Autoimmunity to both proinsulin and IGRP is required for diabetes in nonobese diabetic 8.3 TCR transgenic mice

T cells specific for proinsulin and islet-specific glucose-6-phosphatase catalytic subunit related protein (IGRP) induce diabetes in nonobese diabetic (NOD) mice. TCR transgenic mice with CD8(+) T cells specific for IGRP(206-214) (NOD8.3 mice) develop accelerated diabetes that requires CD4(+) T cell help. We previously showed that immune responses against proinsulin are necessary for IGRP(206-214)-specific CD8(+) T cells to expand. In this study, we show that diabetes development is dramatically reduced in NOD8.3 mice crossed to NOD mice tolerant to proinsulin (NOD-PI mice). This indicates that immunity to proinsulin is even required in the great majority of NOD8.3 mice that have a pre-existing repertoire of IGRP(206-214)-specific cells. However, protection from diabetes could be overcome by inducing islet inflammation either by a single dose of streptozotocin or anti-CD40 agonist Ab treatment. This suggests that islet inflammation can substitute for proinsulin-specific CD4(+) T cell help to activate IGRP(206-214)-specific T cells.     

Prevention of diabetes by manipulation of anti-IGRP autoimmunity: high efficiency of a low-affinity peptide

Antigen therapy may hold great promise for the prevention of autoimmunity; however, most clinical trials have failed, suggesting that the principles guiding the choice of treatment remain ill defined. Here, we examine the antidiabetogenic properties of altered peptide ligands of CD8+ T cells recognizing an epitope of islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP206-214), a prevalent population of autoreactive T cells in autoimmune diabetes. We show that islet-associated CD8+ T cells in nonobese diabetic mice recognize numerous IGRP epitopes, and that these cells have a role in the outcome of protocols designed to induce IGRP206-214-specific tolerance. Ligands targeting IGRP206-214-reactive T cells prevented disease, but only at doses that spared low-avidity clonotypes. Notably, near complete depletion of the IGRP206-214-reactive T-cell pool enhanced the recruitment of subdominant specificities and did not blunt diabetogenesis. Thus, peptide therapy in autoimmunity is most effective under conditions that foster occupation of the target organ lymphocyte niche by nonpathogenic, low-avidity clonotypes.     

Combination of an Antigen-Specific Therapy and an Immunomodulatory Treatment to Simultaneous Block Recurrent Autoimmunity and Alloreactivity in Non-Obese Diabetic Mice

Restoration of endogenous insulin production by islet transplantation is considered a curative option for patients with type 1 diabetes. However, recurrent autoimmunity and alloreactivity cause graft rejection hindering successful transplantation. Here we tested whether transplant tolerance to allogeneic islets could be achieved in non-obese diabetic (NOD) mice by simultaneously tackling autoimmunity via antigen-specific immunization, and alloreactivity via granulocyte colony stimulating factor (G-CSF) and rapamycin (RAPA) treatment. Immunization with insB_9-23 peptide alone or in combination with two islet peptides (IGRP_206-214 and GAD_524-543) in incomplete Freund’s adjuvant (IFA) were tested for promoting syngeneic pancreatic islet engraftment in spontaneously diabetic NOD mice. Treatment with G-CSF/RAPA alone or in combination with insB_9-23/IFA was examined for promoting allogeneic islet engraftment in the same mouse model. InsB_9-23/IFA immunization significantly prolonged syngeneic pancreatic islet survival in NOD mice by a mechanism that necessitated the presence of CD4⁺CD25⁺ T regulatory (Treg) cells, while combination of three islet epitopes was less efficacious in controlling recurrent autoimmunity. G-CSF/RAPA treatment was unable to reverse T1D or control recurrent autoimmunity but significantly prolonged islet allograft survival in NOD mice. Blockade of interleukin-10 (IL-10) during G-CSF/RAPA treatment resulted in allograft rejection suggesting that IL-10-producing cells were fundamental to achieve transplant tolerance. G-CSF/RAPA treatment combined with insB_9-23/IFA did not further increase the survival of allogeneic islets. Thus, insB_9-23/IFA immunization controls recurrent autoimmunity and G-CSF/RAPA treatment limits alloreactivity, however their combination does not further promote allogeneic pancreatic islet engraftment in NOD mice.     

