Breaking of CD8+ T cell tolerance through in vivo ligation of CD40 results in inhibition of chronic graft-versus-host disease and complete donor cell engraftment

In the DBA/2 --> unirradiated (C57BL/6 x DBA/2)F(1) model of chronic graft-vs-host disease (cGVHD), donor CD4(+) T cells play a critical role in breaking host B cell tolerance, while donor CD8(+) T cells are rapidly removed and the remaining cells fall into anergy. Previously we have demonstrated that in vivo ligation of GITR (glucocorticoid-induced TNF receptor-related gene) can activate donor CD8(+) T cells, subsequently converting the disease pattern from cGVHD to an acute form. In this study, we investigated the effect of an agonistic mAb against CD40 on cGVHD. Treatment of anti-CD40 mAb inhibited the production of anti-DNA IgG1 autoantibody and the development of glomerulonephritis. The inhibition of cGVHD occurred because anti-CD40 mAb prevented donor CD8(+) T cell anergy such that subsequently activated donor CD8(+) T cells deleted host CD4(+) T cells and host B cells involved in autoantibody production. Additionally, functionally activated donor CD8(+) T cells induced full engraftment of donor hematopoietic cells and exhibited an increased graft-vs-leukemia effect. However, induction of acute GVHD by donor CD8(+) T cells seemed to be not so apparent. Further CTL analysis indicated that there were lower levels of donor CTL activity against host cells in mice that received anti-CD40 mAb, compared with mice that received anti-GITR mAb. Taken together, our results suggest that a different intensity of donor CTL activity is required for removal of host hematopoietic cells, including leukemia vs induction of acute GVHD.     

Donor IFN-gamma receptors are critical for acute CD4(+) T cell-mediated cardiac allograft rejection.

Recent studies using mouse models demonstrate that CD4(+) T cells are sufficient to mediate acute cardiac allograft rejection in the absence of CD8(+) T cells and B cells. However, the mechanistic basis of CD4-mediated rejection is unclear. One potential mechanism of CD4-mediated rejection is via elaboration of proinflammatory cytokines such as IFN-gamma. To determine whether IFN-gamma is a critical cytokine in CD4-mediated acute cardiac allograft rejection, we studied whether the expression of IFN-gamma receptors on the donor heart was required for CD4-mediated rejection. To investigate this possibility, purified CD4(+) T cells were transferred into immune-deficient mice bearing heterotopic cardiac allografts from IFN-gamma receptor-deficient (GRKO) donors. While CD4(+) T cells triggered acute rejection of wild-type heart allografts, they failed to trigger rejection of GRKO heart allografts. The impairment in CD4-mediated rejection of GRKO hearts appeared to primarily involve the efferent phase of the immune response. This conclusion was based on the findings that GRKO stimulator cells provoked normal CD4 proliferation in vitro and that intentional in vivo challenge of CD4 cells with wild-type donor APC or the adoptive transfer of in vitro primed CD4 T cells failed to provoke acute rejection of GRKO allografts. In contrast, unseparated lymph node cells acutely rejected both GRKO and wild-type hearts with similar time courses, illustrating the existence of both IFN-gamma-dependent and IFN-gamma-independent mechanisms of acute allograft rejection.     

Alloantigen affinity and CD4 help determine severity of graft-versus-host disease mediated by CD8 donor T cells

