Outcome and feasibility of radiotherapy bridging in large B-cell lymphoma patients receiving CD19 CAR T in the UK.

Radiotherapy (RT) has potential synergistic effects with chimeric antigen receptor (CAR) T but is not widely used as bridging therapy due to logistical challenges and lack of standardised protocols. We analysed RT bridging in a multicentre national cohort of large B-cell lymphoma patients approved for 3L axicabtagene ciloleucel or tisagenlecleucel across 12 UK centres. Of 763 approved patients, 722 were leukapheresed, 717 had data available on bridging therapy. 169/717 (24%) received RT bridging, 129 as single modality and 40 as combined modality treatment (CMT). Of 169 patients, 65.7% had advanced stage, 36.9% bulky disease, 86.5% elevated LDH, 41.7% international prognostic index (IPI) ≥3 and 15.2% double/triple hit at the time of approval. Use of RT bridging varied from 11% to 32% between centres and increased over time. Vein-to-vein time and infusion rate did not differ between bridging modalities. RT-bridged patients had favourable outcomes with 1-year progression-free survival (PFS) of 56% for single modality and 47% for CMT (1-year PFS 43% for systemic bridging). This is the largest cohort of LBCL patients receiving RT bridging prior to CAR T reported to date. Our results show that RT bridging can be safely and effectively used even in advanced stage and high-risk disease, with low dropout rates and excellent outcomes.

Shutdown of immunological priming and presentation after in vivo administration of adenovirus.

Adenoviral (Adv) vectors are widely used in both experimental and clinical trials for vaccination and gene therapy. Recombinant Adv can evoke potent innate immune responses and adaptive immune responses to encoded antigens. However, how Adv infection affects the response to subsequently encountered antigens is poorly understood. We show that intravenously administered replication defective (E1 and E3 deleted) Adv educes functional changes in dendritic cells (DC) resulting in impaired priming of cytotoxic T lymphocytes (CTL) more than 7 days after Adv treatment. Generalized DC activation was indicated by transient upregulation of CD86 and reduced endocytosis of fluorescent beads. It is known that CD8+ DC are predominantly responsible for uptake and presentation (cross-presentation) of exogenous antigens to CD8+ CTL. Hence, impaired endocytosis in CD8+, but not CD8-, DC at 7 days after Adv administration provided an explanation for the impaired CTL response to antigen at this time. Shutdown of cross-presentation was confirmed using cytochrome c (cytc), an agent that selectively depletes cross-presenting DC. Adv-infection rendered CD8+ DC resistant to depletion by cytc. As the cross-presentation pathway underlies CD8 T-cell responses to many cancers and to vaccines or viruses that do not directly infect DC, systemic Adv administration may impair these responses.

Without CD4 help, CD8 rejection of pig xenografts requires CD28 costimulation but not perforin killing.

Although CD4 cells are major mediators in cellular rejection of fetal pig pancreas (FPP) in the mouse, rejection still occurs in the absence of CD4 cells, albeit with delayed kinetics. CD4 cell-independent mechanisms of cellular rejection are poorly understood. To investigate the involvement of CD8 T cells in FPP rejection and their activation requirements, we used mice transgenic for anti-CD4 Ab; this is the most complete model of CD4 cell deficiency. We showed that in such mice FPP was infiltrated with CD8 cells starting from 2 wk posttransplantation and FPP was eventually rejected 8 wk posttransplantation. Ab depletion of CD8 cells greatly improved the survival of FPP and reduced cell infiltration at the graft site. This suggests that CD8 cells can mediate the rejection of porcine xenografts in the absence of CD4 cells. This CD8-mediated rejection of FPP is independent of their perforin-mediated lytic function, as graft survival was not affected in mice deficient in perforin. The production of IFN-gamma and IL-5 by the graft infiltrates indicates that CD8 cells may act through cytokine-mediated mechanisms. Remarkably, in the absence of CD4 cells, lymphocyte infiltration at the graft site was absent in mice transgenic for CTLA4Ig such that the islet grafts flourished beyond 24 wk. In contrast, rejection was little affected by CD40 ligand deficiency. Therefore, we show that CD8 cells are activated to mediate FPP rejection independent of perforin and that this CD4-independent activation of CD8 cells critically depends on B7/CD28 costimulation.

Identifying the faces in the mirror: untangling transference and countertransference in self psychology.

