cells and viruses and a main dendritic cell countermeasure is cross-presentation/priming
— id: 47795, year: 2004, vol: 16, page: 147, stat: Journal Article,

Manipulating dendritic cell biology for the active immunotherapy of cancer
O'Neill, David W; Adams, Sylvia; Bhardwaj, Nina
2004 Oct 15;104(8):2235-2246, Blood
Dendritic cells (DCs) are specialized antigen-presenting cells (APCs) that have an unequaled capacity to initiate primary immune responses, including tolerogenic responses. Because of the importance of DCs in the induction and control of immunity, an understanding of their biology is central to the development of potent immunotherapies for cancer, chronic infections, autoimmune disease, and induction of transplantation tolerance. This review discusses recent advances in DC research and the application of this knowledge toward new strategies for the clinical manipulation of DCs for cancer immunotherapy
— id: 47843, year: 2004, vol: 104, page: 2235, stat: Journal Article,

Danger signals: a time and space continuum
Skoberne, Mojca; Beignon, Anne-Sophie; Bhardwaj, Nina
2004 Jun;10(6):251-257, Trends in molecular medicine
— id: 47781, year: 2004, vol: 10, page: 251, stat: Journal Article,

CD8 epitope escape and reversion in acute HCV infection
Timm, Joerg; Lauer, Georg M; Kavanagh, Daniel G; Sheridan, Isabelle; Kim, Arthur Y; Lucas, Michaela; Pillay, Thillagavathie; Ouchi, Kei; Reyor, Laura L; Schulze zur Wiesch, Julian; Gandhi, Rajesh T; Chung, Raymond T; Bhardwaj, Nina; Klenerman, Paul; Walker, Bruce D; Allen, Todd M
2004 Dec 20;200(12):1593-1604, Journal of experimental medicine
In the setting of acute hepatitis C virus (HCV) infection, robust HCV-specific CD8+ cytotoxic T lymphocyte (CTL) responses are associated with initial control of viremia. Despite these responses, 70-80% of individuals develop persistent infection. Although viral escape from CD8 responses has been illustrated in the chimpanzee model of HCV infection, the effect of CD8 selection pressure on viral evolution and containment in acute HCV infection in humans remains unclear. Here, we examined viral evolution in an immunodominant human histocompatibility leukocyte antigen (HLA)-B8-restricted NS3 epitope in subjects with acute HCV infection. Development of mutations within the epitope coincided with loss of strong ex vivo tetramer and interferon gamma enzyme-linked immunospot responses, and endogenous expression of variant NS3 sequences suggested that the selected mutations altered processing and presentation of the variant epitope. Analysis of NS3 sequences from 30 additional chronic HCV-infected subjects revealed a strong association between sequence variation within this region and expression of HLA-B8, supporting reproducible allele-specific selection pressures at the population level. Interestingly, transmission of an HLA-B8-associated escape mutation to an HLA-B8 negative subject resulted in rapid reversion of the mutation. Together, these data indicate that viral escape from CD8+ T cell responses occurs during human HCV infection and that acute immune selection pressure is of sufficient magnitude to influence HCV evolution
— id: 63122, year: 2004, vol: 200, page: 1593, stat: Journal Article,

Type I interferons promote cross-priming: more functions for old cytokines
Beignon, Anne-Sophie; Skoberne, Mojca; Bhardwaj, Nina
2003 Oct;4(10):939-941, Nature immunology
— id: 38687, year: 2003, vol: 4, page: 939, stat: Journal Article,

Immunotherapy for AIDS virus infections: Cautious optimism for cell-based vaccine
Bhardwaj, Nina; Walker, Bruce D
2003 Jan;9(1):13-14, Nature medicine
— id: 33176, year: 2003, vol: 9, page: 13, stat: Journal Article,

Against the self: dendritic cells versus cancer
Cho, Hearn Jay; Bhardwaj, Nina
2003 Jul-Aug;111(7-8):805-817, Apmis
The role of host defense in cancer is highly variable. Although there are cases where spontaneous cures of cancer appear to be mediated by immunologic mechanisms, malignant disease generally progresses even in patients where tumor-specific immunity can be demonstrated. It is apparent that there are complex interactions between tumor cells and dendritic cells, the dominant antigen-presenting cells of the immune system. Through their inhibitory actions upon dendritic cells, tumor cells can negatively regulate priming of tumor-specific immunity. Recent work has also shown that dendritic cells have direct cytotoxic effects upon tumor cells. These interactions may impact on the efficacy of current strategies using dendritic cell-based vaccines for tumor immunotherapy
— id: 38688, year: 2003, vol: 111, page: 805, stat: Journal Article,

Characterization of the MHC class I cross-presentation pathway for cell-associated antigens by human dendritic cells
Fonteneau, Jean Francois; Kavanagh, Daniel G; Lirvall, Margareta; Sanders, Catherine; Cover, Timothy L; Bhardwaj, Nina; Larsson, Marie
2003 Dec 15;102(13):4448-4455, Blood
Major histocompatibility complex (MHC) class I presentation of exogenous antigens is the mechanism enabling professional antigen-presenting cells (APCs) to induce CD8+ T-cell responses against viruses and tumors that do not have access to the classical MHC class I pathway. We have characterized the uptake, processing, and MHC class I cross-presentation by human dendritic cells (DCs) of cell-associated antigens derived from physiologically relevant sources, namely, vaccinia virus-infected apoptotic and necrotic cells. We show that cross-presentation is a rapid process, detectable within 2 to 4 hours after uptake of dead cells, and that proteolysis by cathepsin D in an acidic endosomal compartment is essential for cross-presentation. The presentation is abolished when the phagocytic or macropinocytic functions of the cells are inhibited and is dependent on transporter associated with antigen processing, sensitive to brefeldin A, and requires functional proteasomes. Altogether, these data suggest that antigens derived from apoptotic and necrotic cells require access to the cytosol to intersect with the conventional MHC class I pathway for presentation of cytosolic proteins
— id: 48180, year: 2003, vol: 102, page: 4448, stat: Journal Article,

Activation of influenza virus-specific CD4+ and CD8+ T cells: a new role for plasmacytoid dendritic cells in adaptive immunity
Fonteneau, Jean-Francois; Gilliet, Michel; Larsson, Marie; Dasilva, Ida; Munz, Christian; Liu, Yong-Jun; Bhardwaj, Nina
2003 May 1;101(9):3520-3526, Blood
Plasmacytoid dendritic cells (pDCs) contribute to innate antiviral immune responses by producing type I interferons (IFNs) upon exposure to enveloped viruses. However, their role in adaptive immune responses, such as the initiation of antiviral T-cell responses, is not known. In this study, we examined interactions between blood pDCs and influenza virus with special attention to the capacity of pDCs to activate influenza-specific T cells. pDCs were compared with CD11c(+) DCs, the most potent antigen-presenting cells (APCs), for their capacity to activate T-cell responses. We found that like CD11c(+) DCs, pDCs mature following exposure to influenza virus, express CCR7, and produce proinflammatory chemokines, but differ in that they produce type I IFN and are resistant to the cytopathic effect of the infection. After influenza virus exposure, both DC types exhibited an equivalent efficiency to expand anti-influenza virus cytotoxic T lymphocytes (CTLs) and T helper 1 (TH1) CD4(+) T cells. Our results pinpoint a new role of pDCs in the induction of antiviral T-cell responses and suggest that these DCs play a prominent role in the adaptive immune response against viruses
— id: 48173, year: 2003, vol: 101, page: 3520, stat: Journal Article,

Cross-presentation of cell-associated antigens by dendritic cells
Larsson, M; Fonteneau, J F; Bhardwaj, N
2003 ;276(7-8):261-275, Current topics in microbiology & immunology
There is a strict requirement for professional antigen-presenting cells (APCs) in the generation of immunity toward most viruses. Exogenous pathways of MHC class I-restricted antigen presentation play an important role in the generation of antiviral immunity, particularly in the immune surveillance of virus-infected tissues of nonhematopoietic origin, and to bypass the detrimental effects of direct virus infection on professional APCs. The mechanisms underlying generation of antiviral immunity under these circumstances are discussed
— id: 38689, year: 2003, vol: 276, page: 261, stat: Journal Article,

Dominant effector memory characteristics, capacity for dynamic adaptive expansion, and sex bias in the innate Valpha24 NKT cell compartment
Sandberg, Johan K; Bhardwaj, Nina; Nixon, Douglas F
2003 Mar;33(3):588-596, European journal of immunology
Valpha24 natural killer T (NKT) cells are innate immune cells that recognize self and nonself glycolipids presented by CD1d molecules, and play immunoregulatory roles in autoimmunity and tumor immunity. We have investigated the circulating Valpha24 NKT cells in a large cohort of human subjects. CCR7(-) CD45RO(+) effector memory cells dominated both CD4(+) and CD4() NKT subsets, while a minority displayed a central memory phenotype. CD4(-) central memory NKT cells, however, were atypical in that they largely lacked CD62L expression. Overall, CD4(-) NKT cells displayed a functional phenotype with effector characteristics, while the CD4(+) subset appeared immunoregulatory. Interestingly, NKT cell numbers in blood varied widely between subjects, and elevated numbers of these cells were much more common in women than in men. The CD4(+) subset dominated the NKT cell compartment in both sexes, while circulating NKT cell numbers above 0.1% were associated with an expanded CD4(-) subset. Although NKT cell numbers were generally stable over time, we describe a dynamic fivefold expansion that was associated with a skewing of the NKT CD4(+):CD4(-) ratio that persisted after numbers returned to base line. Thus, the two NKT cell subsets display different properties and dynamics that will influence their function as innate immunoregulatory and effector cells
— id: 38690, year: 2003, vol: 33, page: 588, stat: Journal Article,

Interactions between dead cells and dendritic cells in the induction of antiviral CTL responses
Fonteneau, Jean Francois; Larsson, Marie; Bhardwaj, Nina
2002 Aug;14(4):471-477, Current opinion in immunology
Dendritic cells - professional antigen-presenting cells - are key players for activating adaptive immune responses against viruses. Apoptosis or lytic cell death often accompanies viral infection. Dendritic cells can acquire infected dead or dying cells as exogenous sources of antigens for presentation on MHC class I and II molecules to initiate T cell responses. This pathway of activating T cells may be critical for the development of effective antiviral immunity in vivo
— id: 33182, year: 2002, vol: 14, page: 471, stat: Journal Article,

A division of labor: DC subsets and HIV receptor diversity
Kavanagh, Daniel G; Bhardwaj, Nina
2002 Oct;3(10):891-893, Nature immunology
— id: 33178, year: 2002, vol: 3, page: 891, stat: Journal Article,

Activation of HIV-1 specific CD4 and CD8 T cells by human dendritic cells: roles for cross-presentation and non-infectious HIV-1 virus
Larsson, Marie; Fonteneau, Jean-Francois; Lirvall, Margareta; Haslett, Patrick; Lifson, Jeffrey D; Bhardwaj, Nina
2002 Jul 5;16(10):1319-1329, AIDS
BACKGROUND: The CD4 T cells in mucosal subepithelia are the first cells to become infected during sexual transmission of HIV-1. Dendritic cells (DC) are located in the same area and are known to play a central role in antiviral immune responses. However, extensive viral replication, syncytia formation and cell death follows the interaction between T cells and DC previously exposed to HIV-1. Despite this, anti-HIV responses are generated that control viremia following acute infection. OBJECTIVE: The anti-HIV-1 cellular immune responses observed may be activated by sources other than productively infected DC. HIV-1 induces apoptosis both in cells it infects and in bystander cells. Furthermore, retroviral replication typically generates a predominance of defective particles. We tested whether DC exposed to antigen from either of these sources could elicit anti-HIV specific immune responses. DESIGN AND METHODS: Apoptotic or necrotic monocytes infected with vaccinia virus vectors encoding HIV antigens, a cell line with integrated HIV-1 and apoptotic CD4 T cells pulsed with non-infectious or infectious HIV-1 virus were used as sources of antigens to assess cross presentation by DC. Furthermore, direct DC presentation of antigen from non-infectious and infectious HIV-1 was examined. RESULTS: We find that dead cells expressing HIV-1 antigens as well as non-infectious HIV-1 particles can be acquired and processed by DC, leading to the activation, differentiation and expansion of viral antigen-specific CD4 and CD8 T cells from seropositive individuals. CONCLUSIONS: These sources of antigens may be critical for the generation and maintenance of anti-HIV-1 immunity by DC
— id: 33180, year: 2002, vol: 16, page: 1319, stat: Journal Article,

Amplification of low-frequency antiviral CD8 T cell responses using autologous dendritic cells
Larsson, Marie; Wilkens, David T; Fonteneau, Jean-Francois; Beadle, Thomas J; Merritt, Melissa J; Kost, Rhonda G; Haslett, Patrick A J; Cu-Uvin, Susan; Bhardwaj, Nina; Nixon, Douglas F; Shacklett, Barbara L
2002 Jan 25;16(2):171-180, AIDS
OBJECTIVE: To utilize the potent antigen-presenting capacity of mature dendritic cells (MDC) in order to develop a rapid, sensitive method for quantifying antigen-specific CD8 T cells present at low frequency in peripheral blood. DESIGN: Peripheral blood mononuclear cells (PBMC) were obtained from seven HIV-1-positive individuals with low to moderate CD8 T cell responses, including five on highly active antiretroviral therapy (HAART). IFN-gamma ELISPOT assays were performed using either monocytes or MDC to present antigens expressed by recombinant vaccinia viruses (r-VV). METHODS: Peripheral blood-derived monocytes were cultured for 5-6 days in the presence of IL-4 and granulocyte macrophage colony-stimulating factor, then matured in monocyte-conditioned medium. MDC were infected with r-VV and co-cultured in an ELISPOT assay with autologous monocyte-depleted PBMC. RESULTS: Relative to autologous monocytes, MDC amplified detection of antigen-specific CD8 T cells by 2-30-fold in response to antigens from HIV-1, Epstein-Barr virus and cytomegalovirus. Furthermore, antigenic specificities were revealed that had not been detected using standard ELISPOT of PBMC. CONCLUSION: This assay will prove useful for the detection of memory T cells present at low frequency, and may be of interest for identifying subdominant cytotoxic T lymphocyte epitopes. This method may have broad applications for the detection of antiviral CD8 T cell responses in patient populations in whom such responses have been difficult to detect, including HIV-1-seropositive individuals with advanced disease or undergoing HAART
— id: 33183, year: 2002, vol: 16, page: 171, stat: Journal Article,

