CD4+ T cells orchestrate both amplification and deletion of CD8+ T cells

This review focuses on the role of CD4+ T cells in regulating immune responses, orchestrating both the amplification and deletion of immune cells, particularly CD8+ T cells. These two functions, which represent only an apparent contradiction, appear to be two faces of the same process of regulation. In fact, because the immune response, once activated, needs to be carefully controlled or switched off when the antigenic stimulus is eliminated, the immune system has developed several strategies either to regulate clonal amplification or to avoid useless expansion of activated cells. In particular, we have reported many data demonstrating that CD4+ T cells may be indicated as the regulatory element in the activation as well as the deletion of CD8+ T cells. New data are also reported on the ability of anergic CD4+ T cells to suppress CD8+ T-cell activation through induction of apoptosis, and on the need for CD8+ T cells for antigen recognition in inducing cell death in CD4+ T cells. Moreover, the central role of CD4+ T cells in the maintenance of peripheral tolerance has been widely described.     

HIV-1 directly kills CD4+ T cells by a Fas-independent mechanism

The mechanism by which HIV-1 induces CD4(+) T cell death is not known. A fundamental issue is whether HIV-1 primarily induces direct killing of infected cells or indirectly causes death of uninfected bystander cells. This question was studied using a reporter virus system in which infected cells are marked with the cell surface protein placental alkaline phosphatase (PLAP). Infection by HIV-PLAP of peripheral blood mononuclear cells (PBMCs) and T cell lines leads to rapid depletion of CD4(+) T cells and induction of apoptosis. The great majority of HIV-induced T cell death in vitro involves direct loss of infected cells rather than indirect effects on uninfected bystander cells. Because of its proposed role in HIV-induced cell death, we also examined the Fas (CD95/Apo1) pathway in killing of T cells by HIV-1. Infected PBMCs or CEM cells display no increase in surface Fas relative to uninfected cells. In addition, HIV-1 kills CEM and Jurkat T cells in the presence of a caspase inhibitor that completely blocks Fas-mediated apoptosis. HIV-1 also depletes CD4+ T cells in PBMCs from patients who have a genetically defective Fas pathway. These results suggest that HIV-1 induces direct apoptosis of infected cells and kills T cells by a Fas-independent mechanism.     

Anti-CD4 monoclonal antibody-induced tolerance to MHC-incompatible cardiac allografts maintained by CD4+ suppressor T cells that are not dependent upon IL-4

Anti-CD4 mAb-induced tolerance to transplanted tissues has been proposed as due to down-regulation of Thl cells by preferential induction of Th2 cytokines, especially IL-4. This study examined the role of CD4+ cells and cytokines in tolerance to fully allogeneic PVG strain heterotopic cardiac allografts induced in naive DA rats by treatment with MRC Ox38, a nondepleting anti-CD4 mAb. All grafts survived >100 days but had a minor mononuclear cell infiltrate that increased mRNA for the Thl cytokines IL-2, IFN-gamma, and TNF-beta, but not for Th2 cytokines IL-4 and IL-6 or the cytolytic molecules perforin and granzyme A. These hosts accepted PVG skin grafts but rejected third-party grafts, which were not blocked by anti-IL-4 mAb. Cells from these tolerant hosts proliferated in MLC and produced IL-2, IFN-gamma, and IL-4 at levels equivalent to naive cells. Unfractionated and CD4+ T cells, but not CD8+ T cells, transferred specific tolerance to irradiated heart grafted hosts and inhibited reconstitution of rejection by cotransferred naive cells. This transfer of tolerance was associated with normal induction of IL-2 and delayed induction of IFN-gamma, but not with increased IL-4 or IL-10 mRNA. Transfer of tolerance was also not inhibited by anti-IL-4 mAb. This study demonstrated that tolerance induced by a nondepleting anti-CD4 mAb is maintained by a CD4+ suppressor T cell that is not associated with preferential induction of Th2 cytokines or the need for IL-4; nor is it associated with an inability to induce Th1 cytokines or anergy.     

