Productive infection of neonatal CD8+ T lymphocytes by HIV-1

CD8+ T lymphocytes confer significant but ultimately insufficient protection against HIV infection. Here we report that activated neonatal CD8+ T cells can be productively infected in vitro by macrophage-tropic (M-tropic) HIV-1 isolates, which are responsible for disease transmission, whereas they are resistant to T cell-tropic (T-tropic) HIV strains. Physiological activation of CD8-alpha/beta+ CD4- T cell receptor-alpha/beta+ neonatal T cells, including activation by allogeneic dendritic cells, induces the accumulation of CD4 messenger RNA and the expression of CD4 Ag on the cell surface. The large majority of anti-CD3/B7.1-activated cord blood CD8+ T cells coexpress CD4, the primary HIV receptor, as well as CCR5 and CXCR4, the coreceptors used by M- and T-tropic HIV-1 strains, respectively, to enter target cells. These findings are relevant to the rapid progression of neonatal HIV infection. Infection of primary HIV-specific CD8+ T cells may compromise their survival and thus significantly contribute to the failure of the immune system to control the infection. Furthermore, these results indicate a previously unsuspected level of plasticity in the neonatal immune system in the regulation of CD4 expression by costimulation.     

Functional characterization of porcine CD4+CD8+ extrathymic T lymphocytes

The porcine immune system is unique in that the expression of CD4 and CD8 antigens defines four subpopulations of resting, extrathymic (CD1-) T lymphocytes. In addition to CD4-CD8+ and CD4+CD8- T lymphocytes, CD4-CD8- and CD4+CD8+ lymphocyte subpopulations are prominent in blood as well as in lymphoid tissues. In the present study, a functional comparison was made between CD4+CD8- and CD4+CD8+ T lymphocyte subpopulations. In a primary in vitro immune response against alloantigenic stimulator cells, both subpopulations proliferated without significant differences in their reactivity. Different results were obtained when analyzing the antigen-specific functions of the two CD4+ subpopulations in a secondary response against recall viral antigen; these experiments were performed with T lymphocytes from pseudorabies virus-immunized pigs. The proliferative response against viral antigens could be assigned to the CD4+CD8+ subpopulation, whereas the CD4+CD8- subpopulation remained nonreactive. Further analyses of the virus-specific in vitro immune response revealed a major histocompatibility complex (MHC) class II restricted helper T lymphocyte reaction involving CD4 but not CD8 molecules as restriction elements. Taken together, these results demonstrate that only the extrathymic CD4+CD8+ T lymphocyte subpopulation of swine contains MHC class II-restricted antigen-specific memory T helper cells.     

CD4+ type 1 and CD8+ type 2 T cell subsets in human leishmaniasis have distinct T cell receptor repertoires

The mechanism of protective immunity and immunologic resistance against intracellular pathogens is believed to involve the activation of Ag-specific T cells. The T cells involved in protection/resistance to Leishmania can be studied using localized American cutaneous leishmaniasis (LCL) as a model, because the disease is often self-healing. Our study was undertaken to identify specific T cell populations that had accumulated in LCL lesions on the basis of TCR V beta gene usage. RNA was derived from skin lesions and blood of eight LCL patients, as well as from purified CD4+ and CD8+ subsets from the lesions and blood of three patients. After synthesis of cDNA, V beta gene usage was assessed by polymerase chain reaction. In all eight patients, several V beta gene families were overrepresented in lesions compared to blood. More importantly, the TCR V beta repertoires of both lesional CD4+ and CD8+ subsets were skewed compared to the repertoire of the respective subsets in the blood of the same donor. The overrepresented V beta s in the CD4+ and CD8+ subsets from lesions were in most instances disparate, particularly with the V beta 6 TCR skewed in the lesional CD8+ subset. Not only were the TCR repertoires of the overrepresented V beta in the lesional CD4+ and CD8+ subsets generally distinct, but the cytokine mRNA expressed by these subsets were also discrete. Strikingly, the CD4+ subset was characterized by IFN-gamma mRNA expression and the CD8+ subset by IL-4 and IL-10 mRNA expression. These data indicate that the pathogenesis of human leishmaniasis may be explained by the balance of CD4+ type 1 and CD8+ type 2 T cells, which probably recognize distinct sets of Ag.     

