Apoptotic cells induce tolerance by generating helpless CD8+ T cells that produce TRAIL

The decision to generate a productive immune response or immune tolerance following pathogenic insult often depends on the context in which T cells first encounter Ag. The presence of apoptotic cells favors the induction of tolerance, whereas immune responses generated with necrotic cells promote immunity. We have examined the tolerance induced by injection of apoptotic cells, a system in which cross-presentation of Ag associated with the dead cells induces CD8+ regulatory (or suppressor) T cells. We observed that haptenated apoptotic cells induced CD8+ suppressor T cells without priming CD4+ T cells for immunity. These CD8+ T cells transferred unresponsiveness to naive recipients. In contrast, haptenated necrotic cells stimulated immunity, but induced CD8+ suppressor T cells when CD4+ T cells were absent. We further found that CD8+ T cells induced by these treatments displayed a "helpless CTL" phenotype and suppress the immune response by producing TRAIL. Animals deficient in TRAIL were resistant to tolerance induction by apoptotic cells. Thus, the outcome of an immune response taking place in the presence of cell death can be determined by the presence of CD4+-mediated Th cell function.     

Staphylococcal enterotoxin B induces anergy to conventional peptide in memory T cells

Microbial superantigens can alter host immunity through aberrant activation and subsequent anergy of responding naive T cells. We show here that the superantigen, staphylococcal enterotoxin B (SEB), directly induces tolerance in memory CD4 T cells. Murine naive and memory CD4(+) T cells were labeled with the fluorescent dye CFSE and the cells were exposed to SEB before they were cultured with specific peptide antigen. Memory, but not naive, T cells became anergic and did not respond to their cognate peptide antigen. The extent and duration of T cell receptor (TCR) clustering was similar to promote naive T cell activation and memory T cell anergy, suggesting similar TCR-SEB interactions led to distinct intracellular signaling processes in the two cell types. Like SEB, soluble anti-CD3 mAb does not stimulate memory cell proliferation. However, unlike SEB, soluble anti-CD3 mAbs did not induce anergy to cognate peptide. Anergy was directly visualized in vivo. CD4(+) memory T cells were identified in mice that had been administered SEB. The cells failed to proliferate in response to subsequent immunization with their cognate recall antigen. Hence, one mode of pathogen survival is the modulation of host immunity through selective elimination of memory T cell responses.     

Antigen-experienced T cells undergo a transient phase of unresponsiveness following optimal stimulation

Interaction of the Ag-specific receptor of T lymphocytes with its Ag/MHC ligand can lead either to cell activation or to a state of unresponsiveness often referred to as anergy. It has been generally assumed that anergy develops as a consequence of inadequate stimulation, such as in response to altered peptide ligands or to agonists presented by costimulatory-deficient accessory cells. The present study uncovers an alternative way of inducing an unresponsive state in T cells. Indeed, we demonstrate herein that Ag-stimulation of murine CD4+ Th clones induces cellular activation, characterized by cytokine production and cell proliferation, followed by a state of transient (lasting up to 6 days) unresponsiveness to further antigenic stimulation. This state of activation-induced unresponsiveness 1) is not a consequence of inadequate costimulation, as it occurs when cells are stimulated in the presence of dendritic cells or anti-CD28 Abs; 2) develops after an optimal response to Ag; 3) is not due to cell death/apoptosis or CTLA-4 engagement; 4) down-regulates the proliferation and cytokine production of both Th1- and Th2-like clones; and 5) does not affect the early steps of signal transduction. Finally, naive T cells are not sensitive to this novel form of unresponsiveness, but become gradually susceptible to activation-induced unresponsiveness upon Ag stimulation. Collectively, these data suggest that activation-induced T cell unresponsiveness may represent a regulatory mechanism limiting the clonal expansion and effector cell function of Ag-experienced T cells, thus contributing to the homeostasis of an immune response.     

