Alan B Frey, Ph.D.

Dendritic cells promote pancreatic viability in mice with acute pancreatitis
Bedrosian, Andrea S; Nguyen, Andrew H; Hackman, Michael; Connolly, Michael K; Malhotra, Ashim; Ibrahim, Junaid; Cieza-Rubio, Napoleon E; Henning, Justin R; Barilla, Rocky; Rehman, Adeel; Pachter, H Leon; Medina-Zea, Marco V; Cohen, Steven M; Frey, Alan B; Acehan, Devrim; Miller, George
2011 Nov;141(5):1915-1926.e14, Gastroenterology
BACKGROUND & AIMS: The cellular mediators of acute pancreatitis are incompletely understood. Dendritic cells (DCs) can promote or suppress inflammation, depending on their subtype and context. We investigated the roles of DC in development of acute pancreatitis. METHODS: Acute pancreatitis was induced in CD11c.DTR mice using caerulein or L-arginine; DCs were depleted by administration of diphtheria toxin. Survival was analyzed using Kaplan-Meier method. RESULTS: Numbers of major histocompatibility complex II(+)CD11c(+) DCs increased 100-fold in pancreata of mice with acute pancreatitis to account for nearly 15% of intrapancreatic leukocytes. Intrapancreatic DCs acquired a distinct immune phenotype in mice with acute pancreatitis; they expressed higher levels of major histocompatibility complex II and CD86 and increased production of interleukin-6, membrane cofactor protein-1, and tumor necrosis factor-alpha. However, rather than inducing an organ-destructive inflammatory process, DCs were required for pancreatic viability; the exocrine pancreas died in mice that were depleted of DCs and challenged with caerulein or L-arginine. All mice with pancreatitis that were depleted of DCs died from acinar cell death within 4 days. Depletion of DCs from mice with pancreatitis resulted in neutrophil infiltration and increased levels of systemic markers of inflammation. However, the organ necrosis associated with depletion of DCs did not require infiltrating neutrophils, activation of nuclear factor-kappaB, or signaling by mitogen-activated protein kinase or tumor necrosis factor-alpha. CONCLUSIONS: DCs are required for pancreatic viability in mice with acute pancreatitis and might protect organs against cell stress
— id: 139730, year: 2011, vol: 141, page: 1915, stat: Journal Article,

In hepatic fibrosis, liver sinusoidal endothelial cells acquire enhanced immunogenicity
Connolly, Michael K; Bedrosian, Andrea S; Malhotra, Ashim; Henning, Justin R; Ibrahim, Junaid; Vera, Valery; Cieza-Rubio, Napoleon E; Hassan, Burhan U; Pachter, H Leon; Cohen, Steven; Frey, Alan B; Miller, George
2010 Aug 15;185(4):2200-2208, Journal of immunology
The normal liver is characterized by immunologic tolerance. Primary mediators of hepatic immune tolerance are liver sinusoidal endothelial cells (LSECs). LSECs block adaptive immunogenic responses to Ag and induce the generation of T regulatory cells. Hepatic fibrosis is characterized by both intense intrahepatic inflammation and altered hepatic immunity. We postulated that, in liver fibrosis, a reversal of LSEC function from tolerogenic to proinflammatory and immunogenic may contribute to both the heightened inflammatory milieu and altered intrahepatic immunity. We found that, after fibrotic liver injury from hepatotoxins, LSECs become highly proinflammatory and secrete an array of cytokines and chemokines. In addition, LSECs gain enhanced capacity to capture Ag and induce T cell proliferation. Similarly, unlike LSECs in normal livers, in fibrosis, LSECs do not veto dendritic cell priming of T cells. Furthermore, whereas in normal livers, LSECs are active in the generation of T regulatory cells, in hepatic fibrosis LSECs induce an immunogenic T cell phenotype capable of enhancing endogenous CTLs and generating potent de novo CTL responses. Moreover, depletion of LSECs from fibrotic liver cultures mitigates the proinflammatory milieu characteristic of hepatic fibrosis. Our findings offer a critical understanding of the role of LSECs in modulating intrahepatic immunity and inflammation in fibro-inflammatory liver disease
— id: 111819, year: 2010, vol: 185, page: 2200, stat: Journal Article,

Distinct populations of metastases-enabling myeloid cells expand in the liver of mice harboring invasive and preinvasive intra-abdominal tumor
Connolly, Michael K; Mallen-St Clair, Jon; Bedrosian, Andrea S; Malhotra, Ashim; Vera, Valery; Ibrahim, Junaid; Henning, Justin; Pachter, H Leon; Bar-Sagi, Dafna; Frey, Alan B; Miller, George
2010 Apr;87(4):713-725, Journal of leukocyte biology
The liver is the most common site of adenocarcinoma metastases, even in patients who initially present with early disease. We postulated that immune-suppressive cells in the liver of tumor-bearing hosts inhibit anti-tumor T cells, thereby accelerating the growth of liver metastases. Using models of early preinvasive pancreatic neoplasia and advanced colorectal cancer, aims of this study were to determine immune phenotype, stimulus for recruitment, inhibitory effects, and tumor-enabling function of immune-suppressive cells in the liver of tumor-bearing hosts. We found that in mice with intra-abdominal malignancies, two distinct CD11b(+)Gr1(+) populations with divergent phenotypic and functional properties accumulate in the liver, becoming the dominant hepatic leukocytes. Their expansion is contingent on tumor expression of KC. These cells are distinct from CD11b(+)Gr1(+) populations in other tissues of tumor-bearing hosts in terms of cellular phenotype and cytokine and chemokine profile. Liver CD11b(+)Gr1(+) cells are highly suppressive of T cell activation, proliferation, and cytotoxicity and induce the development of Tregs. Moreover, liver myeloid-derived suppressor cells accelerate the development of hepatic metastases by inactivation of cytotoxic T cells. These findings may explain the propensity of patients with intra-abdominal cancers to develop liver metastases and suggest a promising target for experimental therapeutics
— id: 108918, year: 2010, vol: 87, page: 713, stat: Journal Article,

Tumor-induced disruption of proximal TCR-mediated signal transduction in tumor-infiltrating CD8+ lymphocytes inactivates antitumor effector phase
Vazquez-Cintron, Edwin J; Monu, Ngozi R; Frey, Alan B
2010 Dec 15;185(12):7133-7140, Journal of immunology
The presence in cancer tissue of Ag-specific, activated tumor infiltrating CD8(+) T cells proves that tumors express Ags capable of eliciting immune response. Therefore, in general, tumor escape from immune-mediated clearance is not attributable to immunological ignorance. However, tumor-infiltrating lymphocytes are defective in effector phase function, demonstrating tumor-induced immune suppression that likely underlies tumor escape. Since exocytosis of lytic granules is dependent upon TCR-mediated signal transduction, it is a reasonable contention that tumors may induce defective signal transduction in tumor infiltrating T cells. In this review, we consider the biochemical basis for antitumor T cell dysfunction, focusing on the role of inhibitory signaling receptors in restricting TCR-mediated signaling in tumor-infiltrating lymphocytes
— id: 116210, year: 2010, vol: 185, page: 7133, stat: Journal Article,

In liver fibrosis, dendritic cells govern hepatic inflammation in mice via TNF-alpha
Connolly, Michael K; Bedrosian, Andrea S; Mallen-St Clair, Jon; Mitchell, Aaron P; Ibrahim, Junaid; Stroud, Andrea; Pachter, H Leon; Bar-Sagi, Dafna; Frey, Alan B; Miller, George
2009 Nov;119(11):3213-25, Journal of clinical investigation
Hepatic fibrosis occurs during most chronic liver diseases and is driven by inflammatory responses to injured tissue. Because DCs are central to modulating liver immunity, we postulated that altered DC function contributes to immunologic changes in hepatic fibrosis and affects the pathologic inflammatory milieu within the fibrotic liver. Using mouse models, we determined the contribution of DCs to altered hepatic immunity in fibrosis and investigated the role of DCs in modulating the inflammatory environment within the fibrotic liver. We found that DC depletion completely abrogated the elevated levels of many inflammatory mediators that are produced in the fibrotic liver. DCs represented approximately 25% of the fibrotic hepatic leukocytes and showed an elevated CD11b+CD8- fraction, a lower B220+ plasmacytoid fraction, and increased expression of MHC II and CD40. Moreover, after liver injury, DCs gained a marked capacity to induce hepatic stellate cells, NK cells, and T cells to mediate inflammation, proliferation, and production of potent immune responses. The proinflammatory and immunogenic effects of fibrotic DCs were contingent on their production of TNF-alpha. Therefore, modulating DC function may be an attractive approach to experimental therapeutics in fibro-inflammatory liver disease
— id: 105172, year: 2009, vol: 119, page: 3213, stat: Journal Article,

Signaling defects in anti-tumor T cells
Frey, Alan B; Monu, Ngozi
2008 Apr;222:192-205, Immunological reviews
The immune response to cancer has been long recognized, including both innate and adaptive responses, showing that the immune system can recognize protein products of genetic and epigenetic changes in transformed cells. The accumulation of antigen-specific T cells within the tumor, the draining lymph node, and the circulation, either in newly diagnosed patients or resultant from experimental immunotherapy, proves that tumors produce antigens and that priming occurs. Unfortunately, just as obviously, tumors grow, implying that anti-tumor immune responses are either not sufficiently vigorous to eliminate the cancer or that anti-tumor immunity is suppressed. Both possibilities are supported by current data. In experimental animal models of cancer and also in patients, systemic immunity is usually not dramatically suppressed, because tumor-bearing animals and patients develop T-cell-dependent immune responses to microbes and to either model antigens or experimental cancer vaccines. However, inhibition of specific anti-tumor immunity is common, and several possible explanations of tolerance to tumor antigens or tumor-induced immunesuppression have been proposed. Inhibition of effective anti-tumor immunity results from the tumor or the host response to tumor growth, inhibiting the activation, differentiation, or function of anti-tumor immune cells. As a consequence, anti-tumor T cells cannot respond productively to developmental, targeting, or activation cues. While able to enhance the number and phenotype of anti-tumor T cells, the modest success of immunotherapy has shown the necessity to attempt to reverse tolerance in anti-tumor T cells, and the vanguard of experimental therapy now focuses on vaccination in combination with blockade of immunosuppressive mechanisms. This review discusses several potential mechanisms by which anti-tumor T cells may be inhibited in function
— id: 92674, year: 2008, vol: 222, page: 192, stat: Journal Article,

Skewing the Th cell phenotype toward Th1 alters the maturation of tumor-infiltrating mononuclear phagocytes
Nonaka, Kenichi; Saio, Masanao; Suwa, Tatsuhiko; Frey, Alan B; Umemura, Naoki; Imai, Hisashi; Ouyang, Guan-Feng; Osada, Shinji; Balazs, Margit; Adany, Roza; Kawaguchi, Yoshihiro; Yoshida, Kazuhiro; Takami, Tsuyoshi
2008 Sep;84(3):679-688, Journal of leukocyte biology
Mononuclear phagocytes (MPCs) at the tumor site can be divided into subclasses, including monocyte-lineage myeloid-derived suppressor cells (MDSCs) and the immunosuppressive tumor-infiltrating macrophages (TIMs). Cancer growth coincides with the expansion of MDSCs found in the blood, secondary lymphoid organs, and tumor tissue. These MDSCs are thought to mature into macrophages and to promote tumor development by a combination of growth-enhancing properties and suppression of local antitumor immunoresponses. As little is known about either subset of MPCs, we investigated MPCs infiltrating into murine adenocarcinoma MCA38 tumors. We found that these MPCs displayed immunosuppressive characteristics and a MDSC cell-surface phenotype. Over 70% of the MPCs were mature (F4/80(+)Ly6C(-)) macrophages, and the rest were immature (F480(+) Ly6C(+)) monocytes. MPC maturation was inhibited when the cells infiltrated a tumor variant expressing IL-2 and soluble TNF type II receptor (sTNFRII). In addition, the IL-2/sTNFRII MCA38 tumor microenvironment altered the MPC phenotype; these cells did not survive culturing in vitro as a result of Fas-mediated apoptosis and negligible M-CSFR expression. Furthermore, CD4(+) tumor-infiltrating lymphocytes (TILs) in wild-type tumors robustly expressed IL-13, IFN-gamma, and GM-CSF, and CD4(+) TILs in IL-2/sTNFRII-expressing tumors expressed little IL-13. These data suggest that immunotherapy-altered Th cell balance in the tumor microenvironment can affect the differentiation and maturation of MPCs in vivo. Furthermore, as neither the designation MDSC nor TIM can sufficiently describe the status of monocytes/macrophages in this tumor microenvironment, we believe these cells are best designated as MPCs
— id: 96103, year: 2008, vol: 84, page: 679, stat: Journal Article,

