Differential regulation of peripheral CD4+ T cell tolerance induced by deletion and TCR revision

In Vbeta5 transgenic mice, mature Vbeta5(+)CD4(+) T cells are tolerized upon recognition of a self Ag, encoded by a defective endogenous retrovirus, whose expression is confined to the lymphoid periphery. Cells are driven by the tolerogen to enter one of two tolerance pathways, deletion or TCR revision. CD4(+) T cells entering the former pathway are rendered anergic and then eliminated. In contrast, TCR revision drives gene rearrangement at the endogenous TCR beta locus and results in the appearance of Vbeta5(-), endogenous Vbeta(+), CD4(+) T cells that are both self-tolerant and functional. An analysis of the molecules that influence each of these pathways was conducted to understand better the nature of the interactions that control tolerance induction in the lymphoid periphery. These studies reveal that deletion is efficient in reconstituted radiation chimeras and is B cell, CD28, inducible costimulatory molecule, Fas, CD4, and CD8 independent. In contrast, TCR revision is radiosensitive, B cell, CD28, and inducible costimulatory molecule dependent, Fas and CD4 influenced, and CD8 independent. Our data demonstrate the differential regulation of these two divergent tolerance pathways, despite the fact that they are both driven by the same tolerogen and restricted to mature CD4(+) T cells.     

Evidence for Fas-dependent and Fas-independent mechanisms in the pathogenesis of experimental autoimmune encephalomyelitis

To determine whether Fas or Fas ligand (FasL) plays a role in susceptibility to experimental autoimmune encephalomyelitis (EAE), we bred a TCR transgenic mouse specific for the Ac1-11 peptide of myelin basic protein to mice with inactivating mutations in Fas (lpr) or FasL (gld). Disease induction by peptide immunization in such mice produced similar disease scores, demonstrating that Fas/FasL interactions were not necessary to generate EAE. However, adoptive transfer experiments showed evidence that these interactions can play a role in the pathogenesis of EAE, shown most dramatically by the absence of disease following transfer of cells from a normal myelin basic protein TCR transgenic mouse into a Fas-deficient lpr recipient. Furthermore, transfer of cells lacking FasL (gld) into normal or gld recipients gave a diminished disease score. Thus, Fas/FasL interactions can play a role in the pathogenesis of EAE, but they are not required for disease to occur.     

Comparing the relative role of perforin/granzyme versus Fas/Fas ligand cytotoxic pathways in CD8+ T cell-mediated insulin-dependent diabetes mellitus.

CD8+ cytotoxic T cells play a critical role in initiating insulin-dependent diabetes mellitus. The relative contribution of each of the major cytotoxic pathways, perforin/granzyme and Fas/Fas ligand (FasL), in the induction of autoimmune diabetes remains controversial. To evaluate the role of each lytic pathway in beta cell lysis and induction of diabetes, we have used a transgenic mouse model in which beta cells expressing the influenza virus hemagglutinin (HA) are destroyed by HA-specific CD8+ T cells from clone-4 TCR-transgenic mice. Upon adoptive transfer of CD8+ T cells from perforin-deficient clone-4 TCR mice, there was a 30-fold increase in the number of T cells required to induce diabetes. In contrast, elimination of the Fas/FasL pathway of cytotoxicity had little consequence. When both pathways of cytolysis were eliminated, mice did not become diabetic. Using a model of spontaneous diabetes, which occurs in double transgenic neonates that express both clone-4 TCR and Ins-HA transgenes, mice deficient in either the perforin or FasL/Fas lytic pathway become diabetic soon after birth. This indicates that, in the neonate, large numbers of autoreactive CD8+ T cells can lead to destruction of islet beta cells by either pathway.     

Egr family members regulate nonlymphoid expression of Fas ligand, TRAIL, and tumor necrosis factor during immune responses