Signaling through a CD3 gamma-deficient TCR/CD3 complex in immortalized mature CD4+ and CD8+ T lymphocytes

The biologic role of each CD3 chain and their relative contribution to the signals transduced through the TCR/CD3 complex and to downstream activation events are still controversial: they may be specialized or redundant. We have immortalized peripheral blood CD4+ and CD8+ T lymphocytes from a human selective CD3 gamma deficiency using Herpesvirus saimiri. The accessibility of the mutant TCR/CD3 complex to different Abs was consistently lower in immortalized CD8+ cells when compared with CD4+ cells, relative to their corresponding CD3 gamma-sufficient controls. Several TCR/CD3-induced downstream activation events, immediate (calcium flux), early (cytotoxicity and induction of surface CD69 or CD40L activation markers or intracellular TNF-alpha) and late (proliferation and secretion of TNF-alpha), were normal in gamma-deficient cells, despite the fact that their TCR/CD3 complexes were significantly less accessible than those of controls. In contrast, the accumulation of intracellular IL-2 or its secretion after CD3 triggering was severely impaired in gamma-deficient cells. The defect was upstream of protein kinase C activation because addition of transmembrane stimuli (PMA plus calcium ionophore) completely restored IL-2 secretion in gamma-deficient cells. These results suggest that the propagation of signals initiated at the TCR itself can result in a modified downstream signaling cascade with distinct functional consequences when gamma is absent. They also provide evidence for the specific participation of the CD3 gamma chain in the induction of certain cytokine genes in both CD4+ and CD8+ human mature T cells. These immortalized mutant cells may prove to be useful in isolating cytosolic signaling pathways emanating from the TCR/CD3 complex.     

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.     

Pyk2 is differentially regulated by beta1 integrin- and CD28-mediated co-stimulation in human CD4+ T lymphocytes

Beta1 integrins can provide T cell co-stimulation, but little is known concerning their downstream signaling pathways. We found that Pyk2, a focal adhesion kinase-related tyrosine kinase, is regulated by beta1 integrin signaling in human T cells. Stimulation of Jurkat T cells with the alpha4beta1 integrin ligand VCAM-1 results in Pyk2 tyrosine phosphorylation, and combined stimulation with VCAM-1 and anti-CD3 mAb induces rapid and sustained synergistic Pyk2 phosphorylation. Studies with mAb suggest that in synergistic CD3- and alpha4beta1 integrin-mediated Pyk2 tyrosine phosphorylation, a major contribution of CD3-derived signals is independent of their effects on regulating integrin adhesion. Analysis of resting human CD4+ T cells confirmed the ability of CD3-derived signals to synergize with beta1 integrin-dependent signals in the induction of Pyk2 tyrosine phosphorylation. In addition, although CD28-mediated co-stimulatory signals were able to synergize with CD3-mediated signals in inducing ERK and JNK activation and secretion of IL-2 in the primary T cells, they did not contribute to the induction of Pyk2 phosphorylation. Taken together, these results indicate a potential role for Pyk2 in T cell co-stimulation mediated specifically by beta1 integrins.     

Unequal death in T helper cell (Th)1 and Th2 effectors: Th1, but not Th2, effectors undergo rapid Fas/FasL-mediated apoptosis

T helper cell (Th) 1, but not Th2, effectors undergo rapid Fas/Fas ligand (FasL)-mediated, activation-induced cell death upon restimulation with antigen. Unequal apoptosis is also observed without restimulation, after a longer lag period. Both effectors undergo delayed apoptosis induced by a non-Fas-mediated pathway. When Th1 and Th2 effectors are co-cultured, Th2 effectors survive preferentially, suggesting the responsible factor(s) is intrinsic to each population. Both Th1 and Th2 effectors express Fas and FasL, but only Th2 effectors express high levels of FAP-1, a Fas-associated phosphatase that may act to inhibit Fas signaling. The rapid death of Th1 effectors leading to selective Th2 survival provides a novel mechanism for differential regulation of the two subsets.     