Antigen presentation by epithelial cells induces anergic immunoregulatory CD45RO+ T cells and deletion of CD45RA+ T cells

The immunoregulatory effects of alloantigen presentation by tissue parenchymal cells to resting peripheral blood CD4+ T cells was investigated. Coculture of CD45RO+ (memory) and CD45RA+ (naive) T lymphocytes with primary cultures of MHC class II-expressing epithelial cells rendered both populations of T cells hyporesponsive to a subsequent challenge by the same MHC molecule expressed on EBV-transformed lymphoblastoid B cell lines. However, the mechanisms responsible for the allospecific hyporesponsiveness were distinct. For the CD45RO+ T cells, responsiveness was restored by subsequent culture in the presence of IL-2; the addition of IL-2 had no effect on the reactivity of the CD45RA+ T cells. In contrast, the naive T cells were protected from the induction of nonresponsiveness by the presence of a neutralizing anti-CD95 Ab during the culture with thyroid follicular cells. In addition, the hyporesponsive CD45RO+ T cells effected linked suppression, in that they inhibited proliferation against a third-party DR alloantigen when the third-party alloantigen was coexpressed with the DR Ag against which hyporesponsiveness had been induced. These results suggest that recognition of Ag by T cells on tissue parenchymal cells plays an important role in the maintenance of peripheral T cell tolerance, inducing nonresponsiveness in naive and memory T cells by distinct mechanisms.     

Age-related increase in the fraction of CD27-CD4+ T cells and IL-4 production as a feature of CD4+ T cell differentiation in vivo

The influence of ageing on phenotype and function of CD4+ T cells was studied by comparing young (19-28 years of age) and aged (75-84 years of age) donors that were selected using the SENIEUR protocol to exclude underlying disease. An age-related increase was observed in the relative number of memory cells, not only on the basis of a decreased CD45RA and increased CD45RO expression, but also on the basis of a decrease in the fraction of CD27+CD4+ T cells. Our observation that the absolute number of CD45RO+CD4+ T cells was increased, while absolute numbers of CD27-CD4+ T cells remained unchanged in aged donors, indicates that the latter subset does not merely reflect the size of the CD45RO+CD4+ T cell pool. The increased fraction of memory cells in the aged was functionally reflected in an increased IL-4 production and T cell proliferation, when cells were activated with the combination of anti-CD2 and anti-CD28, whereas IL-2 production was comparable between both groups. No differences were observed with respect to proliferative T cell responses or IL-2 production using plate-bound anti-CD3 or phytohaemagglutinin (PHA). The observation that IL-4 production correlated with the fraction of memory cells in young donors but not in aged donors suggests different functional characteristics of this subset in aged donors.     

Induction of specific T cell tolerance by Fas ligand-expressing antigen-presenting cells

Autocrine interaction of Fas and Fas ligand leads to apoptosis of activated T cells, a process that is critical for the maintenance of peripheral T cell tolerance. Paracrine interactions of Fas ligand with T cells also may play an important role in the maintenance of tolerance, as Fas ligand can create immune-privileged sites and prevent graft rejection by inducing apoptosis in T cells. We surmised that APCs that express Fas ligand might directly induce apoptosis of T cells during presentation of Ag to the T cells, thus inducing Ag-specific, systemic T cell tolerance. Here, we show that profound, specific T cell unresponsiveness to alloantigen was induced by treatment of H-2k mice with H-2b APCs that expressed Fas ligand and that profound T cell unresponsiveness specific for the H-Y Ag was induced by treatment of H-2Db/H-Y TCR transgenic female mice with H-2Db/H-Y APCs that expressed Fas ligand. The induction of this systemic T cell tolerance required the expression of Fas ligand on the APCs as well as the expression of Fas on the T cells. The tolerance was restricted to the Ag presented by the APCs. The rapid and profound clonal deletion of the Ag-specific, peripheral T cells mediated by the Fas ligand-expressing APCs contributed to the induction of tolerance. These findings demonstrate that Ag-specific T cell tolerance can be induced by APCs that express Fas ligand and suggest a novel function for APCs in the induction of T cell apoptosis. Furthermore, they indicate a novel immunointervention strategy for treatment of graft rejection and autoantigen-specific autoimmune diseases.     