The Fas ligand (FasL)/Fas pathway is crucial for homeostasis of the immune system and peripheral tolerance. Peripheral lymphocyte deletion involves FasL/Fas in at least two ways: coexpression of both Fas and its ligand on T cells, leading to activation-induced cell death, and expression of FasL by nonlymphoid cells, such as intestinal epithelial cells (IEC), that kill Fas-positive T cells. We demonstrate here that superantigen Staphylococcus enterotoxin B (SEB) induced a dramatic upregulation of FasL, TRAIL, and TNF mRNA expression and function in IEC from BALB/c and C57BL/6 mice. Using adoptive transfer in which CD4(+) T cells from OT-2 T-cell receptor transgenic mice were transferred into recipients, we observed an induction in IEC of FasL, TRAIL, and TNF mRNA after administration of antigen. Specific Egr-binding sites have been identified in the 5' promoter region of the FasL gene, and Egr-1, Egr-2, and Egr-3 mRNA in IEC from mice treated with SEB and from transgenic OT-2 mice after administration of antigen was upregulated. Overexpression of Egr-2 and Egr-3 induced endogenous ligand upregulation that was inhibited by overexpression of Egr-specific inhibitor Nab1. These results support a role for Egr family members in nonlymphoid expression of FasL, TRAIL, and TNF.     

Induction of allograft tolerance to the H-Y antigen in adult C57BL/6 mice: differential effects on delayed-type hypersensitivity and cytolytic T-lymphocyte activity

This study describes the induction of allograft tolerance to the "male-specific," minor histocompatibility antigen, H-Y, in adult C57BL/6 female mice, and the effects of this tolerance induction on two immune parameters associated with graft rejection: delayed-type hypersensitivity (DTH) and cytolytic T-lymphocytes (CTL). B6 females tolerized to H-Y, by a single iv injection of C57BL/6 male lymphocytes, exhibited prolonged or permanent survival of B6 male tail skin grafts. Graft-induced DTH against H-Y antigen was reduced or abrogated in tolerized females. Delayed onset of graft rejection in partially tolerant females correlated with delayed onset of DTH, and eventual rejection of grafts was accompanied by an increase in H-Y-specific DTH. In contrast, H-Y-specific CTL activity was not consistent with graft status. These data demonstrate a correlation between H-Y-specific DTH and rejection of male skin grafts by B6 female mice and are most consistent with a major effector role for DTH in chronic graft rejection.     

Anti-receptor antibody-induced H-Y-specific delayed-type hypersensitivity responses in nonresponder mice

The present study examines an antiserum prepared against antigen-reactive T cells that induces murine H-Y-specific delayed-type hypersensitivity (DTH) responses. This anti-H-Y receptor antibody (ARA) was raised in C57BL/6 male mice against splenic T lymphocytes from H-Y immune syngeneic females. Subcutaneous administration of ARA to cyclophosphamide-pretreated C57BL/6 females is able to induce H-Y-specific delayed-type footpad swelling responses. The DTH inducing capacity in ARA was selectively retained on rabbit anti-mouse immunoglobulin columns and was absorbed completely by H-Y immune lymphoid cells from C57BL/6 females. The induction of H-Y DTH reactivity was due at least in part to the activation of H-Y antigen-specific T lymphocytes that could adoptively transfer DTH-like responses to naive female mice. ARA induces DTH responses in strains with the same lgh regions, including selected strains of H-Y nonresponders. Therefore, MHC-linked lr genes do not appear to be as critical when responses are triggered by ARA instead of by antigen. Possible mechanisms for the induction of immune responses by ARA are discussed.     

Vasoactive intestinal peptide and pituitary adenylate cyclase-activating polypeptide inhibit antigen-induced apoptosis of mature T lymphocytes by inhibiting Fas ligand expression

Apoptosis in T and B lymphocytes is a major element controlling the immune response. The Ag-induced cell death (AICD) in T cells is a main mechanism for maintaining peripheral tolerance and for limiting an ongoing immune response. AICD is initiated by Ag re-engagement of the TCR and is mediated through Fas/Fas ligand (FasL) interactions. Vasoactive intestinal peptide (VIP) and the structurally related pituitary adenylate cyclase-activating polypeptide (PACAP) are two multifunctional neuropeptides present in the lymphoid microenvironment that act primarily as anti-inflammatory agents. In the present study we investigated whether VIP and PACAP affect AICD in mature peripheral T cells and T cell hybridomas. VIP and PACAP reduce in a dose-dependent manner anti-CD3-induced apoptosis in Con A/IL-2-preactivated peripheral T cells and the murine T hybridomas 2B4.11 and A1.1. A functional study demonstrates that the inhibition of AICD is achieved through the inhibition of activation-induced FasL expression at protein and mRNA levels. VIP/PACAP-mediated inhibition of both AICD and FasL expression is mediated through the specific receptors VPAC1 and VPAC2. Of obvious biological significance is the fact that VIP and PACAP prevent Ag-induced clonal deletion of CD4+ T cells, but not that of CD8+ T cells. By affecting FasL expression, VIP and PACAP may play a physiological role in both the generation of memory T cells and the inhibition of FasL-mediated T cell cytotoxicity.     