Direct activation of human CD8+ cytotoxic T lymphocytes by interleukin-18

Direct activation of human cytotoxic T lymphocytes (CTL) by interleukin (IL)-18 was observed in a system in which CTL effective against autologous tumor cells were generated. Peripheral blood mononuclear cells (PBMC) from tumor-bearing patients, after removal of natural killer (NK) cells, were cultured in a medium containing IL-1, -2, -4, and -6, with or without IL-18, and stimulated with autologous tumor cells. IL-18 increased the activity of the CTL and the proportion of autologous CD8+ T cells present after 28 days in the induction culture. When purified CD8+ T cells were cultured in the presence of IL-18 and IL-2 for 7 days, the CTL showed enhanced cytotoxic activity against autologous tumor cells. Moreover, a purified CD8+ T cell population, which did not exhibit any apparent cytotoxic activity against autologous tumor cells, displayed cytotoxic activity after 7-day incubation with IL-18. These results suggest that IL-18 may be useful to generate autologous CTL in humans and may thereby contribute to adoptive immunotherapy for tumors.     

NKRP1A molecule is involved in transendothelial migration of CD4+ human T lymphocytes

Among human CD4+ T lymphocytes, 5-20% express the C-type lectin molecule NKRP1A. Interestingly, CD4+ NKRP1A+ T lymphocytes express high levels of beta 1 and beta 2 integrins, thus representing a T lymphocyte subset that can possibly adhere and migrate through vascular endothelium. Indeed, resting CD4+ NKRP1A+ lymphocytes, differently from the CD4+ NKRP1A- subset, migrated across endothelial cell monolayers in a Transwell chamber system. This transendothelial migration was strongly reduced after pre-treatment with an anti-NKRP1A monoclonal antibody (mAb). In addition, the NKRP1A negative Jurkatt CD4+ T-cell line that had been stably transfected with NKRP1A cDNA, migrated more rapidly and efficiently than untransfected Jurkatt cells. Finally, mAb-mediated cross-linking of NKRP1A molecule in CD4+ T lymphocytes induced the upregulation of the LFA1 Mg2+ binding site as well as beta 1 and beta 2 integrin chains. Altogether, these findings indicate that NKRP1A molecule is involved in transendothelial migration of resting CD4+ T lymphocytes.     

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.     

Rapid induction of primary human CD4+ and CD8+ T cell responses against cancer-associated MUC1 peptide epitopes

Antigen-specific MHC class II- and class I-restricted helper and cytotoxic T cell responses are important anti-cancer immune responses. MUC1 mucin is a potentially important target for immunotherapy because of its high expression on most human adenocarcinomas. MUC1 peptide-specific type 1 T cell responses were generated in vitro using human peripheral blood lymphocytes (PBL), incubated with liposomes containing synthetic MUC1 lipopeptide antigen. Only two weekly stimulations with the liposomal MUC1 formulation led to the generation of potent anti-MUC1-specific T cell proliferation as well as class I-restricted cytotoxic responses. Thus the use of PBL pulsed with liposome-encapsulated antigen provides an effective approach of rapidly generating effective antigen-presenting cell (APC) function as well as antigen specific T cells in vitro. It may be feasible to use this technology for the rapid and effective generation of APC and/or T cells as cellular vaccines for adenocarcinomas.     

Expression of the CD8alpha beta-heterodimer on CD8(+) T lymphocytes in peripheral blood lymphocytes of human immunodeficiency virus- and human immunodeficiency virus+ individuals