T cells recognizing a peptide contaminant undetectable by mass spectrometry

Synthetic peptides are widely used in immunological research as epitopes to stimulate their cognate T cells. These preparations are never completely pure, but trace contaminants are commonly revealed by mass spectrometry quality controls. In an effort to characterize novel major histocompatibility complex (MHC) Class I-restricted β-cell epitopes in non-obese diabetic (NOD) mice, we identified islet-infiltrating CD8+ T cells recognizing a contaminating peptide. The amount of this contaminant was so small to be undetectable by direct mass spectrometry. Only after concentration by liquid chromatography, we observed a mass peak corresponding to an immunodominant islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP)(206-214) epitope described in the literature. Generation of CD8+ T-cell clones recognizing IGRP(206-214) using a novel method confirmed the identity of the contaminant, further underlining the immunodominance of IGRP(206-214). If left undetected, minute impurities in synthetic peptide preparations may thus give spurious results.     

Role of inflammatory infiltrate in activation and effector function of cloned islet reactive nonobese diabetic CD8+ T cells: involvement of a nitric oxide-dependent pathway

To investigate how CD8+ T cells interact with beta cells and local inflammatory cells in islets, we have isolated CD8+ T cell clones from nonobese diabetic (NOD) spleen that recognize and destroy both islets and the NOD insulinoma cell line NIT-1. The clones destroyed NOD islets with pre-existing inflammation better than islets without signs of inflammation. Islets from NOD-scid mice were destroyed only poorly, but that could be improved by adding IL-7 to the assay. Anti-IFN-gamma Abs inhibited destruction of infiltrated islets. Single islets were effective stimulators of IFN-gamma production by cloned CD8+ T cells, which varied >50-fold depending on the degree of islet infiltration. This effect of the islet mononuclear infiltrate could be mimicked by adding spleen cells to NIT-1 cells, which augmented IFN-gamma production above the level stimulated by NIT-1 cells alone. The enhancing effect of spleen cells could be attributed to their macrophage subpopulation and was not MHC restricted, although recognition of islet Ag by cloned CD8+ T cells and subsequent islet destruction was restricted to islets expressing H-2Db molecules. An inhibitor of inducible NO synthase inhibited destruction of inflamed islets by cloned CD8+ T cells. We propose that macrophages in inflamed islets provide a form of bystander costimulation of beta cell-specific CD8+ T cells. CD8+ T cells respond to Ag and costimulation by producing IFN-gamma that activates macrophages. Activated macrophages facilitate islet destruction by CD8+ T cells through a NO synthesis-dependent pathway.     

Autoantigen Recognition Is Required for Recruitment of IGRP206-214-Autoreactive CD8+ T Cells but Is Dispensable for Tolerance

The progression of autoimmune responses is associated with an avidity maturation process driven by preferential expansion of high avidity clonotypes at the expense of their low avidity counterparts. Central and peripheral tolerance hinder the contribution of high-avidity clonotypes targeting residues 206-214 of islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP(206-214)) during the earliest stages of autoimmune diabetes. In this study, we probe the molecular determinants and biochemical consequences of IGRP(206-214)/K(d) recognition by high-, intermediate-, and low-avidity autoreactive CD8(+) T cells, and we investigate the effects of genetic IGRP(206-214) silencing on their developmental biology. We find that differences in avidity for IGRP(206-214)/K(d) map to CDR1α and are associated with quantitative differences in CD3ε proline-rich sequence exposure and Nck recruitment. Unexpectedly, we find that tolerance of high-avidity CD8(+) T cells, unlike their activation and recruitment into the pancreas, is dissociated from recognition of IGRP(206-214), particularly in adult mice. This finding challenges the view that tolerance of pathogenic autoreactive T cells is invariably triggered by recognition of the peptide-MHC complex that drives their activation in the periphery, indicating the existence of mechanisms of tolerance that are capable of sensing the avidity, hence pathogenicity of autoreactive T cells without the need to rely on local autoantigen availability.     