Osteopontin is linked to p65 and MMP-9 expression in pulmonary adenocarcinoma but not in malignant pleural mesothelioma
Frey, A B; Wali, A; Pass, H; Lonardo, F
2007 May;50(6):720-726, Histopathology
AIMS: Osteopontin (OPN) is a matricellular protein involved in tissue remodelling, cell-mediated immunity and malignant transformation. High OPN serum levels predict poor prognosis in non-small cell carcinoma and set patients with malignant pleural mesothelioma (MM) apart from disease-free asbestos-exposed individuals. Yet neither the spectrum of tissue expression nor the signalling pathways of OPN in MM and pulmonary adenocarcinoma have been characterized, although in vitro evidence links OPN to the epidermal growth factor receptor (EGFR) pathway. The aim of this study was to address these deficiencies. METHODS AND RESULTS: OPN expression was investigated immunohistochemically in 104 adenocarcinomas and 38 MM and correlated with histological features, including tumour type, grade and proliferation and with expression of activated intermediary EGFR signalling pathway molecules p65, p-AKT, p-ERK, p-STAT-3, and of metalloproteinase (MMP)-1, MMP-2 and MMP-9. In MM, OPN expression was widespread (36/38) and independent of the molecular parameters studied. In adenocarcinoma, high OPN expression was correlated with expression of p65, p-ERK and MMP-9. CONCLUSIONS: Frequent OPN expression is typical of, but not specific for MM, whereas it appears to select adenocarcinoma cases with p65 and MMP-9 expression, suggesting a link with EGFR signalling pathways
— id: 110900, year: 2007, vol: 50, page: 720, stat: Journal Article,

Protein kinase Cdelta regulates antigen receptor-induced lytic granule polarization in mouse CD8+ CTL
Ma, Jennifer S Y; Monu, Ngozi; Shen, David T; Mecklenbrauker, Ingrid; Radoja, Nadezda; Haydar, Tarik F; Leitges, Michael; Frey, Alan B; Vukmanovic, Stanislav; Radoja, Sasa
2007 Jun 15;178(12):7814-7821, Journal of immunology
Lytic granule exocytosis is the major pathway used by CD8+ CTL to kill virally infected and tumor cells. Despite the obvious importance of this pathway in adaptive T cell immunity, the molecular identity of enzymes involved in the regulation of this process is poorly characterized. One signal known to be critical for the regulation of granule exocytosis-mediated cytotoxicity in CD8+ T cells is Ag receptor-induced activation of protein kinase C (PKC). However, it is not known which step of the process is regulated by PKC. In addition, it has not been determined to date which of the PKC family members is required for the regulation of lytic granule exocytosis. By combination of pharmacological inhibitors and use of mice with targeted gene deletions, we show that PKCdelta is required for granule exocytosis-mediated lytic function in mouse CD8+ T cells. Our studies demonstrate that PKCdelta is required for lytic granule exocytosis, but is dispensable for activation, cytokine production, and expression of cytolytic molecules in response to TCR stimulation. Importantly, defective lytic function in PKCdelta-deficient cytotoxic lymphocytes is reversed by ectopic expression of PKCdelta. Finally, we show that PKCdelta is not involved in target cell-induced reorientation of the microtubule-organizing center, but is required for the subsequent exocytosis step, i.e., lytic granule polarization. Thus, our studies identify PKCdelta as a novel and selective regulator of Ag receptor-induced lytic granule polarization in mouse CD8+ T cells
— id: 96104, year: 2007, vol: 178, page: 7814, stat: Journal Article,

Suppression of proximal T cell receptor signaling and lytic function in CD8+ tumor-infiltrating T cells
Monu, Ngozi; Frey, Alan B
2007 Dec 1;67(23):11447-11454, Cancer research
CD8(+) tumor-infiltrating lymphocytes (TIL) lack in vivo and in vitro lytic function due to a signaling deficit characterized by failure to flux calcium or activate tyrosine kinase activity upon contact with cognate tumor cells. Although CD3 zeta is phosphorylated by conjugation in vitro with cognate tumor cells, showing that TIL are triggered, PLC gamma-1, LAT, and ZAP70 are not activated and LFA-1 is not affinity-matured, and because p56(lck) is required for LFA-1 activation, this implies that the signaling blockade is very proximal. Here, we show that TIL signaling defects are transient, being reversed upon purification and brief culture in vitro, implying a fast-acting 'switch'. Biochemical analysis of purified nonlytic TIL shows that contact with tumor cells causes transient activation of p56(lck) ( approximately 10 s) which is rapidly inactivated. In contrast, tumor-induced activation of p56(lck) in lytic TIL is sustained coincident with downstream TCR signaling and lytic function. Shp-1 is robustly active in nonlytic TIL compared with lytic TIL, colocalizes with p56(lck) in nonlytic TIL, and inhibition of Shp-1 activity in lytic TIL in vitro blocks tumor-induced defective TIL cytolysis. Collectively, our data support the notion that contact of nonlytic TIL with tumor cells, and not with tumor-infiltrating myeloid-derived suppressor cells, causes activation of Shp-1 that rapidly dephosphorylates the p56(lck) activation motif (Y394), thus inhibiting effector phase functions
— id: 75676, year: 2007, vol: 67, page: 11447, stat: Journal Article,

TNF expressed by tumor-associated macrophages, but not microglia, can eliminate glioma
Nakagawa, Jiro; Saio, Masanao; Tamakawa, Noriyuki; Suwa, Tatsuhiko; Frey, Alan B; Nonaka, Kenichi; Umemura, Naoki; Imai, Hisashi; Ouyang, Guan-Feng; Ohe, Naoyuki; Yano, Hirohito; Yoshimura, Sinichi; Iwama, Toru; Takami, Tsuyoshi
2007 Apr;30(4):803-811, International journal of oncology
It is well known that tumor necrosis factor (TNF) can have both contrary and pleiotropic effects in anti-tumor immune response. In the present study, we prepared two different tumor cell-based immunotherapy models: MCA38 adenocarcinoma and GL261 glioma intracranial interleukin-2 (IL-2)-based. Each tumor was transfected to express IL-2 with or without expression of the soluble form of tumor necrosis factor receptor type II (sTNFRII). Although mice in which TNF is blocked survive longer than IL-2 alone (35.2 versus 26 days), the reverse was observed for GL261 glioma. The differential effect on tumor growth implies enhanced TNF sensitivity of GL261 compared to MCA38. This notion is supported by the observation that TNF induces apoptosis in GL261 but not MCA38 tumors. We further examined tumor infiltrating CD11b+F4/80+ macrophages (or tumor-associated macrophages: TAM) for TNF production in vivo and found that TAM express cell surface TNF implying a role in eliminating glioma cells mediated by the cell surface form of TNF
— id: 96105, year: 2007, vol: 30, page: 803, stat: Journal Article,

Myeloid suppressor cells regulate the adaptive immune response to cancer
Frey, Alan B
2006 Oct;116(10):2587-2590, Journal of clinical investigation
Inflammation resultant from tumor growth, infection with certain pathogens, or in some cases, trauma, can result in systemic release of cytokines, especially GM-CSF, that in turn stimulate the abundant production and activation of a population of immature myeloid cells, termed myeloid suppressor cells (MSCs), that have potent immunosuppressive functions. In this issue of the JCI, Gallina and colleagues have illuminated some complex issues concerning the development, activation, and function of MSCs (see the related article beginning on page 2777). They show that activation of MSCs is initiated in response to IFN-gamma, presumably produced in situ by antitumor T cells in the tumor microenvironment. After this triggering event, MSCs express 2 enzymes involved in l-arginine metabolism, Arginase I and iNOS, whose metabolic products include diffusible and highly reactive peroxynitrites, the ultimate biochemical mediators of T cell immune suppression. The multifaceted regulation of this complex suppressive effector system provides several potential therapeutic targets
— id: 69693, year: 2006, vol: 116, page: 2587, stat: Journal Article,

Effector-phase tolerance: another mechanism of how cancer escapes antitumor immune response
Frey, Alan B; Monu, Ngozi
2006 Apr;79(4):652-662, Journal of leukocyte biology
Growth of cancer in rodent models and in patients elicits immune responses directed toward various antigens expressed by the transformed cell. Clearly though, as most tumors grow, unmanipulated antitumor immune responses are incapable of eliminating cancer. Over the past approximately 15 years, antitumor immunoglobulin and T cells have been used to identify tumor antigens, which in turn, have served as the basis for therapeutic vaccine trials. However, experimental cancer vaccines, although in some patients result in elimination of large tumor burdens, have a low frequency of long-term cancer remission in most patients, ca. <5%. Therefore, as tumors express antigens that distinguish themselves from nontransformed cells in immunological terms (i.e., elicit immune responses to growth of primary tumor and can target tumor cells in vivo), and tumor vaccines prime unsuccessful antitumor immune responses in patients, it is likely that growth of cancer induces immune tolerance to tumor cells. Although there are several types of T cell tolerance, mature, antigen-specific CD8+ T cells isolated from tumors are lytic-defective, implying that the tumor microenvironment inactivates the antitumor effector phase. The nature of the functional local tolerance to antitumor immune response is the subject of this review
— id: 66694, year: 2006, vol: 79, page: 652, stat: Journal Article,

Defective adhesion in tumor infiltrating CD8+ T cells
Koneru, Mythili; Monu, Ngozi; Schaer, David; Barletta, Justine; Frey, Alan B
2006 May 15;176(10):6103-6111, Journal of immunology
CD8(+) tumor-infiltrating lymphocytes (TIL) are defective in cytolysis due to tumor-induced inhibition of proximal TCR-mediated signaling, a defect that is relieved upon purification and brief culture. We show in this study that frequency of conjugation in vitro of nonlytic TIL with tumor cells is low in comparison with their lytic counterparts, and the strength of interaction and duration of conjugation are also reduced. Previous reports show that p56(lck) activation is required for TCR-initiated LFA-1 avidity up-regulation, raising the question: is low LFA-1 avidity the basis of reduced TIL conjugation frequency? When stimulated with phorbol ester, nonlytic TIL bind purified ICAM-1 equivalently as lytic TIL, suggesting that LFA-1 can be activated if proximal TCR signaling is bypassed. However, when treated with phorbol ester, the conjugation frequency of nonlytic TIL does not increase. CD2 and CD8 also mediate T cell adhesion to cognate target cells and are both expressed at lower levels in nonlytic TIL in addition to being excluded from the immune synapse formed upon conjugation. Collectively, these results imply that adhesion defects in nonlytic TIL result from a combination of decreased cell surface levels of adhesion molecules, deficient LFA-1 activation, and the failure to recruit essential adhesion receptors to the membrane contact site formed with cognate target cells
— id: 64588, year: 2006, vol: 176, page: 6103, stat: Journal Article,

T-cell receptor signaling events triggering granule exocytosis
Radoja, Sasa; Frey, Alan B; Vukmanovic, Stanislav
2006 ;26(3):265-290, Critical reviews in immunology
T-cell receptor (TCR) engagement by antigen results in proliferation, differentiation and cytokine secretion. In the CD8+ T-cell subset, TCR triggering also induces granule exocytosis, the directional release of contents of lysosome-like granules toward the target cell presenting the antigen. This process is responsible for immediate death of target cells. The intracellular events required for granule exocytosis are distinct from those of proliferation and cytokine secretion, as the former do not require de novo protein synthesis. Consequently, the key TCR signaling events required for granule exocytosis may be distinct. In this article, we review present knowledge of regulation of granule exocytosis by molecules of the TCR signaling cascade
— id: 67541, year: 2006, vol: 26, page: 265, stat: Journal Article,