Controlling autoreactivity of CD4 T cells by local tolerance induction

We have used a plasmid containing the argB gene to transform an Aspergillus nidulans argB-deleted strain in the presence of restriction enzymes and show a 20- to 60-fold increase in transformation frequency via restriction enzyme-mediated integration (REMI). This procedure was used to try to tag new genes involved in the asexual development of this fungus. More than 2000 transformants isolated following electroporation of conidia and approximately 3700 transformants recovered following protoplast fusion were screened for sporulation defects. Unexpectedly, developmental mutants were obtained only when the protoplast fusion approach was used. Southern blot analysis of these mutants, and of randomly selected transformants obtained by electroporation, was consistent with the occurrence of single plasmid integration events in 33 and 65% of the cases, respectively. The argB marker was shown to be tightly linked to the mutant phenotype in only 62% of the mutants analyzed by sexual crosses. Partial DNA sequencing of a tagged gene, whose mutation delays asexual sporulation and results in a fluffy phenotype, showed no homology to previously reported sequences. Our results indicate that REMI can be used in A. nidulans to increase the transformation frequency and illustrate the advantages and potential problems when using REMI to tag genes of interest in this and other fungi.     

Rejection of cardiac xenografts by CD4+ or CD8+ T cells

We recently showed that brief complement inhibition induces accommodation of hamster cardiac transplants in nude rats. We have reconstituted nude rats carrying an accommodated xenograft with syngeneic CD4+ or CD8+ T cells to investigate the cellular mechanism of xenograft rejection. We show that CD4+ T cells can initiate xenograft rejection (10 +/- 1.7 days) by promoting production of IgG xenoreactive Abs (XAb). These XAb are able to activate complement as well as to mediate Ab-dependent cell-mediated cytotoxicity. Adoptive transfer of these XAb into naive nude rats provoked hyperacute xenograft rejection (38 +/- 13 min). The rejection was significantly (p < 0.001) delayed by cobra venom factor (CVF; 11 +/- 8 h in four of five cases) but was still more rapid than in control nude rats (3.3 +/- 0.5 days). CVF plus NK cell depletion further prolonged survival (>7 days in four of five cases; p < 0.01 vs CVF only). CD8+ T cell-reconstituted nude rats rejected their grafts later (19.4 +/- 5.8 days) and required a larger number of cells for transfer as compared with CD4+ T cell-reconstituted nude rats. However, second xenografts were rejected more rapidly than first xenografts in CD8+ T cell-reconstituted nude rats (9 +/- 2 days), indicating that the CD8+ T cells had been activated. This study demonstrates that CD4+ and CD8+ T cells can both reject xenografts. The CD4+ cells do so at least in part by generation of helper-dependent XAb that act by both complement-dependent and Ab-dependent cell-mediated cytotoxicity mechanisms; the CD8+ cells do so as helper-independent cytotoxic T cells.     

Lysis of CD4+ T cells expressing HIV-1 gag peptides by gag-specific CD8+ cytotoxic T cells

The vast majority of in vitro experiments testing the cytotoxic T lymphocytes (CTL) activity in HIV infection has been performed with target cells consisting of autologous EBV-transformed B lymphoblastoid cell lines (B-LCLs) expressing Human immunodeficiency virus type I (HIV-1) proteins. However data concerning the lysis of primary CD4+ T lymphocytes expressing HIV-1 antigens by CTLs is still lacking. To study the CTL activity against such primary targets, we used a system involving PBMCs of an HIV+ asymptomatic patient (PT) as effector cells and the CD4+ lymphocytes or B-LCLs of his healthy HLA-identical twin brother (HTW) as target cells. These syngeneic targets were either infected with recombinant vaccinia virus containing HIV-1 gag gene (gag-vac), or coated with HIV-1 gag peptides. We demonstrate in this study that PT CTLs (which were CD3+, CD4-, CD8+, TCRalphabeta+, TCRgammadelta-, CD56-) specifically lysed both types of syngeneic target cells expressing gag-vac; however, CD4+ T cells expressing HIV gag proteins were lysed less efficiently than B-LCLs expressing the same HIV epitopes. On the other hand, no specific lysis was detected when the target cells were uninfected or infected by wild-type vaccinia virus.     

Xenospecific CD8+ cytotoxic T lymphocyte generation: accessory function for CD4+ T cells and natural killer 1.1+ cells

BACKGROUND: Controversy exists as to whether natural killer (NK)1.1+ cells additionally support cytotoxic T lymphocyte (CTL) generation. We have previously demonstrated that mice generate a strong in vitro xenospecific CTL response in local popliteal lymph nodes (LN) to footpad immunizations with large numbers of human tumor cells. METHODS: In vivo depletion of various LN subsets using cytotoxic monoclonal antibodies was used to determine their relative importance in stimulating xenospecific CD8+ CTL responses to human Jurkat tumor cells. Depletion of functional NK cells in vivo was evidenced by the relative lack of NK1.1+ cells and NK activity in the spleens and LN of anti-NK1.1 monoclonal antibody-treated mice. CONCLUSION: Depletion of LN subsets indicated that CD4+ T cells were critical in generating an effective xenospecific CD8+ CTL response, but also suggested that NK1.1+ cells play a significant additional accessory role in the development of mouse anti-human xenospecific CTL.     