Deletion of naive CD8 T cells requires persistent antigen and is not programmed by an initial signal from the tolerogenic APC

Activation of naive CD8 T cells in vivo requires the recognition of cognate peptide-MHC complexes on APCs. Depending upon the activation status of the APC, such recognition will promote either a vigorous immune response or T cell tolerance and deletion. Recent studies suggest that the initial signals provided by APCs are sufficient to program the proliferation of naive CD8 T cells and their differentiation into effector cells. In this study, we sought to determine whether an initial encounter with tolerogenic APCs was sufficient to program deletion of naive CD8 T cells. Surprisingly, we find that regardless of whether naive CD8 T cells were stimulated by activated or quiescent APCs, transfer of the activated T cells into an Ag-free host was sufficient to ensure survival. Thus, although the extent of clonal expansion and development of effector function is determined by the activation status of the stimulatory APC, peripheral clonal deletion requires persistent Ag and is not determined by the initial stimulatory event.     

Acquisition of CD80 (B7-1) by T cells

Activation of T cells usually requires two signals. Signal 1 is mediated via a peptide-MHC on the APC; signal 2 is mediated via a costimulatory molecule on the APC surface. We demonstrate here that naive CD4(+) T cells actually acquire the costimulatory molecule CD80 (B7-1) from syngeneic APCs after activation. This phenomenon was demonstrated showing acquisition of CD80 by T cells from CD80/CD86 (B7-2) knockout mice, and by treating T cells with cyclohexamide to further rule out endogenous expression of CD80 by T cells. Moreover, no CD80 mRNA could be detected in T cells that had acquired CD80. The amount of acquisition of CD80 by T cells was shown to be directly related to both the strength of signal 1 and the amount of CD80 on the APC. Specificity of this acquisition was also shown by the lack of acquisition by T cells from CD28 knockout mice (implicating CD28 in this process), the lack of acquisition of CD40 (another molecule on the APC surface) by T cells, and confocal microscopy studies. We demonstrate for the first time that 1) naive T cells, following acquisition of CD80 from APCs, were themselves shown to be capable of acting as APCs; and 2) memory T cells that have acquired CD80 from APCs undergo apoptosis in the presence of increased levels of signal 1. Thus we demonstrate both immunostimulatory and immunoregulatory functions as a result of CD80 acquisition by different T cell populations.     

CD4+CD25+ and CD4+CD25- T cells act respectively as inducer and effector T suppressor cells in superantigen-induced tolerance

The repeated injection of low doses of bacterial superantigens (SAg) is known to induce specific T cell unresponsiveness. We show in this study that the spleen of BALB/c mice receiving chronically, staphylococcal enterotoxin B (SEB) contains SEB-specific CD4(+) TCRBV8(+) T cells exerting an immune regulatory function on SEB-specific primary T cell responses. Suppression affects IL-2 and IFN-gamma secretion as well as proliferation of T cells. However, the suppressor cells differ from the natural CD4(+) T regulatory cells, described recently in human and mouse, because they do not express cell surface CD25. They are CD152 (CTLA-4)-negative and their regulatory activity is not associated with expression of the NF Foxp3. By contrast, after repeated SEB injection, CD4(+)CD25(+) splenocytes were heterogenous and contained both effector as well as regulatory cells. In vivo, CD4(+)CD25(-) T regulatory cells prevented SEB-induced death independently of CD4(+)CD25(+) T cells. Nevertheless, SEB-induced tolerance could not be achieved in thymectomized CD25(+) cell-depleted mice because repeated injection of SEB did not avert lethal toxic shock in these animals. Collectively, these data demonstrate that, whereas CD4(+)CD25(+) T regulatory cells are required for the induction of SAg-induced tolerance, CD4(+)CD25(-) T cells exert their regulatory activity at the maintenance stage of SAg-specific unresponsiveness.     

Programmed cell death of T lymphocytes during acute viral infection: a mechanism for virus-induced immune deficiency.