A clinical grade cocktail of cytokines and PGE(2) results in uniform maturation of human monocyte-derived dendritic cells: implications for immunotherapy
Lee, Andrew W; Truong, Tuan; Bickham, Kara; Fonteneau, Jean-Francois; Larsson, Marie; Da Silva, Ida; Somersan, Selin; Thomas, Elaine K; Bhardwaj, Nina
2002 Dec 19;20 Suppl 4(1):A8-A22, Vaccine
Dendritic cells (DCs) can induce tumor- or pathogen-specific T cell responses in humans. We comprehensively compared the clinically available DC maturation stimuli for their ability to promote uniformly mature DCs that elicit higher levels of T cell responses. We compared the standard maturation stimulus, autologous monocyte-conditioned medium (MCM), with a synthetic double stranded RNA (poly I:C), soluble CD40 ligand trimer, and a defined cocktail of cytokines (TNF-alpha, IL-1beta, IL-6) and PGE(2) to promote mature phenotype and function in human monocyte-derived DCs. The cocktail was the most efficient despite the lack of induction of IL-12p70. While these results support the use of the MCM-mimic cocktail in clinical DC immunotherapy trials, the roles of it's individual constituents remain to be completely defined
— id: 33177, year: 2002, vol: 20 Suppl 4, page: A8, stat: Journal Article,

Residual viral replication during antiretroviral therapy boosts human immunodeficiency virus type 1-specific CD8+ T-cell responses in subjects treated early after infection
Ortiz, Gabriel M; Hu, Jennifer; Goldwitz, Joshua A; Chandwani, Rohit; Larsson, Marie; Bhardwaj, Nina; Bonhoeffer, Sebastian; Ramratnam, Bharat; Zhang, Linqi; Markowitz, Martin M; Nixon, Douglas F
2002 Jan;76(1):411-415, Journal of virology
Human immunodeficiency virus type 1 (HIV-1)-infected subjects treated early after infection have preserved HIV-1-specific CD4+ T-cell function. We studied the effect of highly active antiretroviral therapy (HAART) on the frequency of HIV-1-specific CD8+ T cells in patients treated during early (n = 31) or chronic (n = 23) infection. The degree of viral suppression and time of initiation of treatment influenced the magnitude of the CD8+ T-cell response. HIV-1-specific CD8+ T cells can increase in number after HAART in subjects treated early after infection who have episodes of transient viremia
— id: 26538, year: 2002, vol: 76, page: 411, stat: Journal Article,

Selective loss of innate CD4(+) V alpha 24 natural killer T cells in human immunodeficiency virus infection
Sandberg, Johan K; Fast, Noam M; Palacios, Emil H; Fennelly, Glenn; Dobroszycki, Joanna; Palumbo, Paul; Wiznia, Andrew; Grant, Robert M; Bhardwaj, Nina; Rosenberg, Michael G; Nixon, Douglas F
2002 Aug;76(15):7528-7534, Journal of virology
V alpha 24 natural killer T (NKT) cells are innate immune cells involved in regulation of immune tolerance, autoimmunity, and tumor immunity. However, the effect of human immunodeficiency virus type 1 (HIV-1) infection on these cells is unknown. Here, we report that the V alpha 24 NKT cells can be subdivided into CD4(+) or CD4(-) subsets that differ in their expression of the homing receptors CD62L and CD11a. Furthermore, both CD4(+) and CD4(-) NKT cells frequently express both CXCR4 and CCR5 HIV coreceptors. We find that the numbers of NKT cells are reduced in HIV-infected subjects with uncontrolled viremia and marked CD4(+) T-cell depletion. The number of CD4(+) NKT cells is inversely correlated with HIV load, indicating depletion of this subset. In contrast, CD4(-) NKT-cell numbers are unaffected in subjects with high viral loads. HIV infection experiments in vitro show preferential depletion of CD4(+) NKT cells relative to regular CD4(+) T cells, in particular with virus that uses the CCR5 coreceptor. Thus, HIV infection causes a selective loss of CD4(+) lymph node homing (CD62L(+)) NKT cells, with consequent skewing of the NKT-cell compartment to a predominantly CD4(-) CD62L(-) phenotype. These data indicate that the key immunoregulatory NKT-cell compartment is compromised in HIV-1-infected patients
— id: 33181, year: 2002, vol: 76, page: 7528, stat: Journal Article,

Dendritic cell amplification of HIV type 1-specific CD8+ T cell responses in exposed, seronegative heterosexual women
Shacklett, Barbara L; Means, Robert E; Larsson, Marie; Wilkens, David T; Beadle, Thomas J; Merritt, Melissa J; Bhardwaj, Nina; Palumbo, Paul E; Skurnick, Joan H; Louria, Donald B; Nixon, Douglas F
2002 Jul 20;18(11):805-815, AIDS research & human retroviruses
In the Heterosexual AIDS Transmission Study (HATS), the frequency of high-risk sexual activity and viral load in the seropositive partner were shown to correlate with HIV-1 transmission. However, these parameters could not account for the status of some exposed, seronegative (ESN) individuals who remained uninfected despite years of exposure. To test the hypothesis that antiviral immune responses are a correlate of nontransmission in this cohort, we developed two sensitive methods for assessing HIV-1-specific humoral and cell-mediated responses. To quantify T cell responses, autologous mature dendritic cells (DCs) were used as antigen-presenting cells to elicit HIV-1-specific IFN-gamma production by ELISPOT. Antibody responses to HIV-1 gp120 were assessed by combination immunoprecipitation-Western blot (IP-WB). Previous studies of this cohort, using limiting dilution analysis, did not reveal HIV-1-specific cytotoxic T lymphocyte activity. However, when autologous DCs were used to present HIV-1 antigens, T cells from three of eight ESN women (38%) responded by producing IFN-gamma. T cells from three of four seropositive partners responded to HIV-1 antigens, whereas five negative controls did not. The use of DCs as antigen-presenting cells increased sensitivity by 2- to 30-fold relative to standard ELISPOT. Using IP-WB, low levels of gp120-reactive antibodies were detected in plasma from 1 of 14 ESN women. These results support the hypothesis that HIV-1-specific T cell responses play a role in immune surveillance in this cohort of North American serodiscordant couples. This report also demonstrates the ability of dendritic cells to reveal T cell responses that might be overlooked by other methods
— id: 33179, year: 2002, vol: 18, page: 805, stat: Journal Article,

Immune and clinical responses in patients with metastatic melanoma to CD34(+) progenitor-derived dendritic cell vaccine
Banchereau J; Palucka AK; Dhodapkar M; Burkeholder S; Taquet N; Rolland A; Taquet S; Coquery S; Wittkowski KM; Bhardwaj N; Pineiro L; Steinman R; Fay J
2001 Sep 1;61(17):6451-6458, Cancer research
Immunization to multiple defined tumor antigens for specific immune therapy of human cancer has thus far proven difficult. Eighteen HLA A*0201(+) patients with metastatic melanoma received injections s.c. of CD34(+)progenitor-derived autologous dendritic cells (DCs), which included Langerhans cells. DCs were pulsed with peptides derived from four melanoma antigens [(MelAgs) MelanA/MART-1, tyrosinase, MAGE-3, and gp100], as well as influenza matrix peptide (Flu-MP) and keyhole limpet hemocyanin (KLH) as control antigens. Overall immunological effects were assessed by comparing response profiles using marginal likelihood scores. DC injections were well tolerated except for progressive vitiligo in two patients. DCs induced an immune response to control antigens (KLH, Flu-MP) in 16 of 18 patients. An enhanced immune response to one or more MelAgs was seen in these same 16 patients, including 10 patients who responded to >2 MelAgs. The two patients failing to respond to both control and tumor antigens experienced rapid tumor progression. Of 17 patients with evaluable disease, 6 of 7 patients with immunity to two or less MelAgs had progressive disease 10 weeks after study entry, in contrast to tumor progression in only 1 of 10 patients with immunity to >2 MelAgs. Regression of >1 tumor metastases were observed in seven of these patients. The overall immunity to MelAgs after DC vaccination is associated with clinical outcome (P = 0.015)
— id: 33190, year: 2001, vol: 61, page: 6451, stat: Journal Article,

Processing and presentation of antigens by dendritic cells: implications for vaccines
Bhardwaj N
2001 Sep;7(9):388-394, Trends in molecular medicine
Dendritic cells (DCs) are potent antigen-presenting cells that are specialized in initiation of T-cell immunity. DCs induce promising anti-tumor T-cell and clinical responses, apparently without significant toxicity. Under certain conditions, DCs even silence T-cell immune responses in vivo. This dual capacity to modulate the immune system uniquely positions DCs for the treatment of autoimmunity, cancer and chronic viral infections
— id: 33189, year: 2001, vol: 7, page: 388, stat: Journal Article,

EBNA1-specific CD4+ T cells in healthy carriers of Epstein-Barr virus are primarily Th1 in function
Bickham K; Munz C; Tsang ML; Larsson M; Fonteneau JF; Bhardwaj N; Steinman R
2001 Jan;107(1):121-130, Journal of clinical investigation
The Epstein-Barr virus (EBV) nuclear antigen-1 (EBNA1) maintains the viral episome in all host cells infected with EBV. Recently, EBNA1 was found to be the main EBV latency antigen for CD4+ T cells and could be recognized in cultures from all donors tested. We now identify a polarized Th1 phenotype and obtain evidence for its presence in vivo. When T cells were stimulated with dendritic cells infected with vaccinia vectors expressing EBNA1, 18 of 19 donors secreted IFN-gamma, whereas only two of 19 secreted IL-4. Magnetic selection was then used to isolate cells from fresh blood based on EBNA1-induced cytokine production. Specific IFN-gamma CD4+ cell lines were established from six of six donors and IL-4 lines from three of six. Only the Th1 lines specifically lysed targets expressing three different sources of EBNA1 protein. When the IgG isotype of EBNA1 plasma Ab's was tested, most specific Ab's were IgG1 and of a high titer, confirming a Th1 response to EBNA1 in vivo. Ab's to other microbial antigens generally were not skewed toward IgG1. Given emerging evidence that Th1 CD4+ T cells have several critical roles in host defense to viral infection and tumors, we propose that EBNA1-specific CD4+ Th1 cells contribute to resistance to EBV and EBV-associated malignancies
— id: 33195, year: 2001, vol: 107, page: 121, stat: Journal Article,

Antigen-specific inhibition of effector T cell function in humans after injection of immature dendritic cells
Dhodapkar MV; Steinman RM; Krasovsky J; Munz C; Bhardwaj N
2001 Jan 15;193(2):233-238, Journal of experimental medicine
Immunostimulatory properties of dendritic cells (DCs) are linked to their maturation state. Injection of mature DCs rapidly enhances antigen-specific CD4+ and CD8+ T cell immunity in humans. Here we describe the immune response to a single injection of immature DCs pulsed with influenza matrix peptide (MP) and keyhole limpet hemocyanin (KLH) in two healthy subjects. In contrast to prior findings using mature DCs, injection of immature DCs in both subjects led to the specific inhibition of MP-specific CD8+ T cell effector function in freshly isolated T cells and the appearance of MP-specific interleukin 10-producing cells. When pre- and postimmunization T cells were boosted in culture, there were greater numbers of MP-specific major histocompatibility complex tetramer-binding cells after immunization, but these had reduced interferon production and lacked killer activity. These data demonstrate the feasibility of antigen-specific inhibition of effector T cell function in vivo in humans and urge caution with the use of immature DCs when trying to enhance tumor or microbial immunity
— id: 33192, year: 2001, vol: 193, page: 233, stat: Journal Article,

Mature dendritic cells infected with canarypox virus elicit strong anti-human immunodeficiency virus CD8+ and CD4+ T-cell responses from chronically infected individuals
Engelmayer J; Larsson M; Lee A; Lee M; Cox WI; Steinman RM; Bhardwaj N
2001 Mar;75(5):2142-2153, Journal of virology
Recombinant canarypox virus vectors containing human immunodeficiency virus type 1 (HIV-1) sequences are promising vaccine candidates, as they replicate poorly in human cells. However, when delivered intramuscularly the vaccines have induced inconsistent and in some cases transient antigen-specific cytotoxic T-cell (CTL) responses in seronegative volunteers. An attractive way to enhance these responses would be to target canarypox virus to professional antigen-presenting cells such as dendritic cells (DCs). We studied (i) the interaction between canarypox virus and DCs and (ii) the T-cell responses induced by DCs infected with canarypox virus vectors containing HIV-1 genes. Mature and not immature DCs resisted the cytopathic effects of canarypox virus and elicited strong effector CD8+ T-cell responses from chronically infected HIV+ individuals, e.g., cytolysis, and secretion of gamma interferon (IFN-gamma) and beta-chemokines. Furthermore, canarypox virus-infected DCs were >30-fold more efficient than monocytes and induced responses that were comparable to those induced by vaccinia virus vectors or peptides. Addition of exogenous cytokines was not necessary to elicit CD8+ effector cells, although the presence of CD4+ T cells was required for their expansion and maintenance. Most strikingly, canarypox virus-infected DCs were directly able to stimulate HIV-specific, IFN-gamma-secreting CD4 helper responses from bulk as well as purified CD4+ T cells. Therefore, these results suggest that targeting canarypox virus vectors to mature DCs could potentially elicit both anti-HIV CD8+ and CD4+ helper responses in vivo
— id: 33193, year: 2001, vol: 75, page: 2142, stat: Journal Article,

Dendritic cell-dead cell interactions: implications and relevance for immunotherapy
Fonteneau JF; Larsson M; Bhardwaj N
2001 Jul-Aug;24(4):294-304, Journal of immunotherapy (Hagerstown)
Dendritic cells are a system of antigen-presenting cells with an essential role in the initiation and development of immune responses against infections or tumors. Their unique capacity to stimulate T cells is being adapted for use in immunotherapy. In this review, we focus on their ability to interact with dead cells and, notably, to present exogenous antigens acquired from them to CD8+ T cells. We also discuss the role of this unique antigen presentation pathway for immunotherapeutic development
— id: 33188, year: 2001, vol: 24, page: 294, stat: Journal Article,

Generation of high quantities of viral and tumor-specific human CD4+ and CD8+ T-cell clones using peptide pulsed mature dendritic cells
Fonteneau JF; Larsson M; Somersan S; Sanders C; Munz C; Kwok WW; Bhardwaj N; Jotereau F
2001 Dec 1;258(1-2):111-126, Journal of immunological methods
CD4+ and CD8+ T cells are key components of immune response against tumors and viruses. Many techniques have been used to clone and expand these cells in vitro for purposes of immunotherapy. Here, we describe an improved method to obtain large quantities of tumor and virus-specific human CD4+ and CD8+ T-cell clones. T cells derived from peripheral blood mononuclear cells (PBMCs) of healthy donors were stimulated several times by peptide pulsed monocyte-derived mature dendritic cells (DCs) in the presence of exogenous cytokines. T cells specific for influenza or melanoma antigens were detected by IFN-gamma intracellular staining and were cloned by limiting dilution. Specific polyclonal T-cell populations were derived for all epitopes presented by mature DCs. Nine different populations were cloned and clones were raised from eight of them. Clonality was verified by HLA/peptide tetramer staining. With additional rounds of stimulation after the cloning procedure, it was possible to obtain from 10(9) to 10(12) of each clone. Furthermore, clones could be maintained in culture in the presence of IL-2 for at least 1 month without losing their antigen-specific reactivity (e.g. cytokine secretion, cytolytic activity and proliferation). Importantly, a majority of the CD8+ T-cell clones recognized endogenously processed antigens. This method is of value for the purposes of adoptive anti-virus or anti-tumor immunotherapy
— id: 33186, year: 2001, vol: 258, page: 111, stat: Journal Article,

