Sandra Demaria, M.D.

Radiation and endoplasmic reticulum stress-inducer promote calreticulin translocation, contributing to immunogenic cell death of cancer cells
Golden E.; Demaria S.; Barcellos-Hoff M.H.
2012 ;35(1):100-100, Journal of immunotherapy (Hagerstown)
We hypothesize that cell damage and death from the effect of ionizing radiation (IR) and endoplasmic reticulum (ER) stressinducing agents could: (1) be monitored in vitro; and (2) contribute to an anti-tumor immune response via the induction of mediators of immunogenic cell death (ICD) of cancer cells. ICD promotes the cross-presentation of tumor-derived antigens by dendritic cells (DCs) to T cells (Semin Immunol. 2010;22:113-124). Calreticulin (CRT, an ER chaperone protein) redistribution to the surface of tumor cells acts as a potent 'eat me' signal for DCs involved in tumor associated antigen processing, thereby serving as a key step in ICD. In the clinical setting, IR or ER stress alone may not quantitatively and/or qualitatively achieve tumor cell death in a manner that specifically triggers immune-mediated tumor rejection. Thus, we hypothesized that clinically relevant doses of IR, when combined with thapsigargin (Tg, an ER stress-inducer via sarcoplasmic/ER calcium ATPase inhibition), may intensify CRT translocation to the cell surface. To test this, we employed the poorly immunogenic 4T1 mouse breast cancer cells. 4T1 cells were treated with IR (0, 6, or 20 Gy) followed by 24 hours. culture in the presence or absence of Tg (1 mM). Thereafter, the cells were assayed either via Western blot (WB) or immunofluorescence (IF). Cytotoxicity was determined via MTT assay at 12, 24, and 48 hours. Relative amounts of protein were determined via WB analysis with specific antibodies to phospho-EIF2-a, caspase-8, BAP-31, and PARP. Actin was used as a loading control. CRT redistribution was determined by IF analysis. When combined, IR (6 Gy) + Tg (1mM) triggered elevated phosphorylation of EIF2-a (a marker for ER stress and protein translation inhibition) in 4T1 cells. In addition, IR (6 and 20 Gy) + Tg (1 muM) increased the cleavage of the apoptotic markers caspase-8, BAP-31, and PARP. Finally, we observed that cell death by IR (6 and 10 Gy, single dose) in the presence of Tg (1 muM) was preceded by enhanced CRT translocation to the cell surface. In this in vitro model, IR (6Gy and 10 Gy) alone was unable to incite CRT redistribution. However, in the presence of Tg (1 muM), IR (6 Gy) CRT redistribution occurred and was superior to controls. Taken together, these findings suggest that IR combined with an ER stress-inducing agent is a novel application of radiotherapy that can potently trigger ICD and serve as a strategy to promote immune-mediated tumor rejection in cancer patients
— id: 149974, year: 2012, vol: 35, page: 100, stat: Journal Article,

Expression of cancer testis antigens in human BRCA-associated breast cancers: potential targets for immunoprevention?
Adams, Sylvia; Greeder, Luba; Reich, Elsa; Shao, Yongzhao; Fosina, Denise; Hanson, Nicole; Tassello, Jodie; Singh, Baljit; Spagnoli, Giulio C; Demaria, Sandra; Jungbluth, Achim A
2011 Jul;60(7):999-1007, Cancer immunology immunotherapy
INTRODUCTION: Novel breast cancer risk-reducing strategies for individuals with germline mutations of the BRCA1 and/or BRCA2 genes are urgently needed. Identification of antigenic targets that are expressed in early cancers, but absent in normal breast epithelium of these high-risk individuals, could provide the basis for the development of effective immunoprophylactic strategies. Cancer testis (CT) antigens are potential candidates because their expression is restricted to tumors, and accumulating data suggest that they play important roles in cellular proliferation, stem cell function, and carcinogenesis. The objective of this study was to examine the expression of CT antigens and their frequency in BRCA-associated breast cancers. METHODS: Archived breast cancer tissues (n = 26) as well as morphologically normal breast tissues (n = 7) from women carrying deleterious BRCA 1 and/or 2 mutations were obtained for antigen expression analysis by immunohistochemistry. Expression of the following CT antigens was examined: MAGE-A1, MAGE-A3, MAGE-A4, MAGE-C1.CT7, NY-ESO-1, MAGE-C2/CT10, and GAGE. RESULTS: CT antigens were expressed in 16/26 (61.5%, 95% CI 43-80%) of BRCA-associated cancers, including in situ tumors. Thirteen of twenty-six (50%) breast cancers expressed two or more CT antigens; three cancers expressed all seven CT antigens. MAGE-A was expressed in 13/26 (50%) of cancers, NY-ESO-1 was expressed in 10/26 (38%) of tumors. In contrast, none of the CT antigens were expressed in adjacent or contralateral normal breast epithelium (P = 0.003). CONCLUSIONS: We report a high CT antigen expression rate in BRCA-associated breast cancer as well as the lack of expression of these antigens in benign breast tissue of carriers, identifying CT antigens as potential vaccine targets for breast cancer prevention in these high-risk individuals
— id: 134441, year: 2011, vol: 60, page: 999, stat: Journal Article,

Increased radiosensitivity in vivo following inhibition of transforming growth factor beta in murine model of triple negative breast cancer
Bouquet S.F.; Pilones K.; Demaria S.; DeWyngaert K.J.; Lonning S.; Formenti S.; Barcellos-Hoff M.
2011 ;81(2 SUPPL 1):S714-S715, International journal of radiation oncology biology physics
Purpose/Objective(s): Ionizing radiation triggers activation of transforming growth factor beta1 (TGFbeta), a growth factor that promotes invasion and suppresses immune function. Either genetic or pharmaceutical TGFbeta inhibition prior to IR also inhibits the DNA damage response (DDR) in epithelial cells via blockade of ATM kinase activity (Cancer Res 62:5627, 2002; Cancer Res 66:10861, 2006). Moreover, transient TGFbeta inhibition increases the radiosensitivity of epithelial cancer cells (Bouquet et al. submitted). The current studies test the optimal combination of TGFbeta inhibition using neutralizing antibodies with radiotherapy (RT) in a model of triple negative breast cancer. Materials/Methods: The 4T1 murine mammary tumor model is highly metastatic when grown as subcutaneous tumors. 4T1 cells were injected s.c. in the flank of syngeneic Balb/c mice and about 13 days after implantation approximately 100 mm3 tumors were locally irradiated. An anti-TGFb murine monoclonal antibody, 1D11, was administered i.p. before irradiation. Mice were randomly assigned to four groups receiving control isotype monoclonal antibody, 1D11, RT (8 Gy) and isotype control antibody, or RT and 1D11. Tumor growth, DDR, lymphocyte infiltration, lung metastases and/or survival were evaluated. Results: TGFb TGFb inhibition in vivo reduced DDR as evidenced by gH2AX foci in irradiated tumors. A single injection of 1D11 (5 mg/kg) 24 hr before RT resulted in greater tumor growth delay compared to RT and control antibody (p<0.05). Furthermore, chronic treatment with a higher 1D11 dose (10 mg/kg or 50 mg/kg) in the context of fractionated RT significantly reduced tumor growth rate, decreased visible lung metastases and increased survival (p<0.05). Notably this RT+1D11 protocol increased tumor infiltration of CD8, but not CD4, T lymphocytes, which resulted in a significantly enhanced ratio of CD8+NKG2D+ effector cells to CD4+ T cells in the tumor, suggesting that TGFb inhibition also alters the immunological environment in irradiated tumors. Conclusions: Increased radiosensitivity of 4T1 tumor cells in vitro and in vivo supports the use of TGFb inhibitors as means to increase the response to RT. Moreover, an additional benefit may be that TGFb inhibition promotes an anti-tumor immune response to RT
— id: 150891, year: 2011, vol: 81, page: S714, stat: Journal Article,

TGFbeta1 Inhibition Increases the Radiosensitivity of Breast Cancer Cells In Vitro and Promotes Tumor Control by Radiation In Vivo
Bouquet, Fanny; Pal, Anupama; Pilones, Karsten A; Demaria, Sandra; Hann, Byron; Akhurst, Rosemary J; Babb, Jim S; Lonning, Scott M; Dewyngaert, J Keith; Formenti, Silvia C; Barcellos-Hoff, Mary Helen
2011 Nov 1;17(21):6754-6765, Clinical cancer research
PURPOSE: To determine whether inhibition of TGFbeta signaling prior to irradiation sensitizes human and murine cancer cells in vitro and in vivo. EXPERIMENTAL DESIGN: TGFbeta-mediated growth and Smad phosphorylation of MCF7, Hs578T, MDA-MB-231, and T47D human breast cancer cell lines were examined and correlated with clonogenic survival following graded radiation doses with and without pretreatment with LY364947, a small molecule inhibitor of the TGFbeta type I receptor kinase. The DNA damage response was assessed in irradiated MDA-MB-231 cells pretreated with LY364947 in vitro and LY2109761, a pharmacokinetically stable inhibitor of TGFbeta signaling, in vivo. The in vitro response of a syngeneic murine tumor, 4T1, was tested using a TGFbeta neutralizing antibody, 1D11, with single or fractionated radiation doses in vivo. RESULTS: Human breast cancer cell lines pretreated with TGFbeta small molecule inhibitor were radiosensitized, irrespective of sensitivity to TGFbeta growth inhibition. Consistent with increased clonogenic cell death, radiation-induced phosphorylation of H2AX and p53 was significantly reduced in MDA-MB-231 triple-negative breast cancer cells when pretreated in vitro or in vivo with a TGFbeta type I receptor kinase inhibitor. Moreover, TGFbeta neutralizing antibodies increased radiation sensitivity, blocked gammaH2AX foci formation, and significantly increased tumor growth delay in 4T1 murine mammary tumors in response to single and fractionated radiation exposures. CONCLUSION: These results show that TGFbeta inhibition prior to radiation attenuated DNA damage responses, increased clonogenic cell death, and promoted tumor growth delay, and thus may be an effective adjunct in cancer radiotherapy. Clin Cancer Res; 17(21); 6754-65. (c)2011 AACR
— id: 140531, year: 2011, vol: 17, page: 6754, stat: Journal Article,