CD8(+) T lymphocytes play a pivotal role in controlling human immunodeficiency virus (HIV)-1 replication in vivo. We have performed four-color flow cytometric analysis of CD8(+) peripheral blood lymphocytes (PBL) from 21 HIV-1 seronegative and 103 seropositive individuals to explore the phenotypic heterogeneity of CD8beta-chain expression on CD8(+) T lymphocytes and to clarify how its expression on CD8(+) T lymphocytes may relate to acquired immunodeficiency syndrome (AIDS) clinical progression. We showed that the single monoclonal antibody (MoAb) 2ST8-5H7, directed against the CD8alpha beta-heterodimer, identifies CD8(+) T lymphocytes as effectively as the conventional combination of anti-CD3 and anti-CD8alpha antibodies. However, we detected a significantly lower mean fluorescence (MF) of anti-CD8alpha beta staining on PBL from HIV-1 seropositive donors as compared with seronegative donors. In fact, CD8(+) T lymphocytes from HIV-1-infected individuals with the lowest CD4 counts showed the lowest levels of CD8alpha beta MF. To explore further this change in CD8alpha beta expression, we assessed the expression of 14 different cell surface molecules on CD8alpha beta+ T lymphocytes of PBL from 11 HIV-1 seronegative and 22 HIV-1 seropositive individuals. The MF of anti-CD8alpha beta staining was significantly reduced on CD8(+) T lymphocyte subsets that showed immunophenotypic evidence of activation. The subset of lymphocytes expressing low levels of CD8alpha beta expressed higher levels of activation, adhesion, and cytotoxic-associated molecules and was predominantly CD45RO+ and CD28(-). Finally, we monitored the expression of the CD8alpha beta-heterodimer on PBL of eight HIV-1-infected individuals over a 16-week period after the initiation of highly active antiretroviral therapy (HAART), including zidovudine (ZDV), lamivudine (3TC), and indinavir (IDV), and found a significant increase in the expression of the CD8alpha beta-heterodimer. These results suggest that antibodies recognizing the CD8alpha beta-heterodimer are useful tools to specifically identify CD8(+) T lymphocytes. Moreover, the quantitative monitoring of CD8alpha beta expression allows the detection of discrete CD8(+) T lymphocyte subsets and may be useful for assessing the immune status of individuals infected with HIV-1.     

Naive human T cells develop into Th1 or Th0 effectors and exhibit cytotoxicity early after stimulation with Leishmania-infected macrophages

In MS2 assembly of phage particles results from an interaction between a coat protein dimer and a stem-loop of the RNA genome (the operator hairpin). Amino acid residues Thr45, which is universally conserved among the small RNA phages, and Thr59 are part of the specific RNA binding pocket and interact directly with the RNA; the former through a hydrogen bond, the latter through hydrophobic contacts. The crystal structures of MS2 protein capsids formed by mutants Thr45Ala and Thr59Ser, both with and without the 19 nt wild-type operator hairpin bound, are reported here. The RNA hairpin binds to these mutants in a similar way to its binding to wild-type protein. In a companion paper both mutants are shown to be deficient in RNA binding in an in vivo assay, but in vitro the equilibrium dissociation constant is significantly higher than wild-type for the Thr45Ala mutant. The change in binding affinity of the Thr45Ala mutant is probably a direct consequence of removal of direct hydrogen bonds between the protein and the RNA. The properties of the Thr59Ser mutant are more difficult to explain, but are consistent with a loss of non-polar contact.     

Type 1 and type 2 cytokine-producing mouse CD4+ and CD8+ T cells in acute Schistosoma mansoni infection

CD4+ and CD8+ T cells can be divided based on the cytokines that they secrete into type 1 (Th1, Tc1) and type 2 (Th2, Tc2) subsets. Schistosoma mansoni infection in mice is characterized by a type 2-dominated response. We have used intracellular cytokine staining to demonstrate dramatic changes in the relative numbers of Tc1 and Th2 cells in the spleens of mice during acute schistosome infection. In infected mice prior to egg laying a generalized type 1 response dominated, and was associated with an expansion in the frequency of Tc1 and Th1 cells. By week 7 after infection the cytokine response was of type 2, with an increase in the numbers of Th2 cells and a dramatic reduction in the frequency of Tc1 cells. Following the onset of egg laying there was apoptosis of cells in the spleens of mice, with CD4+ and in particular CD8+ T cells undergoing apoptosis. The loss of CD8+ T cells may in part be attributable to the development of a type 2 environment, following egg laying, with type 2 responses mediating the apoptosis of Tc1 cells. Schistosome regulation of Tc1 during egg laying may be required to prevent type 1 inflammatory responses from exacerbating egg-induced pathology.     

Differential usage of T cell receptor V gene segments in CD4+ and CD8+ subsets of T lymphocytes in monozygotic twins