Dendritic cells resurrect antigens from dead cells
Larsson M; Fonteneau JF; Bhardwaj N
2001 Mar;22(3):141-148, Trends in immunology
Antigens that do not normally access the cytoplasm of antigen-presenting cells, such as certain tumor and viral antigens, become targets of cytotoxic T lymphocytes (CTLs). Over the past 25 years, substantial evidence has emerged for an 'exogenous' pathway for loading MHC class I molecules. Dendritic cells are potent stimulators of T-cell responses and can induce CD8(+) CTLs by phagocytosis of dead tumor or virus-infected cells. Here, Marie Larsson and colleagues discuss the role of dendritic cells in stimulating MHC class I-restricted T-cell responses by exogenous routes
— id: 33191, year: 2001, vol: 22, page: 141, stat: Journal Article,

Efficiency of cross presentation of vaccinia virus-derived antigens by human dendritic cells
Larsson M; Fonteneau JF; Somersan S; Sanders C; Bickham K; Thomas EK; Mahnke K; Bhardwaj N
2001 Dec;31(12):3432-3442, European journal of immunology
Dendritic cells (DC) utilize at least two pathways to process viral antigens onto MHC class I molecules. The conventional endogenous route is used to acquire antigens from both infectious and non-replicating virions. Exogenous pathways are used by DC to acquire and 'cross-present' antigens derived from virus-infected donor cells that by themselves lack the ability to activate T cells directly. We analyzed the role of this pathway for antigens derived from vaccinia, a virus which inhibits DC maturation and causes extensive apoptosis of infected cells, yet is highly immunogenic. Using recombinant vaccinia virus encoding the influenza matrix protein as model vector, DC were shown to cross-present vaccinia-derived antigens from both apoptotic and necrotic infected cells to antigen-specific CD8(+) T cells. Efficient cross presentation required uptake of dead cells by immature DC and exposure to maturation stimuli, especially CD40 ligand. The responding CD8(+) T cells secreted IL-2 and IFN-gamma, proliferated and developed into cytotoxic effectors. Quantification of the cross presentation of vaccinia-derived antigens showed this pathway to be highly efficient, corresponding to a peptide pulse of 10-100 nM. While monocytes also phagocytosed apoptotic and necrotic cells, they were far less efficient at cross-presenting vaccinia-derived antigens to CD8(+) T cells. The ability of DC to cross-present vaccinia-derived antigens from infected apoptotic cells or necrotic cell lysates, bypasses the deleterious effects of direct infection of DC and provides one explanation for this pathogen's immunogenicity
— id: 33184, year: 2001, vol: 31, page: 3432, stat: Journal Article,

Tethering and tickling: a new role for the phosphatidylserine receptor
Somersan S; Bhardwaj N
2001 Nov 12;155(4):501-504, Journal of cell biology
Several receptors are implicated in apoptotic cell (AC) uptake by phagocytic cells; however, their relative dominance in mammalian systems remains to be established. New studies shed light on the role of the phosphatidyl serine (PS) receptor (PSR). Ligation of PSR by PS on AC surfaces is considered essential for signaling uptake of ACs that are tethered to phagocytes via other receptors
— id: 33185, year: 2001, vol: 155, page: 501, stat: Journal Article,

Primary tumor tissue lysates are enriched in heat shock proteins and induce the maturation of human dendritic cells
Somersan S; Larsson M; Fonteneau JF; Basu S; Srivastava P; Bhardwaj N
2001 Nov 1;167(9):4844-4852, Journal of immunology
Upon exposure to lysates or supernatants of necrotic transformed cell lines, human dendritic cells (DCs) undergo maturation. In contrast, DCs exposed to apoptotic transformed cell lines or necrotic lysates of primary cells remain immature. Analysis of supernatants of necrotic transformed cell lines showed them to be enriched in the heat shock proteins (hsp)70 and gp96, in contrast to supernatants of primary cells. Likewise, cells from a variety of primary human tumors contained considerably higher levels of hsp than their normal autologous tissue counterparts. Of the majority of human tumors enriched in hsps (hsp70 and/or gp96), their corresponding lysates matured DCs. The maturation effect of tumor cell lysates was abrogated by treatment with boiling, proteinase K, and geldanamycin, an inhibitor of hsps, suggesting that hsps rather than endotoxin or DNA were the responsible factors. Supporting this idea, highly purified, endotoxin-depleted hsp70, induced DC maturation similar to that seen with standard maturation stimuli LPS and monocyte conditioned medium. These results suggest that the maturation activity inherent within tumor cells and lines is mediated at least in part by hsps. The release of hsps in vivo as a result of cell injury should promote immunity through the maturation of resident DCs
— id: 33187, year: 2001, vol: 167, page: 4844, stat: Journal Article,

Active immunization of humans with dendritic cells
Dhodapkar MV; Bhardwaj N
2000 May;20(3):167-174, Journal of clinical immunology
Dendritic cells (DCs) are potent antigen-presenting cells specialized to initiate T-cell immunity. The development of methods to generate large numbers of DCs has facilitated their application for immunotherapy. Recent studies have demonstrated the safety and immunogenicity of DCs in humans and have begun to outline the durability, kinetics, and nature of the elicited T-cell responses. However, DC-based immunotherapy remains a challenge and several parameters need to be examined to optimize immune responses, in order to maximize clinical efficacy against cancer and infectious diseases
— id: 33196, year: 2000, vol: 20, page: 167, stat: Journal Article,

Mature dendritic cells boost functionally superior CD8(+) T-cell in humans without foreign helper epitopes
Dhodapkar MV; Krasovsky J; Steinman RM; Bhardwaj N
2000 Mar;105(6):R9-R14, Journal of clinical investigation
We have recently shown that a single injection of mature, antigen-pulsed, human dendritic cells (DCs) rapidly elicits CD4(+) and CD8(+) T-cell immunity in vivo. The DCs were pulsed with 2 foreign proteins, keyhole limpet hemocyanin (KLH) and tetanus toxoid (TT), as well as an HLA A2.1-restricted influenza matrix peptide (MP). Responses to all 3 antigens peaked at 30-90 days after immunization and declined thereafter. To determine if the foreign helper proteins (TT and KLH) were essential for CD8(+) T-cell responses to the viral peptide, we reinjected 3 of the HLA-2.1 subjects with mature DCs pulsed with MP alone. All 3 volunteers showed a rapid boost in MP-specific immunity, and freshly sampled blood from 1 contained cytolytic T cells. In all 3 subjects, CD8(+) T-cell responses to booster DCs were faster and of greater magnitude than the responses to the first DC injection. Importantly, the T cells that proliferated after booster DC treatment secreted interferon-gamma upon challenge with much lower doses of viral peptide than those elicited after the first injection, indicating a higher functional avidity for the ligand. These data begin to outline the kinetics of T-cell immunity in response to DCs and demonstrate that booster injections of mature DCs enhance both qualitative and quantitative aspects of CD8(+) T-cell function in humans
— id: 33198, year: 2000, vol: 105, page: R9, stat: Journal Article,

Paucity of functional T-cell memory to melanoma antigens in healthy donors and melanoma patients
Dhodapkar MV; Young JW; Chapman PB; Cox WI; Fonteneau JF; Amigorena S; Houghton AN; Steinman RM; Bhardwaj N
2000 Dec;6(12):4831-4838, Clinical cancer research
The functional characteristics of CD8+ T cells specific for melanoma antigens (MAs) have often been defined after in vitro culture using nonprofessional antigen-presenting cells. We have examined CD8+ T-cell immunity to MAs and a viral antigen (influenza) in uncultured T cells of healthy donors and melanoma patients using autologous, mature, monocyte-derived dendritic cells (DCs) pulsed with peptide antigens and viral vectors. Antigen-specific IFN-gamma-producing T cells reactive with HLA-A*0201-restricted peptides from four melanoma antigens (MelanA/MART-1, MAGE-3, tyrosinase, and gp100) were detected only at low frequencies (<30 per 2 x 10(5) peripheral blood mononuclear cells for each of the MAs) from HLA-A2.1-positive healthy donors (n = 12) and patients with stages III/IV melanoma (n = 8). Detection of MA-specific, but not influenza matrix peptide (Flu-MP)-specific, T cells required a high concentration (10 microg/ml) of the peptide in this assay. Furthermore, these T cells did not recognize endogenously processed antigen on tumor cell lines or cells infected with viral vectors capable of expressing MAs. The use of autologous, mature DCs led to a significant increase in the number of Flu-MP, but not MA-specific, T cells in 16-h ELISPOT assays for both melanoma patients and healthy donors. In 1-week cocultures with DCs pulsed with 10 microg/ml peptide, MelanA/MART-1-specific T cells did not readily proliferate or differentiate into lytic effectors, in contrast to strong influenza-specific lytic responses. Therefore, despite distinct memory responses to influenza antigens, melanoma patients and healthy controls have a paucity of MA-reactive memory T cells, failing to rapidly generate IFN-gamma-secreting lytic effectors in short-term assays, even when stimulated by DCs
— id: 33194, year: 2000, vol: 6, page: 4831, stat: Journal Article,

Identification of subdominant cytotoxic T lymphocyte epitopes encoded by autologous HIV type 1 sequences, using dendritic cell stimulation and computer-driven algorithm
Jin X; Roberts CG; Nixon DF; Safrit JT; Zhang LQ; Huang YX; Bhardwaj N; Jesdale B; DeGroot AS; Koup RA
2000 Jan 1;16(1):67-76, AIDS research & human retroviruses
Conventional analysis of the cytotoxic T lymphocyte (CTL) response to HIV-1 may underestimate the true breadth of CTL epitopes recognized. This underestimation could be due to several reasons, including (1) the use of laboratory-adapted stains of HIV or consensus sequences, which would lead to the identification of only highly conserved epitopes, (2) the use of EBV-transformed B cells (B-LCLs) and vaccinia virus constructs in standard assays that may obscure low level CTL responses due to high EBV or vaccinia reactivity, and (3) relatively insensitive assays wherein PBMCs instead of professional APCs are used to stimulate CTL responses. To address these problems, we first identified an immunodominant HLA-B7-restricted CTL epitope, by standard cloning methods, in a long-term nonprogressor (LTNP). To determine whether the patient had CTLs specific for autologous viral sequences other than the dominant epitope, proviral DNA was cloned and sequenced. A matrix-based epitope algorithm (EpiMatrix) was used to identify the top 2% of peptides from the viral sequences with the highest likelihood of binding to HLA-B7. These 55 peptides were synthesized and tested for HLA-B7 binding in a T2/B7 cell line; 10 peptides were able to stabilize HLA-B7 on the cell surface. By using peptide-pulsed autologous dendritic cells as a more sensitive method of CTL stimulation, we found three additional subdominant CTL epitopes
— id: 33200, year: 2000, vol: 16, page: 67, stat: Journal Article,

Requirement of mature dendritic cells for efficient activation of influenza A-specific memory CD8+ T cells
Larsson M; Messmer D; Somersan S; Fonteneau JF; Donahoe SM; Lee M; Dunbar PR; Cerundolo V; Julkunen I; Nixon DF; Bhardwaj N
2000 Aug 1;165(3):1182-1190, Journal of immunology
It is critical to identify the developmental stage of dendritic cells (DCs) that is most efficient at inducing CD8+ T cell responses. Immature DCs can be generated from monocytes with GM-CSF and IL-4, while maturation is accomplished by the addition of stimuli such as monocyte-conditioned medium, CD40 ligand, and LPS. We evaluated the ability of human monocytes and immature and mature DCs to induce CD8+ effector responses to influenza virus Ags from resting memory cells. We studied replicating virus, nonreplicating virus, and the HLA-A*0201-restricted influenza matrix protein peptide. Sensitive and quantitative assays were used to measure influenza A-specific immune responses, including MHC class I tetramer binding assays, enzyme-linked immunospot assays for IFN-gamma production, and generation of cytotoxic T cells. Mature DCs were demonstrated to be superior to immature DC in eliciting IFN-gamma production from CD8+ effector cells. Furthermore, only mature DCs, not immature DCs, could expand and differentiate CTL precursors into cytotoxic effector cells over 7 days. An exception to this was immature DCs infected with live influenza virus, because of the virus's known maturation effect. Finally, mature DCs pulsed with matrix peptide induced CTLs from highly purified CD8+ T cells without requiring CD4+ T cell help. These differences between DC stages were independent of Ag concentrations or the number of immature DCs. In contrast to DCs, monocytes were markedly inferior or completely ineffective stimulators of T cell immunity. Our data with several qualitatively different assays of the memory CD8+ T cell response suggest that mature cells should be considered as immunotherapeutic adjuvants for Ag delivery
— id: 33197, year: 2000, vol: 165, page: 1182, stat: Journal Article,

Consequences of cell death: exposure to necrotic tumor cells, but not primary tissue cells or apoptotic cells, induces the maturation of immunostimulatory dendritic cells
Sauter B; Albert ML; Francisco L; Larsson M; Somersan S; Bhardwaj N
2000 Feb 7;191(3):423-434, Journal of experimental medicine
Cell death by necrosis is typically associated with inflammation, in contrast to apoptosis. We have identified additional distinctions between the two types of death that occur at the level of dendritic cells (DCs) and which influence the induction of immunity. DCs must undergo changes termed maturation to act as potent antigen-presenting cells. Here, we investigated whether exposure to apoptotic or necrotic cells affected DC maturation. We found that immature DCs efficiently phagocytose a variety of apoptotic and necrotic tumor cells. However, only exposure to the latter induces maturation. The mature DCs express high levels of the DC-restricted markers CD83 and lysosome-associated membrane glycoprotein (DC-LAMP) and the costimulatory molecules CD40 and CD86. Furthermore, they develop into powerful stimulators of both CD4(+) and CD8(+) T cells. Cross-presentation of antigens to CD8(+) T cells occurs after uptake of apoptotic cells. We demonstrate here that optimal cross-presentation of antigens from tumor cells requires two steps: phagocytosis of apoptotic cells by immature DCs, which provides antigenic peptides for major histocompatibility complex class I and class II presentation, and a maturation signal that is delivered by exposure to necrotic tumor cells, their supernatants, or standard maturation stimuli, e.g., monocyte-conditioned medium. Thus, DCs are able to distinguish two types of tumor cell death, with necrosis providing a control that is critical for the initiation of immunity
— id: 33199, year: 2000, vol: 191, page: 423, stat: Journal Article,

