Induction of diabetes in nonobese diabetic mice by Th2 T cell clones from a TCR transgenic mouse

We have produced a panel of cloned T cell lines from the BDC-2.5 TCR transgenic (Tg) mouse that exhibit a Th2 cytokine phenotype in vitro but are highly diabetogenic in vivo. Unlike an earlier report in which T cells obtained from the Tg mouse were cultured for 1 wk under Th2-promoting conditions and were found to induce disease only in NOD.scid recipients, we found that long-term T cell clones with a fixed Th2 cytokine profile can transfer disease only to young nonobese diabetic (NOD) mice and never to NOD.scid recipients. Furthermore, the mechanism by which diabetes is transferred by a Tg Th2 T cell clone differs from that of the original CD4+ Th1 BDC-2.5 T cell clone made in this laboratory. Whereas the BDC-2.5 clone rapidly causes disease in NOD.scid recipients less than 2 wk old, the Tg Th2 T cell clones can do so only when cotransferred with other diabetogenic T cells, suggesting that the Th2 T cell requires the presence of host T cells for initiation of disease.     

Islet allograft rejection in nonobese diabetic mice involves the common gamma-chain and CD28/CD154-dependent and -independent mechanisms

Once nonobese diabetic (NOD) mice become diabetic, they are highly resistant to islet transplantation. The precise mechanism of such resistance remains largely unknown. In the present study we tested the hypothesis that islet allograft survival in the diabetic NOD mouse is determined by the interplay of diverse islet-specific T cell subsets whose activation is regulated by CD28/CD154 costimulatory signals and the common gamma-chain (gammac; a shared signaling element by receptors for IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21). We found that common gammac blockade is remarkably effective in blocking the onset and the ongoing autoimmune diabetes, whereas CD28/CD154 blockade has no effect in suppressing the ongoing diabetes. However, CD28/CD154 blockade completely blocks the alloimmune-mediated islet rejection. Also, a subset of memory-like T cells in the NOD mice is resistant to CD28/CD154 blockade, but is sensitive to the common gammac blockade. Nonetheless, neither common gammac blockade nor CD28/CD154 blockade can prevent islet allograft rejection in diabetic NOD mice. Treatment of diabetic NOD recipients with CD28/CD154 blockade plus gammac blockade markedly prolongs islet allograft survival compared with the controls. However, allograft tolerance is not achieved, and all CTLA-4Ig-, anti-CD154-, and anti-gammac-treated diabetic NOD mice eventually rejected the islet allografts. We concluded that the effector mechanisms in diabetic NOD hosts are inherently complex, and rejection in this model involves CD28/CD154/gammac-dependent and -independent mechanisms.     

Identification of a CD8 T cell that can independently mediate autoimmune diabetes development in the complete absence of CD4 T cell helper functions

Previous work has indicated that an important component for the initiation of autoimmune insulin-dependent diabetes mellitus (IDDM) in the NOD mouse model entails MHC class I-restricted CD8 T cell responses against pancreatic beta cell Ags. However, unless previously activated in vitro, such CD8 T cells have previously been thought to require helper functions provided by MHC class II-restricted CD4 T cells to exert their full diabetogenic effects. In this study, we show that IDDM development is greatly accelerated in a stock of NOD mice expressing TCR transgenes derived from a MHC class I-restricted CD8 T cell clone (designated AI4) previously found to contribute to the earliest preclinical stages of pancreatic beta cell destruction. Importantly, these TCR transgenic NOD mice (designated NOD.AI4alphabeta Tg) continued to develop IDDM at a greatly accelerated rate when residual CD4 helper T cells were eliminated by introduction of the scid mutation or a functionally inactivated CD4 allele. In a previously described stock of NOD mice expressing TCR transgenes derived from another MHC class I-restricted beta cell autoreactive T cell clone, IDDM development was retarded by elimination of residual CD4 T cells. Hence, there is variability in the helper dependence of CD8 T cells contributing to the development of autoimmune IDDM. The AI4 clonotype represents the first CD8 T cell with a demonstrated ability to progress from a naive to functionally activated state and rapidly mediate autoimmune IDDM development in the complete absence of CD4 T cell helper functions.     