Defining the role of the immune system in cancer treatment: highlights from the Immunochemotherapy Conference
Demaria, Sandra
2011 Jun;11(6):841-843, Expert review of anticancer therapy
The Immunochemotherapy: Correcting Immune Escape in Cancer meeting was co-organized by George Prendergast (Lankenau Institute for Medical Research, PA, USA), Guido Kroemer (Gustave Roussy Cancer Institute, Paris, France) and Laurence Zitvogel (Gustave Roussy Cancer Institute) in partnership with Abcam. It brought together investigators with a diverse background in immunology, oncology and cancer biology, and offered a forum for discussion of a new vision that acknowledges the role of the host immune system not only in cancer development and progression, but also as a major determinant of response to treatment. An important implication of this vision is that synergy between conventional cytocidal modalities and immune response modifiers is not only possible, but holds the promise to revolutionize cancer treatment. The emerging evidence that was presented in support of this new concept will be summarized in this article
— id: 134735, year: 2011, vol: 11, page: 841, stat: Journal Article,

Cross-Talk of Breast Cancer Cells with the Immune System
Demaria, Sandra; Pilones, Karsten A; Adams, Sylvia
Breast Cancer : Carcinogenesis, Cell Growth and Signalling Pathways Rijeka, Croatia : InTech, 2011,
— id: 5837, year: 2011, vol: , page: 469, stat: Chapter,

Role of T-lymphocytes for tumour response to radiotherapy
Formenti S.; Encouse G.; Adams S.; Pilones K.; Grazia Ruocco M.; Dustin M.; Demaria S.
2011 ;47:S11-S11, European journal of cancer
Over the past ten years we have developed a clinical translational program based on the rationale of immunizing patients against their own tumour by concomitantly: 1) removing existing 'breaks' in their immune system and 2) harnessing local ionizing radiation (IR) to induce physical and biological perturbations at the irradiated tumour site, to achieve the successful conversion of the original tumour into an immunogenic hub (Formenti, Lancet Oncology 2009). Preclinical investigations have shed some light on the specific role of T cells in these processes. For instance, in the 4T1 syngeneic murine model of metastatic breast cancer targeting regulatory receptors or cells (Treg) by anti-CTLA-4 and anti-CD25 antibodies, respectively, synergized with IR and reduced the number of metastases to the lung (an abscopal effect, defined as a significant growth inhibition of the tumour outside the irradiated field) in a CD8+ T cells dependent way. In the same model IR increased the migration of CD8 CXCR6 activated T cells to tumours. This effect was mediated by IRenhanced secretion by cancer cells of CXCL16, a chemokine that binds to CXCR6 on Th1 and activated CD8 effector T cells. CXCR6-deficient mice showed reduced infiltration of tumours by activated CD8+ T cells and impaired tumour regression following treatment with local IR + CTLA-4 blockade. Interestingly, an abscopal effect, occurred only in mice treated with the combination of 9H10 and fractionated radiotherapy, but not when a single dose of 20 Gy was administered (P < 0.01), as reflected by the frequency of CD8+ T cells showing tumour-specific IFN-gamma production. The contribution of invariant natural killer T (iNKT) cells, a subset with unique regulatory functions, in the response to IR and CTLA-4 blockade was also studied. Growth of 4T1 primary tumours and lung metastases was compared in wild type (WT) and iNKT cells-deficient (iNKT-/-) mice. The response to IR+CTLA-4 blockade was markedly enhanced in the absence of iNKT cells: 50% of iNKT-/- compared to none of the WT mice had complete tumour regression, long-term survival, and resistance to a challenge with 4T1 cells. Finally, intravital microscopy demonstrated that while both IR and CTLA-4 blockade given as monotherapy enhanced the motility of activated CD8 T cells infiltrating 4T1 tumours, IR with anti-CTLA-4 increased the arrest of T cells in contact with tumour cells. The latter required interaction of NKG2D on CD8+ T cells with its ligand retinoic acid early inducible-1 (Rae-1) on the tumour cells, which was up-regulated by IR. Blocking NKG2D-Rae-1 interactions increased markedly the motility of anti-CTLA-4 treated T cells within irradiated tumours inhibiting their contact with tumour cells, and abrogated immune-mediated tumour rejection, suggesting a critical role of radiation-induced NKG2D ligands for the antitumour effects of anti-CTLA-4 in the setting of a poorly immunogenic tumour
— id: 138729, year: 2011, vol: 47, page: S11, stat: Journal Article,

The numbers of FoxP3+ lymphocytes in sentinel lymph nodes of breast cancer patients correlate with primary tumor size but not nodal status
Gupta, Raavi; Babb, James S; Singh, Baljit; Chiriboga, Luis; Liebes, Leonard; Adams, Sylvia; Demaria, Sandra
2011 Jul;29(6):419-425, Cancer investigation
Regulatory T cells, lymphocytes marked by expression of the transcription factor Forkhead Box Protein P3 (FoxP3), inhibit the activation of tumor-specific T cells in tumor-draining lymph nodes. Immunohistochemical analyses of sentinel lymph nodes (SLNs) from 104 breast cancer patients showed a significant association (p = .0028, Pearson correlation) between the number of FoxP3+ cells and the size of primary breast invasive ductal carcinoma. In contrast, there was no correlation between the number of FoxP3+ cells and the presence of SLN metastases, or other clinicopathological parameters. These results suggest the presence of an immune suppressive environment in SLNs of larger tumors
— id: 138850, year: 2011, vol: 29, page: 419, stat: Journal Article,

Immunological targeting of epithelial to mesenchymal transition as a strategy to prevent breast cancer metastases
Kawashima N.; Wang B.; Santori F.; Pilones K.; Pajonk F.; Demaria S.
2011 ;34(9):688-688, Journal of immunotherapy (Hagerstown)
The identification of a subset of breast cancer (BC) cells with properties of cancer stem cells (CSCs), that are responsible for tumorigenesis, metastasis and recurrence, and are intrinsically more resistant to chemotherapy and radiation, provides an unprecedented opportunity to develop new treatment strategies. The transcription factor Twist plays a key role in induction of epithelial to mesenchymal transition (EMT) and acquisition of CSCs properties by BC cells. Twist is also a key regulator of metastasis in the 4T1 mouse model of breast cancer, and has been implicated in resistance to chemotherapy and radiation, and in early disease relapse after adjuvant treatment in patients. Therefore, strategies to target Twist could be effective for the prevention of metastatic BC. Using the SYFPEITHI Epitope Prediction computer-based algorithm we have identified a Twist-derived peptide (pTw9) that forms stable complexes with H2-Kd with DC50 of >6 hour, and was able to sensitize P815 cells to lysis by 4T1 tumor-specific CTL isolated from mice that rejected 4T1 tumor. A pTw9-specific CTL line established by repeated in vitro re-stimulation specifically killed 4T1 cells, confirming that pTw9 is an endogenously produced CTL epitope. Immunization experiments with pTw9 confirmed that this peptide is immunogenic in naive mice. To determine whether CTL targeting pTw9 can prevent the generation of CSCs in vivo and inhibit metastases, we have prepared 4T1 cells expressing ZsGreen-cODC fusion protein, which contains the degron of ornithine decarboxylase leading to rapid proteasome-mediated degradation of the reporter protein. Tumor cells that accumulate ZsGreen-cODC to detectable levels have the characteristics of CSCs and can be imaged in vivo (Vlashi et al., J Natl Cancer Inst. 2009, 101:350-359). 4T1-ZsGreen-cODC cells injected s.c. into RAG2-deficient mice were allowed to spontaneously metastasize to the lungs. Lungs were harvested 30 days later, digested, and analyzed by flow cytometry for the presence of EpCAM+ZsGreen+tumor cells. ZsGreen+cells represented on average 4.4+/-4.9% of EpCAM+tumor cells in the lung (range 0.6% to 15.0%). Since the injected population did not contain detectable ZsGreen+cells, results suggest that 4T1 cells with CSC characteristics are generated in vivo and enriched in the spontaneous lung metastases. Use of 4T1-ZsGreen-cODC cells will allow the tracking of tumor cells with CSCs properties in vivo. This system provides a unique tool to test the hypothesis that immunological targeting of Twist can inhibit EMT and the generation of tumor cells with breast CSCs properties
— id: 141089, year: 2011, vol: 34, page: 688, stat: Journal Article,

Multiplicity of Favorable Effects after Transforming Growth Factor-beta Inhibition in 4T1 Murine Mammary Tumors: Clinical Implications
Barcellos-Hoff, M. H.; Bouquet, S. F.; Pilones, K. A.; Demaria, S.; Formenti, S. C.
2010 OCT 13 ;78(3):S648-S648, International journal of radiation oncology biology physics
— id: 114022, year: 2010, vol: 78, page: S648, stat: Journal Article,