TCR affinity dictates T cell selection in the thymus and also has a high impact on the fate of peripheral T cells. Graft-vs-host disease (GVHD) is a pathological process initiated by activation of donor T cells after adoptive transfer into an allogeneic recipient. How TCR affinity affects the potential of alloreactive T cells to induce GVHD is unclear. Using alloreactive CD4+ and CD8+ TCR transgenic (Tg) T cells, GVHD models are presented that allow for the visualization of how CD8+ alloreactive T cells behave in response to alloantigens with different TCR affinity in the absence or presence of CD4 help. In a nonmyeloablative transplant model where GVHD lethality is due to marrow aplasia, alloreactive CD8+ TCR Tg T cells induced significantly more severe GVHD in the recipients that express an intermediate-affinity alloantigen than in the recipients that express a high-affinity alloantigen. In a myeloablative transplant model where GVHD lethality is due to epithelium injury, CD8+ TCR Tg cells were also more pathogenic in the recipients with an intermediate-affinity alloantigen than in those with a high-affinity alloantigen. The presence of alloreactive CD4+ TCR Tg cells enhanced the potential of CD8+ TCR Tg cells to cause GVHD in recipients with an intermediate-, but not with a high-, affinity alloantigen. These findings underscore that alloantigen affinity and CD4 help control the fate and pathogenicity of alloreactive CD8+ T cells in vivo.     

IL-21 promotes lupus-like disease in chronic graft-versus-host disease through both CD4 T cell- and B cell-intrinsic mechanisms

T cell-driven B cell hyperactivity plays an essential role in driving autoimmune disease development in systemic lupus erythematosus. IL-21 is a member of the type I cytokine family with pleiotropic activities. It regulates B cell differentiation and function, promotes T follicular helper (T(FH)) cell and Th17 cell differentiation, and downregulates the induction of T regulatory cells. Although IL-21 has been implicated in systemic lupus erythematosus, the relative importance of IL-21R signaling in CD4(+) T cells versus B cells is not clear. To address this question, we took advantage of two induced models of lupus-like chronic graft-versus-host disease by using wild-type or IL-21R(-/-) mice as donors in the parent-into-F1 model and as hosts in the Bm12→B6 model. We show that IL-21R expression on donor CD4(+) T cells is essential for sustaining T(FH) cell number and subsequent help for B cells, resulting in autoantibody production and more severe lupus-like renal disease, but it does not alter the balance of Th17 cells and regulatory T cells. In contrast, IL-21R signaling on B cells is critical for the induction and maintenance of germinal centers, plasma cell differentiation, autoantibody production, and the development of renal disease. These results demonstrate that IL-21 promotes autoimmunity in chronic graft-versus-host disease through both CD4(+) T cell- and B cell-intrinsic mechanisms and suggest that IL-21 blockade may attenuate B cell hyperactivity, as well as the aberrant T(FH) cell pathway that contributes to lupus pathogenesis.     

Donor B cells in transplants augment clonal expansion and survival of pathogenic CD4+ T cells that mediate autoimmune-like chronic graft-versus-host disease

We reported that both donor CD4(+) T and B cells in transplants were required for induction of an autoimmune-like chronic graft-versus-host disease (cGVHD) in a murine model of DBA/2 donor to BALB/c recipient, but mechanisms whereby donor B cells augment cGVHD pathogenesis remain unknown. In this study, we report that, although donor B cells have little impact on acute GVHD severity, they play an important role in augmenting the persistence of tissue damage in the acute and chronic GVHD overlapping target organs (i.e., skin and lung); they also markedly augment damage in a prototypical cGVHD target organ, the salivary gland. During cGVHD pathogenesis, donor B cells are activated by donor CD4(+) T cells to upregulate MHC II and costimulatory molecules. Acting as efficient APCs, donor B cells augment donor CD4(+) T clonal expansion, autoreactivity, IL-7Rα expression, and survival. These qualitative changes markedly augment donor CD4(+) T cells' capacity in mediating autoimmune-like cGVHD, so that they mediate disease in the absence of donor B cells in secondary recipients. Therefore, a major mechanism whereby donor B cells augment cGVHD is through augmenting the clonal expansion, differentiation, and survival of pathogenic CD4(+) T cells.     