Specific suppression of human CD4+ Th cell responses to pig MHC antigens by CD8+CD28- regulatory T cells

Evidence that T cells can down-regulate the immune response by producing or consuming certain cytokines or by lysing APCs or Th cells has been provided in various systems. However, the generation and characterization of suppressor T cell lines have met with limited success. Here we show that xenospecific suppressor T cells can be generated by in vitro stimulation of human T cells with pig APCs. Similar to allospecific suppressors, these xenospecific suppressor T cells carry the CD8+CD28- phenotype and react to MHC class I Ags expressed by the APCs used for priming. TCR spectratyping of T suppressor cells showed oligoclonal usage of TCR-Vbeta families, indicating that xenostimulation of CD8+CD28- T cells results in Ag-driven selection of a limited Vbeta repertoire. Xenospecific T suppressor cells prevent the up-regulation of CD154 molecules on the membrane of Th cells, inhibiting their ability to react against the immunizing MHC class II xenoantigens. The mechanism of this suppression, therefore, appears to be blockade of CD154/CD40 interaction required for efficient costimulation of activated T cells.     

The CD7- T cell subset represents the majority of IL-5-secreting cells within CD4+CD45RA- T cells

Absence of CD7 is a stable phenotype in a subset of normal human T cells. Most circulating CD7- T cells express the CD4CD45RO+CD45RA- memory phenotype. We analysed CD4+CD45RA- peripheral blood lymphocytes that were separated into CD7+ and CD7- for their in vitro cytokine secretion in response to different stimuli. The CD4+CD7- subpopulation was found to secrete significantly higher levels of IL-5 compared with the CD4+CD7- subset upon stimulation with ionomycin/phorbol myristate acetate (PMA) plus anti-CD28 MoAbs. In contrast to IL-5 secretion, IL-4 and interferon-gamma (IFN-gamma) secretion was not significantly different in CD7+ and CD7- T cells upon stimulation in vitro. The data indicate that the CD4+CD7- T cell represents the majority of IL-5-secreting cells within the population of CD4+CD45RA- memory T cells. Since CD4+CD7- T cells were found to be enriched in various skin lesions associated with eosinophilic infiltration, the results of our study support the hypothesis that skin-infiltrating CD7- T cells are one of the major sources of IL-5 responsible for the development of eosinophilic inflammation in certain skin diseases.     

CD8+ myelin peptide-specific T cells can chemoattract CD4+ myelin peptide-specific T cells: importance of IFN-inducible protein 10

The demyelination process that occurs in the central nervous system (CNS) of patients with multiple sclerosis (MS) is due, in part, to an inflammatory response in which CD4+ and CD8+ T cells and macrophages infiltrate white matter. While it is thought that the inflammatory and demyelination process in MS is the product of Th1-associated cytokines secreted by CD4+ myelin protein-specific T cells present in the CNS, the mechanisms that are responsible for the recruitment and maintenance of these myelin-reactive CD4+ T cells in the CNS have not been elucidated. We have shown previously that CD8+ CTL that recognize peptides derived from sequences of the myelin proteolipid protein (PLP) presented by HLA class I molecules can be generated in vitro, and that these PLP-specific CD8+ CTL secrete the proinflammatory chemokines macrophage-inflammatory protein-1alpha and -1beta, IL-16, and IP-10. In this study, we demonstrate that soluble products of these PLP-specific CD8+ CTL can chemoattract CD4+ T cells that are specific for a myelin basic protein peptide and a PLP peptide, and that the majority of this chemotactic activity is mediated by IFN-inducible protein 10. These results demonstrate that PLP-specific CD8+ T cells can play a role in the recruitment and retention of myelin-derived peptide-specific CD4+ T cells, and indicate that they may play a proinflammatory role in the pathogenesis of MS.     