Experienced psychotherapists realize that there are many levels of meaning behind much that is said during the therapeutic hour. The challenge for the competent professional is to become wise enough to learn what to ignore and sensitive enough to know what to emphasize. Sorting this out is at the heart of the practice of good psychotherapy. In this article, we will briefly share our thoughts on this rich but complicated interpersonal interaction using Kohutian self psychology theory to understand the role of countertransference in the process of conducting psychotherapy.

Enhanced survival of grafts genetically endowed with the ability to block CD2 and B7.

In a model of transplantation rejection, we have tested whether a graft manipulated to secrete immunomodulators could protect itself from immune destruction. An insulinoma cell line having the NOD genotype but also expressing the neoantigen, SV40 T antigen, was transfected with CTLA4Ig or LFA3Ig to block signals in the co-stimulatory/adhesion pathways. This neoantigen is potent at inducing graft rejection. Secretion of CTLA4Ig and LFA3Ig by transfectants promoted survival of the insulinoma graft in young NOD mice. In immunodeficient mice, cell growth was similar for all transfectants. However, in immunocompetent NOD mice the survival/growth of test grafts was significantly better than that of the controls. Graft survival was enhanced additively, when the two test transfectants were cotransplanted. Endowing the graft the ability to secrete immunomodulators that block individual co-stimulatory/adhesion signals can contribute to transplantation success. Blockade of two signals (CD2 and CD28) in these pathways enhances this success.

Protection of xenografts by a combination of immunoisolation and a single dose of anti-CD4 antibody.

Immunoisolation is the separation of transplanted cells from cells of the immune system using a semipermeable membrane. Using one such immunoisolation capsule-the TheraCyte device-we have assessed the survival of encapsulated xenogeneic tissue in vivo as well as the contribution of CD4+ve T cells to encapsulated xenograft rejection. The foreign body reaction to the TheraCyte capsule in vivo was assessed by transplanting empty capsules into normal mice. These capsules elicit a foreign body response by the host animal. Encapsulated CHO, NIT-1, and PK-15 cells were placed in culture and in immunodeficient mice to investigate their growth characteristics in the TheraCyte device. These cell lines survive both in culture and in immunodeficient SCID mice. Xenogeneic PK cells were also transplanted into normal C57BL/6 mice. These cells do not survive in normal mice despite the absence of direct contact between infiltrating and encapsulated cells. In addition, the survival of encapsulated cells in mice treated with a single dose of anti-CD4 antibody was examined. This was assessed using two systems: 1) histological analysis of capsule sections; 2) a quantitative luciferase reporter system using PK cells transfected to express luciferase. In both cases, anti-CD4 antibody contributed to prolonged encapsulated xenogeneic cell survival. Encapsulated xenogeneic cells survive in immunodeficient mice but not normal mice. Treatment of normal mice with anti-CD4 antibody results in prolonged survival of xenogeneic cells that can be measured using a luciferase reporter system. These results highlight the contribution of CD4+ve T cells to encapsulated xenograft rejection.

Overcoming the poor immunogenicity of a protein by DNA immunization as a fusion construct.

The production of antibodies against poorly immunogenic proteins is problematic. Often there is a failure to generate such antibodies. Furthermore, antibodies against other specificities are frequently induced. We describe a simple approach, analogous to conjugation to a protein carrier, whereby immunization with naked DNA was used to raise antibody to a highly homologous and poorly immunogenic allotypic protein. Deoxyribonucleic acid encoding the protein of interest was fused to DNA encoding the Fc region of a foreign Ig, resulting in increased immunogenicity. The potential applications of this approach include the production of antisera and mAb to allotypic variants, mutant proteins, and proteins that are highly conserved between species.

Nucleic acid vaccines: tasks and tactics.

There are no adequate vaccines against some of the new or reemerged infectious scourges such as HIV and TB. They may require strong and enduring cell-mediated immunity to be elicited. This is quite a task, as the only known basis of protection by current commercial vaccines is antibody. As DNA or RNA vaccines may induce both cell-mediated and humoral immunity, great interest has been shown in them. However, doubt remains whether their efficacy will suffice for their clinical realization. We look at the various tactics to increase the potency of nucleic acid vaccines and divided them broadly under those affecting delivery and those affecting immune induction. For delivery, we have considered ways of improving uptake and the use of bacterial, replicon or viral vectors. For immune induction, we considered aspects of immunostimulatory CpG motifs, coinjection of cytokines or costimulators and alterations of the antigen, its cellular localization and its anatomical localization including the use of ligand-targeting to lymphoid tissue. We also thought that mucosal application of DNA deserved a separate section. In this review, we have taken the liberty to discuss these enhancement methods, whenever possible, in the context of the underlying mechanisms that might argue for or against these strategies.