Opposing effects of ICOS on graft-versus-host disease mediated by CD4 and CD8 T cells

ICOS, a CD28 family member expressed on activated CD4(+) and CD8(+) T cells, plays important roles in T cell activation and effector function. Here we studied the role of ICOS in graft-vs-host disease (GVHD) mediated by CD4(+) or CD8(+) T cells in allogeneic bone marrow transplantation. In comparison of wild-type and ICOS-deficient T cells, we found that recipients of ICOS(-/-) CD4(+) T cells exhibited significantly less GVHD morbidity and delayed mortality. ICOS(-/-) CD4(+) T cells had no defect in expansion, but expressed significantly less Fas ligand and produced significantly lower levels of IFN-gamma and TNF-alpha. Thus, ICOS(-/-) CD4(+) T cells were impaired in effector functions that lead to GVHD. In contrast, recipients of ICOS(-/-) CD8(+) T cells exhibited significantly enhanced GVHD morbidity and accelerated mortality. In the absence of ICOS signaling, either using ICOS-deficient donors or ICOS ligand-deficient recipients, the levels of expansion and Tc1 cytokine production of CD8(+) T cells were significantly increased. The level of expansion was inversely correlated with the level of apoptosis, suggesting that increased ability of ICOS(-/-) CD8(+) T cells to induce GVHD resulted from the enhanced survival and expansion of those cells. Our findings indicate that ICOS has paradoxical effects on the regulation of alloreactive CD4(+) and CD8(+) T cells in GVHD.     

IFN-gamma and CD8+ T cells restore host defenses against Pneumocystis carinii in mice depleted of CD4+ T cells

Host defenses against infection are profoundly compromised in HIV-infected hosts due to progressive depletion of CD4+ T lymphocytes and defective cell-mediated immunity. Although recent advances in antiretroviral therapy can dramatically lower HIV viral load, blood CD4+ T lymphocytes are not restored to normal levels. Therefore, we investigated mechanisms of host defense other than those involving CD4+ T lymphocytes against a common HIV-related opportunistic infection, Pneumocystis carinii (PC) pneumonia. Using CD4-depleted mice, which are permissive for chronic PC infection, we show that up-regulation of murine IFN-gamma by gene transfer into the lung tissue results in clearance of PC from the lungs in the absence of CD4+ lymphocytes. This resolution of infection was associated with a >4-fold increase in recruited CD8+ T lymphocytes and NK cells into the lungs. The role of CD8+ T cells as effector cells in this model was further confirmed by a lack of an effect of IFN-gamma gene transfer in scid mice or mice depleted of both CD4+ and CD8+ T cells. Cytokine mRNA analysis revealed that recruited, lung-derived CD8+ T cells had greater expression of IFN-gamma message in animals treated with the IFN-gamma gene. These results indicate that CD8+ T cells are capable of clearing PC pneumonia in the absence of CD4+ T cells and that this host defense function of CD8+ T cells, as well as their cytokine repertoire, can be up-regulated through cytokine gene transfer.     

CD4+ and CD8+ regulatory T cells generated ex vivo with IL-2 and TGF-beta suppress a stimulatory graft-versus-host disease with a lupus-like syndrome

Regulatory T cells generated ex vivo from conventional mouse T cells have been used to prevent and alter the course of a stimulatory graft-vs-host disease with a lupus-like syndrome. DBA/2 mouse T cells induce this syndrome when injected into (DBA/2 x C57BL/6) F(1) mice. Stimulating DBA/2 T cells with irradiated C57BL/6 in the presence of IL-2 and TGF-beta induced both CD4(+) and CD8(+) cells to develop potent suppressive activity and enhanced their survival. The IL-2 and TGF-beta-treated T cells lost their ability to induce graft-vs-host disease and, instead, prevented other parental T cells from inducing lymphoid hyperplasia, B cell activation, and an immune complex glomerulonephritis. Moreover, a single transfer of TGF-beta-conditioned T cells to animals that had already developed anti-dsDNA Abs decreased the titer, suppressed proteinuria, and doubled survival. This study raises the possibility that autologous regulatory T cells generated ex vivo have the potential to be used as an adoptive immunotherapy to induce allograft tolerance and to control autoimmunity.     