Reciprocal regulation of CD30 expression on CD4+ T cells by IL-4 and IFN-gamma

Prior studies have implicated CD30 as a marker for Th2 cells, but the mechanism that underlies this correlation was unknown. We show here that CD30 was expressed on activated CD4+ T cells in the presence of IL-4. In the absence of endogenously produced IL-4, however, even Th2 lineage cells lost CD30 expression. Thus, CD30 is not an intrinsic marker of Th2 cells, but is inducible by IL-4. CD30 was also found to be down-regulated by IFN-gamma. Committed Th1 effector cells do not express CD30, although differentiating Th1 lineage cells temporarily express CD30. The transient expression of CD30 on differentiating Th1 lineage cells was mainly the result of endogenously produced IL-4 induced by IL-12. Culture of IL-12-primed cells under conditions that reverse the phenotype (Ag plus IL-4) resulted in two cell populations based upon their ability to express CD30. One population responded to IL-4 upon restimulation and became a CD30-positive, Th0-like cell population, while the other remained CD30 negative and synthesized only IFN-gamma. Thus, CD30 expressed on CD4+ T cells reflected the ability of CD4+ T cells to respond to IL-4.     

Position-dependent variegation of a CD4 minigene with targeted expression to mature CD4+ T cells

The CD4 gene follows a complex and highly regulated pattern of expression throughout T cell development. This expression is governed by different regulatory elements that have been partly identified, including a promoter, a proximal enhancer, and a silencer. Here we show that a CD4 minigene comprising a combination of these elements is specifically expressed in mature CD4+ T cells of transgenic mice, but not in CD4+CD8+ double positive thymocytes. The proportion of transgene-expressing CD4+ T cells was constant within a given transgenic line, but varied greatly from one line to another. We demonstrate that this pattern of expression is due to integration of the transgene within or in the vicinity of centromeric heterochromatin. This position-effect variegation demonstrated with a short CD4 transgene has not been observed with larger ones containing additional regulatory sequences, suggesting that the CD4 gene contains a locus control region. Such position-dependent effects must be taken into consideration when developing transgenic models or gene transfer vectors because they can result in the absence of transgene expression in a subpopulation of target cells. Finally, the combination of the CD4 gene silencer, proximal enhancer, and promoter provides an interesting tool to selectively express genes of interest in mature CD4+ T cells of transgenic mice and for the development of gene therapy vectors.     

CD8+ T cells are the effectors of the contact dermatitis induced by urushiol in mice and are regulated by CD4+ T cells

Nitric oxide (NO) reduces platelet aggregation in vitro. However, repeated measurements of platelet aggregation in infants and small children are impossible due to the large blood samples required. Instead, the expression of different platelet receptors mediating platelet adhesion (CD 36 and CD 42b), activation (CD 42b and CD 61) and aggregation (CD 41a) was measured repeatedly by flow cytometry. First, the expression of platelet receptors was quantified in platelet suspensions of 20 healthy volunteers after incubation with different concentrations of NO (0, 25, 100 and 640 ppm) and compared to changes in platelet aggregation and intrathrombocytic cGMP levels. It was then studied in 21 infants and children before, during and up to 3 days after cardiopulmonary bypass surgery. Seven of these patients required NO inhalation postoperatively. The in vitro experiments showed a reduced expression of the CD 41a, CD 42b and CD 61 receptors with increasing doses of NO, predominantly affecting the CD 41a receptor (-11% at 100 ppm and -20% at 640 ppm). This significant effect is in keeping with the observed NO-induced inhibition of platelet aggregation (-44% at 100 ppm) and the rise in platelet cGMP levels (+69% at 100 ppm). In patients without inhaled NO, the expression of CD 41a was slightly attenuated during cardiopulmonary bypass surgery (-15%) but increased significantly afterwards (2 h: +31%, 1st day: +129%, 2nd day: +120%, 3rd day: +111%). Comparable results were obtained regarding the other adhesion molecules CD 36, CD 42b and CD 61. In patients with inhaled NO the same pattern was observed and analysis of variance did not reveal any significant difference between both groups of patients. CONCLUSIONS: NO (> or = 100 ppm) decreases the expression of different platelet adhesion molecules and platelet aggregation, presumably via an increase in intracellular cGMP. However, due to the low dose range used in the clinical setting (1-40 ppm) this is clinically not relevant. Immediately after cardiopulmonary bypass surgery the expression of these adhesion molecules is reduced, but recovers on the 1st postoperative day.     