Dendritic cells are resistant to apoptosis through the Fas (CD95/APO-1) pathway
Ashany D; Savir A; Bhardwaj N; Elkon KB
1999 Nov 15;163(10):5303-5311, Journal of immunology
Immunoregulation of lymphocytes and macrophages in the peripheral immune system is achieved in part by activation-induced cell death. Members of the TNF receptor family including Fas (CD95) are involved in the regulation of activation-induced cell death. To determine whether activation-induced cell death plays a role in regulation of dendritic cells (DCs), we examined interactions between Ag-presenting murine DCs and Ag-specific Th1 CD4+ T cells. Whereas mature bone marrow- or spleen-derived DCs expressed high levels of Fas, these DCs were relatively insensitive to Fas-mediated killing by the agonist mAb, Jo-2, as well as authentic Fas ligand expressed on the CD4+ T cell line, A.E7. The insensitivity to Fas-mediated apoptosis was not affected by priming with IFN-gamma and/or TNF-alpha or by blocking the DC survival signals TNF-related activation-induced cytokine and CD40L. However, apoptosis could be induced with C2-ceramide, suggesting that signals proximal to the generation of ceramide might mediate resistance to Fas. Analysis of protein expression of several anti-apoptotic mediators revealed that expression of the intracellular inhibitor of apoptosis Fas-associated death domain-like IL-1-converting enzyme-inhibitory protein was significantly higher in Fas-resistant DCs than in Fas-sensitive macrophages, suggesting a possible role for Fas-associated death domain-like IL-1-converting enzyme-inhibitory protein in DC resistance to Fas-mediated apoptosis. Our results demonstrate that murine DCs differ significantly from other APC populations in susceptibility to Fas-mediated apoptosis during cognate presentation of Ag. Because DCs are most notable for initiation of an immune response, resistance to apoptosis may contribute to this function
— id: 33203, year: 1999, vol: 163, page: 5303, stat: Journal Article,

Rapid generation of broad T-cell immunity in humans after a single injection of mature dendritic cells
Dhodapkar MV; Steinman RM; Sapp M; Desai H; Fossella C; Krasovsky J; Donahoe SM; Dunbar PR; Cerundolo V; Nixon DF; Bhardwaj N
1999 Jul;104(2):173-180, Journal of clinical investigation
Dendritic cells (DCs) are potent antigen-presenting cells that initiate protective T-cell immunity in mice. To study the immunogenicity of DCs in humans, we injected 9 healthy subjects subcutaneously with a control injection of autologous monocyte-derived, mature DCs, followed 4-6 weeks later by DCs pulsed with keyhole limpet hemocyanin (KLH), HLA-A*0201-positive restricted influenza matrix peptide (MP), and tetanus toxoid (TT). Four more subjects received these antigens without DCs. Injection of unpulsed DCs, or antigens alone, failed to immunize. Priming of CD4(+) T cells to KLH was observed in all 9 subjects injected with KLH-pulsed DCs, and boosting of TT-specific T-cell immunity was seen in 5 of 6 subjects injected with TT-pulsed DCs. Injection of antigen-pulsed DCs led to a severalfold increase in freshly isolated MP-specific, IFN-gamma-secreting CD8(+) T cells in all 6 HLA-A*0201-positive subjects, as early as 7 days after injection. When T cells were boosted in culture, there was an increase in MHC tetramer-binding cells and cytotoxic T cells after DC vaccination. These data provide the first controlled evidence of the immunogenicity of DCs in humans, and demonstrate that a single injection of mature DCs rapidly expands T-cell immunity
— id: 33205, year: 1999, vol: 104, page: 173, stat: Journal Article,

Vaccinia virus inhibits the maturation of human dendritic cells: a novel mechanism of immune evasion
Engelmayer J; Larsson M; Subklewe M; Chahroudi A; Cox WI; Steinman RM; Bhardwaj N
1999 Dec 15;163(12):6762-6768, Journal of immunology
Vaccinia virus employs multiple mechanisms to evade the immune system, yet is highly immunogenic. We studied the interaction between vaccinia and human dendritic cells (DCs), potent APCs. DCs develop from precursor cells in two stages: an immature stage in which Ag uptake and processing occur, and a mature stage in which there is up-regulation of costimulatory and HLA molecules and efficient T cell activation. Vaccinia virus undergoes an abortive replication in both stages of DCs and induces apoptotic cell death. Furthermore, maturation of immature DCs and consequently T cell activation are inhibited. Obstruction of DC maturation may constitute a novel mechanism by which vaccinia attempts to evade the immune response
— id: 33202, year: 1999, vol: 163, page: 6762, stat: Journal Article,

A recombinant vaccinia virus based ELISPOT assay detects high frequencies of Pol-specific CD8 T cells in HIV-1-positive individuals
Larsson M; Jin X; Ramratnam B; Ogg GS; Engelmayer J; Demoitie MA; McMichael AJ; Cox WI; Steinman RM; Nixon D; Bhardwaj N
1999 May 7;13(7):767-777, AIDS
OBJECTIVES: HIV-1-specific CD8 T cells are considered to be critical in anti-HIV responses. It is important to quantify these cells and to determine their antigenic targets. Here quantification of interferon (IFN)-gamma secreting, virus-specific cells was achieved with an enzyme linked immuno spot (ELISPOT) assay. METHODS: Peripheral blood mononuclear cells (PBMC) were infected with recombinant vaccinia vectors expressing HIV-1 genes (gag, pol, env or nef) and added to wells precoated with anti-IFN-gamma monoclonal antibodies. Spot forming cells (SFC), i.e. antigen-specific T cells were detected 24 h later by the addition of biotinylated anti-IFN-gamma monoclonal antibodies, followed by avidin-bound biotinylated horseradish peroxidase. RESULTS: In a cohort of 19 patients, of whom 15 were on highly active antiretroviral therapy, 18 had primed T cells directed against one or more HIV-1 antigens (P < 0.0001). Pol-specific T cells routinely dominated the CD8 response with frequencies up to 2000 SFC per 10(6) PBMC. In HLA A*0201-positive patients, the vaccinia vectors detected much higher frequencies of SFC than haplotype-restricted peptides. Elimination of CD8 T cells resulted in > 90% loss of antigen-specific SFC when vaccinia virus was used as a vector. The number of CD8 SFC exceeded the number of memory cells detected in limiting dilution assays by > 1 log10, whereas a correlation was found between the frequency of effector cells detected by both ELISPOT and MHC class I peptide tetramer assays. CONCLUSIONS: Vaccinia virus vectors used in ELISPOT assays are useful for determining the frequency and specificity of CD8 T cells for individual HIV-1 gene products. The dominance of cytolytic T lymphocytes (CTL) recognizing pol proteins suggests that this antigen should be considered in vaccine strategies
— id: 33206, year: 1999, vol: 13, page: 767, stat: Journal Article,

Airway obstruction caused by transesophageal echocardiography in a patient with double aortic arch and truncus arteriosus
Phoon CK; Bhardwaj N
1999 Jun;12(6):540-540, Journal of the American Society of Echocardiography
— id: 17989, year: 1999, vol: 12, page: 540, stat: Journal Article,

Dendritic cells generated from blood monocytes of HIV-1 patients are not infected and act as competent antigen presenting cells eliciting potent T-cell responses
Sapp M; Engelmayer J; Larsson M; Granelli-Piperno A; Steinman R; Bhardwaj N
1999 Mar;66(1-3):121-128, Immunology letters
The CTL response to HIV-I can be vigorous, but antigen presenting cell requirements have not been studied in detail. To approach this question, we have examined the dendritic cell populations that can be obtained from the blood of HIV-1 infected individuals. We studied 13 asymptomatic patients, who spanned a wide range of plasma viremia and CD4 counts. We show here that sizeable numbers of mature dendritic cells can be generated from nonproliferating progenitors in the blood of HIV + patients using a recently developed approach. The procedure involves two steps. The first step or 'priming' phase is a 7 day culture of T-cell depleted mononuclear cells in medium supplemented with GM-CSF and IL-4. The second step or 'differentiation' phase requires the exposure to monocyte conditioned medium. The yields of DCs from HIV + individuals were comparable to normal blood donors, 0.4 - 3 x 10(6) mature dendritic cells from 50 ml of blood. Strong APC function was evident for both the proliferation of allogeneic T-cells in the MLR, and the generation by syngeneic T-cells of class I restricted, CTL responses to influenza virus. A panel of dendritic cell restricted markers are expressed, including CD83, p55, and perinuclear CD68. By semi-quantitative PCR analysis, the cytokine derived cells did not express HIV-1 DNA. We suggest that these blood derived dendritic cells will be effective for studies of immune responses to HIV-1 antigens and may be considered as adjuvants for active immunotherapy
— id: 33207, year: 1999, vol: 66, page: 121, stat: Journal Article,

Changes in frequency of HIV-1-specific cytotoxic T cell precursors and circulating effectors after combination antiretroviral therapy in children
Spiegel HM; DeFalcon E; Ogg GS; Larsson M; Beadle TJ; Tao P; McMichael AJ; Bhardwaj N; O'Callaghan C; Cox WI; Krasinski K; Pollack H; Borkowsky W; Nixon DF
1999 Aug;180(2):359-368, Journal of infectious diseases
Combination antiretroviral therapy has had a major role in reducing human immunodeficiency virus type 1 (HIV-1) plasma viral loads in HIV-1-infected adults but a variable effect in infants, in whom complete viral suppression appears to be less readily achieved. In adults, after the reduction in plasma viremia, there is a decrease in the numbers of circulating cytotoxic T cell (CTL) effectors and precursors in the majority of patients. This longitudinal study assessed the effect of combination drug therapy on the frequency of HIV-1-specific CTL responses in 8 HIV-1-infected children. Following treatment, the frequency of HIV-1-specific CTL responses initially increased, especially in children with incomplete viral suppression but with increasing CD4+ cell counts. In children with complete viral suppression, the frequency of HIV-1-specific CTL responses decreased, suggesting that viral replication is required to maintain CTL responses in the systemic circulation
— id: 14552, year: 1999, vol: 180, page: 359, stat: Journal Article,

Presentation of epstein-barr virus latency antigens to CD8(+), interferon-gamma-secreting, T lymphocytes
Subklewe M; Chahroudi A; Bickham K; Larsson M; Kurilla MG; Bhardwaj N; Steinman RM
1999 Dec;29(12):3995-4001, European journal of immunology
Epstein-Barr virus (EBV) infects more than 95 % of the human population and causes an asymptomatic life-long infection in the majority of EBV carriers. Cell-mediated immunity provides resistance to EBV, as demonstrated by the occurrence of EBV-induced post-transplant lymphoproliferative disease in immunosuppressed patients. Here we looked for IFN-gamma-producing T lymphocytes in the blood of healthy donors with a rapid enzyme-linked immunospot (ELISPOT) assay, comparing as antigen presenting cells monocytes and dendritic cells (DC) infected with recombinant vaccinia virus (rVV). We found a strong CD8(+) ELISPOT response to one or more of the EBNA 3A, 3B and 3C antigens in the PBMC from 14 / 18 donors. The sensitivity of the overnight ELISPOT assay was increased using DC as antigen-presenting cells, including 3 / 3 individuals who lacked ELISPOT in PBMC. In addition, DC could markedly expand EBV-specific spots after a 7-day culture. In a smaller number of donors, we documented recognition of the subdominant LMP 1, LMP 2 and EBNA 1 antigens that are expressed in a variety of EBV-associated malignancies. Therefore our data provide more evidence for the efficacy of DC in eliciting rapid responses to EBV latency antigens in circulating CD8(+) T cells
— id: 33201, year: 1999, vol: 29, page: 3995, stat: Journal Article,

Induction of Epstein-Barr virus-specific cytotoxic T-lymphocyte responses using dendritic cells pulsed with EBNA-3A peptides or UV-inactivated, recombinant EBNA-3A vaccinia virus
Subklewe M; Chahroudi A; Schmaljohn A; Kurilla MG; Bhardwaj N; Steinman RM
1999 Aug 15;94(4):1372-1381, Blood
Cell-mediated immunity, especially the cytotoxic T lymphocyte (CTL), provides resistance to Epstein-Barr virus (EBV), as is demonstrated by the occurrence of posttransplant lymphoproliferative disease in immunosuppressed patients. We set out to use dendritic cells (DCs) to elicit anti-EBV-specific CTLs in culture. In unselected, HLA-B8(+) donors, monocyte-derived mature DCs were pulsed with the HLA-B8-restricted EBNA-3A peptide, FLRGRAYGL, and added to autologous T cells for 7 days at a DC:T ratio of 1:5 to 1:60. The cultured cells specifically lysed EBNA-3A peptide-pulsed, HLA-B8(+), B-lymphoblastoid cell lines in a 5-hour (51)Cr-release assay. The generation of CTLs did not require the addition of interleukin-2. In comparison, monocytes were weak antigen-presenting cells. DCs were then infected with recombinant vaccinia-EBNA-3A. Vaccinia infection significantly decreased the viability of immature DCs after 3 days of culture (to 25% to 45%) but had a smaller effect on mature DC recovery (40% to 70%). To decrease these cytopathic effects and to expand the potential use of vaccinia vectors for DC therapy in immunocompromised patients, we successfully used psoralen and UV-inactivated virus. Mature DCs pulsed with either live or inactivated vaccinia EBNA-3A virus could elicit strong EBNA-3A-specific CTLs. Therefore, mature DCs are powerful stimulators of EBV-specific CTLs and their major histocompatibility complex class I products can even be charged with UV-inactivated recombinant vaccinia
— id: 33204, year: 1999, vol: 94, page: 1372, stat: Journal Article,

Tumor-specific killer cells in paraneoplastic cerebellar degeneration
Albert ML; Darnell JC; Bender A; Francisco LM; Bhardwaj N; Darnell RB
1998 Nov;4(11):1321-1324, Nature medicine
Models for immune-mediated tumor regression in mice have defined an essential role for cytotoxic T lymphocytes (CTLs); however, naturally occurring tumor immunity in humans is poorly understood. Patients with paraneoplastic cerebellar degeneration (PCD) provide an opportunity to explore the mechanisms underlying tumor immunity to breast and ovarian cancer. Although tumor immunity and autoimmune neuronal degeneration in PCD correlates with a specific antibody response to the tumor and brain antigen cdr2, this humoral response has not been shown to be pathogenic. Here we present evidence for a specific cellular immune response in PCD patients. We have detected expanded populations of MHC class I-restricted cdr2-specific CTLs in the blood of 3/3 HLA-A2.1+ PCD patients, providing the first description, to our knowledge, of tumor-specific CTLs using primary human cells in a simple recall assay. Cross-presentation of apoptotic cells by dendritic cells also led to a potent CTL response. These results indicate a model whereby immature dendritic cells that engulf apoptotic tumor cells can mature and migrate to draining lymph organs where they could induce a CTL response to tissue-restricted antigens. In PCD, peripheral activation of cdr2-specific CTLs is likely to contribute to the subsequent development of the autoimmune neuronal degeneration
— id: 33210, year: 1998, vol: 4, page: 1321, stat: Journal Article,

Immature dendritic cells phagocytose apoptotic cells via alphavbeta5 and CD36, and cross-present antigens to cytotoxic T lymphocytes
Albert ML; Pearce SF; Francisco LM; Sauter B; Roy P; Silverstein RL; Bhardwaj N
1998 Oct 5;188(7):1359-1368, Journal of experimental medicine
Dendritic cells, but not macrophages, efficiently phagocytose apoptotic cells and cross-present viral, tumor, and self-antigens to CD8(+) T cells. This in vitro pathway corresponds to the in vivo phenomena of cross-priming and cross-tolerance. Here, we demonstrate that phagocytosis of apoptotic cells is restricted to the immature stage of dendritic cell (DC) development, and that this process is accompanied by the expression of a unique profile of receptors, in particular the alphavbeta5 integrin and CD36. Upon maturation, these receptors and, in turn, the phagocytic capacity of DCs, are downmodulated. Macrophages engulf apoptotic cells more efficiently than DCs, and although they express many receptors that mediate this uptake, they lack the alphavbeta5 integrin. Furthermore, in contrast to DCs, macrophages fail to cross-present antigenic material contained within the engulfed apoptotic cells. Thus, DCs use unique pathways for the phagocytosis, processing, and presentation of antigen derived from apoptotic cells on class I major histocompatibility complex. We suggest that the alphavbeta5 integrin plays a critical role in the trafficking of exogenous antigen by immature DCs in this cross-priming pathway
— id: 33211, year: 1998, vol: 188, page: 1359, stat: Journal Article,