Cancer and inflammation: promise for biologic therapy
Demaria, Sandra; Pikarsky, Eli; Karin, Michael; Coussens, Lisa M; Chen, Yen-Ching; El-Omar, Emad M; Trinchieri, Giorgio; Dubinett, Steven M; Mao, Jenny T; Szabo, Eva; Krieg, Arthur; Weiner, George J; Fox, Bernard A; Coukos, George; Wang, Ena; Abraham, Robert T; Carbone, Michele; Lotze, Michael T
2010 May;33(4):335-351, Journal of immunotherapy (Hagerstown)
Cancers often arise as the end stage of inflammation in adults, but not in children. As such there is a complex interplay between host immune cells during neoplastic development, with both an ability to promote cancer and limit or eliminate it, most often complicit with the host. In humans, defining inflammation and the presence of inflammatory cells within or surrounding the tumor is a critical aspect of modern pathology. Groups defining staging for neoplasms are strongly encouraged to assess and incorporate measures of the presence of apoptosis, autophagy, and necrosis and also the nature and quality of the immune infiltrate. Both environmental and genetic factors enhance the risk of cigarette smoking, Helicobacter pylori, hepatitis B/C, human papilloma virus, solar irradiation, asbestos, pancreatitis, or other causes of chronic inflammation. Identifying suitable genetic polymorphisms in cytokines, cytokine receptors, and Toll-like receptors among other immune response genes is also seen as high value as genomic sequencing becomes less expensive. Animal models that incorporate and assess not only the genetic anlagen but also the inflammatory cells and the presence of microbial pathogens and damage-associated molecular pattern molecules are necessary. Identifying micro-RNAs involved in regulating the response to damage or injury are seen as highly promising. Although no therapeutic strategies to prevent or treat cancers based on insights into inflammatory pathways are currently approved for the common epithelial malignancies, there remains substantial interest in agents targeting COX2 or PPARgamma, ethyl pyruvate and steroids, and several novel agents on the horizon
— id: 109514, year: 2010, vol: 33, page: 335, stat: Journal Article,

Introduction to the special br-ridge issue
Demaria, Sandra; Williams, Jacqueline P
2010 Apr;173(4):403-405, Radiation research
— id: 108805, year: 2010, vol: 173, page: 403, stat: Journal Article,

Up-regulation of the Pro-inflammatory Chemokine CXCL16 is a Common Response of Tumor Cells to Ionizing Radiation
Matsumura, Satoko; Demaria, Sandra
2010 Apr;173(4):418-425, Radiation research
Abstract We recently showed that mouse and human breast carcinoma cells respond to ionizing radiation therapy by up-regulating the expression and release of the pro-inflammatory chemokine CXCL16, which binds to the CXCR6 receptor expressed by activated T cells. Enhanced recruitment of activated T cells to irradiated mouse 4T1 breast tumors was mediated largely by CXCL16 and was correlated with tumor inhibition in mice treated with the combination of local radiation and immunotherapy. In this study, the expression of CXCL16 and its modulation by radiation were analyzed in mouse melanoma B16/F10, fibrosarcoma MC57, colon carcinoma MCA38, and prostate carcinoma TRAMP-C1 cells. Only TRAMP-C1 cells showed detectable expression of CXCL16, although the level was lower than in 4T1 and 67NR breast carcinoma cells. Ionizing radiation up-regulated CXCL16 expression in all cells except B16/F10, but only TRAMP-C1, 67NR and 4T1 cells released the soluble chemokine in significant quantities. The metalloproteinases ADAM10 and ADAM17, which are responsible for cleaving the chemokine domain from the CXCL16 transmembrane form, were expressed in all cells. Overall, our data indicate that up-regulation of CXCL16 is a common response of tumor cells to radiation, and they have important implications for the use of local radiotherapy in combination with immunotherapy
— id: 108806, year: 2010, vol: 173, page: 418, stat: Journal Article,

Radiotherapy Enhances Antitumor Effect of Anti-CD137 Therapy in a Mouse Glioma Model
Newcomb, Elizabeth W; Lukyanov, Yevgeniy; Kawashima, Noriko; Alonso-Basanta, Michelle; Wang, Shu-Chi; Liu, Mengling; Jure-Kunkel, Maria; Zagzag, David; Demaria, Sandra; Formenti, Silvia C
2010 Apr;173(4):426-432, Radiation research
Abstract Previously, we reported that peripheral vaccination of mice with modified autologous tumor cells secreting granulocyte-macrophage colony-stimulating factor (GM-CSF) combined with ionizing radiation to the whole brain cured 50% of mice using a syngeneic, intracranial model of murine high-grade glioma. Here, we tested the combination of radiotherapy (4 Gy x 2) with an immunotherapeutic approach using an anti-CD137 antibody directed to the co-stimulatory molecule CD137. The CD137 antibody has shown promise in generating effective antitumor responses in several animal models and has demonstrated a favorable toxicity profile in the clinic. The combination of radiation and anti-CD137 therapy resulted in complete tumor eradication and prolonged survival in six of nine (67%) mice with established brain tumors (P = 0.0009). Five of six (83%) long-term survivors in the combination group demonstrated antitumor immunity by rejecting challenge tumors. Antitumor immunity was associated with an increased number of tumor-infiltrating lymphocytes (TILs) in brain tumors and increased tumor-specific production of gammaIFN. In view of the finding that radiation enhanced the antitumor effect of anti-CD137 therapy, this approach should be studied further for clinical translation
— id: 108807, year: 2010, vol: 173, page: 426, stat: Journal Article,

Immunoregulation by Invariant Natural Killer T Cells in a Mouse Model of Metastatic Breast Cancer
Pilones, Karsten A.; Kawashima, Noriko; Babb, James; Demaria, Sandra
2010 OCT ;33(8):888-888, Journal of immunotherapy (Hagerstown)
— id: 114381, year: 2010, vol: 33, page: 888, stat: Journal Article,

Fractionated but not single-dose radiotherapy induces an immune-mediated abscopal effect when combined with anti-CTLA-4 antibody
Dewan, M Zahidunnabi; Galloway, Ashley E; Kawashima, Noriko; Dewyngaert, J Keith; Babb, James S; Formenti, Silvia C; Demaria, Sandra
2009 Sep 1;15(17):5379-5388, Clinical cancer research
PURPOSE: This study tested the hypothesis that the type of dose fractionation regimen determines the ability of radiotherapy to synergize with anti-CTLA-4 antibody. EXPERIMENTAL DESIGN: TSA mouse breast carcinoma cells were injected s.c. into syngeneic mice at two separate sites, defined as a 'primary' site that was irradiated and a 'secondary' site outside the radiotherapy field. When both tumors were palpable, mice were randomly assigned to eight groups receiving no radiotherapy or three distinct regimens of radiotherapy (20 Gy x 1, 8 Gy x 3, or 6 Gy x 5 fractions in consecutive days) in combination or not with 9H10 monoclonal antibody against CTLA-4. Mice were followed for tumor growth/regression. Similar experiments were conducted in the MCA38 mouse colon carcinoma model. RESULTS: In either of the two models tested, treatment with 9H10 alone had no detectable effect. Each of the radiotherapy regimens caused comparable growth delay of the primary tumors but had no effect on the secondary tumors outside the radiation field. Conversely, the combination of 9H10 and either fractionated radiotherapy regimens achieved enhanced tumor response at the primary site (P < 0.0001). Moreover, an abscopal effect, defined as a significant growth inhibition of the tumor outside the field, occurred only in mice treated with the combination of 9H10 and fractionated radiotherapy (P < 0.01). The frequency of CD8+ T cells showing tumor-specific IFN-gamma production was proportional to the inhibition of the secondary tumor. CONCLUSIONS: Fractionated but not single-dose radiotherapy induces an abscopal effect when in combination with anti-CTLA-4 antibody in two preclinical carcinoma models
— id: 101960, year: 2009, vol: 15, page: 5379, stat: Journal Article,

Systemic effects of local radiotherapy
Formenti, Silvia C; Demaria, Sandra
2009 Jul;10(7):718-726, Lancet oncology
Radiotherapy is generally used to treat a localised target that includes cancer. Increasingly, evidence indicates that radiotherapy recruits biological effectors outside the treatment field and has systemic effects. We discuss the implications of such effects and the role of the immune system in standard cytotoxic treatments. Because the effects of chemotherapy and radiotherapy are sensed by the immune system, their combination with immunotherapy presents a new therapeutic opportunity. Radiotherapy directly interferes with the primary tumour and possibly reverses some immunosuppressive barriers within the tumour microenvironment-ideally, recovering the role of the primary tumour as an immunogenic hub. Local radiation also triggers systemic effects that can be used in combination with immunotherapy to induce responses outside the radiation field
— id: 100625, year: 2009, vol: 10, page: 718, stat: Journal Article,

Local Radiotherapy Rescues Responsiveness to Anti-CD137 Immunotherapy in a Mouse Model of Advanced Breast Cancer
Pilones, KA; Kawashima, N; Formenti, SC; Demaria, S
2009 DEC 15 ;69(24):752S-752S, Cancer research
— id: 106455, year: 2009, vol: 69, page: 752S, stat: Journal Article,

Research highlights: Immunotherapy
Pilones, Karsten A; Demaria, Sandra
2009 Sep;1(5):733-736, Immunotherapy
— id: 111389, year: 2009, vol: 1, page: 733, stat: Journal Article,

Invariant natural killer T cells regulate breast cancer response to radiation and CTLA-4 blockade
Pilones, Karsten A; Kawashima, Noriko; Yang, Anne Marie; Babb, James S; Formenti, Silvia C; Demaria, Sandra
2009 Jan 15;15(2):597-606, Clinical cancer research
PURPOSE: Immunoregulatory and suppressive mechanisms represent major obstacles to the success of immunotherapy in cancer patients. We have shown that the combination of radiotherapy to the primary tumor and CTL-associated protein 4 (CTLA-4) blockade induces antitumor immunity, inhibiting metastases and extending the survival of mice bearing the poorly immunogenic and highly metastatic 4T1 mammary carcinoma. Similarly to patients with metastatic cancer, however, mice were seldom cured. Here we tested the hypothesis that invariant natural killer T (iNKT) cells, a subset with unique regulatory functions, can regulate the response to radiotherapy and CTLA-4 blockade. EXPERIMENTAL DESIGN: The growth of 4T1 primary tumors and lung metastases was compared in wild-type and iNKT cell-deficient (iNKT-/-) mice. Treatment was started on day 13 when the primary tumors were palpable. Mice received radiotherapy to the primary tumor in two doses of 12 Gy in combination or not with 9H10 monoclonal antibody against CTLA-4. Response to treatment was assessed by measuring primary tumor growth delay/regression, survival, and number of lung metastases. RESULTS: The response to radiotherapy plus 9H10 was markedly enhanced in the absence of iNKT cells, with 50% of iNKT-/- versus 0% of wild-type mice showing complete tumor regression, long-term survival, and resistance to a challenge with 4T1 cells. Administration of the iNKT cell activator alpha-galactosylceramide did not enhance the response of wild-type mice to radiotherapy plus 9H10. Tumor-infiltrating iNKT cells were markedly reduced in wild-type mice treated with radiotherapy plus 9H10. CONCLUSIONS: iNKT cells play a major role in regulating the response to treatment with local radiotherapy and CTLA-4 blockade
— id: 93549, year: 2009, vol: 15, page: 597, stat: Journal Article,