Comparison of a T cell clone and of T cells from a TCR transgenic mouse: TCR transgenic T cells specific for self-antigen are atypical

It has been widely assumed that T cells from TCR-transgenic (Tg) mice better represent the behavior of T cells from normal mice than do in vitro cultures of T cell clones. We have found that autoreactive T cells arising in the presumably more physiological environment of the BDC-2.5 TCR-Tg mouse, despite being apparently "naive" in surface phenotype, are highly activated functionally and do not resemble CD4(+) T cells from a spontaneously diabetic nonobese diabetic (NOD) mouse or the NOD-derived, diabetogenic CD4(+) T cell clone of origin, BDC-2.5. Our results suggest that autoreactive T cells cloned from the spontaneously diabetic NOD mouse more closely resemble effector T cells arising during the natural disease process.     

Differential effects of B and T lymphocyte attenuator and programmed death-1 on acceptance of partially versus fully MHC-mismatched cardiac allografts

Although fully MHC-mismatched murine cardiac allografts are rapidly rejected, allografts mismatched at only MHC class I or class II alleles survive long term; the immunologic basis for the long-term survival of MHC class I- or II-mismatched allografts is unknown. We examined the roles of two recently described inhibitory receptors, B and T lymphocyte attenuator (BTLA) and programmed death-1 (PD-1), in the survival of partially or fully MHC-mismatched allografts using gene-deficient recipients as well as through use of blocking mAbs in wild-type hosts. Partially MHC-mismatched allografts showed strong induction of BTLA, but not PD-1 mRNA and survived long term in wild-type recipients, whereas targeting of BTLA or its ligand, herpesvirus entry mediator, but not PD-1, prompted their rapid rejection. By contrast, fully MHC-mismatched cardiac allografts were acutely rejected in wild-type recipients despite the induction of both BTLA and PD-1. Targeting of PD-1 in several fully MHC-mismatched models accelerated rejection, whereas targeting of BTLA unexpectedly enhanced PD-1 induction by alloreactive CD4 and CD8 T cells and prolonged allograft survival. In vitro studies using allogeneic dendritic cells and T cells showed that at low levels of T cell activation, BTLA expression was primarily induced, but that with increasing degrees of T cell activation, the expression of PD-1 was strongly up-regulated. These data suggest that BTLA and PD-1 exert distinct inhibitory actions in vivo, with the BTLA/herpesvirus entry mediator pathway appearing to dominate in regulating responses against a restricted degree of allogeneic mismatch.     

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.     

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.     

Loss of invariant chain protects nonobese diabetic mice against type 1 diabetes

The invariant (Ii) chain acts as an essential chaperone to promote MHC class II surface expression, Ag presentation, and selection of CD4(+) T cells. We have examined its role in the development of type 1 diabetes in NOD mice and show that Ii chain-deficient NOD mice fail to develop type 1 diabetes. Surprisingly, Ii chain functional loss fails to disrupt in vitro presentation of islet Ags, in the context of NOD I-A(g7) molecules. Moreover, pathogenic effector cells could be shown to be present in Ii chain-deficient NOD mice because they were able to transfer diabetes to NOD.scid recipients. The ability of these cells to transfer diabetes was markedly enhanced by depletion of CD25 cells coupled with in vivo anti-CD25 treatment of recipient mice. The numbers of CD4(+)CD25(+)Foxp3(+) T cells in thymus and periphery of Ii chain-deficient NOD mice were similar to those found in normal NOD mice, in contrast to conventional CD4(+) T cells whose numbers were reduced. This suggests that regulatory T cells are unaffected in their selection and survival by the absence of Ii chain and that an alteration in the balance of effector to regulatory T cells contributes to diabetes prevention.     