Predominant transgene expression in exocrine pancreas directed by the CMV promoter.

The enhancer/promoter of the human cytomegalovirus gene encoding the major immediate-early protein (CMVp) is reputed to be one of the strongest and most promiscuous regulatory elements for directing transcription of heterologous genes in vitro. However, transgene expression under the promoter in adult transgenic mice is often more restricted. We selected a CMVp segment from position -350 to +59 to control expression of transgenes for two secretory fusion proteins. Expression was analyzed by immunohistology staining and quantified by Northern blot, Western blot, and ELISA of secretions from explanted tissues. In all six lines of transgenic mice, the highest expression of transgenes at the mRNA and protein level was observed in the exocrine tissue of the pancreas, although the levels of expression varied among the lines. The results indicate not only that CMVp is not a universal promoter in vivo but indeed that it can be relatively specific for the exocrine pancreas, where expression of the gene it controlled was consistently very high.

Delayed rejection of fetal pig pancreas in CD4 cell deficient mice was correlated with residual helper activity.

CD4 cells have been shown to play a dominant role in the rejection of xenografts. Depletion of murine CD4 cells by injecting anti-CD4 antibody prolongs the graft survival, but does not prevent its rejection. For a more stable phenotype, we used genetically modified mice. To test whether the delayed rejection is caused by incomplete depletion of CD4 cells, we evaluated the response to fetal pig pancreas (FPP) xenografts in three types of CD4 cell deficient mice. They are MHC class II deficient mice (MHC II(o/o), CD4 deficient mice (CD4(o/o)) and a novel type of CD4 cell deficient mice (designated GK). GK mice were rendered permanently and completely CD4 deficient by transgenic expression of anti-CD4 antibody, whereas both MHC II(o/o) and CD4(o/o) mice have a residual helper cell population. FPP grafts in wild type mice were rejected within a week, whereas FPP grafts survived up to 4 weeks in MHC II(o/o) and CD4(o/o) mice. Survival of grafts in GK mice was even longer (8 weeks). Differences in histology were also noted. Rejecting grafts in MHC II(o/o) and wild-type mice were infiltrated with both eosinophils and mononuclear cells, whereas the infiltrates in CD4(o/o) and GK mice were exclusively mononuclear cells. Immunohistochemistry showed that they were primarily CD8 cells. The immune response to FPP was clearly different in the three types of CD4 cell deficient mice. Splenocytes of MHC II(o/o) 3 weeks post-transplant with FPP produced substantial amounts of IFN-gamma and IL-5, whereas splenocytes of CD4(o/o) mice produced low levels of IFN-gamma but no detectable IL-5. At similar times, these cytokines were not detected in GK mice. Furthermore, CD4(o/o) mice were capable of mounting helper dependent, although reduced, IgG responses to FPP antigens, while GK mice were not. The above results indicate that residual helper activity in some types of CD4 cell deficient mice could still contribute to xenograft rejection. Caution needs to be exercised where such mice are used as models of CD4 cell deficiency. Also, because there is eventual rejection of xenograft FPP in GK mice which lack detectable helper activity, we argue that these mice are a better model to investigate the involvement of CD4-independent rejection mechanisms.

Local production of anti-CD4 antibody by transgenic allogeneic grafts affords partial protection.

BACKGROUND: Immunosuppressive drugs and anti-lymphocyte antibody are used clinically to suppress cellular rejection responses. However, these systemic regimens often led to general immunodeficiency and thus increased susceptibility to opportunistic infection and neoplasia. Immunosuppressive molecules delivered locally may be a way of inhibiting rejection responses, whereas systemic immunity is preserved. To achieve protective local immunosuppression, we produced a graft secreting its own immunomodulator, by deriving transgenic mice expressing a chimeric anti-CD4 antibody (GK2c) in the pancreas. METHODS AND RESULTS: Transgenic mice in bml genetic background expressing a modified anti-mouse CD4 antibody (GK2c) under two promoters have been produced. Tissue expression of GK2c was detected by immunoperoxidase staining. Under the cytomegalovirus promoter, there was abundant GK2c expression in pancreatic exocrine tissue. Under the rat preproinsulin II promoter, there was abundant GK2c expression in pancreatic endocrine tissue only. High-expression transgenic lines had 10-100 microg/ml GK2c in blood plasma. By flow cytometry, these transgenic mice were devoid of CD4+ cells in their peripheral lymphoid organs. To test transgenic mice as donors, fetal pancreata from transgenic mice were grafted into fully allogeneic CBA mice under the kidney capsule, transgenic grafts had prolonged survival compared with control non-transgenic grafts. Furthermore, GK2c transgenic grafts had reduced infiltration with an absence of CD4+ cells at the graft site without any effect on the cell composition in lymphatic tissues. CONCLUSION: Transgenic grafts that secrete anti-CD4 antibody can afford some protection against graft rejection, while only affecting the CD4 population at the graft site.