Defective proximal TCR signaling inhibits CD8+ tumor-infiltrating lymphocyte lytic function
Koneru, Mythili; Schaer, David; Monu, Ngozi; Ayala, Aidee; Frey, Alan B
2005 Feb 15;174(4):1830-1840, Journal of immunology
CD8+ tumor-infiltrating lymphocytes (TIL) are severely deficient in cytolysis, a defect that may permit tumor escape from immune-mediated destruction. Because lytic function is dependent upon TCR signaling, we have tested the hypothesis that primary TIL have defective signaling by analysis of the localization and activation status of TIL proteins important in TCR-mediated signaling. Upon conjugate formation with cognate target cells in vitro, TIL do not recruit granzyme B+ granules, the microtubule-organizing center, F-actin, Wiskott-Aldrich syndrome protein, nor proline rich tyrosine kinase-2 to the target cell contact site. In addition, TIL do not flux calcium nor demonstrate proximal tyrosine kinase activity, deficiencies likely to underlie failure to fully activate the lytic machinery. Confocal microscopy and fluorescence resonance energy transfer analyses demonstrate that TIL are triggered by conjugate formation in that the TCR, p56lck, CD3zeta, LFA-1, lipid rafts, ZAP70, and linker for activation of T cells localize at the TIL:tumor cell contact site, and CD43 and CD45 are excluded. However, proximal TCR signaling is blocked upon conjugate formation because the inhibitory motif of p56lck is rapidly phosphorylated (Y505) and COOH-terminal Src kinase is recruited to the contact site, while Src homology 2 domain-containing protein phosphatase 2 is cytoplasmic. Our data support a novel mechanism explaining how tumor-induced inactivation of proximal TCR signaling regulates lytic function of antitumor T cells
— id: 50292, year: 2005, vol: 174, page: 1830, stat: Journal Article,

Immunocytochemical demonstration of down regulation of HLA class-I molecule expression in human metastatic breast carcinoma
Saio, Masanao; Teicher, Matt; Campbell, Gaynor; Feiner, Helen; Delgado, Yara; Frey, Alan B
2004 ;21(3):243-249, Clinical & experimental metastasis
Deficient expression of HLA class-I molecules observed in many cancers is suggested to influence both disease progression and potential efficacy of T cell-mediated immune therapy. Previous studies have attempted to correlate either primary breast cancer tumor grade with HLA class-I levels or the presence of HLA class-I-deficient cells in metastatic lesions with survival. In this study we evaluated the HLA class-I status of matched primary and secondary breast cancer lesions in order to ask the question: is metastasis of breast cancer associated with down-regulation of HLA class-I expression? Immunocytochemistry analysis shows a definitive correlation between diminished HLA class-I expression and dissemination of breast cancer to tumor-draining lymph nodes: both the total number of HLA class-I+ cells per sample and the levels of expression are dramatically decreased in secondary versus primary tumor lesions. These findings are consistent with the contention that the ability of breast cancer cells to escape the confines of the original tumor lesion requires down-regulation of HLA class-I expression and implies that enhancing HLA class-I expression in secondary breast cancer may have a beneficial effect on T-cell-mediated immunotherapy
— id: 67542, year: 2004, vol: 21, page: 243, stat: Journal Article,

Expression of rat complement control protein Crry on tumor cells inhibits rat natural killer cell-mediated cytotoxicity
Caragine, Theresa A; Imai, Masaki; Frey, Alan B; Tomlinson, Stephen
2002 Nov 1;100(9):3304-3310, Blood
Crry is a rodent membrane-bound inhibitor of complement activation and is a structural and functional analog of the human complement inhibitors decay-accelerating factor and membrane cofactor protein. We found previously that expression of rat Crry on a human tumor cell line enhances tumorigenicity in nude rats. In this study, we investigated the effect that rat Crry expressed on tumor cells has on rat cell-mediated cytotoxicity and antibody-dependent cell-mediated cytotoxicity (ADCC). The expression of rat Crry on the surface of different human tumor cell lines inhibited ADCC mediated by rat natural killer (NK) cells. C3 opsonization is known to enhance NK cell-mediated cytolysis, and a potential mechanism for Crry-mediated inhibition of NK cell lysis is through Crry modulation of C3 deposition on target cells. However, the transfection of tumor cell lines with Crry enhanced their resistance to NK cell-mediated lysis in the absence of exogenous complement. The resistance of Crry-expressing tumor cells to NK cell-mediated ADCC could be reversed by treatment with anti-Crry F(ab)(2). In addition, anti-Crry F(ab)(2) enhanced the susceptibility of 13762 rat mammary adenocarcinoma cells (that endogenously express Crry) to ADCC mediated by allogeneic rat NK cells in the absence of added complement. We found no evidence that rat NK cells were a source of complement for target cell deposition during the in vitro cytolysis assay. These data suggest a novel function for rat Crry in tumor immune surveillance that may be unrelated to complement inhibition
— id: 93833, year: 2002, vol: 100, page: 3304, stat: Journal Article,

A tumor-expressed inhibitor of the early but not late complement lytic pathway enhances tumor growth in a rat model of human breast cancer
Caragine, Theresa A; Okada, Noriko; Frey, Alan B; Tomlinson, Stephen
2002 Feb 15;62(4):1110-1115, Cancer research
Membrane-bound complement inhibitors protect host cells from inadvertent complement attack, and complement inhibitors are often up-regulated on tumors, possibly representing a selective adaptation by tumors to escape elimination by a host antitumor immune response. Relevant in vivo studies using rodent models of human cancer have been hampered by the fact that human complement inhibitors are not effective against rodent complement. Using nude rats and MCF7 cells expressing different rat complement inhibitors, a model of human breast cancer was established to investigate the role of complement and complement inhibitors in tumor progression. Expression of rat CD59, an inhibitor of the terminal cytolytic membrane attack complex of complement, had no effect on the incidence or growth rate of MCF7 tumors. In contrast, expression of rat Crry, an inhibitor of complement activation, dramatically enhanced the tumorigenicity of MCF7 cells. The expression of rat Crry on MCF7 inhibited the in vivo deposition of complement C3 fragments that serve as opsonins for receptors on phagocytes and natural killer cells. These data provide direct in vivo evidence that an inhibitor of complement activation can facilitate tumor growth by modulating C3 deposition. These data indicate an important role for complement opsonization in promoting cell-mediated antitumor immune function, a conclusion further supported by the demonstration that expression of rat Crry, but not rat CD59, on MCF7 cells inhibits rat cell-mediated cytotoxicity in vitro. Rat complement activation on MCF7 tumors was mediated by tumor-reactive antibodies present in the serum of naive nude rats, but there was also an IgM response to MCF7 tumors, a situation with similarities to some human cancers. These data support a hypothesis that blocking complement inhibitor function on tumor cells will not only enhance monoclonal antibody-mediated immunotherapy but may also be effective at enhancing a normally ineffective humoral immune response in the absence of administered antitumor antibody
— id: 91293, year: 2002, vol: 62, page: 1110, stat: Journal Article,

The role of fibroblasts in thymocyte-positive selection
Lilic, Mirjana; Santori, Fabio R; Neilson, Eric G; Frey, Alan B; Vukmanovic, Stanislav
2002 Nov 1;169(9):4945-4950, Journal of immunology
Mice with fibroblast-specific expression of TAP-1 were generated by expressing the TAP-1 transgene under the control of the fibroblast-specific protein (FSP) 1 promoter/enhancer on TAP-1-deficient background. MHC class I expression in primary fibroblast cultures isolated from the resulting strain mimicked that of wild-type counterparts. MHC class I was detected in both types of fibroblasts following treatment with IFN-alphabeta. Positive selection of CD4(-)CD8(+) thymocytes was observed in neither adult nor fetal/neonatal thymus of transgenic mice. IFN-alphabeta-induced expression of MHC class I rescued positive selection of CD4(-)CD8(+) T cells in fetal thymic organ cultures, but not in adult mice. Contrary to previous suggestions, our results indicate a limited role of fibroblasts in promoting positive selection. In addition, the results suggest that positive selection may occur by a different mechanism in fetal vs adult thymus
— id: 34693, year: 2002, vol: 169, page: 4945, stat: Journal Article,

CD8(+) tumor-infiltrating lymphocytes are primed for Fas-mediated activation-induced cell death but are not apoptotic in situ
Radoja S; Saio M; Frey AB
2001 May 15;166(10):6074-6083, Journal of immunology
Induction of Fas-mediated activation-induced cell death in antitumor T cells has been hypothesized to permit tumor escape from immune destruction. Several laboratories have proposed that expression of Fas ligand (L) by tumor is the basis for this form of T cell tolerance. In this study, we characterized murine tumor-infiltrating lymphocytes (TIL) for activation status, cell cycle status, level of apoptosis, cytokine secretion, and proliferative capacity. TILs express multiple activation markers (circa CD69, CD95L, CD122, and LFA-1) and contain IL-2 and IFN-gamma mRNAs, but are neither cycling nor apoptotic in situ. In addition, TIL are dramatically suppressed in proliferative response and do not secrete IL-2 and IFN-gamma. However, upon purification and activation in vitro, TIL secrete high levels of IL-2 and IFN-gamma, enter S phase, and then die by Fas-mediated apoptosis. Activation by injection of anti-TCR Ab or IL-2 into tumor-bearing mice induced TIL entrance into S phase preceding apoptosis, showing that TIL have functional TCR-mediated signal transduction in situ. Our data demonstrate that TIL, not tumor, express both Fas and FasL, are arrested in G(1), do not secrete cytokine in situ, and, upon activation in vitro and in vivo, rapidly die by activation-induced cell death
— id: 20291, year: 2001, vol: 166, page: 6074, stat: Journal Article,

CD8(+) tumor-infiltrating T cells are deficient in perforin-mediated cytolytic activity due to defective microtubule-organizing center mobilization and lytic granule exocytosis
Radoja S; Saio M; Schaer D; Koneru M; Vukmanovic S; Frey AB
2001 Nov 1;167(9):5042-5051, Journal of immunology
Tumor-infiltrating lymphocytes (TIL) are well known to be functionally impaired typified by the inability to lyse cognate tumor cells in vitro. We have investigated the basis for defective TIL lytic function in transplantable murine tumor models. CD8(+) TIL are nonlytic immediately on isolation even though they express surface activation markers, contain effector phase cytokine mRNAs, and contain perforin and granzyme B proteins which are packaged into lytic granules. Ag-specific lytic capability is rapidly recovered if purified TIL are briefly cultured in vitro and tumor lysis is perforin-, but not Fas ligand mediated. In response to TCR ligation of nonlytic TIL in vitro, proximal and distal signaling events are normal; calcium flux is rapid; mitogen-activated protein/extracellular signal-related kinase kinase, extracellular regulatory kinase 2, phosphoinositide-3 kinase, and protein kinase C are activated; and IL-2 and IFN-gamma is secreted. However, on conjugate formation between nonlytic TIL and cognate tumor cells in vitro, the microtubule-organizing center (MTOC) does not localize to the immunological synapse, thereby precluding exocytosis of preformed lytic granules and accounting for defective TIL lytic function. Recovery of TCR-mediated, activation-dependent MTOC mobilization and lytic activity requires proteasome function, implying the existence of an inhibitor of MTOC mobilization. Our findings show that the regulated release of TIL cytolytic granules is defective despite functional TCR-mediated signal transduction
— id: 25552, year: 2001, vol: 167, page: 5042, stat: Journal Article,

Tumor-infiltrating macrophages induce apoptosis in activated CD8(+) T cells by a mechanism requiring cell contact and mediated by both the cell-associated form of TNF and nitric oxide
Saio M; Radoja S; Marino M; Frey AB
2001 Nov 15;167(10):5583-5593, Journal of immunology
We have investigated the ability of different cells present in murine tumors to induce apoptosis of activated CD8(+) T cells in vitro. Tumor cells do not induce apoptosis of T cells; however, macrophages that infiltrate tumors are potent inducers of apoptosis. Tumor macrophages express cell surface-associated TNF, TNF type I (CD120a) and II (CD120b) receptors, and, upon contact with T cells which induces release of IFN-gamma from T cells, secrete nitric oxide. Killing of T cells in vitro is blocked by Abs to IFN-gamma, TNF, CD120a, or CD120b, or N-methyl-L-arginine. In concert with that finding, tumor macrophages isolated from either TNF type I or type II receptor -/- mice are not proapoptotic and do not produce nitric oxide upon contact with activated T cells. Control macrophages do not express TNF receptors or release nitric oxide. Tumor cells or tumor-derived macrophages do not express FasL, and blocking Abs to either Fas or FasL have no effect on macrophage-mediated T cell killing. These results demonstrate that macrophages which infiltrate tumors are highly proapoptotic and may be responsible for elimination of activated antitumor T cells within the tumor bed
— id: 25551, year: 2001, vol: 167, page: 5583, stat: Journal Article,