MHC class II molecules play a role in the selection of autoreactive class I-restricted CD8 T cells that are essential contributors to type 1 diabetes development in nonobese diabetic mice

Development of autoreactive CD4 T cells contributing to type 1 diabetes (T1D) in both humans and nonobese diabetic (NOD) mice is either promoted or dominantly inhibited by particular MHC class II variants. In addition, it is now clear that when co-expressed with other susceptibility genes, some common MHC class I variants aberrantly mediate autoreactive CD8 T cell responses also essential to T1D development. However, it was unknown whether the development of diabetogenic CD8 T cells could also be dominantly inhibited by particular MHC variants. We addressed this issue by crossing NOD mice transgenically expressing the TCR from the diabetogenic CD8 T cell clone AI4 with NOD stocks congenic for MHC haplotypes that dominantly inhibit T1D. High numbers of functional AI4 T cells only developed in controls homozygously expressing NOD-derived H2(g7) molecules. In contrast, heterozygous expression of some MHC haplotypes conferring T1D resistance anergized AI4 T cells through decreased TCR (H2(b)) or CD8 expression (H2(q)). Most interestingly, while AI4 T cells exert a class I-restricted effector function, H2(nb1) MHC class II molecules can contribute to their negative selection. These findings provide insights to how particular MHC class I and class II variants interactively regulate the development of diabetogenic T cells and the TCR promiscuity of such autoreactive effectors.     

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.     

NK cells promote islet allograft tolerance via a perforin-dependent mechanism

Although major histocompatibility complex (MHC) class II-restricted CD4 T cells are well appreciated for their contribution to peripheral tolerance to tissue allografts, little is known regarding MHC class I-dependent reactivity in this process. Here we show a crucial role for host MHC class I-dependent NK cell reactivity for allograft tolerance in mice induced through either costimulation blockade using CD154-specific antibody therapy or by targeting LFA-1 (also known as CD11a). Tolerance induction absolutely required host expression of MHC class I, but was independent of CD8 T cell-dependent immunity. Rather, tolerance required innate immunity involving NK1.1(+) cells, but was independent of CD1d-restricted NKT cells. Therefore, NK cells seem to be generally required for induction of tolerance to islet allografts. Additional studies indicate that CD154-specific antibody-induced allograft tolerance is perforin dependent. Notably, NK cells that are perforin competent are sufficient to restore allograft tolerance in perforin-deficient recipients. Together, these results show an obligatory role for NK cells, through perforin, for induction of tolerance to islet allografts.     

Indirect minor histocompatibility antigen presentation by allograft recipient cells in the draining lymph node leads to the activation and clonal expansion of CD4+ T cells that cause obliterative airways disease

Ag recognition by OVA-reactive OT-II (I-Ab restricted) and DO11.10 (I-Ad restricted) TCR-Tg CD4+ T cells after heterotopic transplantation of OVA transgene-expressing tracheal grafts was examined as a model of minor histocompatibility Ag (mHAg)-induced chronic allograft rejection. In response to airway allotransplantation with grafts expressing the OVA transgene, these TCR-Tg CD4+ T cells expressed the activation markers CD69 and CD44, demonstrated evidence of blastogenesis, underwent multiple rounds of cell division leading to their clonal expansion in the draining lymph node, and proceeded to differentiate to a effector/memory T cell phenotype based on a reduction in the expression of CD45RB. These mHAg-specific TCR-Tg CD4+ T cells responded equally well to fully MHC-mismatched tracheas and to class II-deficient allografts, demonstrating that donor mHAg recognition by recipient CD4+ T cells does not rely on Ag presentation by donor-derived APC. The activation of mHAg-specific TCR-Tg CD4+ T cells after their adoptive transfer into recipient mice given MHC-matched, but mHAg-disparate, airway allografts was associated with their movement into the allograft and the near uniform destruction of the transplanted airway tissue secondary to the development of obliterative airways disease. These results demonstrate that an activation of mHAg-reactive CD4+ T cells in the draining lymph node by recipient APC that indirectly express graft mHAg-derived peptide/class II MHC complexes precedes responder T cell proliferation and differentiation, and leads to the eventual migration of these alloreactive T cells to the transplanted airway tissue and the promotion of chronic graft rejection.     