Transgenic major histocompatibility complex class I antigen expressed in mouse trophoblast affects maternal immature B cells

We have produced transgenic mice using the mouse placental lactogen type II promoter to force and restrict the expression of the mouse major histocompatibility complex (MHC) class I molecule, H-2K(b), to the placenta. We show that the transgenic MHC antigen H-2K(b) is expressed exclusively in trophoblast giant cells from Day 10.5 until the end of gestation. This expression affects neither the fetal development nor the maternal tolerance to the fetus in histoincompatible mothers. We have used the 3.83 B cell receptor (BcR) transgenic mouse line to follow the fate of H-2K(b)-specific maternal B cells in mothers bearing H-2K(b)-positive placentas. Our results suggest that transgenic H-2K(b) molecules on trophoblast giant cells are recognized by 3.83 BcR-transgenic B cells in the bone marrow of pregnant females. This antigen recognition triggers the deletion of a bone marrow B cell subpopulation, including immature and transitional B cells. Their percentage decreases during the second half of gestation and is down to 8% on Day 17.5, compared to 22% in the (3.83 Tg female x Fvb) control group. This deletion might contribute to the process of maternal tolerance of the conceptus.     

Fas ligand is responsible for CXCR3 chemokine induction in CD4+ T cell-dependent liver damage

Immune-mediated hepatic damage has been demonstrated in the pathogenesis of hepatitis C virus (HCV) and other hepatotrophic infections. Fas/Fas ligand (FasL) interaction plays a critical role in immune-mediated hepatic damage. To understand the molecular mechanism(s) of FasL-mediated liver inflammation, we examined the effect of CD4(+) T cells expressing high levels of FasL on the initiation of hepatic damage through analysis of chemokine and chemokine receptor expression in HCV core x TCR (DO11.10) double-transgenic mice. In vivo antigenic stimulation triggers a marked influx of core-expressing Ag-specific CD4(+) T cells into the liver of the immunized core(+) TCR mice but not their core(-) TCR littermates. Strikingly, the inflammatory process in the liver of core(+) TCR mice was accompanied by a dramatic increase in IFN-inducible protein 10 and monokine induced by IFN-gamma production. The intrahepatic lymphocytes were primarily CXCR3-positive and anti-CXCR3 Ab treatment abrogates migration of CXCR3(+) lymphocytes into the liver and hepatic damage. Importantly, the blockade of Fas/FasL interaction reduces the expression of IFN-inducible protein 10 and monokine induced by IFN-gamma and cellular infiltration into the liver. These findings suggest that activated CD4(+) T cells with elevated FasL expression are involved in promoting liver inflammation and hepatic damage through the induction of chemokines.     

Critical role of costimulation in the activation of naive antigen-specific TCR transgenic CD8+ T cells in vitro.

The influence of costimulation on the activation of naive CD8+ T cells and thymocytes was studied in vitro using H-Y-specific TCR-transgenic mice and H-Y antigenic peptide. Using a variety of physiological APC types, the activation of naive CD8+ T cells depended strictly on costimulation, which could not be substituted by high epitope density. T cell activation is known to be regulated by the interactions between CD86/CD80 and CD28/CD152, although it remains unclear whether the B7 isoforms have distinct roles. Addition of soluble anti-CD86 Ab led to profound inhibition of T cell reactivity, further confirming the importance of costimulation in naive CD8+ T cell activation. Finally, TCR engagement in the absence of costimulation had no effect on the subsequent reactivity of peripheral naive transgenic CD8+ T cells, but induced nonresponsiveness in mature CD8+ transgenic thymocytes. Collectively, these results demonstrate the importance of costimulation for naive CD8+ T cell activation, suggest that CD80 and CD86 can mediate opposing effects, possibly due to differential interaction with CD152 and CD28, and indicate differences in the sensitivity of immature vs mature CD8+ T cells to the induction of nonresponsiveness following costimulation-deficient Ag presentation.     

Peripheral deletion of mature CD8+ antigen-specific T cells after in vivo exposure to male antigen.