The TCR confers immunity by the specific recognition of foreign Ag peptides in the context of self-MHC molecules. The mechanisms controlling TCR selection and repertoire generation are not clearly understood and seem to occur in an apparently random, (self) Ag-driven manner. To address the question to what extent the TCR repertoire is randomly shaped or genetically predetermined, we have analyzed the alpha beta TCR repertoire of the CD4+ and CD8+ subsets of peripheral blood lymphocyte cultures of monozygotic twins by using the polymerase chain reaction technique with TCR V region gene family-specific oligonucleotide primers. Our studies demonstrate that there is high concordance in the overall patterns of V gene usage within a pair of twins, particularly in V beta usage (mean V beta CD4+ R2 = 0.869 and CD8+ R2 = 0.833) and to a lesser extent V alpha usage (mean V alpha CD4+ R2 = 0.621 and CD8+ R2 = 0.627); whereas the patterns between unrelated individuals show more variability. This study has also demonstrated that the V alpha and V beta genes are not randomly used within the CD4+ and CD8+ subsets. We observed significant preferential skewing of several V alpha or V beta gene families to either the CD4+ or CD8+ subset in the majority of individuals analyzed (p-value range = 0.0476 to < 0.001). In particular, V alpha 11, 17, 22, and V beta 3, 9, 12, 18 were skewed to the CD4+ subset; whereas V alpha 2, 6, 12, 15, 20 and V beta 7, 14, 17 were skewed to the CD8+ subset. Furthermore, a number of the V genes showed patterns of skewing consistent only within a pair of twins. In three pairs of twins, V beta 2 was skewed to the CD4+ subset, whereas the fourth pair used almost equal frequencies of V beta 2 in both subsets. This observation was made for the V beta 2, 4, 5, 6, 8, 19 and V alpha 7, 16, 18, 21 families. Finally, the ratio of the relative V gene usage frequency that could be observed within an individual was conserved within the sets of twins; for instance, the relative amount of V beta 2 to that of V beta 3 was higher in both individuals of one set of twins, whereas it was lower in all of the other three sets. Together these observations suggest that the predominant influence shaping the TCR repertoire is genetically predetermined, of which, HLA-predicted selection mechanisms exerted during thymic maturation might be contributing factors.     

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.     

Characterization of circulating CD4+ CD8+ lymphocytes in healthy individuals prompted by identification of a blood donor with a markedly elevated level of CD4+ CD8+ lymphocytes

During flow cytometric analysis of lymphocytes from healthy donors, we identified a donor (donor A) with 22% CD4+ CD8+ cells (versus values of < 4% for 65 other controls). To determine if CD4+ CD8+ cells from donor A and other controls were similar, we first defined the phenotypic profile of control CD4+ CD8+ cells. Enriched CD4+ CD8+ cell populations for 10 controls were prepared by a two-step positive selection scheme with anti-CD4-coated magnetic beads and anti-CD8-coated culture flasks; the selected population averaged 69% CD4+ CD8+ cells and 31% CD4+ CD8- cells. For all 10 controls, two subsets of CD4+ CD8+ cells, CD4dim CD8bright and CD4bright CD8dim, were observed. Phenotypic profiles of these two CD4+ CD8+ subsets were defined by pairing anti-CD8 with other monoclonal antibodies, and the profiles were compared with each other and with those of CD4+ CD8-, CD4- CD8bright, and CD4- CD8dim cells. CD8bright and CD4bright CD8dim cells differed in their proportions of CD62-L+ cells and in their levels of CD11a and CD2 expression. Both CD4+ CD8+ subsets resembled CD4+ CD8- cells in CD45RA, CD45RO, and CD25 expression; the comparable CD- CD8+ cells in CD62-L expression; and CD4- CD8bright cells in CD11b, CD11b, CD16/56, and CD28 expression. CD38 expression in both CD4+ CD8+ subsets was decreased compared with those of other cell subsets. Whereas control CD4+ CD8+ cells averaged 33% CD4dim CD8bright, CD4+ CD8+ cells from donor A were > 90% CD4dim CD8bright. Donor A CD4dim CD8bright cells exhibited proportional decreases in CD25 and CD62-L expression and increases in CD11b and CD54 expression compared with those of control CD4dim CD8bright cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

CD8+ T cells are activated during the early Th1 and Th2 immune responses in a murine Lyme disease model

T-helper responses following Borrelia burgdorferi infection in mice determine susceptibility to Lyme arthritis. The ratio of interleukin 4-positive, CD4+ to gamma interferon (IFN-gamma)-positive, CD4+ T cells was significantly greater in infected BALB/cJ mice than in infected C3H/HeJ mice. Increased numbers of IFN-gamma-producing cells predicted greater arthritis severity, and CD8+ T cells were the main source of IFN-gamma in both strains.     

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.