Acute viral infections induce immune deficiencies, as shown by unresponsiveness to mitogens and unrelated antigens. T lymphocytes isolated from mice acutely infected with lymphocytic choriomeningitis virus (LCMV) were found in this study to undergo activation-induced apoptosis upon signalling through the T-cell receptor (TcR)-CD3 complex. Kinetic studies demonstrated that this sensitivity to apoptosis directly correlated with the induction of immune deficiency, as measured by impaired proliferation in response to anti-CD3 antibody or to concanavalin A. Cell cycling in interleukin-2 (IL-2) alone stimulated proliferation of LCMV-induced T cells without inducing apoptosis, but preculturing of T cells from acutely infected mice in IL-2 accelerated apoptosis upon subsequent TcR-CD3 cross-linking. T lymphocytes isolated from mice after the acute infection were less responsive to IL-2, but those T cells, presumably memory T cells, responding to IL-2 were primed in each case to die a rapid apoptotic death upon TcR-CD3 cross-linking. These results indicate that virus infection-induced unresponsiveness to T-cell mitogens is due to apoptosis of the activated lymphocytes and suggest that the sensitization of memory cells by IL-2 induced during infection will cause them to die upon antigen recognition, thereby impairing specific responses to nonviral antigens.     

Presentation of soluble antigens to CD8+ T cells by CpG oligodeoxynucleotide-primed human naive B cells

Naive B lymphocytes are generally thought to be poor APCs, and there is limited knowledge of their role in activation of CD8(+) T cells. In this article, we demonstrate that class I MHC Ag presentation by human naive B cells is enhanced by TLR9 agonists. Purified naive B cells were cultured with or without a TLR9 agonist (CpG oligodeoxynucleotide [ODN] 2006) for 2 d and then assessed for phenotype, endocytic activity, and their ability to induce CD8(+) T cell responses to soluble Ags. CpG ODN enhanced expression of class I MHC and the costimulatory molecule CD86 and increased endocytic activity as determined by uptake of dextran beads. Pretreatment of naive B cells with CpG ODN also enabled presentation of tetanus toxoid to CD8(+) T cells, resulting in CD8(+) T cell cytokine production and granzyme B secretion and proliferation. Likewise, CpG-activated naive B cells showed enhanced ability to cross-present CMV Ag to autologous CD8(+) T cells, resulting in proliferation of CMV-specific CD8(+) T cells. Although resting naive B cells are poor APCs, they can be activated by TLR9 agonists to serve as potent APCs for class I MHC-restricted T cell responses. This novel activity of naive B cells could be exploited for vaccine design.     

Naive T cells are resistant to anergy induction by anti-CD3 antibodies

Anti-CD3 mAbs are potent immunosuppressive agents used in clinical transplantation. It has been generally assumed that one of the anti-CD3 mAb-mediated tolerance mechanisms is through the induction of naive T cell unresponsiveness, often referred to as anergy. We demonstrate in this study that naive T cells stimulated by anti-CD3 mAbs both in vivo and in vitro do not respond to the superantigen staphylococcal enterotoxin B nor to soluble forms of anti-CD3 mAbs and APC, but express increased reactivity to plastic-coated forms of the same anti-CD3 mAbs and to their nominal Ag/class II MHC, a finding that is difficult to rationalize with the concept of anergy. Phenotypic and detailed kinetic studies further suggest that a strong signal 1 delivered by anti-CD3 mAbs in the absence of costimulatory molecules does not lead to anergy, but rather induces naive T cells to change their mitogen responsiveness and acquire features of memory T cells. In marked contrast, Ag-experienced T cells are sensitive to anergy induction under the same experimental settings. Collectively, these studies demonstrate that exposure of naive T cells in vivo and in vitro to a strong TCR stimulus does not induce Ag unresponsiveness, indicating that sensitivity to negative signaling through TCR/CD3 triggering is developmentally regulated in CD4(+) T cells.     