HLA-A*0201-restricted T cells from humanized NOD mice recognize autoantigens of potential clinical relevance to type 1 diabetes

In both humans and NOD mice, particular MHC genes are primary contributors to development of the autoreactive CD4+ and CD8+ T cell responses against pancreatic beta cells that cause type 1 diabetes (T1D). Association studies have suggested, but not proved, that the HLA-A*0201 MHC class I variant is an important contributor to T1D in humans. In this study, we show that transgenic expression in NOD mice of HLA-A*0201, in the absence of murine class I MHC molecules, is sufficient to mediate autoreactive CD8+ T cell responses contributing to T1D development. CD8+ T cells from the transgenic mice are cytotoxic to murine and human HLA-A*0201-positive islet cells. Hence, the murine and human islets must present one or more peptides in common. Islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP) is one of several important T1D autoantigens in standard NOD mice. Three IGRP-derived peptides were identified as targets of diabetogenic HLA-A*0201-restricted T cells in our NOD transgenic stock. Collectively, these results indicate the utility of humanized HLA-A*0201-expressing NOD mice in the identification of T cells and autoantigens of potential relevance to human T1D. In particular, the identified antigenic peptides represent promising tools to explore the potential importance of IGRP in the development of human T1D.     

Cross-priming of diabetogenic T cells dissociated from CTL-induced shedding of beta cell autoantigens

Cross-presentation of self Ags by APCs is key to the initiation of organ-specific autoimmunity. As MHC class I molecules are essential for the initiation of diabetes in nonobese diabetic (NOD) mice, we sought to determine whether the initial insult that allows cross-presentation of beta cell autoantigens in diabetes is caused by cognate interactions between naive CD8(+) T cells and beta cells. Naive splenic CD8(+) T cells from transgenic NOD mice expressing a diabetogenic TCR killed peptide-pulsed targets in the absence of APCs. To ascertain the role of CD8(+) T cell-induced beta cell lysis in the initiation of diabetes, we expressed a rat insulin promoter (RIP)-driven adenovirus E19 transgene in NOD mice. RIP-E19 expression inhibited MHC class I transport exclusively in beta cells and rendered these cells resistant to lysis by CD8(+) (but not CD4(+)) T cells, both in vitro and in vivo. Surprisingly, RIP-E19 expression impaired the accumulation of CD8(+) T cells in islets and delayed the onset of islet inflammation, without affecting the timing or magnitude of T cell cross-priming in the pancreatic lymph nodes, which is the earliest known event in diabetogenesis. These results suggest that access of beta cell autoantigens to the cross-presentation pathway in diabetes is T cell independent, and reveal a previously unrecognized function of MHC class I molecules on target cells in autoimmunity: local retention of disease-initiating clonotypes.     

Tolerance to antigen-presenting cell-depleted islet allografts is CD4 T cell dependent

Pretreatment of pancreatic islets in 95% oxygen culture depletes graft-associated APCs and leads to indefinite allograft acceptance in immunocompetent recipients. As such, the APC-depleted allograft represents a model of peripheral alloantigen presentation in the absence of donor-derived costimulation. Over time, a state of donor-specific tolerance develops in which recipients are resistant to donor APC-induced graft rejection. Thus, persistence of the graft is sufficient to induce tolerance independent of other immune interventions. Donor-specific tolerance could be adoptively transferred to immune-deficient SCID recipient mice transplanted with fresh immunogenic islet allografts, indicating that the original recipient was not simply "ignorant" of donor antigens. Interestingly, despite the fact that the original islet allograft presented only MHC class I alloantigens, CD8+ T cells obtained from tolerant animals readily collaborated with naive CD4+ T cells to reject donor-type islet grafts. Conversely, tolerant CD4+ T cells failed to collaborate effectively with naive CD8+ T cells for the rejection of donor-type grafts. In conclusion, the MHC class I+, II- islet allograft paradoxically leads to a change in the donor-reactive CD4 T cell subset and not in the CD8 subset. We hypothesize that the tolerant state is not due to direct class I alloantigen presentation to CD8 T cells but, rather, occurs via the indirect pathway of donor Ag presentation to CD4 T cells in the context of host MHC class II molecules.     