Dendritic cells acquire antigen from apoptotic cells and induce class I-restricted CTLs
Albert ML; Sauter B; Bhardwaj N
1998 Mar 5;392(6671):86-89, Nature
CD8+ cytotoxic T lymphocytes (CTLs) mediate resistance to infectious agents and tumours. Classically, CTLs recognize antigens that are localized in the cytoplasm of target cells, processed and presented as peptide complexes with class I molecules of the major histocompatibility complex (MHC). However, there is evidence for an exogenous pathway whereby antigens that are not expected to gain access to the cytoplasm are presented on MHC class I molecules. The most dramatic example is the in vivo phenomenon of cross-priming: antigens from donor cells are acquired by bone-marrow-derived host antigen-presenting cells (APCs) and presented on MHC class I molecules. Two unanswered questions concern the identity of this bone-marrow-derived cell and how such antigens are acquired. Here we show that human dendritic cells, but not macrophages, efficiently present antigen derived from apoptotic cells, stimulating class I-restricted CD8+ CTLs. Our findings suggest a mechanism by which potent APCs acquire antigens from tumours, transplants, infected cells, or even self-tissue, for stimulation or tolerization of CTLs
— id: 33213, year: 1998, vol: 392, page: 86, stat: Journal Article,

Utility of transesophageal echocardiography during port-access minimally invasive cardiac surgery
Applebaum RM; Cutler WM; Bhardwaj N; Colvin SB; Galloway AC; Ribakove GH; Grossi EA; Schwartz DS; Anderson RV; Tunick PA; Kronzon I
1998 Jul 15;82(2):183-188, American journal of cardiology
In this study, we sought to determine the use of transesophageal echocardiography (TEE) as the primary imaging technique to assist in the placement of endovascular catheters during minimally invasive, port-access cardiac surgery. The recent development of endovascular catheters that are placed via the femoral artery and vein has enabled patients to be placed on cardiopulmonary bypass without the need for direct visualization of the heart or great vessels via sternotomy. This has allowed cardiac surgery to be performed through smaller thoracotomy incisions. Placement of these catheters has previously been performed with fluoroscopic guidance, which has major imaging limitations. Thirty-six patients underwent port-access cardiac surgery at our institution during the study period. All patients underwent intraoperative TEE. We used TEE to visualize the coronary sinus os, right atrium and superior vena cava, and thoracic aorta to assist with placement of the coronary sinus catheter, venous cannula, and endoaortic clamp. Twenty patients underwent mitral valve surgery, 14 patients coronary artery bypass grafting, 1 patient aortic valve replacement, and 1 patient repair of an atrial septal defect by the port-access approach. TEE was able to adequately visualize the cardiac structures and assist in the placement of the endovascular catheters in all patients. Fluoroscopy was only helpful as an aid to TEE for placement of the coronary sinus catheter. TEE is an excellent imaging modality for the proper placement of these new endovascular catheters, obviating the need for fluoroscopy, except to be on standby and for placement of the coronary sinus catheter
— id: 12089, year: 1998, vol: 82, page: 183, stat: Journal Article,

Transesophageal echocardiography as the guiding imaging technique during port access minimally invasive cardiac surgery
Applebaum, RM; Cutler, WM; Bhardwaj, N; Colvin, SB; Galloway, AC; Ribakove, GH; Grossi, EA; Schwartz, DS; Anderson, RV; Tunick, PA; Kronzon, I
1998 FEB ;31(2):87A-87A, Journal of the American College of Cardiology
— id: 33432, year: 1998, vol: 31, page: 87A, stat: Journal Article,

The distinctive features of influenza virus infection of dendritic cells
Bender A; Albert M; Reddy A; Feldman M; Sauter B; Kaplan G; Hellman W; Bhardwaj N
1998 Mar;198(5):552-567, Immunobiology
CD8+ cytolytic T lymphocytes (CTLs) are considered to be critical mediators for resistance to influenza virus infection. We have previously demonstrated that dendritic cells are potent antigen presenting cells in the development of anti-influenza CTLs. Here we identify distinctive features of the interaction of influenza virus with dendritic cells. Exposure of dendritic cells to influenza virus at MOIs of 2-4:1 leads to > 90% infection, as manifested by the expression of the viral proteins HA and NS1. The infection is non-toxic as viral protein expression is sustained for > 2 days with retention of viability, but little infectious virus is produced. Substantial induction of the anti-viral cytokine IFN-alpha also occurs. Influenza infection of macrophages also results in viral protein expression in a majority of cells, and synthesis of IFN-alpha. In contrast to dendritic cells, macrophages display evidence of apoptosis within 10-12 hours, and the majority of cells die within 24-36 hours. During this interval macrophages synthesize > 10-fold higher levels of virus than dendritic cells. Infected dendritic cells but not macrophages, can induce substantial CTL responses from purified blood CD8+ T cells in the absence of exogenous cytokines such as IL-2. Low levels of infection (MOIs of 0.02) are sufficient to generate potent CTL responses. Influenza virus expressing non-cleaved HA does not elicit CTLs indicating that virus must access the cytoplasm of dendritic cells to utilize traditional class I processing pathways. These observations indicate that DCs are distinct in their handling of influenza virus and for the induction of anti-viral immunity
— id: 33212, year: 1998, vol: 198, page: 552, stat: Journal Article,

Neutralizing monoclonal antibodies block human immunodeficiency virus type 1 infection of dendritic cells and transmission to T cells
Frankel SS; Steinman RM; Michael NL; Kim SR; Bhardwaj N; Pope M; Louder MK; Ehrenberg PK; Parren PW; Burton DR; Katinger H; VanCott TC; Robb ML; Birx DL; Mascola JR
1998 Dec;72(12):9788-9794, Journal of virology
Prevention of the initial infection of mucosal dendritic cells (DC) and interruption of the subsequent transmission of HIV-1 from DC to T cells are likely to be important attributes of an effective human immunodeficiency virus type 1 (HIV-1) vaccine. While anti-HIV-1 neutralizing antibodies have been difficult to elicit by immunization, there are several human monoclonal antibodies (MAbs) that effectively neutralize virus infection of activated T cells. We investigated the ability of three well-characterized neutralizing MAbs (IgG1b12, 2F5, and 2G12) to block HIV-1 infection of human DC. DC were generated from CD14(+) blood cells or obtained from cadaveric human skin. The MAbs prevented viral entry into purified DC and the ensuing productive infection in DC/T-cell cultures. When DC were first pulsed with HIV-1, MAbs blocked the subsequent transmission to unstimulated CD3(+) T cells. Thus, neutralizing antibodies can block HIV-1 infection of DC and the cell-to-cell transmission of virus from infected DC to T cells. These data suggest that neutralizing antibodies could interrupt the initial events associated with mucosal transmission and regional spread of HIV-1
— id: 33209, year: 1998, vol: 72, page: 9788, stat: Journal Article,

Efficient presentation of phagocytosed cellular fragments on the major histocompatibility complex class II products of dendritic cells
Inaba K; Turley S; Yamaide F; Iyoda T; Mahnke K; Inaba M; Pack M; Subklewe M; Sauter B; Sheff D; Albert M; Bhardwaj N; Mellman I; Steinman RM
1998 Dec 7;188(11):2163-2173, Journal of experimental medicine
Cells from the bone marrow can present peptides that are derived from tumors, transplants, and self-tissues. Here we describe how dendritic cells (DCs) process phagocytosed cell fragments onto major histocompatibility complex (MHC) class II products with unusual efficacy. This was monitored with the Y-Ae monoclonal antibody that is specific for complexes of I-Ab MHC class II presenting a peptide derived from I-Ealpha. When immature DCs from I-Ab mice were cultured for 5-20 h with activated I-E+ B blasts, either necrotic or apoptotic, the DCs produced the epitope recognized by the Y-Ae monoclonal antibody and stimulated T cells reactive with the same MHC-peptide complex. Antigen transfer was also observed with human cells, where human histocompatibility leukocyte antigen (HLA)-DRalpha includes the same peptide sequence as mouse I-Ealpha. Antigen transfer was preceded by uptake of B cell fragments into MHC class II-rich compartments. Quantitation of the amount of I-E protein in the B cell fragments revealed that phagocytosed I-E was 1-10 thousand times more efficient in generating MHC-peptide complexes than preprocessed I-E peptide. When we injected different I-E- bearing cells into C57BL/6 mice to look for a similar phenomenon in vivo, we found that short-lived migrating DCs could be processed by most of the recipient DCs in the lymph node. The consequence of antigen transfer from migratory DCs to lymph node DCs is not yet known, but we suggest that in the steady state, i.e., in the absence of stimuli for DC maturation, this transfer leads to peripheral tolerance of the T cell repertoire to self
— id: 33208, year: 1998, vol: 188, page: 2163, stat: Journal Article,

Dendritic cells as immunogens for human CTL responses
Bender A; Sapp M; Feldman M; Reddy A; Seder R; Schuler G; Steinman RM; Bhardwaj N
1997 ;417(6):383-387, Advances in experimental medicine & biology
The cellular requirements for generating potent human CD8+ CTLs to influenza A virus in vitro have been defined. Furthermore, we have developed improved methods for generating large numbers of DCs from non-proliferating progenitors. These developments have enabled the design of new strategies to elicit CTLs in vivo. For example, together with IL-12, antigen-pulsed DCs may be a useful approach for boosting CTL responses against infectious agents and malignancies. Our results also reopen the potential use of inactivated virus preparations as immunogens for CTL responses
— id: 33217, year: 1997, vol: 417, page: 383, stat: Journal Article,

Interactions of viruses with dendritic cells: a double-edged sword
Bhardwaj N
1997 Sep 15;186(6):795-799, Journal of experimental medicine
— id: 33216, year: 1997, vol: 186, page: 795, stat: Journal Article,

Generation of monocyte-derived dendritic cells from precursors in rhesus macaque blood
O'Doherty U; Ignatius R; Bhardwaj N; Pope M
1997 Sep 24;207(2):185-194, Journal of immunological methods
While the dendritic cells (DCs) of mouse and man have been extensively studied, until recently those of non-human primates remained poorly characterized. We present a method for generating large numbers of DCs from precursors in rhesus macaque blood, based on techniques developed for human blood. For 7 days, a T cell-depleted population of mononuclear cells was cultured in 1% human plasma with GM-CSF and IL-4, both to initiate DC differentiation and to inhibit macrophage development. On day 7, 50% of the culture medium was replaced with a monocyte-conditioned medium (MCM), which is required for the final maturation of the DCs into potent stimulators of the allogeneic MLR. Between 0.5 and 1.0 x 10(6) DCs can be generated from 20 ml of rhesus macaque blood. We compared these cytokine-generated DCs to the adherent macrophages present in the same cultures. Cytokine-generated DCs were considerably more potent at stimulating allogeneic T cells than adherent macrophages. Furthermore, the DCs had a distinct morphology and phenotype, with long processes, high levels of p55, and a characteristic perinuclear collection of intracellular CD68. In contrast, adherent macrophages expressed very low levels of p55, and high diffuse levels of CD68. Macaque DCs generated by this method may be useful in vaccine development and for studies of SIV pathogenesis
— id: 33214, year: 1997, vol: 207, page: 185, stat: Journal Article,

A monocyte conditioned medium is more effective than defined cytokines in mediating the terminal maturation of human dendritic cells
Reddy A; Sapp M; Feldman M; Subklewe M; Bhardwaj N
1997 Nov 1;90(9):3640-3646, Blood
Mature human dendritic cells can be generated in substantial numbers from nonproliferating progenitors in human blood using a two-step protocol. T cell-depleted mononuclear cells are first cultured with granulocyte-macrophage colony-stimulating factor and interleukin-4 (IL-4) and then exposed to monocyte conditioned medium (MCM). The dendritic cells generated using this approach are rendered terminally mature and are the most potent antigen presenting cells identified to date in humans. We sought to characterize factors in MCM that induce the terminal differentiation of dendritic cells. MCM contained substantial, although varying, quantities of several factors including tumor necrosis factor-alpha, IL-1beta, IL-6, and interferon-alpha. However, none of the four factors, individually or in various combinations, could fully substitute for the MCM to generate irreversibly differentiated dendritic cells. The yields, percentage of cells expressing the mature phase marker CD83, and mixed leukocyte reaction-stimulatory function were lower when defined cytokines were used in the place of MCM. Therefore, the full maturation of dendritic cells, because it entails changes in many known cell and molecular properties, requires a number of different cytokines that are released in tandem from appropriately stimulated monocytes. We propose that MCM-matured dendritic cells will be the most effective adjuvants for immunotherapy in vivo
— id: 33215, year: 1997, vol: 90, page: 3640, stat: Journal Article,

Improved methods for the generation of dendritic cells from nonproliferating progenitors in human blood
Bender A; Sapp M; Schuler G; Steinman RM; Bhardwaj N
1996 Sep 27;196(2):121-135, Journal of immunological methods
We have investigated an improved method for generating sizable numbers of mature dendritic cells from nonproliferating progenitors in human blood. The procedure uses 1% human plasma in the place of 10% fetal calf serum and involves two steps. The first step or 'priming' phase is a 6-7 day culture of T cell depleted mononuclear cells in medium supplemented with GM-CSF and IL-4. The second step or 'differentiation' phase requires the exposure to macrophage conditioned medium. This medium cannot be replaced by several known cytokines such as TNF-alpha, IL-1, IL-6, IL-12 and IL-15, and cannot be inhibited with neutralizing antibodies to IL-1, TNF-alpha, IL-6 or IL-12 alone, or in combination. Using this two-step approach, we obtain substantial yields. About 1-3 x 10(6) mature dendritic cells are generated from 40 ml of blood vs. < 0.1 x 10(6) from noncytokine treated blood. The dendritic cells derive from progenitors found primarily in a radioresistant population of CD14+ and adherent blood mononuclear cells and have all the features of mature cells. They include a stellate cell shape, nonadherence to plastic, and very strong T cell stimulatory activity. Strong APC function was evident for both the proliferation of allogeneic T cells in the MLR, and the generation by syngeneic T cells of class I restricted, CTL responses to influenza virus. A panel of dendritic cell restricted markers is also expressed, including CD83, p55, and perinuclear CD68. All of these dendritic cell properties are retained for at least 3 days when the cytokines are removed, suggesting that these populations are stable and terminally differentiated. We suggest that these cells will be effective in vivo as adjuvants for active immunotherapy
— id: 33219, year: 1996, vol: 196, page: 121, stat: Journal Article,