Oral tolerance to nickel requires CD4+ invariant NKT cells for the infectious spread of tolerance and the induction of specific regulatory T cells

Previously, oral administration of nickel to C57BL/6 wild-type (WT) mice was shown to render both their splenic T cells and APCs (i.e., T cell-depleted spleen cells) capable of transferring nickel tolerance to naive syngeneic recipients. Moreover, sequential adoptive transfer experiments revealed that on transfer of tolerogenic APCs and immunization, the naive T cells of the recipients differentiated into regulatory T (Treg) cells. Here, we demonstrate that after oral nickel treatment Jalpha18(-/-) mice, which lack invariant NKT (iNKT) cells, were not tolerized and failed to generate Treg cells. However, transfer of APCs from those Jalpha18(-/-) mice did tolerize WT recipients. Hence, during oral nickel administration, tolerogenic APCs are generated that require iNKT cell help for the induction of Treg cells. To obtain this help, the tolerogenic APCs must address the iNKT cells in a CD1-restricted manner. When Jalpha18(-/-) mice were used as recipients of cells from orally tolerized WT donors, the WT Treg cells transferred the tolerance, whereas WT APCs failed to do so, although they proved tolerogenic on transfer to WT recipients. However, Jalpha18(-/-) recipients did become susceptible to the tolerogenicity of transferred WT APCs when they were reconstituted with IL-4- and IL-10-producing CD4(+) iNKT cells. We conclude that CD4(+) iNKT cells are required for the induction of oral nickel tolerance and, in particular, for the infectious spread of tolerance from APCs to T cells. Once induced, these Treg cells, however, can act independently of iNKT cells.     

Uncoupling of IL-2 signaling from cell cycle progression in naive CD4+ T cells by regulatory CD4+CD25+ T lymphocytes

Prior reports have shown that CD4(+)CD25(+) regulatory T cells suppress naive T cell responses by inhibiting IL-2 production. In this report, using an Ag-specific TCR transgenic system, we show that naive T cells stimulated with cognate Ag in the presence of preactivated CD4(+)CD25(+) T cells also become refractory to the mitogenic effects of IL-2. T cells stimulated in the presence of regulatory T cells up-regulated high affinity IL-2R, but failed to produce IL-2, express cyclins or c-Myc, or exit G(0)-G(1). Exogenous IL-2 failed to break the mitotic block, demonstrating that the IL-2 production failure was not wholly responsible for the proliferation defect. This IL-2 unresponsiveness did not require the continuous presence of CD4(+)CD25(+) regulatory T cells. The majority of responder T cells reisolated after coculture with regulatory cells failed to proliferate in response to IL-2, but were not anergic and proliferated in response to Ag. The mitotic block was also dissociated from the antiapoptotic effects of IL-2, because IL-2 still promoted the survival of T cells that had been cocultured with CD4(+)CD25(+) T cells. IL-2-induced STAT5 phosphorylation in the cocultured responder cells was intact, implying that the effects of the regulatory cells were downstream of receptor activation. Our results therefore show that T cell activation in the presence of CD4(+)CD25(+) regulatory T cells can induce an alternative stimulation program characterized by up-regulation of high affinity IL-2R, but a failure to produce IL-2, and uncoupling of the mitogenic and antiapoptotic effects of IL-2.     