An essential contribution by IFN-gamma to CD8+ T cell-mediated rejection of pancreatic islet allografts

CD8+ T cells have long been considered to be the prototypical cytotoxic lymphocyte subpopulation. However, whether alloreactive CD8+ T cells require traditional cytolytic pathways such as perforin and Fas ligand (FasL) to mediate graft rejection has been a controversial issue. In the present studies, we examined the role of varied effector pathways in CD8+ T cell-mediated rejection of pancreatic islet allografts. Our goal was to systematically determine the relative requirements, if any, of perforin and FasL as well as the proinflammatory cytokine IFN-gamma in triggering graft destruction. To study CD8+ T cell effector pathways independently of other lymphocyte populations, purified alloreactive CD8+ T cells were adoptively transferred into severe combined immune-deficient (SCID) recipients bearing established islet allografts. Results indicate that to reject established islet allografts, primed CD8+ T cells do not require the individual action of the conventional cytotoxic effectors perforin and Fas ligand. In contrast, the ability to produce IFN-gamma is critical for efficient CD8+ T cell-mediated rejection of established islet allografts. Furthermore, alloreactive CD8+ TCR transgenic T cells (2C) also show IFN-gamma dependence for mediating islet allograft rejection in vivo. We speculate from these results that the production of IFN-gamma by alloreactive CD8+ T cells is a rate-limiting step in the process of islet allograft rejection.     

Presence of residual beta cells and co-existing islet autoimmunity in the NOD mouse during longstanding diabetes: a combined histochemical and immunohistochemical study

During type 1 diabetes, most beta cells die by immune processes. However, the precise fate and characteristics of beta cells and islet autoimmunity after onset are unclear. Here, the extent of beta cell survival was determined in the non-obese diabetic (NOD) mouse during increasing duration of disease and correlated with insulitis. Pancreata from female NOD mice at diagnosis and at 1, 2, 3 and 4 weeks thereafter were analysed immunohistochemically for insulin, glucagon and somatostatin cells and glucose transporter-2 (glut2) and correlated with the degree of insulitis and islet immune cell phenotypes. Insulitis, although variable, persisted after diabetes and declined with increasing duration of disease. During this period, beta cells also declined sharply whereas glucagon and somatostatin cells increased, with occasional islet cells co-expressing insulin and glucagon. Glut2 was absent in insulin-containing cells from 1 week onwards. CD4 and CD8 T cells and macrophages persisted until 4 weeks, in islets with residual beta cells or extensive insulitis. We conclude that after diabetes onset, some beta cells survive for extended periods, with continuing autoimmunity and expansion of glucagon and somatostatin cells. The absence of glut2 in several insulin-positive cells suggests that some beta cells may be unresponsive to glucose.     

More stringent conditions of plasmid DNA vaccination are required to protect grafted versus endogenous islets in nonobese diabetic mice

Recurrent autoimmune destruction of the insulin-producing beta cells is a key factor limiting successful islet graft transplantation in type I diabetic patients. In this study, we investigated the feasibility of using an Ag-specific plasmid DNA (pDNA)-based strategy to protect pro-islets that had developed from a neonatal pancreas implanted under the kidney capsule of nonobese diabetic (NOD) mice. NOD recipient mice immunized with pDNA encoding a glutamic acid decarboxylase 65 (GAD65)-IgFc fusion protein (JwGAD65), IL-4 (JwIL4), and IL-10 (pIL10) exhibited an increased number of intact pro-islets expressing high levels of insulin 15 wk posttransplant, relative to NOD recipient mice immunized with pDNA encoding a hen egg lysozyme (HEL)-IgFc fusion protein (JwHEL)+JwIL4 and pIL10 or left untreated. Notably, the majority of grafted pro-islets detected in JwGAD65+JwIL4- plus pIL10-treated recipients was free of insulitis. In addition, administration of JwGAD65+JwIL4+pIL10 provided optimal protection for engrafted islets compared with recipient NOD mice treated with JwGAD65+JwIL4 or JwGAD65+pIL10, despite effective protection of endogenous islets mediated by the respective pDNA treatments. Efficient protection of pro-islet grafts correlated with a marked reduction in GAD65-specific IFN-gamma reactivity and an increase in IL-10-secreting T cells. These results demonstrate that pDNA vaccination can be an effective strategy to mediate long-term protection of pro-islet grafts in an Ag-specific manner and that conditions are more stringent to suppress autoimmune destruction of grafted vs endogenous islets.     