It has been well established that T cell tolerance to self Ag occurs primarily via clonal deletion of immature thymocytes in the thymus. Evidence also exists that there are additional mechanisms operative on mature T cells for establishing and maintaining tolerance in the periphery. To follow the fate of mature Ag-specific T cells in vivo, we used female transgenic mice, which contain a large population of male H-Y Ag-specific T cells that can be identified by immunostaining with mAb directed against CD8 and the transgenic TCR. H-Y Ag was introduced into these mice by injecting Ag-bearing male lymphocytes using conditions known to induce CTL precursor response reduction. The number of Ag-reactive CD8+ transgenic T cells in the periphery started to decrease after 2 days of in vivo exposure to male Ag. Decline was maximum (up to 80% of total) by 7 days, and stayed at this level for at least 6 wk. CD4+ cells and those CD8+ cells that did not carry the transgenic TCR were not affected. Most or all of the remaining Ag-reactive CD8+ cells in the periphery were fully responsive when stimulated by male Ag in vitro. Maturation of transgenic T cells in the thymus of injected mice remained the same as that of control animals. Our data provide direct evidence that mature Ag-reactive CD8+ cells are susceptible to clonal deletion in the periphery when exposed to the Ag in vivo. These findings suggest the presence of two types of APC in the periphery: stimulatory APC (e.g., macrophages and dendritic cells) required for initiating an active immune response; and functionally deleting APC (or veto cells) capable of deleting mature T lymphocytes that recognize Ag presented on their surface. Functionally deleting APC that present self Ag to peripheral T cells may provide a fail-safe mechanism against autoreactive cells that escaped deletion during differentiation in the thymus.     

Modulation of CD8+ T cell response to antigen by the levels of self MHC class I

The response of H-Y-specific TCR-transgenic CD8(+) T cells to Ag is characterized by poor proliferation, cytolytic activity, and IFN-gamma secretion. IFN-gamma secretion, but not cytotoxic function, can be rescued by the B7.1 molecule, suggesting that costimulation can selectively enhance some, but not all, effector CD8(+) T cell responses. Although the H-Y epitope binds H-2D(b) relatively less well than some other epitopes, it can induce potent CTL responses in nontransgenic mice, suggesting that the observed poor responsiveness of transgenic CD8(+) T cells cannot be ascribed to the epitope itself. Previously reported reactivity of this TCR to H-2A(b) is also not the cause of the poor responsiveness of the H-Y-specific CD8(+) T cells, as H-Y-specific CD8(+) T cells obtained from genetic backgrounds lacking H-2A(b) also responded poorly. Rather, reducing the levels of H-2(b) class I molecules by breeding the mice to (C57BL/6 x B10.D2)F(1) or TAP1(+/-) backgrounds partially restored cytotoxic activity and enhanced proliferative responses. These findings demonstrate that the self MHC class I gene dosage may regulate the extent of CD8(+) T cell responsiveness to Ag.     

Proteasome inhibitors block Ras/ERK signaling pathway resulting in the downregulation of Fas ligand expression during activation-induced cell death in T cells

Activation-induced cell death (AICD) plays a critical role in the maintenance of homeostasis and peripheral tolerance in the immune system, and is mediated by Fas ligand (FasL) expression and the interaction between Fas and FasL. In the present study, we examined the role of the ubiquitin-proteasome system in AICD using T cell hybridoma N3-6-71 cells. The peptidyl aldehyde proteasome inhibitor carbobenzoxyl-Ile-Glu(O-t-butyl)-Ala-leucinal (PSI) blocked T cell receptor (TCR) stimulation-induced apoptosis in the T cell hybridoma. Fas and FasL gene expression and mouse FasL promoter activity following TCR stimulation were suppressed by PSI pretreatment. Deletion or point mutation of the kappaB site in the FasL promoter region did not suppress inducible FasL promoter activity effectively. PSI blocked extracellular signal-regulated kinase (ERK) activity induced by TCR stimulation, but had no effect on c-jun N-terminal kinase activation. ERK activation was essential for FasL expression and AICD. The initial tyrosine phosphorylation steps following TCR stimulation, i.e., phosphorylation of CD3zeta and Vav, were not altered by PSI. These data suggest that the ubiquitin-proteasome system has some regulatory function at an intermediate step between the initial tyrosine phosphorylation steps and ERK activation in AICD.     