Both CD4(+) T cells and CD8(+) T cells are required for iodine accelerated thyroiditis in NOD mice

The nonobese diabetic (NOD) mouse, a spontaneous animal model for insulin-dependent diabetes mellitus, displays a tendency in common with human diabetic populations to develop autoimmune thyroiditis although incidence and severity of thyroid lesions vary widely among different colonies around the world. A congenic strain of NOD mice bearing I-Ak on a NOD background (NOD-H2(h4)) has recently been derived and displays a much greater tendency to develop thyroiditis and autoantibodies to mouse thyroglobulin (MTg) although it is free of diabetes. Both thyroid infiltrates and autoantibody formation are accelerated and enhanced in NOD-H2(h4) mice by increased iodine intake. The effect of increased iodine intake on NOD mice themselves has not been directly investigated although a recent study of these animals given high or low doses of iodine showed no follicular destruction unless the mice were first rendered goitrous by iodine deprivation. We found that dietary iodine increased both the incidence and the severity of thyroid lesions in our NOD mice although autoantibodies to MTg were absent. NOD background genes appear to be essential for the development of these lesions, which were maximal after 4 weeks of iodine administration and showed no significant regression when the iodine was stopped. Furthermore, our studies show for the first time that both CD4(+) and CD8(+) T cells are necessary for the development of this accelerated but essentially spontaneous murine thyroid disease.     

Alloimmune response results in expansion of autoreactive donor CD4+ T cells in transplants that can mediate chronic graft-versus-host disease

Chronic graft-versus-host disease (cGVHD) is considered an autoimmune-like disease mediated by donor CD4(+) T cells, but the origin of the autoreactive T cells is still controversial. In this article, we report that the transplantation of DBA/2 donor spleen cells into thymectomized MHC-matched allogeneic BALB/c recipients induced autoimmune-like cGVHD, although not in control syngeneic DBA/2 recipients. The donor-type CD4(+) T cells from the former but not the latter recipients induced autoimmune-like manifestations in secondary allogeneic BALB/c as well as syngeneic DBA/2 recipients. Transfer of donor-type CD4(+) T cells from secondary DBA/2 recipients with disease into syngeneic donor-type or allogeneic host-type tertiary recipients propagated autoimmune-like manifestations in both. Furthermore, TCR spectratyping revealed that the clonal expansion of the autoreactive CD4(+) T cells in cGVHD recipients was initiated by an alloimmune response. Finally, hybridoma CD4(+) T clones derived from DBA/2 recipients with disease proliferated similarly in response to stimulation by syngeneic donor-type or allogeneic host-type dendritic cells. These results demonstrate that the autoimmune-like manifestations in cGVHD can be mediated by a population of donor CD4(+) T cells in transplants that simultaneously recognize Ags presented by both donor and host APCs.     

CD4+ T cells generated de novo from donor hemopoietic stem cells mediate the evolution from acute to chronic graft-versus-host disease

Acute and chronic graft-versus-host disease (GVHD) remain the major complications limiting the efficacy of allogeneic hemopoietic stem cell transplantation. Chronic GVHD can evolve from acute GVHD, or in some cases may overlap with acute GVHD, but how acute GVHD evolves to chronic GVHD is unknown. In this study, in a classical CD8+ T cell-dependent mouse model, we found that pathogenic donor CD4+ T cells developed from engrafted hemopoietic stem cells (HSCs) in C57BL/6SJL(B6/SJL, H-2(b)) mice suffering from acute GVHD after receiving donor CD8+ T cells and HSCs from C3H.SW mice (H-2(b)). These CD4+ T cells were activated, infiltrated into GVHD target tissues, and produced high levels of IFN-gamma. These in vivo-generated CD4+ T cells caused lesions characteristic of chronic GVHD when adoptively transferred into secondary allogeneic recipients and also caused GVHD when administered into autologous C3H.SW recipients. The in vivo generation of pathogenic CD4+ T cells from engrafted donor HSCs was thymopoiesis dependent. Keratinocyte growth factor treatment improved the reconstitution of recipient thymic dendritic cells in CD8+ T cell-repleted allogeneic hemopoietic stem cell transplantation and prevented the development of pathogenic donor CD4+ T cells. These results suggest that de novo-generated donor CD4+ T cells, arising during acute graft-versus-host reactions, are key contributors to the evolution from acute to chronic GVHD. Preventing or limiting thymic damage may directly ameliorate chronic GVHD.     