IL-12 in conjunction with dendritic cells enhances antiviral CD8+ CTL responses in vitro
Bhardwaj N; Seder RA; Reddy A; Feldman MV
1996 Aug 1;98(3):715-722, Journal of clinical investigation
CD8+ cytolytic T lymphocytes (CTLs) are important mediators for resistance to infections and malignant diseases. IL-12 enhances proliferative and cytolytic responses by killer cells, but its function in the generation of human antiviral CD8+ T cell responses has not been defined. We therefore evaluated the role of IL-12 in the generation of CTLs to influenza-infected dendritic cells. IL-12 was not detectable in supernatants of infected-dendritic cells, or during CTL generation. Furthermore, anti-IL-12 antibody did not block CTL generation. However, exogenous IL-12 (30-300 pg/ml) enhanced CD8+ T cell proliferative and cytolytic responses. The effect was greatest in individuals with weak reactivity to influenza virus or at antigen-presenting cell (APC):T cell ratios of 1:100 or less. IL-12 augmented interferon-gamma production during CTL generation. The CTL enhancing effects of the cytokine, however, could not be blocked by neutralizing anti-interferon-gamma antibody. Together with IL-12, antigen-pulsed dendritic cells may be a useful approach for boosting CTL responses against infectious agents and malignancies
— id: 33221, year: 1996, vol: 98, page: 715, stat: Journal Article,

Efficient interaction of HIV-1 with purified dendritic cells via multiple chemokine coreceptors
Granelli-Piperno A; Moser B; Pope M; Chen D; Wei Y; Isdell F; O'Doherty U; Paxton W; Koup R; Mojsov S; Bhardwaj N; Clark-Lewis I; Baggiolini M; Steinman RM
1996 Dec 1;184(6):2433-2438, Journal of experimental medicine
HIV-1 actively replicates in dendritic cell (DC)-T cell cocultures, but it has been difficult to demonstrate substantial infection of purified mature DCs. We now find that HIV-1 begins reverse transcription much more efficiently in DCs than T cells, even though T cells have higher levels of CD4 and gp120 binding. DCs isolated from skin or from blood precursors behave similarly. Several M-tropic strains and the T-tropic strain IIIB enter DCs efficiently, as assessed by the progressive formation of the early products of reverse transcription after a 90-min virus pulse at 37 degrees C. However, few late gag-containing sequences are detected, so that active viral replication does not occur. The formation of these early transcripts seems to follow entry of HIV-1, rather than binding of virions that contain viral DNA. Early transcripts are scarce if DCs are exposed to virus on ice for 4 h, or for 90 min at 37 degrees C, conditions which allow virus binding. Also the early transcripts once formed are insensitive to trypsin. The entry of a M-tropic isolates is blocked by the chemokine RANTES, and the entry of IIIB by SDF-1. RANTES interacts with CCR5 and SDF-1 with CXCR4 receptors. Entry of M-tropic but not T-tropic virus is ablated in DCs from individuals who lack a functional CCR5 receptor. DCs express more CCR5 and CXCR4 mRNA than T cells. Therefore, while HIV-1 does not replicate efficiently in mature DCs, viral entry can be active and can be blocked by chemokines that act on known receptors for M- and T-tropic virus
— id: 33218, year: 1996, vol: 184, page: 2433, stat: Journal Article,

Allelic variants of human TCR BV17S1 defined by restriction fragment length polymorphism, single strand conformation polymorphism, and amplification refractory mutation system analyses
Li Y; Sun GR; Zheng Q; Yoo DH; Bhardwaj N; Posnett DN; Crow MK; Friedman SM
1996 Sep 1;49(2):85-95, Human immunology
Several human TCR BV gene subfamilies, including BV3, BV14, and BV17S1, are single member genes but are overutilized among activated CD4+ synovial T cells in the rheumatoid arthritis (RA). To define the role of these TCR BV genes in the pathogenesis of disease, it is critical to characterize the genomic organization and the allelic variations of these genes. In this study we describe allelic variations of BV17S1 defined by restriction fragment length polymorphism (RFLP), single strand conformation polymorphism (SSCP), and amplification refractory mutation system (ARMS) analyses. A single nucleotide replacement (C/T) results in an amino acid substitution (F/L) in the leader and distinguishes BV17S1*1 from BV17S1*2. This nucleotide substitution was found to create a BsmAI restriction enzyme recognition site in BV17S1*2. Therefore genotypic analyses can be performed either by the SSCP or RFLP method. The analyses of 75 unrelated individuals show that the frequency for allele BV17S1*1 is 52.7% and for allele BV17S1*2 is 47.3%. Both alleles are functionally expressed and are distributed within CD4+/CD8+ T cell subsets. Another point mutation in the CDR2 region of BV17S1, which results in the amino acid replacement of Gln by His, originally identified form a cDNA clone, has now been confirmed as an allele by ARMS analysis using genomic DNA preparations and designated to as BV17S1*3. Screening of this CDR2 related variant among normal populations indicates that this is a rare allele (1 of 75). Although this variant may be of functional significance, the genotypic analysis and functional studies are difficult due to the low frequency of BV17S1*3. In an attempt to define a correlation between BV17S1 allelic usage and susceptibility to RA, the germline distribution of BV17S1 alleles *1 and *2 has been examined in a small number of RA patients and no skewed usage has been identified
— id: 33220, year: 1996, vol: 49, page: 85, stat: Journal Article,

Inactivated influenza virus, when presented on dendritic cells, elicits human CD8+ cytolytic T cell responses
Bender A; Bui LK; Feldman MA; Larsson M; Bhardwaj N
1995 Dec 1;182(6):1663-1671, Journal of experimental medicine
Inactivated or subunit virus preparations have been excellent vaccines for inducing antibody responses. Generation of cytolytic T cell responses, however, is thought to require replicating virus, primarily to provide sufficiently large amounts of cytoplasmic proteins for processing and presentation on major histocompatibility complex class I molecules by antigen-presenting cells. Potent human CD8+ cytolytic T cell responses to live replicating influenza A virus are generated when dendritic cells are used as the antigen-presenting cells. Here, we demonstrate that dendritic cells pulsed with poorly replicating, heat- or ultraviolet-inactivated influenza virus, induce equally strong CD8+ cytolytic T lymphocyte responses. The cytotoxic T lymphocytes are generated in the apparent absence of CD4+ helper cells or exogenous cytokines. Active viral protein synthesis is not required to charge class I molecules on dendritic cells. When pulsed with inactivated virus, < 1% of dendritic cells express nonstructural protein 1, which is only synthesized in the infectious cycle. To be optimally effective, however, the inactivated virus must retain its fusogenic activity, and presumably access the cytoplasm of dendritic cells. The data indicate, therefore, that dendritic cells require only small amounts of viral protein to charge class I molecules, most likely via traditional class I processing pathways. These results reopen the potential use of inactivated virus preparations as immunogens for cytotoxic T lymphocyte responses
— id: 33222, year: 1995, vol: 182, page: 1663, stat: Journal Article,

Stimulation of human anti-viral CD8+ cytolytic T lymphocytes by dendritic cells
Bhardwaj N; Bender A; Gonzalez N; Bui LK; Garrett MC; Steinman RM
1995 ;378(9):375-379, Advances in experimental medicine & biology
— id: 33223, year: 1995, vol: 378, page: 375, stat: Journal Article,

Influenza virus-infected dendritic cells stimulate strong proliferative and cytolytic responses from human CD8+ T cells
Bhardwaj N; Bender A; Gonzalez N; Bui LK; Garrett MC; Steinman RM
1994 Aug;94(2):797-807, Journal of clinical investigation
Antigen-specific, CD8+, cytolytic T lymphocytes (CTLs) could potentially provide resistance to several infectious and malignant diseases. However, the cellular requirements for the generation of specific CTLs in human lymphocyte cultures are not well defined, and repetitive stimulation with antigen is often required. We find that strong CD8+ CTL responses to influenza virus can be generated from freshly isolated blood T cells, as long as dendritic cells are used as antigen presenting cells (APCs). Small numbers of dendritic cells (APC:T cell ratio of 1:50-1:100) induce these CTL responses from most donors in 7 d of culture, but monocytes are weak or inactive. Whereas both dendritic cells and monocytes are infected with influenza virus, the former serve as effective APCs for the induction of CD8+ T cells while the latter act as targets for the CTLs that are induced. The strong CD8+ response to influenza virus-infected dendritic cells is accompanied by extensive proliferation of the CD8+ T cells, but the response can develop in the apparent absence of CD4+ helpers or exogenous lymphokines. CD4+ influenza virus-specific CTLs can also be induced by dendritic cells, but the cultures initially must be depleted of CD8+ cells. These findings should make it possible to use dendritic cells to generate human, antigen-specific, CD8+ CTLs to other targets. The results illustrate the principle that efficient T cell-mediated responses develop in two stages: an afferent limb in which dendritic cells are specialized APCs and an efferent limb in which the primed T cells carry out an immune response to many types of presenting cells
— id: 33224, year: 1994, vol: 94, page: 797, stat: Journal Article,

Human blood contains two subsets of dendritic cells, one immunologically mature and the other immature
O'Doherty U; Peng M; Gezelter S; Swiggard WJ; Betjes M; Bhardwaj N; Steinman RM
1994 Jul;82(3):487-493, Immunology
Two subsets of dendritic cells, differing in T-cell stimulatory function, have been purified directly from human blood. Both subsets are positive for major histocompatibility complex (MHC) class II expression and negative for lineage-specific antigens (e.g. CD3, CD14, CD16, CD19 negative), but are separated by exploiting differences in expression of the beta 2-integrin, CD11c. The CD11c-negative subset is functionally immature, requiring monocyte-derived cytokines to develop into typical dendritic cells. The CD11c-positive subset has potent T-cell stimulating activity and expresses the activation antigen CD45RO, unlike its immature counterpart. However, these mature cells only develop typical dendritic morphology and high levels of MHC proteins and adhesins after a period of culture independent of exogenous cytokines. Although the freshly isolated mature dendritic cells resemble monocytes in cytospin preparations, the former lack CD14 and have a much stronger primary T-cell stimulatory capacity. We hypothesize that the CD11c-negative immature cells are marrow-derived precursors to tissue dendritic cells, such as epidermal Langerhans' cells, while the CD11c-positive cells are derived from tissues where they have been activated by antigen, and are en route to the spleen or lymph nodes to stimulate T-cell responses there
— id: 33225, year: 1994, vol: 82, page: 487, stat: Journal Article,

Small amounts of superantigen, when presented on dendritic cells, are sufficient to initiate T cell responses
Bhardwaj N; Young JW; Nisanian AJ; Baggers J; Steinman RM
1993 Aug 1;178(2):633-642, Journal of experimental medicine
Dendritic cells are potent antigen-presenting cells for several primary immune responses and therefore provide an opportunity for evaluating the amounts of cell-associated antigens that are required for inducing T cell-mediated immunity. Because dendritic cells express very high levels of major histocompatibility complex (MHC) class II products, it has been assumed that high levels of ligands bound to MHC products ('signal one') are needed to stimulate quiescent T cells. Here we describe quantitative aspects underlying the stimulation of human blood T cells by a bacterial superantigen, staphylococcal enterotoxin A (SEA). The advantages of superantigens for quantitative studies of signal one are that these ligands: (a) engage MHC class II and the T cell receptor but do not require processing; (b) are efficiently presented to large numbers of quiescent T cells; and (c) can be pulsed onto dendritic cells before their application to T cells. Thus one can relate amounts of dendritic cell-associated SEA to subsequent lymphocyte stimulation. Using radioiodinated SEA, we noted that dendritic cells can bind 30-200 times more superantigen than B cells and monocytes. Nevertheless, this high SEA binding does not underlie the strong potency of dendritic cells to present antigen to T cells. Dendritic cells can sensitize quiescent T cells, isolated using monoclonals to appropriate CD45R epitopes, after a pulse of SEA that occupies a maximum of 0.1% of surface MHC class II molecules. This corresponds to an average of 2,000 molecules per dendritic cell. At these low doses of bound SEA, monoclonal antibodies to CD3, CD4, and CD28 almost completely block T cell proliferation. In addition to suggesting new roles for MHC class II on dendritic cells, especially the capture and retention of ligands at low external concentrations, the data reveal that primary T cells can generate a response to exceptionally low levels of signal one as long as these are delivered on dendritic cells
— id: 33228, year: 1993, vol: 178, page: 633, stat: Journal Article,

Dendritic cells freshly isolated from human blood express CD4 and mature into typical immunostimulatory dendritic cells after culture in monocyte-conditioned medium
O'Doherty U; Steinman RM; Peng M; Cameron PU; Gezelter S; Kopeloff I; Swiggard WJ; Pope M; Bhardwaj N
1993 Sep 1;178(3):1067-1076, Journal of experimental medicine
A procedure has been developed to isolate dendritic cells to a high degree of purity from fresh blood. Prior enrichment methods have relied upon an initial 1-2-d culture period. Purified fresh isolates lack the characteristic morphology, phenotype, and immunostimulatory function of dendritic cells. The purified cells have the appearance of medium sized lymphocytes and express substantial levels of CD4, but lack the T cell molecules CD3, CD8, and T cell receptor. When placed in culture, the cells mature in a manner resembling the previously described, cytokine-dependent maturation of epidermal dendritic cells (Langerhans cells). The cells enlarge and exhibit many cell processes, express much higher levels of major histocompatibility complex class II and a panel of accessory molecules for T cell activation, and become potent stimulators of the mixed leukocyte reaction. Among the many changes during this maturation process are a fall in CD4 and the appearance of high levels of B7/BB1, the costimulator for enhanced interleukin 2 production in T cells. These changes are not associated with cell proliferation, but are dependent upon the addition of monocyte-conditioned medium. We suggest that the freshly isolated CD4-positive blood dendritic cells are recent migrants from the bone marrow, and that subsequent maturation of the lineage occurs in tissues in situ upon appropriate exposure to cytokines
— id: 16505, year: 1993, vol: 178, page: 1067, stat: Journal Article,

Tolerizing mice to human leukocytes: a step toward the production of monoclonal antibodies specific for human dendritic cells
O'Doherty U; Swiggard WJ; Inaba K; Yamaguchi Y; Kopeloff I; Bhardwaj N; Steinman RM
1993 ;329(3):165-172, Advances in experimental medicine & biology
Despite several attempts to isolate a mAb specific for human dendritic cells, none currently exists. Recent attempts have utilized an improved dendritic cell purification method to prepare immunogens and a rapid two-color flow cytometric screening procedure that allows large numbers of hybridoma supernatants to be examined in each fusion. Yet these improvements have also failed, yielding only hybridomas that bind 'shared' antigens expressed by both dendritic cells and other leukocytes. Dendritic cells express many shared antigens, including CD45 [leukocyte common antigen], CD40, leukocyte [beta 2] integrins CD11a and CD11c, CD54 [ICAM-1], CD44 [Pgp-1], CD58 [LFA-3], and the B7/BB1 antigen. Therefore, we are attempting to bias the immune response toward rarer, dendritic cell-specific clones by tolerizing or immunosuppressing our animals to shared antigens. In one approach, adult mice held in barrier cages are injected with 'nondendritic' cells and cyclophosphamide [CP], in order to ablate responding 'nonspecific' B cell clones. Fifteen days after the last dose of CP, they are challenged with nondendritic cells. A week later they are bled, and serum antibody titers against nondendritic cells are determined by FACS, in order to demonstrate tolerance compared to controls injected with CP alone. In the second approach, neonatal mice are injected with human T lymphoblasts at birth, followed by boosting at 1 week. In adulthood, they are challenged sequentially with sheep erythrocytes [sRBC], then with T blasts, to demonstrate that they can respond to unrelated cells but not to tolerogenic cells. One week after each kind of challenge, mice are bled and serum antibody levels are determined for treated and sham-injected mice. When these two approaches were compared, CP led only to nonspecific immunosuppression, while neonatal injections produced selective, antigen-specific nonresponsiveness to the tolerizing T blasts
— id: 16506, year: 1993, vol: 329, page: 165, stat: Journal Article,