Protection against tick-transmitted Lyme disease in dogs vaccinated with a multiantigenic vaccine
Straubinger, RK; Rao, TD; Davidson, E; Summers, BA; Jacobson, RH; Frey, AB
2001 Oct 12;20(1-2):181-193, Vaccine
In an effort to develop a safe and effective vaccine for the prevention of Lyme borreliosis that addresses concerns raised over currently available vaccines, dogs were vaccinated twice with a multiantigenic preparation of Borrelia burgdorferi, strain N40, on days 0 and 20 of the experiment. About 70 and 154 days after the first immunization, dogs were challenged by exposing them to field-collected Ixodes scapularis ticks harboring B. burgdorferi. Vaccinated dogs were completely protected from infection by all criteria utilized to assess infection, developed high-titer anti-B. burgdorferi serum antibodies and growth inhibitory activity which persisted for over 200 days, and did not demonstrate any untoward consequence of vaccination. Serum absorption experiments revealed that borreliacidal and most likely protective antibodies in dogs receiving the multiantigenic preparation were not only elicited against the OspA antigen, but were also produced against additional yet to be determined targets on B. burgdorferi organisms. These data demonstrate that a multiantigenic vaccine is effective in preventing Lyme disease transmitted via the natural vector. (C) 2001 Elsevier Science Ltd. All rights reserved
— id: 28209, year: 2001, vol: 20, page: 181, stat: Journal Article,

Type I IFN modulates innate and specific antiviral immunity
Durbin JE; Fernandez-Sesma A; Lee CK; Rao TD; Frey AB; Moran TM; Vukmanovic S; Garcia-Sastre A; Levy DE
2000 Apr 15;164(8):4220-4228, Journal of immunology
IFNs protect from virus infection by inducing an antiviral state and by modulating the immune response. Using mice deficient in multiple aspects of IFN signaling, we found that type I and type II IFN play distinct although complementing roles in the resolution of influenza viral disease. Both types of IFN influenced the profile of cytokines produced by T lymphocytes, with a significant bias toward Th2 differentiation occurring in the absence of responsiveness to either IFN. However, although a Th1 bias produced through inhibition of Th2 differentiation by IFN-gamma was not required to resolve infection, loss of type I IFN responsiveness led to exacerbated disease pathology characterized by granulocytic pulmonary inflammatory infiltrates. Responsiveness to type I IFN did not influence the generation of virus-specific cytotoxic lymphocytes or the rate of viral clearance, but induction of IL-10 and IL-15 in infected lungs through a type I IFN-dependent pathway correlated with a protective response to virus. Combined loss of both IFN pathways led to a severely polarized proinflammatory immune response and exacerbated disease. These results reveal an unexpected role for type I IFN in coordinating the host response to viral infection and controlling inflammation in the absence of a direct effect on virus replication
— id: 11766, year: 2000, vol: 164, page: 4220, stat: Journal Article,

Distinct requirements for IFNs and STAT1 in NK cell function
Lee CK; Rao DT; Gertner R; Gimeno R; Frey AB; Levy DE
2000 Oct 1;165(7):3571-3577, Journal of immunology
NK cell functions were examined in mice with a targeted mutation of the STAT1 gene, an essential mediator of IFN signaling. Mice deficient in STAT1 displayed impaired basal NK cytolytic activity in vitro and were unable to reject transplanted tumors in vivo, despite the presence of normal numbers of NK cells. IL-12 enhanced NK-mediated cytolysis, but poly(I:C) did not, and a similar phenotype occurred in mice lacking IFNalpha receptors. Molecules involved in activation and lytic function of NK cells (granzyme A, granzyme B, perforin, DAP10, and DAP12) were expressed at comparable levels in both wild-type and STAT1(-/-) mice, and serine esterase activity necessary for CTL function was normal, showing that the lytic machinery was intact. NK cells with normal cytolytic activity could be derived from STAT1(-/-) bone marrow progenitors in response to IL-15 in vitro, and enhanced NK lytic activity and normal levels of IFN-gamma were produced in response to IL-12 treatment in vivo. Despite these normal responses to cytokines, STAT1(-/-) mice could not reject the NK-sensitive tumor RMA-S, even following IL-12 treatment in vivo. Whereas in vitro NK cytolysis was also reduced in mice lacking both type I and type II IFN receptors, these mice resisted tumor challenge. These results demonstrate that both IFN-alpha and IFN-gamma are required to maintain NK cell function and define a STAT1-dependent but partially IFN-independent pathway required for NK-mediated antitumor activity
— id: 20292, year: 2000, vol: 165, page: 3571, stat: Journal Article,

Cancer-induced defective cytotoxic T lymphocyte effector function: another mechanism how antigenic tumors escape immune-mediated killing [In Process Citation]
Radoja S; Frey AB
2000 Jun;6(6):465-479, Molecular medicine
BACKGROUND: The notion that a deficit in immune cell functions permits tumor growth has received experimental support with the discovery of several different biochemical defects in T lymphocytes that infiltrate cancers. Decreased levels of enzymes involved with T-cell signal transduction have been reported by several laboratories, suggesting that tumors or host cells recruited to the tumor site actively down-regulate antitumor T-cell immune response. This permits tumor escape from immune-mediated killing. The possibility that defects in T-cell signal transduction can be reversed, which would potentially permit successful vaccination or adoptive immunotherapy, motivates renewed interest in the field. Summarizing the literature concerning tumor-induced T-cell dysfunction, we focus on the end stage of immune response to human cancer, that of defective cytotoxic T lymphocyte killing function. Based on the data from several laboratories, we hypothesize a biochemical mechanism that accounts for the unusual phenotype of antitumor T-cell accumulation in tumors, but with defective killing function
— id: 11514, year: 2000, vol: 6, page: 465, stat: Journal Article,

Mice bearing late-stage tumors have normal functional systemic T cell responses in vitro and in vivo
Radoja S; Rao TD; Hillman D; Frey AB
2000 Mar 1;164(5):2619-2628, Journal of immunology
Immune suppression in tumor-bearing hosts is considered to be one factor causally associated with the growth of antigenic tumors. Support for this hypothesis has come from reports that spleen T cells in tumor-bearing mice are deficient in either priming or effector phase functions. We have reexamined this hypothesis in detail using multiple murine tumor models, including transplantable adenocarcinoma, melanoma, sarcoma, and thymoma, and also a transgenic model of spontaneous breast carcinoma. In both in vitro and in vivo assays of T cell function (proliferation, cytokine production, induction of CD8+ alloreactive CTL, and development of anti-keyhole limpet hemocyanin CD4+ T cells, rejection of allogeneic or syngeneic regressor tumors, respectively) we show that mice bearing sizable tumor burdens are not systemically suppressed and do not have diminished T cell functions. Therefore, if immune suppression is a causal function in the growth of antigenic tumor, the basis for escape from immune destruction is likely to be dependent upon tumor-induced T cell dysfunction at the site of tumor growth
— id: 8539, year: 2000, vol: 164, page: 2619, stat: Journal Article,

NKT cell cytokine imbalance in murine diabetes mellitus
Frey AB; Rao TD
1999 ;29(3):201-214, Autoimmunity
A minor subset of murine MHC class I-restricted T cells which express both the alphabeta form of the T cell receptor and a NK lineage marker, termed NKT cells, is capable of secreting significant amounts of Interleukin-4 and Interferon-y upon activation. As such NKT cells may play a role in development of Th1 and Th2 cells during T cell ontogeny or expansion of T cells expressing a dominant cytokine pattern in the effector phase. We have studied the role of NKT cells in a murine model of disease multidose streptozotocin induced diabetes mellitus (MDSDM). In MDSDM thymic and splenic NKT cells are present at normal levels but have greatly reduced capacity to secrete Interleukin-4 upon stimulation with anti-TCR antibody compared to control mice; conversely, Interferon-y secretion is maintained. By analysis of cytokine RNA production we found that treatment of several strains of mice with streptozotocin changes the peripheral helper T cell phenotype elicited after immunization with Keyhole Limpet Hemocyanin from a mixed Th1- and Th2-type cytokine pattern (characterized by IFN-gamma and IL-4 and IL-5 expressions, respectively) to predominately Th1-type. Furthermore, susceptibility to MDSDM is significantly enhanced when NKT cells are selectively eliminated in vivo by administration of depleting anti-CD122 antibody TMbeta-1. In addition, antibody depletion of NKT cells from non-obese diabetic mice significantly accelerates onset of disease. Collectively these data support a model for development of murine diabetes mellitus in which NKT cell cytokine expression influences the development of Th1-type diabetogenic T cells
— id: 6171, year: 1999, vol: 29, page: 201, stat: Journal Article,

Protection from Lyme neuroborreliosis in nonhuman primates with a multiantigenic vaccine
Pachner AR; Delaney E; Zhang WF; O'Neill T; Major E; Frey AB; Davidson E
1999 Jun;91(3):310-313, Clinical immunology
In an effort to develop an effective and safe vaccine for lyme disease, rhesus macaques were injected with a multiantigenic preparation of Borrelia burgdorferi, strain N40. One month later animals were boosted before intradermal challenge with infectious B. burgdorferi. Challenges were performed at 1 and again at 5 months after the booster vaccination. Vaccinated and control nonvaccinated animals were monitored for development of systemic infection by measurement of serum anti-spirochetal antibodies by ELISA and Western blotting, and neurological involvement was monitored by testing of cerebrospinal fluid (CSF) and PCR analysis of central nervous system (CNS) tissue obtained at necropsy. Two control nonhuman primates (NHPs), given saline injections instead of vaccine and then challenged with B. burgdorferi, developed CSF pleocytosis, PCR positivity of the brain, and high levels of specific anti-B. burgdorferi antibody in the serum and CSF. Necropsy studies revealed widespread invasion of the CNS of one of these animals by the spirochete. In contrast, none of the four vaccinated animals developed evidence of invasion of the CNS after either of two challenge inoculations with infectious B. burgdorferi. In addition to resisting infection, no vaccinated animal demonstrated any untoward consequence of vaccination. These data demonstrate that a multiantigenic vaccine is effective in preventing systemic infection and lyme neuroboreliosis in NHPs and suggest that a successful vaccine could be developed in humans which would prevent lyme disease
— id: 8508, year: 1999, vol: 91, page: 310, stat: Journal Article,

Multidose streptozotocin induction of diabetes in BALB/cBy mice induces a T cell proliferation defect in thymocytes which is reversible by interleukin-4
Wood SC; Rao TD; Frey AB
1999 Feb 25;192(1):1-12, Cellular immunology
Thymic T cell function in streptozotocin-treated (STZ) diabetic mice has been examined. STZ administration suppresses thymic T cell proliferation in response to mitogen stimulation in vitro. Secretion of IL-4 was dramatically reduced; however, secretion of IL-2 or IFN-gamma was not significantly inhibited. RT-PCR analysis of thymocyte RNA revealed that levels of IL-4 mRNA were dramatically decreased in STZ-treated mice. Levels of mRNA encoding IFN-gamma were similar, but the appearance was delayed in thymocytes derived from STZ-treated mice, implying differential regulation of IL-4 and IFN-gamma. Defective thymocyte proliferation was partially restored by exposure to IL-2 in vitro; however, IL-4 completely reversed the STZ-induced defect. Administration in vivo of IL-4 before STZ treatment reversed the STZ-induced thymocyte proliferation defect and prevented both pancreatic islet destruction and hyperglycemia. Thymocyte cell surface differentiation markers were not appreciably different from control mice. Collectively these experiments suggest that STZ treatment of mice reduces expression of IL-4 which is associated with development of autoimmune diabetes.
— id: 6056, year: 1999, vol: 192, page: 1, stat: Journal Article,