Revealing the in vivo behavior of CD4+ T cells specific for an antigen expressed in Escherichia coli

The clonal expansion and anatomic location of microbe-specific CD4+ Th cells was studied by tracking the fate of adoptively transferred DO11.10 TCR transgenic T cells specific for OVA peptide 323-339/I-Ad in BALB/c mice infected s.c. with Escherichia coli expressing a MalE-OVA fusion protein. After infection, the DO11.10 T cells accumulated in the T cell-rich paracortical regions of the draining lymph nodes, proliferated there for several days, and then moved into the B cell-rich follicles before they slowly disappeared from the lymph nodes. These changes occurred despite the fact that viable organisms were never found in the lymph nodes. The DO11.10 T cells also accumulated in the s.c. infection site, but about 1 day later than in the draining lymph nodes. Injection of purified MalE-OVA fusion protein alone induced a transient accumulation of DO11.10 T cells in the paracortical regions, but these T cells never entered follicles and the mice did not produce anti-OVA antibodies. The DO11.10 T cells that survived in animals injected with MalE-OVA alone were hyporesponsive to in vitro Ag restimulation and did not produce IL-2 and IFN-gamma, whereas DO11.10 T cells from mice infected with MalE-OVA-expressing bacteria produced both lymphokines. These results suggest that Ag-specific T cells are first activated in secondary lymphoid organs following primary bacterial infection and then migrate to the infection site. Furthermore, productive activation of the T cells during the primary response is dependent on bacterial components other than the Ag itself.     

Enrichment for Th1 cells in the Mel-14+ CD4+ T cell fraction in aged mice

CD4+ T cells from young and aged mice were sorted into Mel-14+ cells which are regarded as naive cells and Mel-14- cells which are regarded as memory cells. These subsets were stimulated in short-time cultures with anti-CD3 or anti-CD3/anti-CD28 in order to determine the presence of Th1 and/or Th2 cytokines. Based on the simultaneous production of IL-2, IL-4, IL-10, and IFN-gamma upon anti-CD3 stimulation by Mel-14- cells from young and aged mice, it is concluded that this cell population comprises Th1, Th2, and/or Th0 cells. Mel-14+ cells from young mice only secrete substantial amounts of IL-2 in the presence of anti-CD28 as a costimulatory signal and can therefore be regarded as Th precursor cells. By contrast, Mel-14+ cells from aged mice responded to anti-CD3 alone, not only by the production of IL-2 but also by the production of high amounts of IFN-gamma and minute amounts of IL-4 and IL-10, suggesting that these "naive" cells in aged mice are enriched for Th1 cells. This was not due to lack of CD28 triggering since anti-CD28 enhanced IFN-gamma as well as IL-4 and IL-10 to a similar extent. Our data therefore indicate that Mel-14 is not exclusively expressed on naive CD4+ T cells.     

IL-10 impacts autoimmune diabetes via a CD8+ T cell pathway circumventing the requirement for CD4+ T and B lymphocytes

IL-10 is essential for an early phase of diabetes in nonobese diabetic (NOD) mice, but later becomes protective against its development. The mechanism by which IL-10 mediates the pathway to diabetes in these mice is unknown. Herein, we dissected the cellular and costimulation requirements for diabetes in transgenic (tg) NOD mice that expressed IL-10 in their pancreatic islets (IL-10-NOD mice). We found that IL-10 alone did not cause diabetes because the offspring (IL-10-NOD-scid mice) from back-crosses of IL-10-NOD mice with NOD-scid mice had no diabetes. Moreover, these IL-10-NOD-scid mice were free of lymphocytic infiltration. Treatment of IL-10-NOD mice with depleting anti-CD4 mAb or control mAb had no effect on diabetes. Surprisingly, depletion of CD8+ T cells by treatment with the corresponding mAb inhibited diabetes without attenuating insulitis, demonstrating a critical role for CD8+ T cells in the disease process. Interestingly, B cell-deficient IL-10-NOD mice readily developed diabetes with kinetics and incidence similar to those observed in wild-type mice, demonstrating that B lymphocytes as APCs were not required in the disease process. Administration of anti-CD40 ligand (CD40L) mAb did not prevent disease, indicating that CD40/CD40L costimulation is not required for diabetes in IL-10-NOD mice. Immunization of IL-10-NOD mice with CFA or heat-shock protein 65, known to block diabetes in NOD mice, had no effect on their diabetes. We demonstrate that IL-10 contributes early to the pathology of diabetes via a CD8+ T cell pathway, eliminating the requirement for B lymphocytes and CD40-CD40L costimulation. Our findings provide a mechanism for the participation of IL-10 in the early development of diabetes.