FRESH HUMAN BLOOD CONTAINS PRESUMPTIVE DENDRITIC CELLS THAT EXPRESS CD4
ODOHERTY, U; CAMERON, P; KOPELOFF, I; GEZELTER, S; PENG, M; POPE, M; BHARDWAJ, N; STEINMAN, RM
1993 MAR 29 ;43(4):35-35, Journal of cellular biochemistry
— id: 54211, year: 1993, vol: 43, page: 35, stat: Journal Article,

Sequestration from immune CD4+ T cells of mycobacteria growing in human macrophages
Pancholi P; Mirza A; Bhardwaj N; Steinman RM
1993 May 14;260(5110):984-986, Science
CD4+ helper T cells mediate resistance to tuberculosis, presumably by enhancing the antimicrobial activity of macrophages within which the Mycobacterium tuberculosis organism grows. A first step in resistance should be the presentation of mycobacterial antigens by macrophages to CD4+ T cells. However, when the antigenic stimulus is limited to organisms growing in human monocytes, the organisms become sequestered from immune CD4+ T cells. This block in presentation is selective for growing mycobacteria and not for other stimuli. Sequestration would allow replicating organisms to persist in infected individuals and may contribute to virulence
— id: 33229, year: 1993, vol: 260, page: 984, stat: Journal Article,

Presentation of mycobacterial antigens by human dendritic cells: lack of transfer from infected macrophages
Pancholi P; Mirza A; Schauf V; Steinman RM; Bhardwaj N
1993 Dec;61(12):5326-5332, Infection & immunity
When exposed to a challenge of 10 Mycobacterium bovis BCG cells per antigen-presenting cell, most human monocytes engulf several organisms. In contrast, blood dendritic cells which are potent antigen-presenting cells for several antigens are not detectably phagocytic for mycobacteria. We investigated the possibility that infected macrophages might regurgitate antigens for presentation by populations of human blood dendritic cells. Macrophages were infected with M. bovis BCG, mixed with uninfected dendritic cells, and added to immune T cells, either bulk T cells or cloned populations from BCG vaccinees or patients recovering from tuberculosis. The macrophages were from donors who were mismatched to the T cells so that transfer of antigen to major histocompatibility complex-matched dendritic cells could be evaluated. As we describe, there was no evidence for the transfer of mycobacterial antigens from macrophages to dendritic cells in a form that was stimulatory for the T cells
— id: 33226, year: 1993, vol: 61, page: 5326, stat: Journal Article,

Interaction of Mycoplasma arthritidis superantigen with human T cells
Posnett DN; Hodtsev AS; Kabak S; Friedman SM; Cole BC; Bhardwaj N
1993 Aug;17 Suppl 1(3):S170-S175, Clinical infectious diseases
To define the interaction site of T cell antigen receptors (TCRs) with the superantigen of Mycoplasma arthritidis (MAM), we characterized the TCRs of human MAM-reactive T cell lines and T cell clones. Most MAM-reactive T cells express the V beta 17 gene segment; however, other V beta may also be used and not all V beta 17+ TCRs are MAM-reactive. Alignment of MAM-reactive and MAM-nonreactive V beta sequences suggests that MAM does not interact with a single site encoded by a V beta segment but that several sites may be involved or that other TCR elements contribute to formation of the MAM recognition site
— id: 33227, year: 1993, vol: 17 Suppl 1, page: S170, stat: Journal Article,

Dendritic cells are potent antigen-presenting cells for microbial superantigens
Bhardwaj N; Friedman SM; Cole BC; Nisanian AJ
1992 Jan 1;175(1):267-273, Journal of experimental medicine
Dendritic cells are a small subset of human blood mononuclear cells that are potent stimulators of several T cell functions. Here we show they are 10-50-fold more potent than monocytes or B cells in inducing T cell responses to a panel of superantigens. Furthermore, dendritic cells can present femtomolar concentrations of superantigen to T cells even at numbers where other antigen-presenting cells (APCs) are inactive. Although dendritic cells express very high levels of the major histocompatibility complex products that are required to present superantigens, it is only necessary to pulse these APCs for 1 hour with picomolar levels of one superantigen, staphylococcal enterotoxin B, to maximally activate T cells. Our results suggest that very small amounts of superantigen will be immunogenic in vivo if presented on dendritic cells
— id: 33231, year: 1992, vol: 175, page: 267, stat: Journal Article,

Dendritic cells efficiently immunoselect mycobacterial-reactive T cells in human blood, including clonable antigen-reactive precursors
Pancholi P; Steinman RM; Bhardwaj N
1992 Jun;76(2):217-224, Immunology
Given the persistence of tuberculosis throughout the world, the delineation of mechanisms that lead to protective immunity to Mycobacterium tuberculosis is important. We have evaluated the presenting function of human dendritic cells for mycobacterial antigens, since these antigen-presenting cells (APC) are particularly effective in initiating antigen-specific T-cell responses. Dendritic cells from blood prove to be active APC for mycobacteria-specific proliferative responses by CD4+ T cells from bacillus Calmette-Guerin (BCG)-vaccinated individuals. In the first 24-48 hr of the response, dendritic cells that have been pulsed with mycobacterial antigens, including live BCG, effectively bind T cells forming discrete cell clusters. The clusters represent about 1% of the applied T cells. Clusters are highly enriched in mycobacterial reactivity while the non-clusters are depleted. Clustered T cells can be used as a starting point to expand antigen-specific cell lines. Mitogen and allogeneic feeder cells were used as APC to expand the mycobacterial-reactive lines, because the antigen-specific T cells had been preselected by virtue of their binding to antigen-pulsed dendritic cells. We discuss the advantages of obtaining antigen-reactive T cells by using dendritic cells as immunoadsorbents. These lines should help delineate the range of mycobacterial antigens and T-cell responses that participate in host responses to mycobacteria
— id: 33230, year: 1992, vol: 76, page: 217, stat: Journal Article,

An approach to isolating T cell lines that react to antigens presented on the surface of dendritic cells
Pancholi P; Steinman RM; Bhardwaj N
1991 Aug;85(2):349-356, Clinical & experimental immunology
We describe an approach that might be useful for identifying antigens on surfaces of antigen presenting cells. It is known that dendritic cells carry antigens in situ and are efficient at clustering antigen-specific T cells. Using the human mixed lymphocyte reaction (MLR) system, we have shown that alloreactive CD4+ T cells can be selected by their capacity to cluster with dendritic cells in the first 2 days of the MLR. Small numbers of clustered cells, 1-10/culture well, could then be expanded as antigen-specific lines in presence of either antigen or mitogen, sodium periodate. Few antigen-specific lines could be isolated from the nonclustered fraction. When T cell lines derived from the dendritic T cell clusters were maintained without antigen, i.e. using second party (syngeneic antigen-presenting cells (APC] or irrelevant antigen bearing APC, i.e. third-party (HLA-mismatched) stimulator cells plus mitogen, the T cells retained their specificity for the original stimulating alloantigen over the time course tested, several weeks to months. These findings show that by using dendritic cells as immunoadsorbents one can prepare antigen-specific cell lines and maintain the specificity of the lines without the need for adding exogeneous antigen during either immunoselection or cloning. We discuss the possible use of dendritic cells as a means for raising T cell lines and clones that recognize antigens being carried by APC and which might be pertinent to protective immunity and autoimmunity
— id: 33232, year: 1991, vol: 85, page: 349, stat: Journal Article,

Dendritic cells in human blood and synovial exudates
Freudenthal PS; Bhardwaj N
1990 ;6(2-3):103-116, International reviews of immunology
Dendritic cells from human blood and synovial exudates are distinct from other leukocytes and are homogeneous by several criteria. Morphologically, their most prominent feature is numerous veils. Phenotypically, dendritic cells lack the surface antigens that identify monocytes, T cells, B cells, and NK cells. Human dendritic cells strongly express class I and class II MHC products, and have a distinct array of integrin and adhesin molecules. In many systems, dendritic cells are potent stimulators of T cell function. In the allogeneic mixed leukocyte reaction, for example, dendritic cells are 30-100 times more efficient than other cells in presenting transplantation antigens, for the induction of DNA synthesis, cytokine release, and generation of cytotoxic T cells. In addition, dendritic cells can induce the long-term clonal growth of T lymphocytes. Although dendritic cells are a minor subpopulation in human blood, new isolation protocols are available that permit efficient isolation and enrichment to > 90%
— id: 33233, year: 1990, vol: 6, page: 103, stat: Journal Article,

Interleukin 1 production during accessory cell-dependent mitogenesis of T lymphocytes
Bhardwaj N; Lau LL; Friedman SM; Crow MK; Steinman RM
1989 Mar 1;169(3):1121-1136, Journal of experimental medicine
We have studied the control and significance of IL-1 production in human leukocyte cultures during accessory cell-dependent, T lymphocyte mitogenesis using sensitive bioassays and immunolabeling techniques. In primary antigen-dependent systems like the MLR, IL-1 production was not detected in accessory cells (monocytes, dendritic cells) or T cells, suggesting that it is not an early product in these responses. However, monocytes could be induced to make IL-1 after interacting with sensitized antigen-specific T cells. Both alloreactive T cell clones or freshly prepared lymphoblasts induced IL-1 provided the monocytes carried the HLA-DR antigens to which the T cells were initially sensitized. Even in these circumstances, dendritic cells and B cells failed to make IL-1. The mechanism whereby activated T cells induce IL-1 in monocytes was explored. Supernatants from cocultures of monocytes and T cells or several recombinant cytokines induced little or no IL-1. A more potent antigen independent pathway of IL-1 induction was identified. IL-1 could be induced in third-party HLA-DR nonspecific monocytes in cocultures of alloreactive T cell clones or blasts and HLA-DR-specific dendritic cells. The induction was factor independent since dendritic cells and T blasts placed in a chamber separate from third-party monocytes by a semipermeable membrane did not induce monocyte IL-1. These results suggest that a cell contact mechanism rather than an IL-1-inducing factor leads to IL-1 production. The role of IL-1 in T cell proliferation was tested with a polyclonal anti-IL-1 antibody. The antibody failed to block the proliferation of primary T cells, or alloreactive T cell clones and blasts stimulated with HLA-specific monocytes or dendritic cells, even though IL-1 in the medium was neutralized
— id: 33235, year: 1989, vol: 169, page: 1121, stat: Journal Article,

IL-6/IFN-beta 2 in synovial effusions of patients with rheumatoid arthritis and other arthritides. Identification of several isoforms and studies of cellular sources
Bhardwaj N; Santhanam U; Lau LL; Tatter SB; Ghrayeb J; Rivelis M; Steinman RM; Sehgal PB; May LT
1989 Oct 1;143(7):2153-2159, Journal of immunology
We have looked for IL-6, a cytokine that has immunomodulating and inflammation-associated activities, in joint exudates (fluid and mononuclear cells) from patients with rheumatoid arthritis and other arthritides using both biologic and biochemical assays. IL-6 was assessed by its ability to stimulate alpha 1-antichymotrypsin secretion from the human hepatoma cell line Hep3B clone 2, an activity which is blocked by an antiserum to Escherichia coli derived IL-6, and by the growth of the IL-6-dependent murine hybridoma 7TD1 cell line. IL-6 isoforms in synovial fluid were characterized by immunoaffinity chromatography followed by Western blotting. The presence of IL-1 in synovial fluids and its production by synovial fluid mononuclear cells was monitored by Western blotting and indirect immunofluorescence with polyclonal anti-IL-1 beta antisera. In an analysis of 30 effusions from 27 rheumatoid patients with acutely inflamed joints, abundant quantities of IL-6 (greater than 2 ng/ml) were detected in 23 by the alpha 1-antichymotrypsin bioassay. Several rheumatoid synovial fluids also had elevated IL-6 levels in the 7TD1 bioassay. Seven of nine nonrheumatoid effusions also contained high levels of IL-6 (greater than 2 ng/ml). No IL-1 (less than 0.25 ng/ml) could be detected by Western blotting in 10 rheumatoid effusions even though eight of these contained high levels of IL-6. The IL-6 activity could be neutralized with a rabbit antiserum to rIL-6. Multiple IL-6 isoforms (25, 30, 45 kDa) were present in two rheumatoid and one traumatic effusion studied. Fresh mononuclear cells isolated from various synovial effusions did not appear to make IL-6 constitutively, as no IL-6 could be detected in the media of cells cultured for 12 to 18 h after isolation. Similarly, there was no constitutive production of IL-1 by these cells. However, synovial fluid mononuclear cells could be induced to secrete both IL-6 and IL-1 after stimulation with LPS. The LPS-responsive cells were monocytes and not lymphocytes or dendritic cells. These findings suggest that IL-6 is involved in inflammatory joint disease. However, the primary cells synthesizing it may be located in the synovial lining instead of the joint exudate
— id: 33234, year: 1989, vol: 143, page: 2153, stat: Journal Article,

Multiple forms of IFN-beta 2/IL-6 in serum and body fluids during acute bacterial infection
Helfgott DC; Tatter SB; Santhanam U; Clarick RH; Bhardwaj N; May LT; Sehgal PB
1989 Feb 1;142(3):948-953, Journal of immunology
Many of the major alterations in plasma proteins characteristic of the hepatic acute phase response are regulated by IFN-beta 2/IL-6. Using a specific bioassay for IFN-beta 2/IL-6, which relies on the induction of the hepatic acute phase plasma protein alpha 1-antichymotrypsin in the human hepatoma cell line Hep3B clone 2 and its inhibition by anti-rIFN-beta 2/IL-6 antiserum, we have detected high levels of IFN-beta 2/IL-6 in the body fluids of patients with acute bacterial infections. Cerebrospinal fluid from four patients with acute bacterial meningitis (Streptococcus pneumoniae, Staphylococcus aureus, two cases of Listeria monocytogenes) all had high levels of IFN-beta 2/IL-6 (up to 500 ng/ml). Two of these patients with concomitant bacteremia had lower concentrations of IFN-beta 2/IL-6 in the serum (5 to 70 ng/ml). Three additional patients with Escherichia coli, Pseudomonas aeruginosa, and Neisseria meningitidis bacteremia had high levels of serum IFN-beta 2/IL-6, as did the ankle fluid of a patient with Streptococcus canis arthritis. Normal cerebrospinal fluid and serum had little detectable IFN-beta 2/IL-6. A combination of immunoaffinity chromatography and immunoblotting procedures were used to characterize the IFN-beta 2/IL-6 species present in a representative sampling of serum and cerebrospinal fluids. Multiple immunoreactive species of IFN-beta 2/IL-6 in the size range 23 to 30 kDa as well as immunoreactive complexes in the range 60 to 70 kDa were detected in human body fluids. This is the first demonstration that previous descriptions of heterogeneity in human IFN-beta 2/IL-6 species produced in cell culture correspond to observations in the infected host
— id: 33236, year: 1989, vol: 142, page: 948, stat: Journal Article,