Protection of human breast cancer cells from complement-mediated lysis by expression of heterologous CD59
Yu J; Caragine T; Chen S; Morgan BP; Frey AB; Tomlinson S
1999 Jan;115(1):13-18, Clinical & experimental immunology
CD59, decay accelerating factor (DAF) and membrane cofactor protein (MCP) are widely expressed cell surface glycoproteins that protect host cells from the effects of homologous complement attack. Complement inhibitory activity of these proteins is species-selective. We show that the human breast cancer cell line MCF7 is relatively resistant to lysis by human complement, but is effectively lysed by rat or mouse complement. CD59, DAF and MCP were all shown to be expressed by MCF7. The species-selective nature of CD59 activity was used to demonstrate directly the effectiveness of CD59 at protecting cancer cells from complement-mediated lysis. cDNAs encoding rat and mouse CD59 were separately transfected into MCF7 cells, and cell populations expressing high levels of the rodent CD59 were isolated by cell sorting. Data show that rat and mouse CD59 were highly effective at protecting transfected MCF7 cells from lysis by rat and mouse complement, respectively. Data further reveal that rat CD59 is not effective against mouse complement, whereas mouse CD59 is effective against both mouse and rat complement. These studies establish a model system for relevant in vivo studies aimed at determining the effect of complement regulation on tumourigenesis, and show that for effective immunotherapy using complement-activating anti-tumour antibodies, the neutralization of CD59 and/or other complement inhibitory molecules will probably be required
— id: 7468, year: 1999, vol: 115, page: 13, stat: Journal Article,

Oncogenes and immunity: immune responses against products of transforming genes and the effects of oncogene activity on immunogenicity
Appleman L; Frey AB
Molecular approaches to tumor immunotherapy Singapore : World Scientific, 1998,
— id: 3588, year: 1998, vol: , page: ?, stat: Chapter,

Rat NKR-P1+ CD3+ T cells: selective proliferation in interleukin-2, diverse T-cell-receptor-Vbeta repertoire and polarized interferon-gamma expression
Badovinac V; Boggiano C; Trajkovic V; Frey AB; Vujanovic NL; Gold DP; Mostarica-Stojkovic M; Vukmanovic S
1998 Sep;95(1):117-125, Immunology
Cells expressing markers of both natural killer and T lymphocytes (NK T cells) in humans and mice express a restricted T-cell receptor (TCR) repertoire, are of CD4- CD8- or CD4+ CD8- phenotype, and upon anti-CD3 stimulation secrete large amounts of interleukin-4 (IL-4) and interferon-gamma (IFN-gamma). NK T cells may be the primary source of IL-4-promoting T helper type 2 (Th2) responses and/or they might be involved in regulating the balance between Th1- and Th2-type immune responses, and may consequently affect susceptibility to autoimmune diseases associated with a skewed Th phenotype. We show that rat NK T cells selectively proliferate to IL-2, and use this fact to analyse cytokine production by NK T cells in two rat strains differentially susceptible to Th1- or Th2-type autoimmune diseases. Analysis by reverse transcription-polymerase chain reaction revealed that, in contrast to mouse, rat NK T cells secrete exclusively IFN-gamma and not IL-4 after anti-CD3 stimulation, and use a wider TCR-Vbeta repertoire, suggesting that rat NK T cells are not essential for the development of Th2-type CD4+ T-cell responses
— id: 7999, year: 1998, vol: 95, page: 117, stat: Journal Article,

Repression of interleukin-2 mRNA translation in primary human breast carcinoma tumor-infiltrating lymphocytes
Lopez CB; Rao TD; Feiner H; Shapiro R; Marks JR; Frey AB
1998 Dec 15;190(2):141-155, Cellular immunology
Human breast carcinoma tumor-infiltrating lymphocytes (TIL) express activation antigens in situ indicative of ongoing immune response-CD28, CD45RO, CD69, CD71, and DR. However, interleukin 2 (IL-2) receptor was poorly expressed: CD25 was detected in only 1/24 samples and CD122 in only 2/24 samples. Furthermore, isolated breast cancer TIL were defective in proliferative response but recover when treated with recombinant IL-2. Nineteen of 24 tumor samples expressed B7-1, B7-2, and CD28 protein, showing that absence of costimulator proteins or counter ligand was not the basis for TIL proliferative deficit. Expression of IL-2 activity was not detected; however, mRNA encoding IL-2 was produced and translatable in vitro. These findings show that human breast cancer tumor-induced repression of IL-2 RNA translation is the basis of failure of TIL to express the IL-2 receptor and subsequent T cell hyporesponsiveness.
— id: 7394, year: 1998, vol: 190, page: 141, stat: Journal Article,

Administration of silica sensitizes lipopolysaccharide responsiveness of murine macrophages but inhibits T and B cell priming by inhibition of antigen presenting function
Rao TD; Frey AB
1998 May;27(3):181-199, Immunological investigations
Macrophages play a key role in natural host defense against infection by a variety of pathogens. In addition, macrophages initiate the development of acquired immunity via antigen processing and presentation. The role of macrophages in resistance to pathogens, the development of autoimmune diseases and the induction of acquired immunity has been studied by treatment of rodents with reagents which are cytotoxic. We have studied the effects of one such reagent, silica, on the function of spleen macrophages and peritoneal exudate cells (PEC). Intraperitoneal administration of silica caused the accumulation of spleen macrophages and neutrophils, reduction in the number of B cells and had a modest effect on T cell abundance. The percentage of CD11b+ PEC was not affected by silica treatment but total PEC recovery was diminished 5-8 fold. Silica treatment did not cause release of TNF-alpha or IL-1-beta but, when stimulated with lipopolysaccharide (LPS) in vitro after silica treatment, PEC or spleen macrophages produced elevated levels of both cytokines compared to controls. In contrast, release of IL-12 from non-LPS treated PEC was stimulated 4-5 fold by silica treatment. In addition, sensitivity to LPS toxicity in vivo was significantly enhanced by silica. The ability of macrophages to present antigen to a T cell clone in vitro was found to be dramatically inhibited by silica treatment, as was the ability to prime antigen-specific T cells and B cells by antigen injection. Collectively these data demonstrate that silica treatment enhances macrophage sensitivity to LPS exposure but inhibits antigen processing and presentation
— id: 7751, year: 1998, vol: 27, page: 181, stat: Journal Article,

Soluble proteins isolated from Borrelia burgdorferi by extraction with Triton X-114 confer resistance to experimental infection
Rao TD; Frey AB
1998 Oct;89(1):94-104, Clinical immunology & immunopathology
Fractionation of Borrelia burgdorferi was made by extraction of infectious spirochetes using the detergent Triton X-114. Gel electrophoresis analysis of hydrophilic and hydrophobic proteins demonstrated that detergent extraction resulted in two populations of proteins with nonoverlapping electrophoretic profiles. Immunoblot analysis with monoclonal antibodies reactive with two abundant membrane proteins demonstrated that hydrophilic proteins were uncontaminated with hydrophobic proteins. In addition, assay of thymidine incorporation into and secretion of tumor necrosis factor-alpha from splenocytes cocultured in vitro with either detergent or aqueous phase proteins showed that lymphocyte mitogenic and macrophage activation activities of B. burgdorferi were completely absent from the hydrophilic phase proteins. The Triton X-114 aqueous and detergent phase proteins were used to immunize BALB/c and separately microMT/microMT (B cell knockout) mice that were subsequently challenged with infectious B. burgdorferi. The hydrophilic phase proteins were able to induce protective resistance to infection in either strain of mice demonstrating that potential candidate vaccine antigens are contained in the biochemical class of antigens which is devoid of both lymphocyte mitogen activity and major outer surface proteins. Furthermore, the ability to vaccinate B cell knockout mice suggests that the humoral antispirochete immune response is not the exclusive basis for protective immunity.
— id: 7431, year: 1998, vol: 89, page: 94, stat: Journal Article,

Study of immune response to tumors in the rat
Frey AB
1997 Jun;12(2):173-188, Methods
Use of the rat as host for the study of cancer has become popular for several reasons. The larger body size compared to mice is especially convenient for lines of experiments involving surgical manipulation, transplantation, or biochemical purification of molecules of interest. Immune response to cancer is also studied in rat models, and this article focuses on the methodological aspects of in vivo and in vitro protocols related to rat tumor immunology
— id: 7149, year: 1997, vol: 12, page: 173, stat: Journal Article,

Killing of rat adenocarcinoma 13762 in situ by adoptive transfer of CD4+ anti-tumor T cells requires tumor expression of cell surface MHC class II molecules
Frey AB; Cestari S
1997 May 25;178(1):79-90, Cellular immunology
CD4+ anti-tumor T cells reactive with rat adenocarcinoma 13762 kill tumor in vitro and cause regression of tumor in vivo. The role of various host immune cells in CD4+ T-cell-mediated tumor elimination in vivo was investigated by adoptive transfer of anti-tumor T cell clones to recipients that were selectively depleted of individual immune cell types. By these means, macrophages and NK cells were found to be required for tumor killing. Depletion of host CD4+ T cells, CD8+ T cells, or neutrophils was without effect on tumor elimination by anti-tumor T cells. An essential role for antigen receptor-negative NK cells is likely dependent upon secretion of IFN-gamma from NK cells since treatment of tumor recipients with anti-IFN-gamma antibody prior to adoptive transfer and tumor challenge abrogated T cell killing, resulting in progressive tumor growth. Viability of adenocarcinoma 13762 or anti-tumor T cells was unaffected by treatment with either IFN-gamma or anti-IFN-gamma antibody in vitro, but cell surface MHC class II expression was induced in tumor cells by exposure to IFN-gamma. In addition, tumor cells were isolated from tumor-bearing animals by absorption using anti-MHC class II antibody, demonstrating that 13762 tumor expresses cell surface MHC class II antigens in situ. However, if hosts were depleted of NK cells before tumor challenge, MHC class II+ tumor was not recovered. Collectively these results suggest that adenocarcinoma 13762 is eliminated by MHC class II-restricted CD4+ T cells by direct tumor killing
— id: 7150, year: 1997, vol: 178, page: 79, stat: Journal Article,

Tumors antigens encoded by oncogenes and the impact of oncogenes upon the immune responses
Appleman LJ; Frey AB
1996 May 25;170(1):1-10, Cellular immunology
— id: 8048, year: 1996, vol: 170, page: 1, stat: Journal Article,

Role of host antigen receptor-bearing and antigen receptor-negative cells in immune response to rat adenocarcinoma 13762
Frey AB
1996 May 15;156(10):3841-3849, Journal of immunology
Involvement of individual immune cell populations in the immune response to rat breast adenocarcinoma 13762 was studied by selective depletion treatments in vivo. Depletion of Ag nonspecific host defense cells from naive animals before tumor challenge resulted in statistically significant acceleration of tumor growth (p less than 0.01 or less), whereas depletion of either CD4+ T cells or CD8+ T cells had no effect on the incidence or kinetics of tumor development. In contrast to naive animals treated before tumor challenge, animals actively immunized by injection of irradiated tumor before depleting treatments were shown to require CD4+ T cells to reject tumorigenic challenge. Depletion of either macrophages or neutrophils from immune animals also increased tumor development, whereas NK cells were not involved. Depletion of CD8+ T cells from immune animals permitted transient growth of tumors that were subsequently rejected, implying a role in tumor rejection. Transfer of immune antiserum to naive animals at the time of tumor challenge was without effect on tumor development. Depletion of CD4+ T cells, neutrophils, or macrophages in the priming phase of antitumor immune response abrogated tumor immunity, but depletion of CD8+ T cells or NK cells was without effect on the ability to prime animals by immunization with irradiated cells. Collectively, these data suggest that host natural defense cells that do not express Ag receptor are primarily responsible for resistance of adenocarcinoma 13762 growth in naive animals. In contrast, tumor immunity induced by active immunization requires Ag receptor-bearing CD4+ T cells and involves participation of CD8+ T cells, neutrophils, or macrophages in elimination of tumor
— id: 6995, year: 1996, vol: 156, page: 3841, stat: Journal Article,