MHC class I-restricted determinants on the glutamic acid decarboxylase 65 molecule induce spontaneous CTL activity

CD4(+) T cell responses to glutamic acid decarboxylase (GAD65) spontaneously arise in nonobese diabetic (NOD) mice before the onset of insulin-dependent diabetes mellitus (IDDM) and may be critical to the pathogenic process. However, since both CD4(+) and CD8(+) T cells are involved in autoimmune diabetes, we sought to determine whether GAD65-specific CD8(+) T cells were also present in prediabetic NOD mice and contribute to IDDM. To refine the analysis, putative K(d)-binding determinants that were proximal to previously described dominant Th determinants (206-220 and 524-543) were examined for their ability to elicit cytolytic activity in young NOD mice. Naive NOD spleen cells stimulated with GAD65 peptides 206-214 (p206) and 546-554 (p546) produced IFN-gamma and showed Ag-specific CTL responses against targets pulsed with homologous peptide. Conversely, several GAD peptides distal to the Th determinants, and control K(d)-binding peptides did not induce similar responses. Spontaneous CTL responses to p206 and p546 were mediated by CD8(+) T cells that are capable of lysing GAD65-expressing target cells, and p546-specific T cells transferred insulitis to NOD.scid mice. Young NOD mice pretreated with p206 and p546 showed reduced CTL responses to homologous peptides and a delay in the onset of IDDM. Thus, MHC class I-restricted responses to GAD65 may provide an inflammatory focus for the generation of islet-specific pathogenesis and beta cell destruction. This report reveals a potential therapeutic role for MHC class I-restricted peptides in treating autoimmune disease and revisits the notion that the CD4- and CD8-inducing determinants on some molecules may benefit from a proximal relationship.     

Identical beta cell-specific CD8(+) T cell clonotypes typically reside in both peripheral blood lymphocyte and pancreatic islets

A major issue regarding T cell responses in autoimmunity is how the repertoire compares between the periphery and target organ. In type 1 diabetes, the status of at-risk or diabetic individuals can be monitored by measuring beta cell-specific T cells isolated from PBL, but whether these T cells accurately reflect the repertoire residing in the pancreatic islets is unclear. The TCR repertoire of disease-relevant, tetramer-sorted CD8(+) T cells was examined at the single-cell level in PBL, pancreatic lymph nodes (PLN), and the islets of individual NOD mice. CDR3alpha and CDR3beta sequences demonstrated that the same repertoire of T cells in PBL was detected in the islets and PLN, although the frequency of specific clonotypes varied. Albeit infrequent, clonotypes that were prevalent in the islets but not found in PBL were also detected. beta cell Ag immunization expanded immunodominant PBL clonotypes present in the islets and PLN. These results show that insight into repertoire profiles of islet-infiltrating T cells can be obtained from PBL.     