CD4 help-independent induction of cytotoxic CD8 cells to allogeneic P815 tumor cells is absolutely dependent on costimulation.

Mice made transgenic (Tg) for a rat anti-mouse CD4 Ab (GK mice) represent a novel CD4-deficient model. They not only lack canonical CD4 cells in the periphery, but also lack the residual aberrant Th cells that are found in CD4-/- mice and MHC class II-/- mice. To analyze the role of CD4 help and costimulation for CTL induction against alloantigens, we have assessed the surface and functional phenotype of CD8 cells in vivo (e.g., clearance of allogeneic P815 cells) and in vitro. In our CD4-deficient GK mice, CTL responses to allogeneic P815 cells were induced, albeit delayed, and were sufficient to eliminate P815 cells. Induction of CTL and elimination of allogeneic P815 cells were inhibited both in the presence and absence of CD4 cells by temporary CD40 ligand blockade. This indicated that direct interaction of CD40/CD40L between APCs and CD8 cells may be an accessory signal in CTL induction (as well as the indirect pathway via APC/CD4 interaction). Furthermore, whereas in CTLA4Ig single Tg mice P815 cells were rejected promptly, in the double Tg GK/CTLA4Ig mice CTL were not induced and allogeneic P815 cells were not rejected. These findings suggest that CD40/CD40L is involved in both CD4-dependent and CD4-independent pathways, and that B7/CD28 is pivotal in the CD4-independent pathway of CTL induction against allogeneic P815 cells.

Prolonged allograft survival in anti-CD4 antibody transgenic mice: lack of residual helper T cells compared with other CD4-deficient mice.

BACKGROUND: Investigations of the role of CD4 T lymphocytes in allograft rejection and tolerance have relied on the use of mouse models with a deficiency in CD4 cells. However, in mice treated with depleting monoclonal antibody (mAb) and in MHC class II knockout (KO) mice, there are residual populations of CD4 cells. CD4 KO mice had increased CD4- CD8-TCRalphabeta+ helper T cells, and both strains of KO mice could reject skin allografts at the normal rate. In this study, transgenic mice with no peripheral CD4 cells were the recipients of skin and heart allografts. Results were compared with allograft survival in CD4 and MHC class II KO mice. METHODS: GK5 (C57BL/6 bml mice transgenic for a chimeric anti-CD4 antibody) had no peripheral CD4 cells. These mice, and CD4 and class II KO mice, received BALB/c or CBA skin or cardiac allografts. Some GK5 mice were treated with anti-CD8 mAb to investigate the role of CD8 cells in rejection. CD4 and CD8 cells were assessed by FACS and immunohistochemistry. RESULTS: BALB/c skin on GK5 mice had a mean survival time +/- SD of 24+/-6 days, compared with 9+/-2 days in wild-type mice. Anti-CD8 mAb prolonged this to 66+/-7 days. BALB/c skin survived 10+/-2 days on class II KO and 14+/-2 days on CD4 KO, both significantly less than the survival seen on GK5 recipients (P<0.001). BALB/c hearts survived >100 days in GK5 recipients and in wild-type recipients treated with anti-CD4 mAb at the time of grafting, in contrast to a mean survival time of 10+/-2 days in untreated wild-type mice. Immunohistochemistry revealed that long-term surviving heart allografts from the GK5 recipients had CD8 but no CD4 cellular infiltrate. These hearts showed evidence of transplant vasculopathy. CONCLUSIONS: The GK5 mice, with a complete absence of peripheral CD4 cells, provide the cleanest available model for investigating the role of CD4 lymphocytes in allograft rejection. Prolonged skin allograft survival in these mice compared with CD4 and MHC class II KO recipients was clearly the result of improved CD4 depletion. Nevertheless, skin allograft rejection, heart allograft infiltration, and vascular disease, mediated by CD8 cells, developed in the absence of peripheral CD4 T cells.