Cutting edge: Rac GTPases sensitize activated T cells to die via Fas

In activated CD4(+) T cells, TCR restimulation triggers apoptosis that depends on interactions between the death receptor Fas and its ligand, FasL. This process, termed restimulation-induced cell death (RICD), is a mechanism of peripheral immune tolerance. TCR signaling sensitizes activated T cells to Fas-mediated apoptosis, but what pathways mediate this process are not known. In this study we identify the Rho GTPases Rac1 and Rac2 as essential components in restimulation-induced cell death. RNA interference-mediated knockdown of Rac GTPases greatly reduced Fas-dependent, TCR-induced apoptosis. The ability of Rac1 to sensitize T cells to Fas-induced apoptosis correlated with Rac-mediated cytoskeletal reorganization, dephosphorylation of the ERM (ezrin/radixin/moesin) family of cytoskeletal linker proteins, and the translocation of Fas to lipid raft microdomains. In primary activated CD4(+) T cells, Rac1 and Rac2 were independently required for maximal TCR-induced apoptosis. Activating Rac signaling may be a novel way to sensitize chronically stimulated lymphocytes to Fas-induced apoptosis, an important goal in the treatment of autoimmune diseases.     

Involvement of the SHP-1 tyrosine phosphatase in regulation of T cell selection.

The selection events shaping T cell development in the thymus represent the outcome of TCR-driven intracellular signaling cascades evoked by Ag receptor interaction with cognate ligand. In view of data indicating TCR-evoked thymocyte proliferation to be negatively modulated by the SHP-1 tyrosine phosphatase, a potential role for SHP-1 in regulating selection processes was investigated by analysis of T cell development in H-Y TCR transgenic mice rendered SHP-1 deficient by introduction of the viable motheaten mutation or a dominant negative SHP-1-encoding transgene. Characterization of thymocyte and peripheral T cell populations in H-Y TCR-viable motheaten mice revealed TCR-evoked proliferation as well as the positive and negative selection of H-Y-specific thymocytes to be enhanced in these mice, thus implicating SHP-1 in the negative regulation of each of these processes. T cell selection processes were also augmented in H-Y TCR mice carrying a transgene driving lymphoid-restricted expression of a catalytically inert, dominant-negative form of SHP-1. SHP-1-negative effects on thymocyte TCR signaling were not influenced by co-cross-linking of the CD28 costimulatory and/or CTLA-4 inhibitory receptors and appear, accordingly, to be realized independently of these comodulators. These observations indicate that SHP-1 raises the signaling threshold required for both positive and negative selection and reveal the inhibitory effects of SHP-1 on TCR signaling to be cell autonomous. The demonstrated capacity for SHP-1 to inhibit TCR-evoked proliferation and selection indicate SHP-1 modulatory effects on the magnitude of TCR-generated signal to be a key factor in determining the cellular consequences of TCR-ligand interaction.     

Immunology of pregnancy: renewed interest

The long-standing question of pregnancy immunological paradox has been generating renewed interest. Recent insights have emerged from studies in pregnant mice and humans demonstrating a number of mechanisms that prevent potentially harmful effects of maternal anti-paternal allo-antibodies (complement inhibition, partial deletion of maternal B cells specific of paternal antigens), cytotoxic CD8+ T cells (lack of HLA-A and HLA-B expression on trophoblast, local immunosuppressive molecules, transient tolerance of paternal allo-antigens specific T cells) and uterine NK cells directed against fetal-derived trophoblast cells (limited NK cytotoxic potential, trophoblast resistance to NK killing). Interestingly, it appears that not only decidual NK cell/trophoblast interactions are not harmful for the fetus but are beneficial for the placental vascularization and its subsequent development. A recent report has indeed demonstrated that during pregnancy most of the combinations of uterine KIR (killer cell immunoglobulin-like receptor) NK cell receptors and fetal HLA-C molecules expressed by trophoblast led to normal pregnancies, whereas mothers lacking activating KIR of the AA genotype when the fetus possessed HLA-C of the C2 group were at a greatly increased risk of severe preeclampsia pathology.     