Thymic and postthymic regulation of diabetogenic CD8 T cell development in TCR transgenic nonobese diabetic (NOD) mice

Natural development of diabetes in nonobese diabetic (NOD) mice requires both CD4 and CD8 T cells. Transgenic NOD mice carrying alphabeta TCR genes from a class I MHC (Kd)-restricted, pancreatic beta cell Ag-specific T cell clone develop diabetes significantly faster than nontransgenic NOD mice. In these TCR transgenic mice, a large fraction of T cells express both transgene derived and endogenous TCR beta chains. Only T cells expressing two TCR showed reactivity to the islet Ag. Development of diabetogenic T cells is inhibited in mice with no endogenous TCR expression due to the SCID mutation. These results demonstrate that the expression of two TCRs is necessary for the autoreactive diabetogenic T cells to escape thymic negative selection in the NOD mouse. Further analysis with MHC congenic NOD mice revealed that diabetes development in the class I MHC-restricted islet Ag-specific TCR transgenic mice is still dependent on the presence of the homozygosity of the NOD MHC class II I-Ag7.     

Granzyme B is dispensable in the development of diabetes in non-obese diabetic mice

Pancreatic beta cell destruction in type 1 diabetes is mediated by cytotoxic CD8(+) T lymphoctyes (CTL). Granzyme B is an effector molecule used by CTL to kill target cells. We previously showed that granzyme B-deficient allogeneic CTL inefficiently killed pancreatic islets in vitro. We generated granzyme B-deficient non-obese diabetic (NOD) mice to test whether granzyme B is an important effector molecule in spontaneous type 1 diabetes. Granzyme B-deficient islet antigen-specific CD8(+) T cells had impaired homing into islets of young mice. Insulitis was reduced in granzyme B-deficient mice at 70 days of age (insulitis score 0.043±0.019 in granzyme B-deficient versus 0.139±0.034 in wild-type NOD mice p<0.05), but was similar to wild-type at 100 and 150 days of age. We observed a reduced frequency of CD3(+)CD8(+) T cells in the islets and peripheral lymphoid tissues of granzyme B-deficient mice (p<0.005 and p<0.0001 respectively), but there was no difference in cell proportions in the thymus. Antigen-specific CTL developed normally in granzyme B-deficient mice, and were able to kill NOD islet target cells as efficiently as wild-type CTL in vitro. The incidence of spontaneous diabetes in granzyme B-deficient mice was the same as wild-type NOD mice. We observed a delayed onset of diabetes in granzyme B-deficient CD8-dependent NOD8.3 mice (median onset 102.5 days in granzyme B-deficient versus 57.50 days in wild-type NOD8.3 mice), which may be due to the delayed onset of insulitis or inefficient priming at an earlier age in this accelerated model of diabetes. Our data indicate that granzyme B is dispensable for beta cell destruction in type 1 diabetes, but is required for efficient early activation of CTL.     

Characterization of primary T cell subsets mediating rejection of pancreatic islet grafts

The cellular mechanisms by which pancreatic islet grafts are rejected have not been clearly defined. In order to address the roles of CD4+ and CD8+ T cells in pancreatic islet rejection, we used an adoptive transfer model in which H-2b nude mice were reconstituted with negatively selected H-2b CD4+ or CD8+ T cell subpopulations and engrafted with fully allogeneic pancreatic islet grafts. We found that primary (unprimed) CD4+ T cells mediated the rejection of pancreatic islet grafts, whereas, primary CD8+ T cells failed to do so, even though both T cell subpopulations were competent to reject skin allografts. These data indicate that primary CD4+ T cells are necessary for rejection of allogeneic pancreatic islet grafts, whereas primary CD8+ T lymphocytes are not. Implications concerning the nature of the APC involved in the initiation of the rejection response to islet allografts and the expression of MHC Ag by pancreatic islet cells are discussed.     

Cutting edge: accelerated autoimmune diabetes in the absence of LAG-3

Lymphocyte activation gene-3 (LAG-3; CD223) is a CD4 homolog that is required for maximal regulatory T cell function and for the control of CD4(+) and CD8(+) T cell homeostasis. Lag3(-)(/)(-) NOD mice developed substantially accelerated diabetes with 100% incidence. Adoptive transfer experiments revealed that LAG-3 was primarily responsible for limiting the pathogenic potential of CD4(+) T cells and, to a lesser extent, CD8(+) T cells. Lag3(-)(/)(-) mice exhibited accelerated, invasive insulitis, corresponding to increased CD4(+) and CD8(+) T cell islet infiltration and intraislet proliferation. The frequencies of islet Ag-reactive chromogranin A-specific CD4(+) T cells and islet specific glucose-6-phosphatase-specific CD8(+) T cells were significantly increased in the islets of Lag3(-)(/)(-) mice, suggesting an early expansion of pathogenic clones that is normally restrained by LAG-3. We conclude that LAG-3 is necessary for regulating CD4(+) and CD8(+) T cell function during autoimmune diabetes, and thus may contribute to limiting autoimmunity in disease-prone environments.     