Local secretion of a chimeric anti-CD4 antibody protects against graft rejection in the NOD mouse.

BACKGROUND: Engineering a graft to secrete its own immunosuppressive antibodies may minimize the risks associated with current high dose systemic immunosuppression. METHODS AND RESULTS: A beta cell insulinoma cell line (NIT-1) was transfected with genes encoding a chimeric anti-CD4 antibody. The NIT-1 cells secreted functional chimeric anti-CD4 antibody that bound to the CD4 molecule on mouse thymocytes and inhibited in vitro proliferation of CD4+ve T cells. Both test and control transfected cell lines grew at a similar rate in immunodeficient mice. In immunocompetent NOD mice, NIT-1 cells are normally rejected by a cellular immune response against the SV40 T antigen. Although control transfected NIT-1 cells were rapidly rejected by NOD mice, anti-CD4 secreting NIT-1 cells grew significantly better and were able to form tumors at the site of injection. CONCLUSIONS: The local secretion of chimeric anti-CD4 antibody from transfected cells can contribute to graft survival in our transplantation model.

Protective effect of CTLA4Ig secreted by transgenic fetal pancreas allografts.

BACKGROUND: Pancreas allotransplantation offers a cure for insulin-dependent diabetes mellitus. Systemic immunosuppression used to prevent immune destruction of the graft has side-effects, including increased susceptibility to infection and neoplasia. These unwanted effects may be limited by engineering the graft to secrete immunomodulatory molecules, to achieve local immunosuppression. Several studies have shown that transient local CTLA4Ig results in partial protection of allogeneic grafts. Our intent has been to determine whether sustained secretion of transgenic CTLA4Ig from pancreatic islets is able to protect against allograft rejection. METHODS AND RESULTS: Mouse CTLA4 (test=CTLA4Ig) or CD5 leader sequence (control=CD5LIg) was fused to the Fc of mouse IgG2c, and expressed transgenically under the control of the rat insulin promoter in C57BL/6 mice carrying the bml mutation of H-2K(b) (B6.C-H-2(bm1)). This resulted in expression in pancreatic islets. We used ELISA quantification of transgene products secreted into the supernatants of cultured fetal pancreata to select high (CTLA4Ig(hi)) and low (CTLA4Ig(lo)) expresser transgenic mice. Cultured fetal pancreata were transplanted under the kidney capsule of wholly allogeneic CBA recipient mice. CTLA4Ig(hi) but not CTLA4Ig(lo) expresser grafts showed enhanced survival compared with control CD5LIg grafts at 6 weeks posttransplant, provided the recipient mice were transiently depleted of CD4 T cells (by a single low-dose injection of GK1.5) before transplantation. CONCLUSIONS: Sustained local secretion of CTLA4Ig from transgenic grafts in combination with transient systemic CD4 T-cell depletion can enhance allograft acceptance.

A fusion DNA vaccine that targets antigen-presenting cells increases protection from viral challenge.

Improving the immunological potency, particularly the Ab response, is a serious hurdle for the protective efficacy and hence broad application of DNA vaccines. We examined the immunogenicity and protective efficacy of a hemagglutinin-based influenza DNA vaccine that was targeted to antigen-presenting cells (APCs) by fusion to CTLA4. The targeted vaccine was shown to induce an accelerated and increased Ab response (as compared with those receiving the nontargeted control) that was predominated by IgG1 and recognized conformationally dependent viral epitopes. Moreover, mice receiving the APC-targeted DNA vaccine had significantly reduced viral titers (100-fold) after a nonlethal virus challenge. The increased protective efficacy was most likely because of increased Ab responses, as cytotoxic T lymphocyte responses were not enhanced. Targeting was demonstrated by direct binding studies of CTLA4 fusion proteins to the cognate ligand (B7; expressed on APCs in vivo). In addition, a targeted protein was detected at 4-fold higher levels in draining lymph nodes within 2-24 h of administration. Therefore, this study demonstrates that targeting DNA-encoded antigen to APCs results in enhanced immunity and strongly suggests that this approach may be useful in improving the protective efficacy of DNA vaccines.