CD8+ Foxp3+ regulatory T cells are induced during graft-versus-host disease and mitigate disease severity

Regulatory T cells (Tregs), in particular CD4(+) Foxp3(+) T cells, have been shown to play an important role in the maintenance of tolerance after allogeneic stem cell transplantation. In the current study, we have identified a population of CD8(+) Foxp3(+) T cells that are induced early during graft-versus-host disease (GVHD), constitute a significant percentage of the entire Treg population, and are present in all major GVHD target organs. These cells expressed many of the same cell surface molecules as found on CD4(+) Tregs and potently suppressed in vitro alloreactive T cell responses. Induction of these cells correlated positively with the degree of MHC disparity between donor and recipient and was significantly greater than that observed for CD4(+)-induced Tregs (iTregs) in nearly all tissue sites. Mice that lacked the ability to make both CD8(+) and CD4(+) iTregs had accelerated GVHD mortality compared with animals that were competent to make both iTreg populations. The absence of both iTreg populations was associated with significantly greater expansion of activated donor T cells and increased numbers of CD4(+) and CD8(+) T cells that secreted IFN-γ and IL-17. The presence of CD8(+) iTregs, however, was sufficient to prevent increased GVHD mortality in the complete absence of CD4(+) Tregs, indicating at least one functional iTreg population was sufficient to prevent an exacerbation in GVHD severity, and that CD8(+) iTregs could compensate for CD4(+) iTregs. These studies define a novel population of CD8(+) Tregs that play a role in mitigating the severity of GVHD after allogeneic stem cell transplantation.     

IL-12 and type-I IFN synergize for IFN-gamma production by CD4 T cells, whereas neither are required for IFN-gamma production by CD8 T cells after Listeria monocytogenes infection

Differentiation of Ag-specific T cells into IFN-gamma producers is essential for protective immunity to intracellular pathogens. In addition to stimulation through the TCR and costimulatory molecules, IFN-gamma production is thought to require other inflammatory cytokines. Two such inflammatory cytokines are IL-12 and type I IFN (IFN-I); both can play a role in priming naive T cells to produce IFN-gamma in vitro. However, their role in priming Ag-specific T cells for IFN-gamma production during experimental infection in vivo is less clear. In this study, we examine the requirements for IL-12 and IFN-I, either individually or in combination, for priming Ag-specific T cell IFN-gamma production after Listeria monocytogenes (Lm) infection. Surprisingly, neither individual nor combined defects in IL-12 or IFN-I signaling altered IFN-gamma production by Ag-specific CD8 T cells after Lm infection. In contrast, individual defects in either IL-12 or IFN-I signaling conferred partial ( approximately 50%) reductions, whereas combined deficiency in both IL-12 and IFN-I signaling conferred more dramatic (75-95%) reductions in IFN-gamma production by Ag-specific CD4 T cells. The additive effects of IL-12 and IFN-I signaling on IFN-gamma production by CD4 T cells were further demonstrated by adoptive transfer of transgenic IFN-IR(+/+) and IFN-IR(-/-) CD4 T cells into normal and IL-12-deficient mice, and infection with rLm. These results demonstrate an important dichotomy between the signals required for priming IFN-gamma production by CD4 and CD8 T cells in response to bacterial infection.     