Synergy of Radiation and Immune Therapy in Tumor Eradication
Ruocco, MG; Kawashima, N; Huang, J; Formenti, S; Dustin, ML; Demaria, S
2009 NOV-DEC ;32(9):995-995, Journal of immunotherapy (Hagerstown)
— id: 105633, year: 2009, vol: 32, page: 995, stat: Journal Article,

Abscopal response in irradiated patients: Results of a proof of principle trial
Formenti, SC; Friedman, K; Chao, K; Adams, S; Fenton-Kerimian, M; Donach, ME; Demaria, S
2008 AUG ;72(1):S6-S7, International journal of radiation oncology biology physics
— id: 86793, year: 2008, vol: 72, page: S6, stat: Journal Article,

Effects of chemoradiation on tumor-host interactions: the immunologic side
Formenti, Silvia C; Demaria, Sandra
2008 Mar 20;26(9):1562-1563, Journal of clinical oncology
— id: 76645, year: 2008, vol: 26, page: 1562, stat: Journal Article,

Local control by radiotherapy: is that all there is?
Formenti, Silvia C; Demaria, Sandra
2008 ;10(6):215-215, Breast cancer research
Radiotherapy is a local treatment modality employed in breast cancer to reduce local recurrence following surgery. The observed association of optimal local control with improved survival was not expected in a disease characterized by early systemic spread. The underlying mechanisms whereby the application of ionizing radiation to the primary tumor site can have systemic effects remain unclear and are the subject of much debate. In the present article we discuss the hypothesis that radiotherapy has unique biological effects and that, in addition to killing residual neoplastic cells after surgery is performed, it might favorably alter the microenvironment at the primary tumor site during the process of wound healing and the development of antitumor immune responses
— id: 93552, year: 2008, vol: 10, page: 215, stat: Journal Article,

Quantification of regulatory T-cells in sentinel lymph nodes of breast cancer patients
Gupta, R; Singh, B; Chiriboga, L; Demaria, S
2008 JAN ;21(2):34A-34A, Modern pathology
— id: 75904, year: 2008, vol: 21, page: 34A, stat: Journal Article,

Quantification of regulatory T-cells in sentinel lymph nodes of breast cancer patients
Gupta, R; Singh, B; Chiriboga, L; Demaria, S
2008 JAN ;88(2):34A-34A, Laboratory investigation
— id: 75926, year: 2008, vol: 88, page: 34A, stat: Journal Article,

Radiation-induced CXCL16 release by breast cancer cells attracts effector T cells
Matsumura, Satoko; Wang, Baomei; Kawashima, Noriko; Braunstein, Steve; Badura, Michelle; Cameron, Thomas O; Babb, James S; Schneider, Robert J; Formenti, Silvia C; Dustin, Michael L; Demaria, Sandra
2008 Sep 1;181(5):3099-3107, Journal of immunology
Recruitment of effector T cells to inflamed peripheral tissues is regulated by chemokines and their receptors, but the factors regulating recruitment to tumors remain largely undefined. Ionizing radiation (IR) therapy is a common treatment modality for breast and other cancers. Used as a cytocidal agent for proliferating cancer cells, IR in combination with immunotherapy has been shown to promote immune-mediated tumor destruction in preclinical studies. In this study we demonstrate that IR markedly enhanced the secretion by mouse and human breast cancer cells of CXCL16, a chemokine that binds to CXCR6 on Th1 and activated CD8 effector T cells, and plays an important role in their recruitment to sites of inflammation. Using a poorly immunogenic mouse model of breast cancer, we found that irradiation increased the migration of CD8(+)CXCR6(+) activated T cells to tumors in vitro and in vivo. CXCR6-deficient mice showed reduced infiltration of tumors by activated CD8 T cells and impaired tumor regression following treatment with local IR to the tumor and Abs blocking the negative regulator of T cell activation, CTLA-4. These results provide the first evidence that IR can induce the secretion by cancer cells of proinflammatory chemotactic factors that recruit antitumor effector T cells. The ability of IR to convert tumors into 'inflamed' peripheral tissues could be exploited to overcome obstacles at the effector phase of the antitumor immune response and improve the therapeutic efficacy of immunotherapy
— id: 81352, year: 2008, vol: 181, page: 3099, stat: Journal Article,

Optimizing the tumor response to ionizing radiation and immunotherapy by blocking VEGF
Pilones, KA; Formenti, SC; Demaria, S
2008 ;72(1):S168-S168, International journal of radiation oncology biology physics
— id: 109261, year: 2008, vol: 72, page: S168, stat: Journal Article,

Exploiting breast cancer cells stress response to ionizing radiation to improve the effectiveness of immunotherapy
Demaria, S; Wang, B; Badura, M; Matsumura, S; Kawashima, N; Cameron, T; Dustin, M; Schneider, RJ; Formenti, SC
2007 JAN ;69(3):S597-S597, International journal of radiation oncology biology physics
— id: 87199, year: 2007, vol: 69, page: S597, stat: Journal Article,

Immunotherapy against metastatic breast cancer with a twist
Demaria, S; Wang, B; Yang, AM; Santori, F; Kawashima, N; Matsumura, S
2007 DEC ;106(1):S31-S31, Breast cancer research & treatment
— id: 75799, year: 2007, vol: 106, page: S31, stat: Journal Article,

Sensors of ionizing radiation effects on the immunological microenvironment of cancer
Demaria, Sandra; Formenti, Silva C
2007 Nov-Dec;83(11-12):819-825, International journal of radiation biology
PURPOSE: When cancer develops in an immunocompetent host it represents the result of a successful deception of the immune system as to the nature of the danger and the type of response needed to reject the neoplastic tissue. We will briefly review some of the recently emerged evidence that irradiation of the tumor and its microenvironment can induce essential molecular signals required for an effective response of the immune system to the tumor. CONCLUSIONS: The subversion of a highly organized tissue architecture is a hallmark of cancer, and results in uneven distribution of oxygen and nutrients, interstitial pressure gradients and areas of patchy necrosis and inflammation. In this microenvironment, cancer cells that carry mutations favoring survival rather than cell death in response to stress find a selection advantage. Importantly, the signals derived from the disruption of orderly physiology within tissues are also what the immune system has evolved to respond to. The type of response is tuned to be adequate to the cause of the disruption. An infectious organism will carry or elicit from the involved tissue a number of 'danger signals' leading to development of cell mediated and humoral responses to both eliminating the invader and preventing future infections. In contrast, a simple wound will call for a repair response. The sensors of the type of damage are complex molecular interactions between the damaged organ and cells of the innate and adaptive immune system. Progress in the identification of these interactions elucidates which pathways are specifically altered in cancer. It also provides a novel understanding of the radiation-induced effects on tumor immunogenicity. We propose that specific radiation-induced effects could be successfully exploited to improve the effectiveness of immunotherapy
— id: 76330, year: 2007, vol: 83, page: 819, stat: Journal Article,

Pre-clinical assessment of CD137 (4-1BB)-mediated co-stimulation in combination with whole brain radiation therapy in the 61,261 glioma model
Newcomb, EW; Demaria, S; Lukyanov, Y; Schnee, T; Kawashima, N; Formenti, SC
2007 JAN ;69(3):S152-S152, International journal of radiation oncology biology physics
— id: 87194, year: 2007, vol: 69, page: S152, stat: Journal Article,

The combination of ionizing radiation and peripheral vaccination produces long-term survival of mice bearing established invasive GL261 gliomas
Newcomb, Elizabeth W; Demaria, Sandra; Lukyanov, Yevgeniy; Shao, Yongzhao; Schnee, Tona; Kawashima, Noriko; Lan, Li; Dewyngaert, J Keith; Zagzag, David; McBride, William H; Formenti, Silvia C
2006 Aug 1;12(15):4730-4737, Clinical cancer research
PURPOSE: High-grade glioma treatment includes ionizing radiation therapy. The high invasiveness of glioma cells precludes their eradication and is responsible for the dismal prognosis. Recently, we reported the down-regulation of MHC class I (MHC-I) products in invading tumor cells in human and mouse GL261 gliomas. Here, we tested the hypothesis that whole-brain radiotherapy (WBRT) up-regulates MHC-I expression on GL261 tumors and enhances the effectiveness of immunotherapy. EXPERIMENTAL DESIGN: MHC-I molecule expression on GL261 cells was analyzed in vitro and in vivo by flow cytometry and immunohistochemistry, respectively. To test the response of established GL261 gliomas to treatment, mice with measurable (at CT imaging) brain tumors were randomly assigned to four groups receiving (a) no treatment, (b) WBRT in two fractions of 4 Gy, (c) vaccination with irradiated GL261 cells secreting granulocyte-macrophage colony-stimulating factor, or (d) WBRT and vaccination. Endpoints were tumor response and survival. RESULTS: An ionizing radiation dose of 4 Gy maximally up-regulated MHC-I molecules on GL261 cells in vitro. In vivo, WBRT induced the expression of the beta2-microglobulin light chain subunit of the MHC class I complex on glioma cells invading normal brain and increased CD4+ and CD8+ T cell infiltration. However, the survival advantage obtained with WBRT or vaccination alone was minimal. In contrast, WBRT in combination with vaccination increased long-term survival to 40% to 80%, compared with 0% to 10% in the other groups (P < 0.002). Surviving animals showed antitumor immunity by rejecting challenge tumors. CONCLUSION: Ionizing radiation can be successfully combined with peripheral vaccination for the treatment of established high-grade gliomas
— id: 67436, year: 2006, vol: 12, page: 4730, stat: Journal Article,