Interferon-gamma and antibiotics fail to act synergistically to kill Legionella pneumophila in human monocytes
Bhardwaj N; Horwitz MA
1988 Jun;8(3):283-293, Journal of interferon research
Legionella pneumophila, the agent of Legionnaires' disease, is a gram-negative facultative intracellular bacterial pathogen that multiplies in human blood monocytes and alveolar macrophages. Interferon-gamma (IFN-gamma)-activated human monocytes inhibit the intracellular multiplication of L. pneumophila but fail to kill the organism. Similarly, erythromycin and rifampin, the drugs of choice in the treatment of Legionnaires' disease, inhibit the growth of L. pneumophila within monocytes without exerting a cidal effect. In this study, we examined the combined effects of IFN-gamma and antibiotics (erythromycin, rifampin, and clindamycin) to determine whether these independently acting agents would synergistically mediate the killing of intracellular L. pneumophila. Each agent alone or in combination was effective in inhibiting the intracellular multiplication of L. pneumophila. However, IFN-gamma and antibiotics together were unable to kill intracellular L. pneumophila, regardless of the sequence in which they were administered to monocytes. Like erythromycin and rifampin, clindamycin, which is highly concentrated in human alveolar macrophages, was capable of inhibiting the intracellular multiplication of L. pneumophila but failed to kill the bacteria in nonactivated or IFN-gamma-activated monocytes. These results demonstrate that intracellular L. pneumophila are highly resistant to the bactericidal effects of both activated monocytes and antibiotics, alone or in combination
— id: 33238, year: 1988, vol: 8, page: 283, stat: Journal Article,

Interleukin-1 production by mononuclear cells from rheumatoid synovial effusions
Bhardwaj N; Lau LL; Rivelis M; Steinman RM
1988 Jul;114(2):405-423, Cellular immunology
The polypeptide interleukin-1 (IL-1) is a cytokine that may mediate inflammation and connective tissue damage in rheumatoid arthritis (RA). We examined cytokine production by normal blood and by rheumatoid synovial mononuclear cells with sensitive (picomolar) assays. The assays were immunolabeling and immunoblotting with rabbit anti-IL-1 beta sera, and proliferation of the murine D10 cell line to IL-1. Little or no cytokine was detected in rheumatoid joint fluid or in exudate mononuclear cells from patients with acute rheumatoid flares. The mononuclear cells could be induced to make IL-1 upon stimulation with lipopolysaccharide (LPS). The responsive cells were monocytes, since all could be double-labeled with anti-IL-1 and the monocyte-specific CD14 antibody. More than 80% of the synovial fluid monocytes made IL-1 beta after 24 hr in 2 ng/ml LPS. Other agents failed to induce IL-1 from enriched populations of monocytes including interferon gamma (IFN-gamma), poly (I/C), phorbol myristate acetate (PMA), concanavalin A (Con A), phytohemagglutinin (PHA), and anti-CD3 antibodies. Relatively high levels of dendritic cells (DC) were present in RA effusions, but these did not produce IL-1 in response to any of the above stimuli. Blood dendritic cells also did not make IL-1, whereas blood monocytes responded comparably to synovial exudate cells. The data indicate that rheumatoid exudate monocytes make very little IL-1 during acute flares of arthritis and that this cytokine is primarily a macrophage rather than a dendritic cell product
— id: 33237, year: 1988, vol: 114, page: 405, stat: Journal Article,

Phosphorylation of secreted forms of human beta 2-interferon/hepatocyte stimulating factor/interleukin-6
May LT; Santhanam U; Tatter SB; Bhardwaj N; Ghrayeb J; Sehgal PB
1988 May 16;152(3):1144-1150, Biochemical & biophysical research communications
'Beta 2-Interferon/hepatocyte stimulating factor/interleukin-6' (IFN-beta 2) has emerged as a major mediator of the plasma protein response to tissue injury (the acute phase response) in addition to its numerous effects on cells of the immune system. Human fibroblasts and monocytes induced with tumor necrosis factor, interleukin-1, bacterial lipopolysaccharide (endotoxin) or virus infection secrete multiple forms of differentially glycosylated IFN-beta 2 polypeptides: at least a doublet of molecular mass approximately 25 kD and a triplet of mass approximately 30 kD. We report that immunoprecipitation analyses of medium from [32P]orthophosphate- labeled cultures of induced fibroblasts carried out using a rabbit polyclonal antibody to recombinant E. coli-derived human IFN-beta 2 reveal that the secreted gp23-25 and gp28-30 forms of IFN-beta 2 are phosphorylated. IFN-beta 2 gp23-25 secreted by induced monocytes is phosphorylated whereas the monocytic gp28-30 is poorly labeled with [32P]orthophosphate suggesting tissue-specific differences in IFN-beta 2 phosphorylation. Phosphoamino acid analyses indicate that all of the detected phosphate is in phosphoserine residues. Furthermore, IFN-beta 2 can be completely dephosphorylated by alkaline phosphatase (E.C. No. 3.1.3.1); thus all of the phosphate label is in readily accessible sites. These observations suggest the possibility that differential phosphorylation of IFN-beta 2 forms may be a mechanism to modulate its functions in a tissue-specific manner
— id: 33239, year: 1988, vol: 152, page: 1144, stat: Journal Article,

The sensitization phase of T-cell-mediated immunity
Steinman RM; Koide S; Witmer M; Crowley M; Bhardwaj N; Freudenthal P; Young J; Inaba K
1988 ;546(3):80-90, Annals of the New York Academy of Sciences
Many cell-mediated immune responses appear to develop in two phases: A sensitization phase in which unprimed or memory T cells interact with dendritic cells to become active lymphoblasts, and an effector phase in which the T lymphoblasts and other presenting cells interact to eliminate the antigen. Antigen presentation is essential to both phases. Here we review several features that are pertinent to the special sensitization role of dendritic cells. First, dendritic cells from lymphoid tissues, blood, and lymph (lymphoid dendritic cells) express very high levels of class I and II MHC products, and these levels cannot be increased by exposure to cytokines such as immune interferon. Second, dendritic cells efficiently cluster antigen-specific T cells during primary responses. Other presenting cells, like macrophages and B lymphocytes, do not form clusters but do bind to sensitized T lymphoblasts. Dendritic-T-cell binding is not inhibited by mAb to CD4 and LFA-1 antigens. It is suggested that a dendritic-cell-specific molecule is required. Third, it is not yet clear if dendritic cells make a 'lymphocyte activating factor.' However, IL-1 is not produced, even when dendritic cells are in contact with responding T cells. Fourth, dendritic cells have the capacity to migrate from the tissues and move to T-dependent areas. Epidermal Langerhans cells represent a reservoir of tissue dendritic cells but seem to be immunologically immature. The viability and accessory function of the Langerhans cell greatly depend on a single cytokine, granulocyte-macrophage colony stimulating factor (GM-CSF), leading to the proposal that GM-CSF is critical in mobilizing active dendritic cells at the onset of a cell-mediated immune response
— id: 33240, year: 1988, vol: 546, page: 80, stat: Journal Article,

Interferon-gamma-activated human monocytes inhibit the intracellular multiplication of Legionella pneumophila
Bhardwaj N; Nash TW; Horwitz MA
1986 Oct 15;137(8):2662-2669, Journal of immunology
We have examined the interaction between interferon-gamma (IFN-gamma)-activated human monocytes and Legionella pneumophila, the agent of Legionnaires' disease. Human monocytes activated with human recombinant IFN-gamma inhibit the intracellular multiplication of L. pneumophila. The degree of inhibition is proportional to the concentration of IFN-gamma, and maximal inhibition consistently occurs with greater than or equal to 2 micrograms/ml. Monoclonal anti-IFN-gamma antibody completely neutralizes the capacity of IFN-gamma to activate monocytes. Monocytes infected 24 hr after explantation maximally inhibit L. pneumophila multiplication if treated with IFN-gamma before infection or up to 2 hr after infection; treatment 6 hr or more after infection results in submaximal inhibition. Monocytes infected 48 hr after explantation inhibit L. pneumophila multiplication maximally if treated with IFN-gamma up to 12 hr before infection, but submaximally if treated at the time of infection. Once activated, monocytes inhibit L. pneumophila multiplication in the absence of IFN-gamma in the culture. Strikingly, monocytes maximally inhibit L. pneumophila multiplication after treatment with IFN-gamma for as briefly as 1 hr before infection. In the absence of anti-L. pneumophila antibody, neither IFN-gamma-activated monocytes nor nonactivated monocytes kill L. pneumophila. In the presence of specific antibody and complement, IFN-gamma-activated monocytes kill a proportion (0.5 log) of an inoculum but not more than nonactivated monocytes. L. pneumophila forms a specialized phagosome in IFN-gamma-activated monocytes that does not differ ultrastructurally from the L. pneumophila phagosome in nonactivated monocytes. These results demonstrate that IFN-gamma can activate human monocytes to exert a potent antimicrobial effect against a highly virulent intracellular bacterial pathogen. These findings extend previous observations on interactions between activated mononuclear phagocytes and L. pneumophila, and additionally support the hypothesis that cell-mediated immunity plays a major role in host defense against L. pneumophila
— id: 33241, year: 1986, vol: 137, page: 2662, stat: Journal Article,

Transfer of cutaneous delayed type hypersensitivity in Balb/c mice with murine dialyzable leukocyte extracts
Bhardwaj, Nina
[S.l. : s.n.], 1982,
Thesis (Ph.D.) - New York University, 1982
— id: 2102, year: 1982, vol: , page: , stat: ,

Antigen-specific activity of murine leukocyte dialysates containing transfer factor on human leukocytes in the leukocyte migration inhibition (LMI) assay
Borkowsky W; Suleski P; Bhardwaj N; Lawrence HS
1981 Jan;126(1):80-82, Journal of immunology
We report on the extension of the direct leukocyte migration inhibition (LMI) test as an assay for antigen-specific activity in human leukocyte dialysates (DLE) containing transfer factor to an evaluation of antigen-specific activity in DLE prepared from inbred mice. Murine DLE was observed to cause antigen-dependent and antigen-specific effects on the inhibition of migration of nonimmune human leukocyte populations. Pulsing of nonimmune human leukocyte with DLE preparations from BALB/c and SJL mice immunized with Candida, diphtheria toxoid, and SK-SD resulted in their inhibition of migration in the presence of the respective antigens. The antigen-specific activity in murine DLE was found to be present in lymph node cell preparations and to be absent from spleen cell preparations of the same donors. The activity of DLE in lymph node cells was found to be present in the theta-cell enriched subpopulation of nonadherent lymphocytes after passage through nylon wool columns. The antigen-specific activity of murine DLE, as we have reported for human DLE, was found to reside in the < 3500 dalton dialysis fraction and not in the < 3500 dalton fraction. We conclude that nonimmune human leukocytes in the LMI test provide a suitable assay for the detection of antigen-specific activity in murine DLE as well as that in human DLE. Additionally, murine DLE is active across species barriers and appears to share properties with human DLE
— id: 14589, year: 1981, vol: 126, page: 80, stat: Journal Article,

A method for eliciting indurated DTH reactions to soluble protein antigens in the flank skin of mice: correlation of visual measurements with 125I-UdR uptake indices
Brummer E; Bhardwaj N; Lawrence HS
1981 ;64(3):266-276, International archives of allergy & applied immunology
A new technique for eliciting specific indurated delayed type hypersensitivity (DTH) reactions to soluble protein antigens in the flank skin of mice is reported here. We show that immunization with soluble antigens emulsified in complete Freund's adjuvant emulsion allows elicitation of indurated reactions upon intracutaneous test of sensitized mice with antigen on alum precipitates. With this method it is possible to measure simultaneously the gross induration of the cutaneous DTH reactions and correlate it with the degree of mononuclear cell infiltration as quantitated by the cPM of 125I-UdR uptake in mononuclear cells at biopsied reaction sites and confirmed by histological examination
— id: 33242, year: 1981, vol: 64, page: 266, stat: Journal Article,

MITOGENIC AND ADJUVANT PROPERTIES OF THE LPS-LIKE COMPONENT OF LISTERIA-MONOCYTOGENES
Oppenheim, JD; Nachbar, MS; Bhardwaj, N; Wexler, H
1981 ;10(1):25-28, FEMS microbiology letters
— id: 30224, year: 1981, vol: 10, page: 25, stat: Journal Article,

ANTIGEN-SPECIFIC ACTIVITY IN MURINE LEUKOCYTE DIALYSATES CONTAINING TRANSFER-FACTOR ASSAYED ON MIGRATING HUMAN-LEUKOCYTES
BORKOWSKY, W; BHARDWAJ, N; LAWRENCE, HS
1980 ;28(2):A500-A500, Clinical research
— id: 98663, year: 1980, vol: 28, page: A500, stat: Journal Article,

Interaction of subpopulations of murine lymph node lymphocytes in antigen-induced [14C]-thymidine incorporation: T and B cell synergy in the response to antigen
Brummer E; Bhardwaj N; Lawrence HS
1979 Oct;38(2):301-310, Immunology
The antigen-induced [14C]-thymidine incorporation of murine lymph node cells (LNC) that were non-adherent (NAD) or adherent (AD) to nylon wool was studied. In contrast to NAD-LNC, AD-LNC responded like unfractionated LNC, and these responses were T lymphocyte dependent. By co-culturing NAD-LNC with subpopulations of AD-LNC the cellular requirements and interactions necessary for maximal incorporation of [14C]-thymidine were determined. A synergistic effect was observed when NAD-LNC and AD-LNC were co-cultured. Synergism was not dependent on T lymphocytes or macrophages in the AD-LNC population but was associated with the B lymphocyte subpopulation. These results indicate that the number of B lymphocytes present in a population of LNC can significantly influence the magnitude of the response to antigen
— id: 33243, year: 1979, vol: 38, page: 301, stat: Journal Article,

Stimulation of limb regeneration in the adult Notopthalamus viridescens by suppression of the cyclic AMP-adenyl cyclase system
Bhardwaj, Nina
[S.l. : s.n.], 1975,
Honors thesis -- Wellesley College
— id: 2101, year: 1975, vol: , page: , stat: ,

A rapid and accurate procedure for assaying DNA polymerase, RNA polymerase, or ribosome dependent protein synthesis
Weinstein BI; Bhardwaj N; Li HC
1975 Sep;68(1):62-69, Analytical biochemistry
— id: 33244, year: 1975, vol: 68, page: 62, stat: Journal Article,