CD4 T cells are required for CD8 T cell survival during both primary and memory recall responses

The role of CD4 T cell help in primary and secondary CD8 T cell responses to infectious pathogens remains incompletely defined. The primary CD8 T response to infections was initially thought to be largely independent of CD4 T cells, but it is not clear why some primary, pathogen-specific CD8 T cell responses are CD4 T cell dependent. Furthermore, although the generation of functional memory CD8 T cells is CD4 T cell help dependent, it remains controversial when the help is needed. In this study, we demonstrated that CD4 T cell help was not needed for the activation and effector differentiation of CD8 T cells during the primary response to vaccinia virus infection. However, the activated CD8 T cells showed poor survival without CD4 T cell help, leading to a reduction in clonal expansion and a diminished, but stable CD8 memory pool. In addition, we observed that CD4 T cell help provided during both the primary and secondary responses was required for the survival of memory CD8 T cells during recall expansion. Our study indicates that CD4 T cells play a crucial role in multiple stages of CD8 T cell response to vaccinia virus infection and may help to design effective vaccine strategies.     

CD4+ T cells in the absence of the CD8+ cytotoxic T cells are critical and sufficient for NKT cell-dependent tumor rejection

NKT cells perform crucial roles in tumor surveillance, functioning as regulators of early host response. In this study, we have assessed the effects of NKT activation at the time of tumor Ag immunization, and have evaluated the contributions of CD4+ and CD8+ T cells in tumor rejection during adaptive immune response against live tumor cells. Our data indicate that CD4+ T cells play critical roles, not only in assisting CTL, but also in the orchestration of host response against the tumor. The CD4+ T cells were found to reject the transplanted tumor cells very efficiently under conditions in which the CTLs were removed either genetically, or via the action of anti-CD8 Ab in mice that had been immunized with tumor extracts and alpha-galactosylceramide. Immunization resulted in an NKT cell-dependent antitumor adaptive immune response, which was associated with both CD4+ T cells and cytokine IFN-gamma.     

CD28 is required for T cell activation and IFN-gamma production by CD4+ and CD8+ T cells in response to Trypanosoma cruzi infection

In the present study we evaluated the mechanisms behind the implication of the costimulatory molecule CD28 for the immune response against the intracellular protozoan parasite Trypanosma cruzi. Our results reveal a critical role for CD28 in the activation of both CD4+ and CD8+ T cells and induction of the effector mechanisms that ultimately mediate the control of parasite growth and pathogenesis in infected mice. CD28-deficient (CD28-/-) mice are highly susceptible to T. cruzi infection, presenting higher parasitemia and tissue parasitism, but less inflammatory cell infiltrate in the heart than C57Bl/6 wild-type (WT) mice. All the infected WT mice survived acute infection, whereas 100% of CD28-/- mice succumbed to it. The increased susceptibility of the CD28-/- mice was associated with a dramatic decrease in the production of IFN-gamma by both CD4+ and CD8+ T cells resulting in a diminished capacity to produce nitric oxide (NO) and mediate parasite killing. T cell activation was also profoundly impaired in CD28-/- mice, which presented decreased lymphoproliferative response after the infection compared to WT mice. Together, these data represent the first evidence that CD28 is critical for efficient CD4+ T cell activation in response to T. cruzi infection in mice.     

Superantigen-induced CD4 T cell tolerance mediated by myeloid cells and IFN-gamma