Dominant-negative p53 can restore tumorigenicity of L-929 cells in immunocompetent mice
Appleman LJ; Frey AB
1995 Nov 15;63(4):576-583, International journal of cancer
To study the effect of a transforming allele of the tumor suppressor p53 upon the anti-tumor immune response, antigenic L-929 cells were transfected with the dominant-negative valine135 mutant of murine p53. Several p53val135-expressing transfectants formed non-regressing tumors in immunocompetent hosts. The growth rates of tumorigenic and non-tumorigenic clones were equivalent in vitro in sublethally irradiated C3H/HeN mice and in nude mice. Tumorigenic and non-tumorigenic p53val135-expressing L-929 clones expressed equivalent levels of cell surface class I major histocompatibility complex (MHC) glycoproteins. Immunization with a tumorigenic Lp53val135 clone protected mice from subsequent challenge and primed MHC class I-restricted cytotoxic T-lymphocytic precursors. Secretion of an immunosuppressive cytokine, transforming growth factor beta-1 and sensitivity to tumor necrosis factor-alpha were equivalent from tumorigenic and non-tumorigenic cell lines. These data suggest that expression of a transforming allele of p53 can allow L-929 cells to escape the host immune system
— id: 7930, year: 1995, vol: 63, page: 576, stat: Journal Article,

Mouse embryo fibroblasts transformed by activated ras or dominant-negative p53 express cross-reactive tumor rejection antigens
Appleman LJ; Uyeki J; Frey AB
1995 Jun 9;61(6):887-894, International journal of cancer
To study the immune response against oncogene-transformed tumors, C3H/HcN mouse embryo fibroblasts (MEF) were transfected with an activated allele of the H-ras proto-oncogene VaII2 and a dominant-negative allele of the murine p53 tumor suppressor gene VaII35. Transformed cell lines were derived and found to be tumorigenic in syngeneic mice. Immunization with irradiated p53 + ras-transformed MEF, but not primary MEF or unrelated syngeneic cells, protected mice from subsequent challenge with live tumor cells. The role of different immune cell subsets in the effector phase of anti-tumor immunity induced by immunization with p53 + ras-transformed MEF was investigated by in vivo antibody depletion experiments. Immunized mice depleted of CD8+ T, NK or B cells were resistant, but depletion of CD4+ T cells rendered mice susceptible to tumorigenic challenge. In contrast to the tumor-specific immune responses mounted against most chemically or UV-induced tumors, a series of independently derived p53 + ras-transformed MEF were cross-reactive in tumor rejection assays. In addition, immunization with C3H-derived L-929 cell lines expressing single gene products H-ras or p53 did not protect mice against tumorigenic challenge with p53 + ras-transformed tumors. However, MEF transformed by expression of either H-ras or p53 were cross-protective in vivo. Our data suggest that the p53 + ras-transformed MEF share tumor rejection antigens which are also induced by single gene transformation of the parental primary cell but are not the products of oncogenic ras or p53 protein
— id: 12762, year: 1995, vol: 61, page: 887, stat: Journal Article,

Rat mammary adenocarcinoma 13762 expressing IFN-gamma elicits antitumor CD4+ MHC class II-restricted T cells that are cytolytic in vitro and tumoricidal in vivo
Frey AB
1995 May 1;154(9):4613-4622, Journal of immunology
Rat adenocarcinoma 13762 was modified by transfection to express IFN-gamma, and the tumor-forming potential of cytokine-producing cells was found to be dramatically impaired. Animals resistant to inocula of IFN-gamma-modified tumor were resistant to subsequent challenge with unmodified 13762 tumor. Induced immunity was tumor specific in that syngeneic but non-cross-reactive tumor grew with normal kinetics in animals injected with IFN-gamma-producing 13762 tumor. Antitumor T cells were derived from animals primed with IFN-gamma-producing 13762 tumor and expanded into a cell line by coculture in vitro with IFN-gamma-producing 13762 cells. Anti-13762-gamma T cells were cytotoxic in vitro toward IFN-gamma-producing 13762 tumor and were not reactive with other syngeneic tumors or spleen B cells. Anti-13762-gamma T cells were determined to be CD4+ by Ab staining and flow cytometric analysis, and recognition of 13762-gamma in vitro was inhibited by anti-MHC class II Ab. Anti-13762-gamma T cells were not reactive in vitro with wild-type 13762 tumor unless treated with exogenous rIFN-gamma, which induced expression of cell surface MHC class II. However, adoptively transferred anti-13762-gamma T cells could effect regression of wild-type 13762 tumor or dramatically inhibit progressive growth in animals carrying significant tumor burden, and the antitumor phenotype did not require CD8+ T cells in vivo. These experiments demonstrate that although antitumor T cells elicited against cytokine-modified tumor may fail to demonstrate reactivity with unmodified tumor in vitro, antitumor properties may be manifest in vivo
— id: 12776, year: 1995, vol: 154, page: 4613, stat: Journal Article,

Expression of activated H-rasval12 in nontumorigenic and non-cross-reactive syngeneic cells induces tumor antigens cross-reactive with rat mammary adenocarcinoma 13762
Frey AB; Cestari S
1995 Nov 15;155(10):4783-4789, Journal of immunology
Adenocarcinoma 13762 expresses tumor Ags that can induce protective immunity to tumorigenic challenge. Syngeneic fibroblast Rat1 cells transformed by expression of H-rasval12 (Rat1/ras) but not parental Rat1 cells, and p53-transformed Rat1 cells, or other syngeneic cells or tumors, can immunize rats against tumorigenic challenge of 13762 tumor. Coincident with induced resistance to 13762 tumors, immunization of rats with Rat1/ras tumor induces CD4+ T cells that cross-react with adenocarcinoma 13762 in vitro and transfer protective immunity to tumorigenic 13762 challenge in vivo. Cross-reactive tumor Ags expressed in Rat1/ras tumor are not derived from ras protein, because immunization with purified H-rasval12 protein induces protective immunity to challenge by Rat1/ras tumor but not to adenocarcinoma 13762. In addition, immunization with H-rasval12 protein induces anti-ras CD4+ T cells that are uniquely reactive with Rat1/ras tumor: anti-ras T cells are not reactive with 13762 tumor in vitro and do not confer protective immunity to challenge with 13762 tumor in vivo. Tumor Ags expressed in Ras-transformed Rat1 cells that elicit cross-protective immunity likely arise as a consequence of transformation mediated by activated ras oncogene but are not derived from the Ras protein sequence
— id: 6825, year: 1995, vol: 155, page: 4783, stat: Journal Article,

Single exposure of mice to Borrelia burgdorferi elicits immunoglobulin G antibodies characteristic of secondary immune response without production of interleukin-4 by immune T cells
Frey AB; Rao TD
1995 Jul;63(7):2596-2603, Infection & immunity
Borrelia burgdorferi antigen can elicit immunoglobulins (Igs) characteristic of the primary and secondary immune responses without the contribution of an interleukin-4-producing helper T-cell population. Single exposure of mice to soluble B. burgdorferi antigen elicited both Th1-type and Th2-type antispirochete antibodies. Production of the Ig classes showed different patterns with increasing time postinjection (IgM levels decreased; IgG1, IgG2a, IgG2b, and IgG3 levels increased; IgE was not detected), and Ig patterns were similar to those produced in infected mice. Upon infectious challenge, immunized mice achieved maximal titers of all antispirochete IgG subclasses more quickly than unimmunized mice did. In contrast to the antibody responses which showed both Th1- and Th2-type patterns, T-cell immune response to either immunization or infection was characterized by interleukin-2 and gamma interferon production; interleukin-4 and interleukin-5 were undetectable. Injection with whole spirochetes induced a pattern of antibodies and cytokine production similar to those obtained by injection with soluble antigen. In addition, mouse strains of different major histocompatibility complex backgrounds produced similar patterns of Ig in response to immunization. None of the various parameters of immunization tested resulted in detectable interleukin-4 production by primary or secondary immune T cells. The production of both IgM and IgG1 at early times following a single exposure to spirochete antigen clearly differs from immune responses to haptens or model protein antigens. Production of similar Ig classes in infected and immune mice implies that antigen-specific antibody is responsible for passive immunizing activity found in immune sera
— id: 6623, year: 1995, vol: 63, page: 2596, stat: Journal Article,

Apparent split tolerance of CD8+ T cells from beta 2-microglobulin-deficient (beta 2m-/-) mice to syngeneic beta 2m+/+ cells
Jhaver KG; Rao TD; Frey AB; Vukmanovic S
1995 Jun 15;154(12):6252-6261, Journal of immunology
beta 2-microglobulin-deficient (beta 2m-/-) mice express reduced levels of MHC class I molecules and, consequently, have impaired positive selection of CD8+ T lymphocytes in the thymus. However, small numbers of CD8+ CTLs can be found in beta 2m-/- mice after immunization with allogeneic as well as syngeneic beta 2m+/+ tumor or spleen cells. It has been proposed, therefore, that because of the low ligand density in beta 2m-/- mice, negative selection does not remove cells capable of recognizing syngeneic MHC class I expressed at normal levels. We report here that beta 2m-/- CD8+ T cells are partially tolerant to syngeneic beta 2m+/+ cells. Despite the ability of beta 2m-/- mice to raise CD8+ CTLs against syngeneic beta 2m+/+ cells, these CD8+ cells do not proliferate and do not secrete IFN-gamma or IL-3/granulocyte-macrophage-CSF upon in vitro stimulation with syngeneic beta 2m+/+ cells. In contrast, all of these cellular responses are displayed by the beta 2m-/- CD8+ cells upon recognition of the allogeneic MHC class I. These in vitro findings of partial responsiveness to syngeneic and of full responsiveness to allogeneic MHC class I correlate well with the ability of beta 2m-/- mice to reject allogeneic, but not syngeneic, tumors in vivo. It appears, thus, that the significantly reduced levels of MHC class I molecules found in beta 2m-/- mice, although not capable of inducing deletion of all reactive clones, can induce deletion of high affinity clones and, therefore, maintain tolerance to self-MHC class I, even when expressed at much higher (beta 2m+/+) levels
— id: 7900, year: 1995, vol: 154, page: 6252, stat: Journal Article,

Protective resistance to experimental Borrelia burgdorferi infection of mice by adoptive transfer of a CD4+ T cell clone
Rao TD; Frey AB
1995 May;162(2):225-234, Cellular immunology
The anti-spirochete T cell immune response to immunization with Borrelia burgdorferi was investigated. The cellular immune response to vaccination of immunocompetent BALB/c mice was characterized initially in vitro by assay of the proliferative response of primary lymph node cells to B. burgdorferi sonicate. Subsequently, an anti-spirochete T cell line (RBN2.1) and clone (97.1) were derived from lymph node cells of BALB/c mice primed with B. burgdorferi antigen. Both the line and clone were CD4+ by flow cytometric analysis. Significantly, RBN2.1 and clone 97.1 were able to transfer resistance to infection to syngeneic naive recipients. Assay of antigen-specific interleukin-2, interleukin-4, tumor necrosis factor-alpha, and interferon-gamma production demonstrated that clone 97.1 was of the Th2 subclass. When B. burgdorferi sonicate was fractionated on SDS-PAGE and then electroeluted, clone 97.1 was reactive exclusively to a spirochete protein of approximately 21 kDa. These data suggest that T-cell-mediated protective immunity to infection by B. burgdorferi can be elicited by active immunization
— id: 12773, year: 1995, vol: 162, page: 225, stat: Journal Article,

T_h1 T cell immune response is produced in mice either by infection or immunization with <i>Borrelia burgdorferi</i>
Rao, T. D.; Frey, A. B.
1995 ;5(1):27-39, Immunology & infectious diseases
The nature of immune response to immunization or infection of mice by <i>B. burgdorferi</i> was investigated. T cell immune response to either immunization or infection was determined to be predominantly T_h1, as judged by interleukin-2, interleukin-4 and interferon- gamma production by anti-spirochaete T cells. Parameters of immunization currently thought to be able to influence T_h phenotype were evaluated, including mouse major histocompatibility complex background, antigen physical form, the dose, route and schedule of immunization, and the influence of adjuvant. None of the parameters tested could alter the T_h1 phenotype and the authors postulate that, like several other pathogenic microorganisms, T cell immune response to <i>B. burgdorferi</i> is inherently determined by the nature of spirochaete antigens
— id: 98792, year: 1995, vol: 5, page: 27, stat: Journal Article,