Transgenic anti-CD4 monoclonal antibody secretion by mouse segmental pancreas allografts promotes long term survival.

To compare the effectiveness of transgenic and systemic monoclonal antibody therapy for pancreas transplantation, vascularised segmental pancreas allografts from wild-type or transgenic pancreatic tissue that secreted monoclonal anti-CD4 were placed in CBA recipients in which diabetes had been induced chemically by streptozotocin (STZ, non-autoimmune diabetes). In untreated CBA recipients, wild-type BALB/c or C57BL/6 bml pancreas transplants were rejected in a mean survival time (MST) of 27 and 30 days, respectively. BALB/c and C57BL/6 graft survival improved when recipients were given a short course of T cell depleting monoclonal anti-CD4 antibody, (GK 1.5, 2 mg total on days -1, 0, 1, 2 with grafting on day 0) with MST +/- S.D. of 71 +/- 29 and 44 +/- 36 days, respectively. Thus, transient depletion of CD4 was effective in delaying pancreas allograft rejection in these strain combinations. The use of C57BL/6 bml mice transgenic for a rat anti-CD4 antibody (GK5 mice) as pancreas donors provided allografts that secreted sufficient anti-CD4 antibody to cause CD4 T cell depletion in the recipients (CD4 cells decreased from 30 to < 5% of small lymphocytes). This degree of depletion was not sustained and the CD4 recovery inversely correlated with graft survival. Mice with > 20% CD4 cells in the splenic lymphocyte population 4 weeks post-transplant rejected their grafts (3 of 10 mice). However, in 7 of 10 mice CD4 cells remained low (< 15%) and allografts survived for > 80 days. The GK5 allografts survived significantly longer than those from non-transgenic bml controls (MST 83 +/- 32 days, compared with 30 days, P < 0.0005). This survival time was similar to that of BALB/c allografts in CBA recipients treated with a high dose of anti-CD4 antibody. Thus, transgenic secretion of anti-CD4 antibody by the pancreas allograft was very effective in prolonging its survival.

Additive efficacy of CTLA4Ig and OX40Ig secreted by genetically modified grafts.

BACKGROUND: The use of systemic immunosuppressive drugs have been paramount in the success in transplantation, but there are serious deleterious effects. Genetic modification of grafts to secrete immunomodulators locally may be a way to reduce the need for systemic immunosuppression. METHODS AND RESULTS: An insulinoma cell line, NIT, having the nonobese diabetic (NOD) genotype but also expressing the SV40 large T Ag, was transfected with CTLA4Ig or OX40Ig in an attempt to block signals in the costimulatory/adhesion pathways. The extracellular domains of these molecules have been fused to the Fc of IgG2c derived from the NOD mouse strain. This resulted in secreted and dimerized proteins. SV40 T Ag is potent at inducing graft rejection. Test and control transfectants were transplanted subcutaneously into young NOD mice to determine whether secretion of CTLA4Ig and OX40Ig would promote survival of the insulinoma graft. In immunodeficient mice, cell growth was similar for all transfectants. However, in immunocompetent NOD mice, the survival/growth of test grafts was significantly better than that of controls. By combining test transfectants, we found that graft survival was enhanced in an additive and significant fashion. In vitro, there was a significant reduction in immune responses-compared with control-when purified fusion proteins were added to mixed leukocyte reaction cultures. CONCLUSIONS: We conclude that blockade of individual costimulatory/adhesion signals by graft manipulation can contribute to transplantation success and that blockade of combinations of signals in these pathways enhances this success. Successful immunomodulation by the graft itself can be achieved.

Transgenic overexpression of human Bcl-2 in islet beta cells inhibits apoptosis but does not prevent autoimmune destruction.

Insulin-dependent diabetes mellitus results when > 90% of the insulin-producing beta cells in the pancreatic islets are killed as a result of autoimmune attack by T cells. During the progression to diabetes, islet beta cells die as a result of different insults from the immune system. Agents such as perforin and granzymes, CD95 ligand and tumor necrosis factor-alpha, or cytokines and free-radicals have all been shown to cause beta cell apoptosis. The anti-apoptotic protein, Bcl-2, might protect against some of these stimuli. We have therefore generated transgenic mice expressing human Bcl-2 in their islet beta cells. Although Bcl-2 was able to prevent apoptosis induced by cytotoxic agents against beta cells in vitro, Bcl-2 alone could not prevent or ameliorate cytotoxic or autoimmune beta cell damage in vivo.