CXCR6 and CXCL16 are expressed by breast cancer cells and may play a dual role in tumor progression
Wang, B; Badura, M; He, C; Cameron, T; Dustin, M; Formenti, SC; Schneider, RJ; Demaria, S
2006 FEB ;100(2):S299-S299, Breast cancer research & treatment
— id: 71013, year: 2006, vol: 100, page: S299, stat: Journal Article,

The combination of ionizing radiation and peripheral vaccination produces long-term survival of mice bearing invasive GL261 glioma
Demaria, S; Newcomb, EW; Zagzag, D; Lukyanov, E; Schnee, T; Kawashima, N; Devitt, M; Formenti, SC
2005 NOV 16 ;63(2):S171-S171, International journal of radiation oncology biology physics
— id: 58991, year: 2005, vol: 63, page: S171, 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,

Immune-mediated inhibition of metastases after treatment with local radiation and CTLA-4 blockade in a mouse model of breast cancer
Demaria, Sandra; Kawashima, Noriko; Yang, Anne Marie; Devitt, Mary Louise; Babb, James S; Allison, James P; Formenti, Silvia C
2005 Jan 15;11(2 Pt 1):728-734, Clinical cancer research
PURPOSE: Ionizing radiation therapy (RT) is an important component in the management of breast cancer. Although the primary tumor can be successfully treated by surgery and RT, metastatic breast cancer remains a therapeutic challenge. Here we tested the hypothesis that the combination of RT to the primary tumor with CTLA-4 blockade can elicit antitumor immunity inhibiting the metastases.EXPERIMENTAL DESIGN: The poorly immunogenic metastatic mouse mammary carcinoma 4T1 was used as a model. Mice were injected s.c. with 4T1 cells, and treatment was started 13 days later when the primary tumors measured 5 mm in average diameter. Mice were randomly assigned to four treatment groups receiving: (1) control IgG (IgG), (2) RT + IgG, (3) 9H10 monoclonal antibody against CTLA-4, (4) RT + 9H10. RT was delivered to the primary tumor by one or two fractions of 12 Gy. 9H10 and IgG were given i.p. thrice after RT.RESULTS: Consistent with the fact that 4T1 is poorly immunogenic, 9H10 alone did not have any effect on primary tumor growth or survival. RT was able to delay the growth of the primary irradiated tumor, but in the absence of 9H10 survival was similar to that of control mice. In contrast, mice treated with RT + 9H10 had a statistically significant survival advantage. The increased survival correlated with inhibition of lung metastases formation and required CD8+ but not CD4+ T cells.CONCLUSIONS: The combination of local RT with CTLA-4 blockade is a promising new immunotherapeutic strategy against poorly immunogenic metastatic cancers
— id: 52626, year: 2005, vol: 11, page: 728, stat: Journal Article,

Select forms of tumor cell apoptosis induce dendritic cell maturation
Demaria, Sandra; Santori, Fabio R; Ng, Bruce; Liebes, Leonard; Formenti, Silvia C; Vukmanovic, Stanislav
2005 Mar;77(3):361-368, Journal of leukocyte biology
Dendritic cells (DC) play a crucial role in initiating immune responses to tumors. DC can efficiently present antigens from apoptotic tumor cells, but apoptotic cells are thought to lack the inflammatory signals required to induce DC maturation. Here, we show that apoptosis of 67NR mouse carcinoma cells via the Fas (CD95) pathway or induced by the anticancer drug bortezomib (PS-341) but not by ultraviolet irradiation is associated with the production of maturation signals for DC. These data have important implications for the effects of chemotherapy on antitumor immunity in solid and hematologic malignancies
— id: 48257, year: 2005, vol: 77, page: 361, stat: Journal Article,

Beta-catenin induces a population of radio-resistant alveolar stem/progenitors that progress to form hormone-independent breast tumors in mice
Formenti, SC; Hiremath, M; Yang, A; Demaria, S; Cowin, P
2005 ;63(2):2396-2396, International journal of radiation oncology biology physics
— id: 109265, year: 2005, vol: 63, page: 2396, stat: Journal Article,

Downregulation of major histocompatibility complex antigens in invading glioma cells: stealth invasion of the brain
Zagzag, David; Salnikow, Konstantin; Chiriboga, Luis; Yee, Herman; Lan, Li; Ali, M Aktar; Garcia, Roberto; Demaria, Sandra; Newcomb, Elizabeth W
2005 Mar;85(3):328-341, Laboratory investigation
Invasion into surrounding brain tissue is a fundamental feature of gliomas and the major reason for treatment failure. The process of brain invasion in gliomas is not well understood. Differences in gene expression and/or gene products between invading and noninvading glioma cells may identify potential targets for new therapies. To look for genes associated with glioma invasion, we first employed Affymetrix microarray Genechip((R)) technology to identify genes differentially expressed in migrating glioma cells in vitro and in invading glioma cells in vivo using laser capture microdissection. We observed upregulation of a variety of genes, previously reported to be linked to glioma cell migration and invasion. Remarkably, major histocompatiblity complex (MHC) class I and II genes were significantly downregulated in migrating cells in vitro and in invading cells in vivo. Decreased MHC expression was confirmed in migrating glioma cells in vitro using RT-PCR and in invading glioma cells in vivo by immunohistochemical staining of human and murine glioblastomas for beta2 microglobulin, a marker of MHC class I protein expression. To the best of our knowledge, this report is the first to describe the downregulation of MHC class I and II antigens in migrating and invading glioma cells, in vitro and in vivo, respectively. These results suggest that the very process of tumor invasion is associated with decreased expression of MHC antigens allowing glioma cells to invade the surrounding brain in a 'stealth'-like manner.Laboratory Investigation (2005) 85, 328-341, advance online publication, 31 January 2005; doi:10.1038/labinvest.3700233
— id: 48256, year: 2005, vol: 85, page: 328, stat: Journal Article,

Molecular identification of a novel gene product involved in soluble HLA-release by the metalloproteinase pathway
Bushkin, Y; Watanabe, H; Lieskovska, J; Tolias, P; Demaria, S
2004 JUL ;4(7):317-317, American journal of transplantation
— id: 46603, year: 2004, vol: 4, page: 317, stat: Journal Article,

Combination of local radiation with targeted immunomodulation improves systemic tumor control
Demaria, Sandra; Kawashima, Noriko; Devitt, Mary Louise; Babb, James S.; Allison, James P.; Formenti, Silvia C.
2004 ;45(6):505-505, Proceedings (American Association for Cancer Research)
— id: 109230, year: 2004, vol: 45, page: 505, stat: Journal Article,

Ionizing radiation inhibition of distant untreated tumors (abscopal effect) is immune mediated
Demaria, Sandra; Ng, Bruce; Devitt, Mary Louise; Babb, James S; Kawashima, Noriko; Liebes, Leonard; Formenti, Silvia C
2004 Mar 1;58(3):862-870, International journal of radiation oncology biology physics
PURPOSE: Ionizing radiation can reduce tumor growth outside the field of radiation, known as the abscopal effect. Although it has been reported in multiple malignancies, the abscopal effect remains a rare and poorly understood event. Ionizing radiation generates inflammatory signals and, in principle, could provide both tumor-specific antigens from dying cells and maturation stimuli that are necessary for dendritic cells' activation of tumor-specific T cells. We therefore tested the hypothesis that the abscopal effect elicited by radiation is immune mediated. This was directly tested by enhancing the number of available dendritic cells using the growth factor Flt3-Ligand (Flt3-L). METHODS AND MATERIALS: Mice bearing a syngeneic mammary carcinoma, 67NR, in both flanks were treated with Flt3-L daily for 10 days after local radiation therapy (RT) to only 1 of the 2 tumors at a single dose of 2 or 6 Gy. The second nonirradiated tumor was used as indicator of the abscopal effect. Data were analyzed using repeated measures regression. RESULTS: RT alone led to growth delay exclusively of the irradiated 67NR tumor, as expected. Surprisingly, growth of the nonirradiated tumor was also impaired by the combination of RT and Flt3-L. As control, Flt3-L had no effect without RT. Importantly, the abscopal effect was shown to be tumor specific, because growth of a nonirradiated A20 lymphoma in the same mice containing a treated 67NR tumor was not affected. Moreover, no growth delay of nonirradiated 67NR tumors was observed when T cell deficient (nude) mice were treated with RT plus Flt3-L. CONCLUSIONS: These results demonstrate that the abscopal effect is in part immune mediated and that T cells are required to mediate distant tumor inhibition induced by radiation
— id: 42588, year: 2004, vol: 58, page: 862, stat: Journal Article,

HLA-A2-restricted CD8+-cytotoxic-T-cell responses to novel epitopes in Mycobacterium tuberculosis superoxide dismutase, alanine dehydrogenase, and glutamine synthetase
Dong, Yuzhi; Demaria, Sandra; Sun, Xuming; Santori, Fabio R; Jesdale, Bill M; De Groot, Anne S; Rom, William N; Bushkin, Yuri
2004 Apr;72(4):2412-2415, Infection & immunity
Major histocompatibility complex class I-restricted CD8(+) cytotoxic T lymphocytes (CTL) are implicated in protective Th1 immunity to Mycobacterium tuberculosis infection. We report the identification of three novel HLA-A*0201-restricted CTL epitopes within mycobacterial superoxide dismutase (SodA), L-alanine dehydrogenase (AlaDH), and L-glutamine synthetase (GlnS) proteins
— id: 44727, year: 2004, vol: 72, page: 2412, stat: Journal Article,

Combination of local radiation with CTLA4 blockade: a new approach to the immunotherapy of breast cancer
Demaria S; Kawashima N; Devitt M; Allison JP; Formenti SC
2003 Oct 1;57(2 Suppl):S259-S259, International journal of radiation oncology biology physics
— id: 39082, year: 2003, vol: 57, page: S259, stat: Journal Article,