Rat adenocarcinoma 13762 expresses tumor rejection antigens but tumor-bearing animals exhibit tumor-specific immunosuppression
Frey AB; Appleman LJ
1993 Nov;69(2):223-233, Clinical immunology & immunopathology
Rat adenocarcinoma 13762 was adapted to continuous growth in culture and used in a variety of experiments to investigate the immune response to inoculation of animals with replication-defective tumor cells. The results demonstrate that 13762 cells express tumor-specific tumor rejection antigens that elicit protective immunity to tumorigenic challenge. By several criteria there is no apparent humoral component of the anti-tumor immunity; however, anti-tumor immunity is characterized by nylon-wool nonadherent spleen T cells. Anti-tumor T cells demonstrate tumoricidal activity in local adoptive transfer assays and are not found in spleens of naive animals or animals immunized against either nontumorigenic Rat 1 cells or a syngeneic fibrosarcoma. Despite the expression of tumor rejection antigens 13762 tumor, and the demonstrable ability of injection of irradiated tumor to induce anti-tumor immunity, tumors elicited in unimmunized syngeneic animals grow progressively. The reasons for growth of antigenic tumor are unknown but are shown not to be due to defective antigen expression in 13762 tumor since, in addition to being able to elicit T cell immune response in immunized animals, 13762 tumor expresses MHC Class I molecules and can be a target for allogeneic T cell recognition in vitro. These data suggest that in tumor-bearing animals an effective anti-tumor immune response is either not initiated or down-regulated. Since animals bearing 13762 tumors can be immunized against an unrelated syngeneic sarcoma, can produce humoral responses to several protein antigens, and can produce delayed type hypersensitivity response against dinitrofluorobenzene, the immune response to 13762-induced tumors appears specifically suppressed. In support of this contention, 13762 cells express high levels of transforming growth factor beta 1 in vitro which is postulated to impact upon the nascent anti-tumor immune response
— id: 13053, year: 1993, vol: 69, page: 223, stat: Journal Article,

Regulated replication of an episomal simian virus 40 origin plasmid in COS7 cells
Chittenden T; Frey A; Levine AJ
1991 Nov;65(11):5944-5951, Journal of virology
The replication of a simian virus 40 (SV40) origin-containing plasmid, pSLneo, stably transfected COS7 cells has been studied. pSLneo contains the SV40 origin of replication and encodes the positive selectable marker for G418 resistance. In transient replication assays, pSLneo replicates to a high copy number in COS7 cells. Uncontrolled SV40 plasmid replication has been reported to be lethal to such transfected cells. Thus, it was anticipated that extensive plasmid replication would preclude isolation of permanent cell lines containing pSLneo. However, significant number of G418-resistant colonies arose after transfection of COS7 cells with pSLneo. Cell lines established from these drug-resistant colonies contained between 100 and 1,000 extrachromosomal pSLneo copies per cell. Episomal plasmid DNA in pSLneo/COS7 lines was stably maintained after 2 months of continuous culture in selective medium. Bromodeoxyuridine labeling and density shift experiments demonstrated that replication of pSLneo closely paralleled that of cellular DNA. On average, plasmid DNA did not replicate more than once during a single cell generation period. Regulation of pSLneo replication appeared to be negatively controlled by a cis-acting mechanism. Endogenous copies of episomal pSLneo remained at a stable low copy number during the simultaneous, high-level replication of a newly transfected plasmid encoding SV40 large T antigen in the same cells. These results indicate that regulated replication of an SV40 origin plasmid can be acquired in a cell and does not require the presence of additional genetic elements. The molecular mechanism by which cells enforce this regulation on extrachromosomal SV40 plasmids remains to be defined
— id: 55867, year: 1991, vol: 65, page: 5944, stat: Journal Article,

Differential expression of MAG isoforms during development
Pedraza L; Frey AB; Hempstead BL; Colman DR; Salzer JL
1991 Jun;29(2):141-148, Journal of neuroscience research
The myelin-associated glycoproteins (MAG) mediate the cell interactions of oligodendrocytes and Schwann cells with axons that are myelinated. MAG exists in two developmentally regulated isoforms: large MAG (L-MAG) and small MAG (S-MAG). In this paper, we have studied the tissue-specific and developmentally regulated alternative splicing of these isoforms using monospecific antibodies that recognize epitopes common to both isoforms or that are present only on L-MAG. In the central nervous system (CNS), L-MAG is the major form synthesized early in development, and it persists as a significant proportion of the MAG present in the adult. In the peripheral nervous system (PNS), L-MAG is expressed at modest levels during development; it is virtually absent in the adult. Thus, the expression of L-MAG is not limited to the CNS, as was formerly believed, suggesting that it plays a common role during the early stages of myelin formation by both oligodendrocytes and Schwann cells. In both the CNS and PNS, S-MAG is the predominant isoform in the adult. A higher-molecular-weight form of MAG is present in the PNS at low abundance, that is developmentally regulated, and appears to be a glycosylation variant. An analysis of the carbohydrate residues on MAG demonstrates that it contains both N-linked and O-linked sugars that could be modulated during development. These results suggest a possible mechanism for the regulation of MAG function during myelinogenesis via the expression of alternative isoforms and carbohydrate modifications
— id: 14013, year: 1991, vol: 29, page: 141, stat: Journal Article,

PROTEIN ZERO OF PERIPHERAL-NERVE MYELIN - BIOSYNTHESIS, MEMBRANE INSERTION, AND EVIDENCE FOR HOMOTYPIC INTERACTION
Durso, D; Brophy, PJ; Staugaitis, SM; Gillespie, CS; Frey, AB; Stempak, JG; Colman, DR
1990 Mar;4(3):449-460, Neuron
— id: 32091, year: 1990, vol: 4, page: 449, stat: Journal Article,

Epstein-Barr virus DNA replication
Frey A; Chittenden T; Levine AJ
1989 ;144:227-232, Current topics in microbiology & immunology
— id: 55869, year: 1989, vol: 144, page: 227, stat: Journal Article,

p53-plus-ras-transformed rat embryo fibroblasts express tumor-specific transplantation antigen activity which is shared by independently transformed cells
Frey AB; Levine AJ
1989 Dec;63(12):5440-5444, Journal of virology
p53-plus-ras-transformed rat cell lines express a tumor-specific transplantation antigen that is common to a number (85%) of independently derived p53-plus-ras-transformed cell lines. This has been shown by immunizing rats with irradiated p53-plus-ras-transformed cells and demonstrating protection of these animals by subsequent live-cell tumor challenge. Several c-myc-plus-ras-transformed cell lines (54% of the lines tested) and one adenovirus E1a-plus-ras-transformed cell line (9% of those tested) were shown to share a common tumor-specific transplantation antigen by their ability to immunize a rat against a p53-plus-ras cell line challenge. Several experimental approaches have been used to fractionate and identify the antigen common to these cell lines. The experimental results reported here make it clear that the p53 protein common to most of these transformed cell lines is not likely to be the tumor-specific transplantation antigen
— id: 55868, year: 1989, vol: 63, page: 5440, stat: Journal Article,

Purification of complexes of nuclear oncogene p53 with rat and Escherichia coli heat shock proteins: in vitro dissociation of hsc70 and dnaK from murine p53 by ATP
Clarke CF; Cheng K; Frey AB; Stein R; Hinds PW; Levine AJ
1988 Mar;8(3):1206-1215, Molecular & cellular biology
Oligomeric protein complexes containing the nuclear oncogene p53 and the simian virus 40 large tumor antigen (D. I. H. Linzer and A. J. Levine, Cell 17:43-51, 1979), the adenovirus E1B 55-kilodalton (kDa) tumor antigen, and the heat shock protein hsc70 (P. Hinds, C. Finlay, A. Frey, and A. J. Levine, Mol. Cell. Biol. 7:2863-2869, 1987) have all been previously described. To begin isolating, purifying, and testing these complexes for functional activities, we have developed a rapid immunoaffinity column purification. p53-protein complexes are eluted from the immunoaffinity column by using a molar excess of a peptide comprising the epitope recognized by the p53 monoclonal antibody. This mild and specific elution condition allows p53-protein interactions to be maintained. The hsc70-p53 complex from rat cells is heterogeneous in size, with some forms of this complex associated with a 110-kDa protein. The maximum apparent molecular mass of such complexes is 660,000 daltons. Incubation with micromolar levels of ATP dissociates this complex in vitro into p53 and hsc70 110-kDa components. Nonhydrolyzable substrates of ATP fail to promote this dissociation of the complex. Murine p53 synthesized in Escherichia coli has been purified 660-fold on the same antibody affinity column and was found to be associated with an E. coli protein of 70 kDa. Immunoblot analysis with specific antisera demonstrated that this E. coli protein was the heat shock protein dnaK, which has extensive sequence homology with the rat hsc70 protein. Incubation of the immunopurified p53-dnaK complex with ATP resulted in the dissociation of the p53-dnaK complex as it did with the p53-hsc70 complex. This remarkable conservation of p53-heat shock protein interactions and the specificity of dissociation reactions suggest a functionally important role for heat shock proteins in their interactions with oncogene proteins
— id: 55870, year: 1988, vol: 8, page: 1206, stat: Journal Article,

Determination of the membrane topology of the phenobarbital-inducible rat liver cytochrome P-450 isoenzyme PB-4 using site-specific antibodies
De Lemos-Chiarandini C; Frey AB; Sabatini DD; Kreibich G
1987 Feb;104(2):209-219, Journal of cell biology
Fifteen peptides, ranging in length from 6 to 31 amino acids and corresponding in sequence to portions of the major phenobarbital-inducible form of rat liver cytochrome P-450 (P-450 PB-4), were previously synthesized chemically and used to prepare site-specific rabbit antibodies (Frey, A. B., D.J. Waxman, and G. Kreibich, 1985, J. Biol. Chem., 260:15253-15265). The antipeptide antibodies were affinity purified using Sepharose resins derivatized with the respective peptides and 14 preparations were obtained that in an ELISA assay showed affinities to immobilized P-450 judged to be adequate for binding studies on intact rat liver microsomes. The binding of these antibodies to rough microsomes from the livers of phenobarbital treated rats was assessed using 125I-labeled IgG and by immunoelectron microscopy employing protein A-gold as a marker. It was found that many of the antibodies bound to the cytoplasmic surface of the membrane but none bound to the luminal face of ruptured or inverted microsomal vesicles or to contaminating membranes of other organelles present in the preparations. These observations eliminate previously proposed models for the transmembrane disposition of P-450 that postulate the existence of multiple transmembrane domains and the exposure of several polar segments of the polypeptide on the luminal side of the membrane. The fact that an antibody raised to the first 31 residues of P-450 bound well to the purified P-450 but very poorly to rough microsomes, whereas an antibody to a peptide comprising residues 24-38 showed relatively strong binding to intact microsomes, is consistent with the proposal that the amino terminal segment of P-450 extending approximately to residue 20 is embedded in the phospholipid bilayer and the immediately following segment is exposed on the cytoplasmic surface of the membrane. All these results favor a model in which the cytochrome P-450 molecule is largely exposed on the cytoplasmic surface of the endoplasmic reticulum membrane to which it is anchored by its short amino terminal hydrophobic segment
— id: 18421, year: 1987, vol: 104, page: 209, stat: Journal Article,