Ex vivo analysis of thymic CD4 T cells in nonobese diabetic mice with tetramers generated from I-A(g7)/class II-associated invariant chain peptide precursors

The MHC determines susceptibility and resistance to type 1 diabetes in humans and nonobese diabetic (NOD) mice. To investigate how a disease-associated MHC molecule shapes the T cell repertoire in NOD mice, we generated a series of tetramers from I-A(g7)/class II-associated invariant chain peptide precursors by peptide exchange. No CD4 T cell populations could be identified for two glutamic acid decarboxylase 65 peptides, but tetramers with a peptide mimetic recognized by the BDC-2.5 and other islet-specific T cell clones labeled a distinct population in the thymus of young NOD mice. Tetramer-positive cells were identified in the immature CD4(+)CD8(low) population that arises during positive selection, and in larger numbers in the more mature CD4(+)CD8(-) population. Tetramer labeling was specific based on the use of multiple control tetramers, including one with a single amino acid analog peptide in which a critical TCR contact residue was substituted. The T cell population was already present in the thymus of 2-wk-old NOD mice before the typical onset of insulitis and was detected in B10 mice congenic for the NOD MHC locus, but not B10 control mice. These results demonstrate that a T cell population can expand in the thymus of NOD mice to levels that are at least two to three orders of magnitude higher than estimated for a given specificity in the naive T cell pool. Based on these data, we propose a model in which I-A(g7) confers susceptibility to type 1 diabetes by biasing positive selection in the thymus and later presenting peptides from islet autoantigens to such T cells in the periphery.     

I-Ag7-mediated antigen presentation by B lymphocytes is critical in overcoming a checkpoint in T cell tolerance to islet beta cells of nonobese diabetic mice.

B cell-deficient nonobese diabetic (NOD) mice are protected from the development of spontaneous autoimmune diabetes, suggesting a requisite role for Ag presentation by B lymphocytes for the activation of a diabetogenic T cell repertoire. This study specifically examines the importance of B cell-mediated MHC class II Ag presentation as a regulator of peripheral T cell tolerance to islet beta cells. We describe the construction of NOD mice with an I-Ag7 deficiency confined to the B cell compartment. Analysis of these mice, termed NOD BCIID, revealed the presence of functionally competent non-B cell APCs (macrophages/dendritic cells) with normal I-Ag7 expression and capable of activating Ag-reactive T cells. In addition, the secondary lymphoid organs of these mice harbored phenotypically normal CD4+ and CD8+ T cell compartments. Interestingly, whereas control NOD mice harboring I-Ag7-sufficient B cells developed diabetes spontaneously, NOD BCIID mice were resistant to the development of autoimmune diabetes. Despite their diabetes resistance, histologic examination of pancreata from NOD BCIID mice revealed foci of noninvasive peri-insulitis that could be intentionally converted into a destructive process upon treatment with cyclophosphamide. We conclude that I-Ag7-mediated Ag presentation by B cells serves to overcome a checkpoint in T cell tolerance to islet beta cells after their initial targeting has occurred. Overall, this work indicates that the full expression of the autoimmune potential of anti-islet T cells in NOD mice is intimately regulated by B cell-mediated MHC class II Ag presentation.     

Blockade of late stages of autoimmune diabetes by inhibition of the receptor for advanced glycation end products

Ligation of the receptor for advanced glycation end products (RAGE) occurs during inflammation. Engagement of RAGE results in enhanced expression of addressins and it is therefore, not surprising that previous studies have shown a role of RAGE/ligand interactions in immune responses including cell/cell contact but the role of RAGE in spontaneous autoimmunity has not been clearly defined. To study the role of RAGE/ligand interactions in autoimmune diabetes, we tested the ability of soluble RAGE, a scavenger of RAGE ligands, in late stages of diabetes development in the NOD mouse-disease transferred with diabetogenic T cells and recurrent disease in NOD/scid recipients of syngeneic islet transplants. RAGE expression was detected on CD4(+), CD8(+), and B cells from diabetic mice and transferred to NOD/scid recipients. RAGE and its ligand, S100B, were found in the islets of NOD/scid mice that developed diabetes. Treatment of recipient NOD/scid mice with soluble RAGE prevented transfer of diabetes and delayed recurrent disease in syngeneic islet transplants. RAGE blockade was associated with increased expression of IL-10 and TGF-beta in the islets from protected mice. RAGE blockade reduced the transfer of disease with enriched T cells, but had no effect when diabetes was transferred with the activated CD4(+) T cell clone, BDC2.5. We conclude that RAGE/ligand interactions are involved in the differentiation of T cells to a mature pathogenic phenotype during the late stages of the development of diabetes.     