Chemotherapy can induce apoptosis of cancer cells coupled to the production of maturation signals for dendritic cells
Demaria, Sandra; Ng, Bruce; Santori, Fabio R.; Liebes, Leonard; Vukmanovic, Stanislav; Formenti, Silvia C.
2002 ;43(6):678-678, Proceedings (American Association for Cancer Research)
— id: 109236, year: 2002, vol: 43, page: 678, stat: Journal Article,

Pre-clinical studies of concomitant PS-341 and ionizing radiation therapy: local and systemic anti-tumor effects
Formenti, S; Demaria, S; Liebes, L; Ng, B; Devitt, M; Babbs, J; Muggia, F
2002 NOV ;38(4):34-A256, European journal of cancer
— id: 98237, year: 2002, vol: 38, page: 34, stat: Journal Article,

Development of tumor-infiltrating lymphocytes in breast cancer after neoadjuvant paclitaxel chemotherapy
Demaria S; Volm MD; Shapiro RL; Yee HT; Oratz R; Formenti SC; Muggia F; Symmans WF
2001 Oct;7(10):3025-3030, Clinical cancer research
PURPOSE: Neoadjuvant chemotherapy for breast cancer creates new possibilities for the analysis of biological factors in the tumor and/or host, which may play a role in the response to treatment. In this study we analyzed whether changes in local antitumor immunity take place after neoadjuvant paclitaxel therapy and if they correlate with response to treatment. Experimental Design: Neoadjuvant chemotherapy (paclitaxel, 200 mg/m2 q2w, 4 treatments) was followed by definitive surgical management. Histological sections from the pre- and post-treatment surgical specimens of 25 patients were analyzed for the extent of lymphocytic infiltration and presence of tumor infiltrating lymphocytes (TILs). The cumulative apoptotic response in the tumor after the first dose of paclitaxel was also studied in 10 of 25 patients. RESULTS: Pretreatment lymphocytic infiltrate in the tumor was minimal in the majority of patients and showed no relationship with clinical response. In the patients without TILs before treatment, development of TILs after treatment was noted in 0/3 (0%) patients with stable disease, 3/12 (25%) patients with clinical partial response, and 4/6 (67%) patients with clinical complete response and pathological residual disease. These correlated with the tumor cell apoptotic response to the first dose of paclitaxel. CONCLUSIONS: These results suggest that development of TILs after treatment correlates with clinical response to neoadjuvant paclitaxel therapy. The possible mechanism(s) whereby neoadjuvant chemotherapy may lead to induction of antitumor T cells is discussed. Immunological processes may influence the response of breast cancer patients to neoadjuvant treatment
— id: 24141, year: 2001, vol: 7, page: 3025, stat: Journal Article,

Serial fine needle aspirations during neoadjuvant chemotherapy: Assessment of apoptotic responses in breast cancer
Symmans, W. F.; Volm, M.; Shapiro, R. L.; Demaria, S.; Yee, H.; Formenti, S. C.; Muggia, F.
2001 ;8(Supplement 1):S46-S46, International journal of molecular medicine
— id: 109251, year: 2001, vol: 8, page: S46, stat: Journal Article,

Peptide loading of nascent MHC class I molecules
Vukmanovic S; Lilic M; Santori FR; Demaria S; Kulig K
2001 ;49(3):195-201, Archivum immunologii & therapiae experimentalis
A critical molecular interaction during assembly of the major histocompatibility complex (MHC) class I molecules takes place between the heavy chain and the transporter-associated with antigen-processing (TAP) complex. The recent mapping of regions of the heavy chain involved in the binding to TAP suggests a complex molecular interaction essential for the cell surface expression of the MHC class I. The advances made in understanding the TAP-MHC class I interaction are reviewed and discussed here
— id: 24142, year: 2001, vol: 49, page: 195, stat: Journal Article,

Soluble HLA proteins with bound peptides are released from the cell surface by the membrane metalloproteinase
Demaria S; Bushkin Y
2000 Dec;61(12):1332-1338, Human immunology
The metalloproteinase (MPase)-mediated pathway of MHC class I processing is a distinct cellular mechanism that generates soluble HLA proteins. It has been implicated in modulation of immune responses induced during transplantation events. It is, therefore, important to define the characteristics of soluble HLA species produced by the MPase pathway. We have previously shown that some mutant peptide-conformed beta(2)-microglobulin (beta(2)m) free heavy chains (HC) with lower affinity for beta(2)m can be released into supernatants by the MPase. These soluble conformed beta(2)m-free HC intermediates can re-associate with beta(2)m in solution giving rise to beta(2)m-associated HC. We now demonstrate that also nonmutant soluble conformed beta(2)m-free HC can be detected in supernatants of activated cells. These soluble HC intermediates appear to have bound peptides and readily re-associate with exogenous beta(2)m producing beta(2)m-associated HC that are stable at physiologic temperature. Thus, generation of peptide-conformed beta(2)m-free HC intermediates is an important step, which precedes generation of both soluble beta(2)m-free and beta(2)m-associated HC by the MPase pathway operating in activated cells
— id: 24143, year: 2000, vol: 61, page: 1332, stat: Journal Article,

Increase in the lymphocytic infiltrate in breast cancer after neoadjuvant paclitaxel chemotherapy
Demaria, Sandra; Volm, M. D.; Shapiro, R. L.; Yee, H. T.; Oratz, R.; Formenti, S.; Muggia, F.; Symmans, W. F.
2000 ;43(41):334-334, Proceedings (American Association for Cancer Research)
— id: 109238, year: 2000, vol: 43, page: 334, stat: Journal Article,

Paclitaxel-induced apoptosis and mitotic arrest assessed by serial fine-needle aspiration: implications for early prediction of breast cancer response to neoadjuvant treatment
Symmans WF; Volm MD; Shapiro RL; Perkins AB; Kim AY; Demaria S; Yee HT; McMullen H; Oratz R; Klein P; Formenti SC; Muggia F
2000 Dec;6(12):4610-4617, Clinical cancer research
The extent of tumor reduction from neoadjuvant chemotherapy for breast cancer correlates with outcome. We investigated whether the initial cellular responses to paclitaxel are related to the extent of tumor reduction. Eleven women with breast cancer received paclitaxel (every 2 weeks for 4 cycles) as neoadjuvant treatment. Serial fine-needle aspirations (FNA; 25-gauge, 1 pass) were obtained before treatment and at 24, 48, 72, and 96 h after the first paclitaxel dose. Microscopic counts of apoptotic and mitotic indices were performed. The change in cancer volume from treatment was determined using radiological measurements with allowance for change in the histopathological amount of cancer. Apoptotic and mitotic responses usually subsided within 4 days. The duration of the initial apoptotic response was different for women with different treatment results. Cumulative apoptotic response for the first 4 days inversely correlated with the proportion of residual cancer after neoadjuvant treatment. FNA is a versatile clinical method to obtain breast cancer cells for therapy response studies. Apoptotic response to the first dose of paclitaxel is almost complete within 4 days, implying that more frequent (weekly) paclitaxel dosing might be beneficial. The apoptotic response to the first dose of paclitaxel appeared to predict the amount of cancer reduction from this treatment. This is a promising start toward the development of an early chemopredictive assay for paclitaxel treatment of breast cancer
— id: 22642, year: 2000, vol: 6, page: 4610, stat: Journal Article,

Multilocular thymic cyst with follicular lymphoid hyperplasia in a male infected with HIV. A case report with fine needle aspiration cytology
Chhieng DC; Demaria S; Yee HT; Yang GC
1999 Nov-Dec;43(6):1119-1123, Acta cytologica
BACKGROUND: Multilocular thymic cyst with follicular lymphoid hyperplasia is a rare complication in HIV-infected patients, causing pseudotumorous enlargement of the anterior mediastinum. There have been six reported cases, all with only histologic findings. This paper reports another such case and includes perhaps the first cytologic findings on this rare entity. CASE: A 35-year-old, HIV-infected male intravenous drug abuser, who complained of worsening central chest discomfort and pain on deep inspiration, was found to have a large, septated anterior mediastinal mass. Computed tomography-guided fine needle aspiration biopsy was performed. The cytologic presentation mimicked that of thymoma, with cystic degeneration and a dual population of epithelial cells and lymphocytes as well as large aggregates of 'epithelial' cells intermixed with lymphocytes in a background of macrophages and cyst fluid. Histologic examination of the resected mass revealed a multilocular thymic cyst with follicular lymphoid hyperplasia. HIV-1 core protein p24 was localized immunohistochemically in the dendritic follicular cells of the germinal centers. In retrospect, the quantity of epithelium derived from the cyst lining was too scanty for thymoma, and the presence of plasma cells and lymphohistiocytic aggregates suggested follicular lymphoid hyperplasia. CONCLUSION: Multilocular thymic cyst with follicular lymphoid hyperplasia should be considered in the differential diagnosis of an anterior mediastinal mass in HIV-infected patients after lymphoma and tuberculosis
— id: 6247, year: 1999, vol: 43, page: 1119, stat: Journal Article,

Peptide-conformed beta2m-free class I heavy chains are intermediates in generation of soluble HLA by the membrane-bound metalloproteinase
Demaria S; DeVito-Haynes LD; Salter RD; Burlingham WJ; Bushkin Y
1999 Dec;60(12):1216-1226, Human immunology
Molecular mechanisms of soluble HLA-release by a membrane-bound metalloproteinase (MPase) are not defined. We have investigated the possibility that certain beta2-microglobulin (beta2m)-free heavy chains (HC) retain peptide-induced conformations before and after the cleavage by using mutant HLA-A2.242K HC with reduced affinity for beta2m. We show that dissociation of HC/beta2m complexes on the surface of C1R lymphoblastoid cells generates both conformed and non-conformed beta2m-free HC recognized by conformation-dependent antibodies. Conformed HC, having bound the HLA-A2-specific peptide HTLV-1 tax 11-19, can retain their proper conformations after dissociation of beta2m. Further, conformed and non-conformed surface beta2m-free HC are cleaved by the MPase, and some released HC preserve their conformations. Exogenous beta2m binds only to conformed HC, and protects them from cleavage as effectively as the MPase inhibitor BB-2116. We propose that soluble HLA-release requires generation of peptide-conformed beta2m-free HC intermediates on the cell surface, which are then cleaved by the MPase and in solution may reassociate with beta2m. Given the role of soluble HLA in the indirect allorecognition, the activity of this MPase may be important in transplant rejection
— id: 14603, year: 1999, vol: 60, page: 1216, stat: Journal Article,