Dual function of the extracellular matrix: stimulatory for cell cycle progression of naive T cells and antiapoptotic for tissue-derived memory T cells

Tissue T cells encounter Ag in a distinct microenvironment, where they are embedded in the interstitial extracellular matrix (ECM). In contrast, while naive T cells are exposed to Ag in the lymph node, immediately after naive T cells are activated they must extravasate into the ECM to function effectively. Because integrin-mediated adhesion to the ECM modulates cell cycle progression and survival in adherent nonimmune cells, we hypothesize that blood and tissue-derived T cells have similarly adapted their behavior to their first or continued encounter with ECM. T cells from peripheral blood (PBT) and tissue (the intestinal lamina propria T cell (LPT)) were stimulated with anti-CD3-coated beads in the presence or absence of native ECM derived from intestinal fibroblasts, plate-immobilized fibronectin, or collagen type I. Native ECM and collagen, but not fibronectin, induced in anti-CD3 activated PBT a 4- to 5-fold increase in the entry, progression, and completion of the cell cycle over that triggered by anti-CD3 alone. Neutralizing beta1 integrin Abs abrogated this increase. None of these ECM proteins stimulated cell cycle progression in LPT. In contrast, anti-CD3 activation of LPT in the presence of native ECM and fibronectin reduced activation-induced cell death by 40%. These results demonstrate that naive and effector/memory T cells respond differently upon exposure to specific ECM components. When naive PBT encounter Ag in the context of ECM, their progression through the cell cycle is enhanced, favoring clonal expansion; while tissue T cell longevity may be mediated by interactions with the ECM.     

Regulatory T cells maintain long-term tolerance to myelin basic protein by inducing a novel, dynamic state of T cell tolerance

The pathogenesis of multiple sclerosis involves a breakdown in T cell tolerance to myelin proteins like myelin basic protein (MBP). Most MBP-specific T cells are eliminated by central tolerance in adult mice, however, the developmentally regulated expression of MBP allows MBP-specific thymocytes in young mice to escape negative selection. It is not known how these T cells that encounter MBP for the first time in the periphery are regulated. We show that naive MBP-specific T cells transferred into T cell-deficient mice induce severe autoimmunity. Regulatory T cells prevent disease, however, suppression of the newly transferred MBP-specific T cells is abrogated by activating APCs in vivo. Without APC activation, MBP-specific T cells persist in the periphery of protected mice but do not become anergic, raising the question of how long-term tolerance can be maintained if APCs presenting endogenous MBP become activated. Our results demonstrate that regulatory T cells induce naive MBP-specific T cells responding to nonactivated APCs to differentiate into a unique, tolerized state with the ability to produce IL-10 and TGF-beta1 in response to activated, but not nonactivated, APCs presenting MBP. This tolerant response depends on continuous activity of regulatory T cells because, in their absence, these uniquely tolerized MBP-specific T cells can again induce autoimmunity.     

Differential susceptibility to staphylococcal superantigen (SsAg)-induced apoptosis of CD4+ T cells from atopic dermatitis patients and healthy subjects: the inhibitory effect of IL-4 on SsAg-induced apoptosis

This study had two aims: 1) to determine whether there are differences between atopic dermatitis (AD) patients and healthy subjects in staphylococcal superantigen (SsAg)-induced CD4(+) T cell activation, cytokine production, chemokine receptor expression, and apoptosis; and 2) to investigate the effect of IL-4 on SsAg-induced apoptosis. By using immunofluorescence and annexin V staining, we analyzed PBMC with or without staphylococcal enterotoxin B (SEB) stimulation in the presence or absence of rIL-4 or anti-IL-4-neutralizing Abs in 15 healthy subjects and 27 AD patients. We found that SEB preferentially induced production of Th1 cytokine in SEB-reactive (TCRVbeta3(+) or Vbeta12(+) or Vbeta17(+)) CD4(+) T cells from healthy subjects and Th2 cytokine in those from AD patients. SEB induced up-regulation of CXCR3(+) cells in SEB-reactive CD4(+) T cells from healthy subjects and CCR4(+) cells in those from AD patients. SEB-reactive CD4(+) T cells from AD patients were more resistant to SEB-induced apoptosis than those from healthy subjects. There was no significant difference between AD and healthy subjects in SEB-induced activation of CD4(+) T cells. CXCR3(+) CD4(+) T cells were more susceptible to SEB-induced apoptosis than CCR4(+) CD4(+) T cells in healthy subjects. Exogenously added IL-4 inhibited SEB-induced apoptosis of SEB-reactive CD4(+) and CXCR3(+) CD4(+) T cells but not of CCR4(+) CD4(+) T cells in healthy subjects. Inhibition of endogenous IL-4 increased SEB-induced apoptosis of SEB-reactive CD4(+) T cells from AD patients. These results might provide new clues to the mechanism that SsAgs contribute to the persistence and exacerbation of allergic skin inflammation in AD.     