IFN-gamma promotes Fas ligand- and perforin-mediated liver cell destruction by cytotoxic CD8 T cells

To study liver cell damage by CTL, CD8 T cells from P14 TCR transgenic (tg) mice specific for the gp33 epitope of lymphocytic choriomeningitis virus with either deficiency in IFN-gamma (P14.IFN-gamma(null)), functional Fas ligand (P14.gld), or perforin (P14.PKO) were transferred into H8 tg mice ubiquitously expressing gp33 Ag. Treatment of H8 recipient mice with agonistic anti-CD40 Abs induced vigorous expansion of the transferred P14 T cells and led to liver cell destruction determined by increase of glutamate dehydrogenase serum levels and induction of caspase-3 in hepatocytes. Liver injury was mediated by the Fas/Fas ligand (FasL) pathway and by perforin, because P14.gld and P14.PKO T cells failed to induce increased glutamate dehydrogenase levels despite strong in vivo proliferation. In addition, H8 tg mice lacking Fas were resistant to the pathogenic effect of P14 T cells. Besides FasL and perforin, IFN-gamma was also required for liver cell damage, because P14.IFN-gamma(null) T cells adoptively transferred into H8 mice failed to induce disease. Moreover, Fas expression on hepatocytes from H8 recipient mice was increased after transfer of wild-type compared with P14.IFN-gamma(null) T cells, and wild-type P14 T cells expressed higher levels of FasL than P14 T cells lacking IFN-gamma. Thus, our data suggest that IFN-gamma released by activated CD8 T cells upon Ag contact facilitates liver cell destruction.     

Inhibition of H-Y cell-mediated cytolysis by monoclonal H-Y-specific antibody

Assays of H-Y-specific, cell-mediated cytolysis (CMC) in vitro were carried out with B6 female effector cells and B6 male target cells. Monoclonal H-Y antibody was added to the lytic assay to test whether the antigenic determinant(s) involved in H-Y-specific CMC was distinct from the serologically detected H-Y antigen. Significant blocking was observed, suggesting that the H-Y antigen detectable serologically is similar to H-Y antigen recognized by cytotoxic T cells.Abbreviations used in this paper B6 C57BL/6 - BALB BALB/c - CMC cell-mediated cytolysis - E effector cells - T target cells     

Dendritic cells genetically engineered to express Fas ligand induce donor-specific hyporesponsiveness and prolong allograft survival

Polarization of an immune response toward tolerance or immunity is dictated by the interactions between T cells and dendritic cells (DC), which in turn are modulated by the expression of distinct cell surface molecules, and the cytokine milieu in which these interactions are taking place. Genetic modification of DC with genes coding for specific immunoregulatory cell surface molecules and cytokines offers the potential of inhibiting immune responses by selectively targeting Ag-specific T cells. In this study, the immunomodulatory effects of transfecting murine bone marrow-derived DC with Fas ligand (FasL) were investigated. In this study, we show that FasL transfection of DC markedly augmented their capacity to induce apoptosis of Fas+ cells. FasL-transfected DC inhibited allogeneic MLR in vitro, and induced hyporesponsiveness to alloantigen in vivo. The induction of hyporesponsiveness was Ag specific and was dependent on the interaction between FasL on DC and Fas on T cells. Finally, we show that transfusion of FasL-DC significantly prolonged the survival of fully MHC-mismatched vascularized cardiac allografts. Our findings suggest that DC transduced with FasL may facilitate the development of Ag-specific unresponsiveness for the prevention of organ rejection. Moreover, they highlight the potential of genetically engineering DC to express other genes that affect immune responses.     

Uptake of apoptotic antigen-coupled cells by lymphoid dendritic cells and cross-priming of CD8(+) T cells produce active immune unresponsiveness

The induction of immunologic unresponsiveness by i.v. administration of Ag-coupled lymphoid cells has been studied extensively, but the mechanisms remain unclear. We have further explored this model by examining the role of Fas/Fas ligand (FasL)-mediated apoptosis. Using i.v. injection of trinitrophenyl-coupled splenocytes (TNP-spl) as tolerogen, we found that Fas signaling for apoptosis in the spleen cells delivered by FasL in the recipient is the critical event. The requirement for Fas and FasL was overcome by prior induction of apoptosis in TNP-spl, making the tolerogen 100 times more potent. Prevention of apoptosis by a caspase inhibitor blocks tolerance. Interestingly, while blocking CD40/CD40 ligand interaction does not prevent tolerance induction, an agonist anti-CD40 Ab turns tolerogenic TNP-spl into an immunizing Ag. Studies further showed that tolerance is induced through cross-presentation of Ag in a class I MHC-dependent manner by CD8(+)CD11c(+) lymphoid-derived dendritic cells to regulatory T cells. The results provide a mechanism for a well-established method of inducing immunologic unresponsiveness.