We have previously shown that systemic staphylococcal enterotoxin A (SEA) injections cause CD4 T cells in TCR-transgenic mice to become tolerant to subsequent ex vivo restimulation. An active IFN-gamma-dependent mechanism of suppression was responsible for the apparent unresponsiveness of the CD4 T cells. In this study, we analyze the response of CD4 T cells isolated throughout the first 10 days of the in vivo response to injected SEA. We show that CD4 T cells isolated at the peak of the in vivo response undergo very little activation-induced cell death after sterile FACS sorting or restimulation in the presence of neutralizing Abs to IFN-gamma. We also show that the IFN-gamma-dependent tolerance develops soon after SEA injection in the spleens of both normal and TCR-transgenic mice. This suppression is dependent upon myeloid cells from the SEA-treated mice and is optimal when inducible NO synthase activity and reactive oxygen intermediates are both present. The data indicate that IFN-gamma, myeloid cells, and a combination of NO and reactive oxygen intermediates all contribute to a common pathway of T cell death that targets activated or responding CD4 T cells. Sorted Gr-1(+) cells from SEA-treated mice also directly suppress the response of naive CD4 T cells in mixed cultures, indicating that this tolerance mechanism may play a role in down-regulating other vigorous immune responses.     

TNF enhances CD4+ T cell alloproliferation,IFN-gamma responses,and intestinal graft-versus-host disease by IL-12-independent mechanisms

Inhibition of TNF/TNFR2 interactions ameliorates intestinal graft-vs-host disease (GVHD) and Th1 cytokine responses induced by transfer of B6 CD4(+) spleen cells into irradiated MHC class II disparate B6.C-H-2(bm12) (bm12) x B6 F(1) recipients. The present studies examined whether these effects of TNF are IL-12 dependent. T cell proliferative responses of B6.129S1-IL-12rb2(tm1Jm) (B6.IL-12R(-/-)) responder spleen cells were found to be comparable to those of control B6 spleen cells. TNF inhibition reduced T cell proliferation and IFN-gamma production in supernatants of MLC using either B6.IL-12R(-/-) or control B6 responder cells. GVHD induced wasting disease in recipients of B6.IL-12R(-/-) CD4(+) spleen cells that received a TNF inhibitor-encoding adenovirus (5.4 +/- 6.5% weight loss (n = 7)) was significantly reduced compared with levels of weight loss observed in recipients that had received a control adenovirus (25.7 +/- 12.2% weight loss (n = 11), p = 0.001). Furthermore, TNF inhibition was associated with a reduction in colonic GVHD scores (p = 0.039) and in the percentage of the splenic CD4(+) T cells that expressed IFN-gamma (16 vs 6%). These findings indicate that TNF promotes CD4(+) T cell alloproliferation, IFN-gamma responses, and intestinal GVHD by IL-12-independent mechanisms.     

Memory phenotype of CD8+ T cells in MHC class Ia-deficient mice

B6.K(b-)D(b-) mice are devoid of class Ia but express normal levels of class Ib molecules. They have low levels of CD8 T cells in both the thymus as well as peripheral T cell compartments. Although the percentage of splenic CD8 alpha alpha T cells is increased in these animals, approximately 90% of CD8 T cells are CD8 alpha beta. In contrast to B6 animals, most of the CD8 T cells from these mice have a memory phenotype (CD44(high)CD122(high) CD62L(low)) including both CD8 alpha beta and CD8 alpha alpha subsets. In the thymus of B6.K(b-)D(b-) animals, there is a decrease in the percentage of SP CD8 T cells, although most are CD44(low), similar to that seen in B6 mice. The spleens from day 1-old B6 and B6.K(b-)D(b-) mice have a relatively high proportion of CD44(high)CD62L(low) CD8 T cells. However, by day 28 most CD8 T cells in B6 mice have a naive phenotype while in B6.K(b-)D(b-) mice the memory phenotype remains. Unlike CD44(high) cells that are found in B6 animals, most CD44(high) cells from B6.K(b-)D(b-) mice do not secrete IFN-gamma rapidly upon activation. The paucity of CD8 T cells in B6.K(b-)D(b-) mice might be due in part to their inability to undergo homeostatic expansion. Consistent with this, we found that CD8 T cells from these animals expand poorly in X-irradiated syngeneic hosts compared with B6 CD8 T cells that respond to class Ia Ags. We examined homeostatic expansion of B6 CD8 T cells in single as well as double class Ia knockout mice and were able to estimate the fraction of cells reactive against class Ia vs class Ib molecules.