Homeostasis of naive and memory CD4+ T cells: IL-2 and IL-7 differentially regulate the balance between proliferation and Fas-mediated apoptosis

Cytokines play a crucial role in the maintenance of polyclonal naive and memory T cell populations. It has previously been shown that ex vivo, the IL-7 cytokine induces the proliferation of naive recent thymic emigrants (RTE) isolated from umbilical cord blood but not mature adult-derived naive and memory human CD4(+) T cells. We find that the combination of IL-2 and IL-7 strongly promotes the proliferation of RTE, whereas adult CD4(+) T cells remain relatively unresponsive. Immunological activity is controlled by a balance between proliferation and apoptotic cell death. However, the relative contributions of IL-2 and IL-7 in regulating these processes in the absence of MHC/peptide signals are not known. Following exposure to either IL-2 or IL-7 alone, RTE, as well as mature naive and memory CD4(+) T cells, are rendered only minimally sensitive to Fas-mediated cell death. However, in the presence of the two cytokines, Fas engagement results in a high level of caspase-dependent apoptosis in both RTE as well as naive adult CD4(+) T cells. In contrast, equivalently treated memory CD4(+) T cells are significantly less sensitive to Fas-induced cell death. The increased susceptibility of RTE and naive CD4(+) T cells to Fas-induced apoptosis correlates with a significantly higher IL-2/IL-7-induced Fas expression on these T cell subsets than on memory CD4(+) T cells. Thus, IL-2 and IL-7 regulate homeostasis by modulating the equilibrium between proliferation and apoptotic cell death in RTE and mature naive and memory T cell subsets.     

Microglia-mediated nitric oxide cytotoxicity of T cells following amyloid beta-peptide presentation to Th1 cells

Alzheimer's disease is marked by progressive accumulation of amyloid beta-peptide (Abeta) which appears to trigger neurotoxic and inflammatory cascades. Substantial activation of microglia as part of a local innate immune response is prominent at sites of Abeta plaques in the CNS. However, the role of activated microglia as Abeta APCs and the induction of adaptive immune responses has not been investigated. We have used primary microglial cultures to characterize Abeta-Ag presentation and interaction with Abeta-specific T cells. We found that IFN-gamma-treated microglia serve as efficient Abeta APCs of both Abeta1-40 and Abeta1-42, mediating CD86-dependent proliferation of Abeta-reactive T cells. When cultured with Th1 and Th2 subsets of Abeta-reactive T cells, Th1, but not Th2, cells, underwent apoptosis after stimulation, which was accompanied by increased levels of IFN-gamma, NO, and caspase-3. T cell apoptosis was prevented in the presence of an inducible NO synthase type 2 inhibitor. Microglia-mediated proliferation of Abeta-reactive Th2 cells was associated with expression of the Th2 cytokines IL-4 and IL-10, which counterbalanced the toxic levels of NO induced by Abeta. Our results demonstrate NO-dependent apoptosis of T cells by Abeta-stimulated microglia which may enhance CNS innate immune responses and neurotoxicity in Alzheimer's disease. Secretion of NO by stimulated microglia may underlie a more general pathway of T cell death in the CNS seen in neurodegenerative diseases. Furthermore, Th2 type T cell responses may have a beneficial effect on this process by down-regulation of NO and the proinflammatory environment.     