Cutting edge: myelin basic protein-specific cytotoxic T cell tolerance is maintained in vivo by a single dominant epitope in H-2k mice.

Multiple sclerosis (MS) is believed to be an autoimmune disease mediated by T cells specific for CNS Ags. MS lesions contain both CD4+ and CD8+ T lymphocytes. The contribution of CD4+ T cells to CNS autoimmune disease has been extensively studied in an animal model of MS, experimental autoimmune encephalomyelitis. However, little is known about the role of autoreactive CD8+ cytotoxic T cells in MS or experimental autoimmune encephalomyelitis. We demonstrate here that myelin basic protein (MBP) is processed in vivo by the MHC class I pathway leading to a MBP79-87/Kk complex. The recognition of this complex by MBP-specific cytotoxic T cells leads to a high degree of tolerance in vivo. This study is the first to show that the pool of self-reactive lymphocytes specific for MBP contain MHC class I-restricted T cells whose response is regulated in vivo by the induction of tolerance.     

Targeting of a T cell agonist peptide to lysosomes by DNA vaccination induces tolerance in the nonobese diabetic mouse

CD4 T cells are crucial effectors in the pathology of type 1 diabetes (T1D). Successful therapeutic interventions for prevention and cure of T1D in humans are still elusive. Recent research efforts have focused on the manipulation of T cells by treatment with DNA. In this paper, we studied the effects of a DNA treatment strategy designed to target antigenic peptides to the lysosomal compartment on a monospecific T cell population termed 2.5mi(+) T cells that shares reactivity with the diabetogenic T cell clone BDC-2.5 in the NOD mouse. MHC class II tetramer analysis showed that repeated administrations were necessary to expand 2.5mi(+) T cells in vivo. This expansion was independent of Ag presentation by B cells. A single peptide epitope was sufficient to induce protection against T1D, which was not due to Ag-specific T cell anergy. Typical Th2 cytokines such as IL-10 or IL-4 were undetectable in 2.5mi(+) T cells, arguing against a mechanism of immune deviation. Instead, the expanded 2.5mi(+) T cell population produced IFN-γ similar to 2.5mi(+) T cells from naive mice. Protection against T1D by DNA treatment was completely lost in NOD.CD28(-/-) mice which are largely deficient of natural regulatory T cells (Treg). Although Ag-specific Foxp3(+) Treg did not expand in response to DNA treatment, diabetes onset was delayed in Treg-reconstituted and DNA-treated NOD.SCID mice. These observations provide evidence for a Treg-mediated protective mechanism that is independent of the expansion or de novo generation of Ag-specific Treg.     

Induction of robust diabetes resistance and prevention of recurrent type 1 diabetes following islet transplantation by gene therapy

We have previously shown that the development of type 1 diabetes (T1D) can be prevented in nonobese diabetic (NOD) mice by reconstitution with autologous hemopoietic stem cells retrovirally transduced with viruses encoding MHC class II I-A beta-chain molecules associated with protection from the disease. In this study we examined whether a blockade of the programmed death-1 (PD-1)-programmed death ligand-1 (PD-L1) pathway, a major pathway known to control diabetes occurrence, could precipitate T1D in young NOD mice following reconstitution with autologous bone marrow retrovirally transduced with viruses encoding protective MHC class II I-A beta-chain molecules. In addition, we examined whether the expression of protective MHC class II alleles in hemopoietic cells could be used to prevent the recurrence of diabetes in mice with pre-existing disease following islet transplantation. Protection from the occurrence of T1D diabetes in young NOD mice by the expression of protective MHC class II I-A beta-chain molecules in bone marrow-derived hemopoietic cells was resistant to induction by PD-1-PD-L1 blockade. Moreover, reconstitution of NOD mice with pre-existing T1D autologous hemopoietic stem cells transduced with viruses encoding protective MHC class II I-A beta-chains allowed for the successful transplantation of syngeneic islets, resulting in the long-term reversal of T1D. Reversal of diabetes was resistant to induction by PD-1-PDL-1 blockade and depletion of CD25(+) T cells. These data suggest that expression of protective MHC class II alleles in bone marrow-derived cells establishes robust self-tolerance to islet autoantigens and is sufficient to prevent the recurrence of autoimmune diabetes following islet transplantation.     

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.