Immunological evidence for the association of p53 with a heat shock protein, hsc70, in p53-plus-ras-transformed cell lines
Hinds PW; Finlay CA; Frey AB; Levine AJ
1987 Aug;7(8):2863-2869, Molecular & cellular biology
A rabbit antiserum was prepared against the C-terminal peptide of 21 amino acids from the human heat shock protein hsp70. These antibodies were shown to be specific for this highly inducible heat shock protein (72 kilodaltons [kDa] in rat cells), and for a moderately inducible, constitutively expressed heat shock protein, hsc70 (74 kDa). In six independently derived rat cell lines transformed by a murine cDNA-genomic hybrid clone of p53 plus an activated Ha-ras gene, elevated levels of p53 were detected by immunoprecipitation by using murine-specific anti-p53 monoclonal antibodies. In all cases, the hsc70, but not the hsp70, protein was coimmunoprecipitated with the murine p53 protein. Similarly, antiserum to heat shock protein coimmunoprecipitated p53. Western blot (immunoblot) analysis demonstrated that the hsc70 and p53 proteins did not share detectable antigenic epitopes. The results provide clear immunological evidence for the specific association of a single heat shock protein, hsc70, with p53 in p53-plus-ras-transformed cell lines. A p53 cDNA clone, p11-4, failed to produce clonable cell lines from foci of primary rat cells transfected with p11-4 plus Ha-ras. A mutant p53 cDNA clone derived from p11-4, SVKH215, yielded a 2- to 35-fold increase in the number of foci produced after transfection of rat cells with SVKH215 plus Ha-ras. When cloned, 87.5% of these foci produced transformed cell lines. SVKH215 encodes a mutant p53 protein that binds preferentially to the heat shock proteins of 70 kDa compared with binding by the parental p11-4 p53 gene product. These data suggest that the p53-hsc70 protein complex could have functional significance in these transformed cells
— id: 55871, year: 1987, vol: 7, page: 2863, stat: Journal Article,

3-(Trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine photolabels a substrate-binding site of rat hepatic cytochrome P-450 form PB-4
Frey AB; Kreibich G; Wadhera AB; Clarke L; Waxman DJ
1986 Aug 26;25(17):4797-4803, Biochemistry
Hepatic microsomes isolated from untreated male rats or from rats pretreated with phenobarbital (PB) or 3-methylcholanthrene (3-MC) were labeled with the hydrophobic, photoactivated reagent 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine ([125I]TID). [125I]TID incorporation into 3-MC- and PB-induced liver microsomal protein was enhanced 5- and 8-fold, respectively, relative to the incorporation of [125I]TID into uninduced liver microsomes. The major hepatic microsomal cytochrome P-450 forms inducible by PB and 3-MC, respectively designated P-450s PB-4 and BNF-B, were shown to be the principal polypeptides labeled by [125I]TID in the correspondingly induced microsomes. Trypsin cleavage of [125I]TID-labeled microsomal P-450 PB-4 yielded several radiolabeled fragments, with a single labeled peptide of Mr approximately 4000 resistant to extensive proteolytic digestion. The following experiments suggested that TID binds to the substrate-binding site of P-450 PB-4. [125I]TID incorporation into microsomal P-450 PB-4 was inhibited in a dose-dependent manner by the P-450 PB-4 substrate benzphetamine. In the absence of photoactivation, TID inhibited competitively about 80% of the cytochrome P-450-dependent 7-ethoxycoumarin O-deethylation catalyzed by PB-induced microsomes with a Ki of 10 microM; TID was a markedly less effective inhibitor of the corresponding activity catalyzed by microsomes isolated from uninduced or beta-naphthoflavone-induced livers.(ABSTRACT TRUNCATED AT 250 WORDS)
— id: 18423, year: 1986, vol: 25, page: 4797, stat: Journal Article,

The structure of phenobarbital-inducible rat liver cytochrome P-450 isoenzyme PB-4. Production and characterization of site-specific antibodies
Frey AB; Waxman DJ; Kreibich G
1985 Dec 5;260(28):15253-15265, Journal of biological chemistry
Fifteen peptides corresponding in sequence to segments of the major phenobarbital-inducible forms of rat hepatic cytochrome P-450 (termed P-450 PB-4 and P-450 PB-5) were chemically synthesized, conjugated to carrier proteins, and used to prepare site-specific rabbit and/or mouse antipeptide antibodies. Four of the synthetic peptides were recognized by rabbit heterosera raised against purified P-450 PB-4. The titer of these heterosera measured against P-450 PB-4 was only partially reduced upon complete adsorption of antipeptide activity suggesting that these peptides represent minor antigenic determinants. Each of the antipeptide antibodies recognized purified P-450 PB-4 and the highly homologous P-450 PB-5 as demonstrated by a solid-phase enzyme-linked immunosorbent assay. Although each antipeptide immunoprecipitated both purified 125I-labeled P-450 PB-4 and also in vitro-synthesized apo-P-450 PB-4, the yields of immunoprecipitation were low relative to that obtained using anti-P-450 heterosera. Only one of the antipeptide antibodies gave a good signal in an immunoblot analysis of either microsomal or purified P-450s PB-4 and PB-5. Three antipeptide antibodies raised against hydrophilic segments located in the amino-terminal one-third of P-450 PB-4 markedly inhibited the P-450 PB-4-catalyzed O-deethylation of the model substrate 7-ethoxycoumarin. Four of the antipeptide antibodies were found to cross-react with P-450 beta NF-B, the major aromatic hydrocarbon-inducible rat hepatic P-450, suggesting that certain amino acid sequences or regions of secondary structure are conserved between the major phenobarbital-induced and polycyclic-induced rat liver P-450 isoenzymes. These studies demonstrate the utility of antipeptide antibodies for evaluation of antigenic sites exposed in native P-450 PB-4, for identification of specific amino acid sequences important for the interaction of P-450 PB-4 with its substrate and/or with cytochrome P-450 reductase in a reconstituted system and for elucidation of structural and immunochemical homologies between P-450 PB-4 and other P-450 isoenzymes present in rat liver endoplasmic reticulum
— id: 18424, year: 1985, vol: 260, page: 15253, stat: Journal Article,

Induction of cytochrome P-450 isozymes in rat hepatoma-derived cell cultures
Frey AB; Rosenfeld MG; Dolan WJ; Adesnik M; Kreibich G
1984 Aug;120(2):169-180, Journal of cellular physiology
We have investigated the responsiveness of established rat hepatocyte cell cultures to inducers of cytochrome P-450. One Reuber hepatoma-derived line (Fu5-C8), which under normal culture conditions produces no detectable cytochrome P-450(MC) or cytochrome P-450(PB)--the major cytochrome P-450 isozymes induced by 3-methylcholanthrene and phenobarbital, respectively--was tested for the ability to accumulate either cytochrome P-450 isozyme in response to treatment with various xenobiotics. By immune-precipitation from [35S]-methionine-labeled cell extracts, using monospecific anticytochrome P-450(MC) antibody or monoclonal anticytochrome P-450(PB) antibody, it was demonstrated that these cells possess the capability to synthesize cytochrome P-450(MC) in response to 3-methylcholanthrene treatment, while none of the drug treatments caused the synthesis of detectable quantities of cytochrome P-450(PB). RNA extracted from Fu5-C8 cells directed the in vitro synthesis of immune-precipitable cytochrome P-450(MC) only after treatment of the cells with 3-methylcholanthrene. Kinetic analysis of the response of these cells to 3-methylcholanthrene induction revealed detectable levels of immune-precipitable cytochrome P-450(MC) 2 h after drug treatment with maximal induction occurring between 12 and 16 h of exposure. Another cell line (HF 1.5), obtained originally by hybridization of Fao X H5 variants of a Reuber H35 hepatoma, produces cytochrome P-450(MC) and also cytochrome P-450(PB) constitutively, as determined by specific immune-precipitation from labeled cell extracts. Exposure of confluent monolayers to either phenobarbital or 3-methylcholanthrene resulted in an induction of cytochrome P-450(PB) or cytochrome P-450(MC), respectively. Double-labeling immunofluorescence studies indicate that all cells in the culture produce albumin and most of the cells produce cytochrome P-450(MC), but only a subset of cells synthesize cytochrome P-450(PB). Our results demonstrate that some continuously dividing hepatocyte cell cultures retain the capacity to respond to xenobiotics, including phenobarbital, a response which is typically exhibited by fully differentiated liver cells. Such established hepatocyte cell cultures should prove useful for investigating the mechanism of induction of cytochrome P-450(PB)
— id: 18429, year: 1984, vol: 120, page: 169, stat: Journal Article,

Studies on the subunit composition of rat liver glutathione S-transferases
Frey AB; Friedberg T; Oesch F; Kreibich G
1983 Sep 25;258(18):11321-11325, Journal of biological chemistry
Native glutathione S-transferases are composed of subunits with apparent molecular weights of 25,000, 23,500, or 22,000 which form either homo- or heterodimers. Glutathione S-transferases A, C, and X which contain two subunits with molecular weights of 23,500 yielded similar but nonidentical proteolytic fragmentation patterns. Fragments unique to the subunits of the homodimers A and X were present in decreased intensities in the patterns of form C. Two-dimensional electrophoresis under denaturing conditions showed single nonoverlapping spots for transferases A and X, while form C yielded two spots corresponding in position to those obtained from forms A and X. Renaturation of dissociated glutathione S-transferase C yielded enzymatically active transferases A, C, and X. These results indicate that form C is a heterodimer composed of one subunit from the homodimeric transferases A and X. This was substantiated by NH2-terminal sequence analysis showing extensive NH2-terminal homology amongst all three forms. However, in the positions where forms A and X yielded different residues, both amino acids were detected in the sequence of form C, indicating that the two subunits of Mr = 23,500 are the products of two different genes. NH2-terminal sequence analysis of the heterodimeric glutathione S-transferase B which is composed of subunits with molecular weights of 22,000 and 25,000 revealed a single unique sequence which bore no resemblance to the sequences of either forms A or X. Despite the identical NH2-terminal sequences, proteolytic fragmentation of the separated subunits showed markedly different fragmentation patterns. This indicates that two different mRNAs code for these two subunits
— id: 18430, year: 1983, vol: 258, page: 11321, stat: Journal Article,

Synthesis and incorporation of myelin polypeptides into CNS myelin
Colman DR; Kreibich G; Frey AB; Sabatini DD
1982 Nov;95(2 Pt 1):598-608, Journal of cell biology
The distribution of newly synthesized proteolipid protein (PLP, 23 kdaltons) and myelin basic proteins (MBPs, 14-21.5 kdaltons) was determined in microsomal and myelin fractions prepared from the brainstems o1 10-30 d-old rats sacrificed at different times after an intracranial injection of 35S-methionine. Labeled MBPs were found in the myelin fraction 2 min after the injection, whereas PLP appeared first in the rough microsomal fraction and only after a lag of 30 min in the myelin fraction. Cell-free translation experiments using purified mRNAs demonstrated that PLP and MBPs are synthesized in bound and free polysomes, respectively. A mechanism involving the cotranslational insertion into the ER membrane and subsequent passage of the polypeptides through the Golgi apparatus is consistent with the lag observed in the appearance of the in vivo-labeled PLP in the myelin membrane. Newly synthesized PLP and MBPs are not proteolytically processed, because the primary translation products synthesized in vitro had the same electrophoretic mobility and N-terminal amino acid sequence as the mature PLP and MBP polypeptides. It was found that crude myelin fractions are highly enriched in mRNAs coding for the MBPs but not in mRNA coding for PLP. This suggests that whereas the bound polysomes synthesizing PLP are largely confined to the cell body, free polysomes synthesizing MBPs are concentrated in oligodendrocyte processes involved in myelination, which explains the immediate incorporation of MBPs into the developing myelin sheath
— id: 18436, year: 1982, vol: 95, page: 598, stat: Journal Article,

Studies on the biosynthesis of microsomal membrane proteins. Site of synthesis and mode of insertion of cytochrome b5, cytochrome b5 reductase, cytochrome P-450 reductase and epoxide hydrolase
Okada Y; Frey AB; Guenthner TM; Oesch F; Sabatini DD; Kreibich G
1982 Feb;122(2):393-402, European journal of biochemistry
— id: 18440, year: 1982, vol: 122, page: 393, stat: Journal Article,

BIOSYNTHESIS OF CNS MYELIN MEMBRANE-PROTEINS
Colman, DR; Kreibich, G; Frey, AB; Sabatini, DD
1981 ;91(2):A404-A404, Journal of cell biology
— id: 30541, year: 1981, vol: 91, page: A404, stat: Journal Article,

Location of a gelatin-binding region of human plasma fibronectin
Furie MB; Frey AB; Rifkin DB
1980 May 25;255(10):4391-4394, Journal of biological chemistry
— id: 42396, year: 1980, vol: 255, page: 4391, stat: Journal Article,