Infectious nickel tolerance: a reciprocal interplay of tolerogenic APCs and T suppressor cells that is driven by immunization

Previously, we reported that tolerance to nickel, induced by oral administration of Ni(2+) ions, can be adoptively transferred to naive mice with only 10(2) splenic T cells. Here we show that 10(2) T cell-depleted spleen cells (i.e., APCs) from orally tolerized donors can also transfer nickel tolerance. This cannot be explained by simple passive transfer of the tolerogen. The APCs from orally tolerized donors displayed a reduced allostimulatory capacity, a tolerogenic phenotype, and an increased expression of CD38 on B cells. In fact, it was B cells among the APCs that carried the thrust of tolerogenicity. Through serial adoptive transfers with Ly5.1(+) donors and two successive sets of Ly5.2(+) recipients, we demonstrated that nickel tolerance was infectiously spread from donor to host cells. After the transfer of either T cells or APCs from orally tolerized donors, the spread of tolerance to the opposite cell type of the recipients (i.e., APCs and T cells, respectively) required recipient immunization with NiCl(2)/H(2)O(2). For the spread of tolerance from a given donor cell type, T cell or APC, to the homologous host cell type, the respective opposite cell type in the host was required as intermediate. We conclude that T suppressor cells and tolerogenic APCs induced by oral administration of nickel are part of a positive feedback loop that can enhance and maintain tolerance when activated by Ag associated with a danger signal. Under these conditions, APCs and T suppressor effector cells infectiously spread the tolerance to naive T cells and APCs, respectively.     

Anti-CD3 epsilon F(ab')2 prevents graft-versus-host disease by selectively depleting donor T cells activated by recipient alloantigens

Transplantation tolerance is facilitated by activation-induced apoptosis of peripheral T cells triggered by specific AG: Abs specific for the nonpolymorphic CD3 component of the TCR complex bind to APCs through Fc-FcR interactions, mimic MHC-peptide, and activate polyclonal T cells. In contrast, F(ab')(2) of anti-CD3epsilon Abs do not activate naive T cells but induce apoptosis of Ag-activated, cycling T cells. Here, we report that treatment with anti-CD3epsilon F(ab')(2) can selectively induce apoptosis of donor T cells that recognize a recipient alloantigen, thereby preventing graft-vs-host disease initiated by a TCR-transgenic T cell population. The selective elimination of Ag-activated T cells by non-FcR-binding anti-CD3epsilon Abs could serve as an ideal strategy to prevent graft-vs-host disease and allograft rejection or to treat autoimmune disorders.     

Impairment of immunological synapse formation in adaptively tolerant T cells

Adaptive tolerance is a hyporesponsive state in which lymphocyte Ag receptor signaling becomes desensitized after prolonged in vivo encounter with Ag. The molecular mechanisms underlying this hyporesponsive state in T cells are not fully understood, although a major signaling block has been shown to be present at the level of ZAP70 phosphorylation of linker for activation of T cells (LAT). In this study, we investigated the ability of adaptively tolerant mouse T cells to form conjugates with Ag-bearing APCs and to translocate signaling molecules into the interface between the T cells and APCs. Compared with naive or preactivated T cells, adaptively tolerant T cells showed no dramatic impairment in their formation of conjugates with APCs. In contrast, there was a large impairment in immunological synapse formation. Adaptively tolerant T cells were defective in their translocation of signaling molecules, such as ZAP70, LAT, and phospholipase C γ1, into the T cell-APC contact sites. Although Ag-induced activation of VAV1 was normal, VAV's recruitment into the synapse was also impaired. Interestingly, expressions of both IL-2-inducible T cell kinase and growth factor receptor-bound protein 2-related adaptor downstream of SHC were decreased by 60-80% in adaptively tolerant T cells. These decreases, in addition to the impairment in LAT phosphorylation by ZAP70, appear to be the major impediments to the phosphorylation of SLP76 (SRC homology 2 domain-containing leukocyte protein of 76 kDa) and the recruitment of VAV1, which are important for stable immunological synapse formation.