Pancreatic islets engineered with SA-FasL protein establish robust localized tolerance by inducing regulatory T cells in mice

Allogeneic islet transplantation is an important therapeutic approach for the treatment of type 1 diabetes. Clinical application of this approach, however, is severely curtailed by allograft rejection primarily initiated by pathogenic effector T cells regardless of chronic use of immunosuppression. Given the role of Fas-mediated signaling in regulating effector T cell responses, we tested if pancreatic islets can be engineered ex vivo to display on their surface an apoptotic form of Fas ligand protein chimeric with streptavidin (SA-FasL) and whether such engineered islets induce tolerance in allogeneic hosts. Islets were modified with biotin following efficient engineering with SA-FasL protein that persisted on the surface of islets for >1 wk in vitro. SA-FasL-engineered islet grafts established euglycemia in chemically diabetic syngeneic mice indefinitely, demonstrating functionality and lack of acute toxicity. Most importantly, the transplantation of SA-FasL-engineered BALB/c islet grafts in conjunction with a short course of rapamycin treatment resulted in robust localized tolerance in 100% of C57BL/6 recipients. Tolerance was initiated and maintained by CD4(+)CD25(+)Foxp3(+) regulatory T (Treg) cells, as their depletion early during tolerance induction or late after established tolerance resulted in prompt graft rejection. Furthermore, Treg cells sorted from graft-draining lymph nodes, but not spleen, of long-term graft recipients prevented the rejection of unmodified allogeneic islets in an adoptive transfer model, further confirming the Treg role in established tolerance. Engineering islets ex vivo in a rapid and efficient manner to display on their surface immunomodulatory proteins represents a novel, safe, and clinically applicable approach with important implications for the treatment of type 1 diabetes.     

Beta-cell apoptosis in an accelerated model of autoimmune diabetes.

BACKGROUND: The non-obese diabetic (NOD) mouse is a model of human type 1 diabetes in which autoreactive T cells mediate destruction of pancreatic islet beta cells. Although known to be triggered by cytotoxic T cells, apoptosis has not been unequivocally localized to beta cells in spontaneously diabetic NOD mice. We created a model of accelerated beta-cell destruction mediated by T cells from spontaneously diabetic NOD mice to facilitate the direct detection of apoptosis in beta cells. MATERIALS AND METHODS: NOD.scid (severe combined immunodeficiency) mice were crossed with bm1 mice transgenically expressing the costimulatory molecule B7-1 (CD80) in their beta cells, to generate B7-1 NOD.scid mice. Apoptosis in islet cells was measured as DNA strand breakage by the TdT-mediated-dUTP-nick end labeling (TUNEL) technique. RESULTS: Adoptive transfer of splenocytes from spontaneously diabetic NOD mice into B7-1 NOD.scid mice caused diabetes in recipients within 12-16 days. Mononuclear cell infiltration and apoptosis were significantly greater in the islets of B7-1 NOD.scid mice than in nontransgenic NOD.scid mice. Dual immunolabeling for TUNEL and either B-7 or insulin, or the T cell markers CD4 and CD8, and colocalization by confocal microscopy clearly demonstrated apoptosis in beta cells as well in a relatively larger number of infiltrating T cells. The clearance time of apoptotic beta cells was estimated to be less than 6 min. CONCLUSIONS: B7-1 transgenic beta cells undergo apoptosis during their accelerated destruction in response to NOD mouse effector T cells. Rapid clearance implies that beta cells undergoing apoptosis would be detected only rarely during more protracted disease in spontaneously diabetic NOD mice.