Fine needle aspiration of primary pleomorphic liposarcoma of the breast. A case report
Demaria S; Yee HT; Cangiarella J; Cohen JM; Chhieng DC
1999 Nov-Dec;43(6):1131-1136, Acta cytologica
BACKGROUND: Primary liposarcoma of the breast is an extremely rare lesion. Only two cases describing the aspiration biopsy findings have been reported in the literature. We report the cytologic findings in an additional case, stressing the cytologic clues necessary to distinguish this neoplasm from a primary adenocarcinoma. CASE: A 53-year-old female presented to the emergency room with bleeding from a 20-cm, ulcerating mass in the right breast. Four months earlier she had been seen at another institution, where a diagnosis of poorly differentiated carcinoma was made by aspiration biopsy. Computed tomography had been negative for metastatic disease, and the patient refused further evaluation. Aspiration biopsy of the breast mass was repeated at our institution and interpreted as consistent with a poorly differentiated carcinoma. Histologic, immunophenotypic and ultrastructural evaluation of the mastectomy specimen revealed a pleomorphic liposarcoma. CONCLUSION: With increasing utilization of fine needle aspiration to evaluate breast lesions, it can be anticipated that unusual entities, including liposarcomas, will be encountered increasingly in breast aspirates. Therefore, it is important to consider liposarcoma in the differential diagnosis of aspirates showing isolated spindle and polygonal cells with vacuolated cytoplasm, nuclear scalloping and pleomorphism to avoid a misdiagnosis of carcinoma
— id: 6248, year: 1999, vol: 43, page: 1131, stat: Journal Article,

The metalloproteinase-mediated pathway is essential for generation of soluble HLA class I proteins by activated cells in vitro: proposed mechanism for soluble HLA release in transplant rejection
DeVito-Haynes LD; Demaria S; Bushkin Y; Burlingham WJ
1998 Jul;59(7):426-434, Human immunology
We and others have found donor-derived soluble beta2m-associated HLA class I proteins (sHLA/beta2m) in the serum of allograft recipients with acute and chronic rejection. Whether appearance of sHLA/beta2m and upregulated expression of donor cell-bound HLA/beta2m during allograft rejection are related events is unknown. Activation-induced upregulation of in vitro HLA/beta2m expression correlates with the surface expression of another form of HLA class I, namely beta2m-free HLA heavy chains (beta2m-free HC). We have shown that beta2m-free HC, but not beta2m-associated HC, are then cleaved by a specific membrane-bound metalloproteinase and released into supernatants as soluble 36 kDa proteins. We show now that activated peripheral blood lymphocytes produce predominantly the 36 kDa form of sHLA proteins which is present in supernatants as both beta2m-free HC and sHLA/beta2m. Importantly, the metalloprotease inhibitor BB-94 blocked not only the release of soluble beta2m-free HC, but also the appearance of sHLA/beta2m in cell supernatants. Low levels of 36 kDa beta2m-free HC were also present in human plasma of healthy donors. These data suggest an important role for the HLA class I-specific metalloproteinase in vivo in healthy individuals and during allograft rejection in the generation of soluble beta2m-free and beta2m-associated HLA proteins
— id: 14605, year: 1998, vol: 59, page: 426, stat: Journal Article,

Soluble CD4 induces the binding of human immunodeficiency virus type 1 to cells via the V3 loop of glycoprotein 120 and specific sites in glycoprotein 41
Demaria S; Bushkin Y
1996 Mar 1;12(4):281-290, AIDS research & human retroviruses
We have previously reported that incubation of human immunodeficiency virus type 1 (HIV-1) at 4 degrees C with soluble CD4 (sCD4) does not block but increases the binding of virions to CD4-positive H9 cells. In this study, we investigated the mechanism of this effect. It appears that sCD4 can induce the binding of HIV-1IIIB to CD4-negative human cells and to H9 cells with downregulated expression of CD4 at both 4 and 37 degrees C. The binding is proportional to the amount of sCD4 associated with virions, and requires the presence of heparan sulfate proteoglycans on the surface of cells. Monoclonal antibody (MAb) 9284 directed at an epitope overlapping with a putative heparin binding motif in the V3 loop of gp120 almost completely blocked the sCD4-induced binding of virions, while MAbs recognizing other sites of V2 or V3 loops had no effect. The binding of sCD4-coated virions to cells was also inhibited by MAbs 50-69 and 98-6 directed at extracellular epitopes of gp41, whose exposure is increased on binding of sCD4 to virions. Therefore, sCD4 potentiates the binding of HIV-1IIIB virions to cells by inducing conformational changes that enable envelope gp120 and gp41 to interact with cell surface components other than the CD4 receptor
— id: 14606, year: 1996, vol: 12, page: 281, stat: Journal Article,

Bathophenanthroline disulfonate and soluble CD4 as probes for early events of HIV type 1 entry
Demaria S; Tilley SA; Pinter A; Bushkin Y
1995 Jan;11(1):127-139, AIDS research & human retroviruses
We report here that a metalloprotease inhibitor, bathophenanthroline disulfonate (Bphe-ds), neutralizes both laboratory-adapted and primary strains of HIV-1. Presaturation of Bphe-ds with zinc does not alter its neutralizing activity, suggesting that the metal-chelating ability of Bphe-ds is not required for neutralization. Bphe-ds blocks infection of CD4+ cells at the stage of viral entry, not through a direct viricidal effect, but by interfering with both binding and postbinding events. This drug interacts with HIV-1 envelope, blocking almost completely the binding of three MAbs that recognize epitopes overlapping the CD4-binding site on gp120, but has no effect on the binding of MAbs directed to the cellular receptor CD4. The exposure of epitopes in the V2 and V3 but not C5 domains of gp120 is partially decreased in the presence of Bphe-ds, suggesting that the drug induces conformational changes in the envelope glycoprotein(s). Binding of both virions and soluble gp120 to CD4+ cells is inhibited by this drug in a dose-dependent manner. This contrasted with the effects of soluble CD4, which actually increased binding of virions to cells at 4 degrees C, while inhibiting the binding of soluble gp120. Bphe-ds also increases shedding of gp120 from cells infected with HIV-1IIIB. Thus, Bphe-ds appears to be an envelope-directed inhibitor of HIV-1 that neutralizes HIV-1 infectivity via multiple mechanisms
— id: 14607, year: 1995, vol: 11, page: 127, stat: Journal Article,

Soluble beta 2-microglobulin-free class I heavy chains are released from the surface of activated and leukemia cells by a metalloprotease
Demaria S; Schwab R; Gottesman SR; Bushkin Y
1994 Mar 4;269(9):6689-6694, Journal of biological chemistry
Regulation of the expression of major histocompatibility complex (MHC) class I heavy chains not associated with beta 2-microglobulin (beta 2m) on freshly isolated and in vitro cultured human B and T leukemia cells was analyzed. These beta 2m-free class I heavy chains originate from surface beta 2m-associated MHC class I molecules and are expressed as integral membrane glycoproteins on activated, but not resting, cells. We found that the levels of beta 2m-free class I heavy chains can be regulated by proteolytic cleavage and release into the medium of soluble molecules containing the extracellular domains. The release is mediated by a Zn(2+)-dependent, membrane-bound metalloprotease that does not cleave HLA-DR, CD4, and CD71 surface receptors and can be activated by phorbol myristate acetate. Specific cleavage by the metalloprotease occurs at a site close to the papain cleavage site in the alpha 3 domain of class I heavy chains. This site is not accessible to the metalloprotease in beta 2m-associated MHC class I molecules. The dissociation of beta 2m-associated MHC class I molecules and subsequent cleavage of beta 2m-free class I heavy chains may be partially responsible for controlling the levels of MHC class I molecules on the surface of activated cells
— id: 14609, year: 1994, vol: 269, page: 6689, stat: Journal Article,

CD8 and beta 2-microglobulin-free MHC class I molecules in T cell immunoregulation
Demaria S; Bushkin Y
1993 ;23(2):61-69, International journal of clinical & laboratory research
Intracellular assembly of MHC class I heavy chains with beta 2-microglobulin occurs prior to the expression of the antigen-presenting complex on the cell surface. The association of beta 2-microglobulin with newly synthesized class I heavy chains is thought to be a strict prerequisite for their transport to the cell surface. However, MHC class I molecules not associated with beta 2-microglobulin (beta 2-microglobulin-free class I heavy chains) have been detected on the surface of activated lymphoid cells. These molecules have different conformations. Therefore, their interactions with other membrane proteins and biological functions may be different from those assigned to beta 2-microglobulin-associated MHC class I molecules. The two forms of MHC class I molecules on the surface of activated cells can self-associate and also form complexes with distinct proteins. Upon interaction with the appropriate ligands these molecular complexes transduce signals regulating cell activation. The ligand for beta 2-microglobulin-free class I heavy chains appears to be soluble CD8. A model is presented describing a novel mechanism of immunoregulation mediated by both soluble and membrane-bound forms of CD8 and beta 2-microglobulin-free class I heavy chains
— id: 14610, year: 1993, vol: 23, page: 61, stat: Journal Article,

The origin and fate of beta 2m-free MHC class I molecules induced on activated T cells
Demaria S; Schwab R; Bushkin Y
1992 Jun;142(1):103-113, Cellular immunology
We report here that the expression of major histocompatibility complex (MHC) class I heavy chains not associated with beta 2-microglobulin is induced on resting human T cells by a variety of stimuli. These beta 2m-free class I heavy chains are not transported as such from the endoplasmic reticulum but originate from surface beta 2m-associated MHC class I molecules. beta 2m-free class I heavy chains are spontaneously released from the surface of activated cells. Cross-linking of beta 2m-free class I heavy chains with specific monoclonal antibodies results in the rapid down-regulation and internalization of these molecules. In contrast, beta 2m-associated MHC class I molecules display a different pattern of modulation. Previously, we reported that beta 2m-free class I heavy chains interact with CD8 molecules expressed on the same activated T cells. We propose that interactions between these molecules are involved in a mechanism regulating the function of activated T cells
— id: 14611, year: 1992, vol: 142, page: 103, stat: Journal Article,

Activation of human CD8-positive T cells via the CD8/HLA class I complex
Bushkin Y; Demaria S; Mohagheghpour N; Le JM
1990 Mar;126(1):185-195, Cellular immunology
Cross-linking of CD8 and HLA class I molecules with appropriate monoclonal antibodies (mAb) and goat anti-mouse Ig (GaMIg) antibody resulted in a marked proliferation of resting human CD8 cells in the presence of interleukin-2 (IL-2). These cells also expressed IL-2 receptor (IL-2R), transferrin receptor, HLA-DR and -DQ antigens. Activation of the cross-linked CD8 cells is apparently independent of accessory monocytes. Various anti-CD8 and anti-HLA class I mAb recognizing nonpolymorphic antigenic determinants were examined for the efficacy of activating CD8 cells. Among mAb specific for HLA class I molecules, PA2.6, MB40.5, BB7.7, A1.4, and W6/32 mAb markedly stimulated the proliferation of cross-linked CD8 cells, whereas BBM.1, Q1/28, and HC10 mAb were found inactive. Footprinting analysis of HLA class I molecules suggested that the activity of these anti-HLA class I mAb appeared to be related to the corresponding peptides they protect from enzymatic digestion. In contrast to the anti-HLA class I mAb, all anti-CD8 mAb examined (C8, OKT8A, and anti-Leu-2a) induced the proliferation of CD8-HLA class I cross-linked cells with similar efficacy. These results suggest that physical interaction between CD8 and at least one specific region of HLA class I molecules can trigger the activation of resting human CD8 cells
— id: 14612, year: 1990, vol: 126, page: 185, stat: Journal Article,

Increased density of ecto 5' nucleotidase antigen on leukemic T cells from patients with cutaneous T-cell lymphoma and adult T-cell leukemia/lymphoma
Fukunaga Y; Evans SS; Yamamoto M; Ueda Y; Tamura K; Takakuwa T; Gebhard D; Allopenna J; Demaria S; Clarkson B; et al.
1989 Nov 15;74(7):2486-2492, Blood
Malignant CD4+ T cells in adult T-cell leukemia/lymphoma (ATL) and cutaneous T-cell lymphoma (CTCL) express a number of cell surface molecules that are upregulated on normal T cells activated by foreign antigen. In this report we describe an interesting exception to the parallel phenotypic features of activated T cells and malignant CD4+ T cells. A monoclonal antibody (MoAb; termed 27.2) that was raised to HTLV-1+, CD4+25+ leukemic T cells stained weakly 25% of peripheral T cells, including approximately 50% of CD8+ T cells and 20% of CD4+ T cells. Flow cytometry analysis indicated that the surface density of the 27.2 antigen was unchanged or diminished when normal T cells were activated by antigen. However, 3/4 Sezary cases and 4/8 cases of ATL had relatively high densities of the 27.2 antigen. Immunoprecipitation and sodium dodecylsulfate polyacrylamide gel electrophoresis of the NP-40-solubilized membranes of surface-iodinated ATL cells indicated that MoAb 27.2 reacted with a 75 Kd molecule. The size and distribution of the 27.2 antigen on T cell subsets suggested that it might be the enzyme ecto-5' nucleotidase (NT), a phosphatidylinositol-linked enzyme that catalyzes dephosphorylation of monophosphate nucleotides to their respective nucleosides. This was confirmed by demonstrating that lymphocyte ecto-5'NT activity was blocked partially and inhibited completely by preincubating cells with MoAb 27.2 for 1 hour at 4 degrees C and 24 hours at 37 degrees C, respectively. When used with a second MoAb (27.1) to a novel T cell activation antigen found on all CTCL and ATL leukemias examined, 27.2 was found to discriminate between normal and leukemic T cells in two patients with ATL. These studies suggest that ecto-5'NT has diagnostic value in T cell malignancies and may be aberrantly expressed in some cases of ATL and CTCL
— id: 24144, year: 1989, vol: 74, page: 2486, stat: Journal Article,

Physical association between the CD8 and HLA class I molecules on the surface of activated human T lymphocytes
Bushkin Y; Demaria S; Le JM; Schwab R
1988 Jun;85(11):3985-3989, Proceedings of the National Academy of Sciences of the United States of America
Immune recognition by cytotoxic effector T cells requires participation of the CD8 and major histocompatibility complex class I antigens. We found that the CD8 molecule is noncovalently associated with the HLA class I heavy chain on the surface of human T cells activated by Con A. Accordingly, anti-CD8 monoclonal antibodies precipitated a heterodimer containing polypeptides of 32 and 43 kDa from the lysates of activated T cells. The 43-kDa chain of this heterodimer can be adsorbed from cell lysates with anti-HLA-A, -B, and -C antibodies. Endoglycosidase F treatment and chymotryptic peptide mapping identified a structural similarity between this 43-kDa molecule and the HLA class I heavy chain precipitated by the anti-HLA-A, -B, and -C antibody W6/32. Analysis of anti-CD8 precipitates under nonreducing and reducing conditions indicated a lack of interchain disulfide bonding between the CD8 and HLA heavy chain molecules. The CD8-HLA heavy chain complex was also detected in mixed lymphocyte cultures and a cloned cytotoxic T-lymphocyte line but not in purified natural killer cells. The present study indicates that CD8 is complexed with HLA heavy chain on the same cells, and the complex may have functional relevance in the T-cell recognition process
— id: 14615, year: 1988, vol: 85, page: 3985, stat: Journal Article,

Role of cell surface receptors in the regulation of non adaptive immune response by the large granular lymphocytes
De Maria S; Funaro A; Dellabona P; Alessio M; Demonte L; Bellone G; Perotto S; Cappa AP; Bushkin Y; Malavasi F
1988 ;24(1):129-136, Annali dell'Istituto Superiore di Sanita
— id: 14616, year: 1988, vol: 24, page: 129, stat: Journal Article,

Definition by CB12 monoclonal antibody of a differentiation marker specific for human monocytes and their bone marrow precursors
Malavasi F; Funaro A; Bellone G; Caligaris-Cappio F; Semenzato G; Cappa AP; Ferrero E; Novelli F; Alessio M; Demaria S; et al.
1986 Feb;97(2):276-285, Cellular immunology
The CB12 monoclonal antibody, which reacts with a molecule expressed on monocytes, was characterized using human embryonic material as immunizer. Analysis of the monoclonal antibody at the phenotypic, molecular, and functional levels indicates that its reactivity is restricted to circulating monocytes and their precursors in the bone marrow, whereas it is undetectable on tissue macrophages. CB12 displays a pattern of reactivity compatible with that of a marker of monocyte differentiation. Preliminary data indicate a possible receptor role for the CB12 molecule
— id: 24145, year: 1986, vol: 97, page: 276, stat: Journal Article,

Murine monoclonal antibodies as probes for the phenotypical, functional, and molecular analysis of a discrete peripheral blood lymphocyte population exerting natural killer activity in vitro
Malavasi F; Bellone G; Matera L; Milanese C; Ferrero E; Funaro A; Demaria S; Caligaris-Cappio F; Camussi G; Dellabona P
1985 Oct;14(2):87-102, Human immunology
Two monoclonal antibodies (AB8.28 and A10) reacting with large granular lymphocytes were extensively studied and characterized. The two peripheral blood lymphocyte subsets positive for the expression of AB8.28 and A10 determinants were isolated by cell sorting and the phenotype analyzed using a panel of anti-lymphocyte reagents. Both subsets displayed the characteristics of 'null cells.' Moreover, these subsets encompassed a significant amount of the natural killer activity, since preparations of peripheral blood lymphocytes deprived of AB8.28+ and A10+ cells showed a remarkable reduction of such activity. The analysis of the distribution of the AB8.28 and A10 epitopes has been carried out using a variety of cells, i.e., normal tissues, tumor cells, established cell lines, and preparations obtained from patients with different leukemic disorders. The structure bearing the epitopes recognized by the two monoclonal antibodies was characterized immunologically (immunoprecipitation, SDS-PAGE analysis, immunomodulation, and competition with other antibodies) and by various functional assays. On the basis of inhibition tests, the AB8.28 molecule seems to be related functionally and/or structurally with the IgG Fc receptor. By contrast, the A10 structure does not share this activity and so far has eluded any precise biological characterization
— id: 24147, year: 1985, vol: 14, page: 87, stat: Journal Article,

Functional and molecular characterization by the CB04 monoclonal antibody of a cell surface structure exerting C3-complement receptor activity
Malavasi F; Funaro A; Bellone G; Caligaris-Cappio F; Berti E; Tetta C; Dellabona P; DeMaria S; Campogrande M; Cappa AP
1985 Nov;5(6):412-420, Journal of clinical immunology
CB04 monoclonal antibody which reacts with an epitope of a surface molecule expressed on human monocytes has been elicited using peripheral blood lymphocytes as immunizer. The characterization of the monoclonal antibody examined at the phenotypic, molecular, and functional levels indicates that the CB04 antibody defines a structure present on monocytes, tissue macrophages, B cells, polymorphonucleates, and erythrocytes. The molecular weight (220 kD), the tissue distribution in health and disease conditions, and the involvement in relevant biological processes indicate that the CB04 structure is the receptor for the C3b fragment of the complement. The binding of the antibody to the cell surface induces inhibition of the C3bi receptorial function
— id: 24146, year: 1985, vol: 5, page: 412, stat: Journal Article,