Dendritic cell motility and T cell activation requires regulation of Rho-cofilin signaling by the Rho-GTPase activating protein myosin IXb.

Directed migration of stimulated dendritic cells (DCs) to secondary lymphoid organs and their interaction with Ag-specific T cells is a prerequisite for the induction of primary immune responses. In this article, we show that murine DCs that lack myosin IXB (Myo9b), a motorized negative regulator of RhoA signaling, exhibit increased Rho signaling activity and downstream acto-myosin contractility, and inactivation of the Rho target protein cofilin, an actin-depolymerizing factor. On a functional level, Myo9b(-/-) DCs showed impaired directed migratory activity both in vitro and in vivo. Moreover, despite unaltered Ag presentation and costimulatory capabilities, Myo9b(-/-) DCs were poor T cell stimulators in vitro in a three-dimensional collagen matrix and in vivo, associated with altered DC-T cell contact dynamics and T cell polarization. Accordingly, Myo9b(-/-) mice showed an attenuated ear-swelling response in a model of contact hypersensitivity. The impaired migratory and T cell stimulatory capacity of Myo9b(-/-) DCs was restored in large part by pharmacological activation of cofilin. Taken together, these results identify Myo9b as a negative key regulator of the Rho/RhoA effector Rho-kinase [Rho-associated coiled-coil-forming kinase (ROCK)]/LIM domain kinase signaling pathway in DCs, which controls cofilin inactivation and myosin II activation and, therefore may control, in part, the induction of adaptive immune responses.

LFA-1 activity state on dendritic cells regulates contact duration with T cells and promotes T-cell priming.

A key event in the successful induction of adaptive immune responses is the antigen-specific activation of T cells by dendritic cells (DCs). Although LFA-1 (lymphocyte function-associated antigen 1) on T cells is considered to be important for antigen-specific T-cell activation, the role for LFA-1 on DCs remains elusive. Using 2 different approaches to activate LFA-1 on DCs, either by deletion of the αL-integrin cytoplasmic GFFKR sequence or by silencing cytohesin-1-interacting protein, we now provide evidence that DCs are able to make use of active LFA-1 and can thereby control the contact duration with naive T cells. Enhanced duration of DC/T-cell interaction correlates inversely with antigen-specific T-cell proliferation, generation of T-helper 1 cells, and immune responses leading to delayed-type hypersensitivity. We could revert normal interaction time and T-cell proliferation to wild-type levels by inhibition of active LFA-1 on DCs. Our data further suggest that cytohesin-1-interacting protein might be responsible for controlling LFA-1 deactivation on mature DCs. In summary, our findings indicate that LFA-1 on DCs needs to be in an inactive state to ensure optimal T-cell activation and suggest that regulation of LFA-1 activity allows DCs to actively control antigen-driven T-cell proliferation and effective immune responses.

Galectin-2 suppresses contact allergy by inducing apoptosis in activated CD8+ T cells.

Galectins, a family of structurally related beta-galactoside-binding proteins, are expressed by various cells of the immune systems and seem to be important for the regulation of immune responses and immune cell homeostasis. Since it has been demonstrated that galectin-2 regulates cell-mediated inflammatory bowel disease and colitis in mice, we intended to investigate the role of galectin-2 in inflammatory cutaneous T cell-mediated immune responses. To address this issue, groups of naive mice were sensitized to the contact allergen 2,4-dinitro-1-fluorobenzene and systemically treated with galectin-2 to analyze the effects of galectin-2 on contact allergy. Here we show that galectin-2 is expressed in murine skin and is up-regulated upon cutaneous inflammation. Interestingly, treatment of mice with galectin-2 significantly reduced the contact allergy response. This effect was long-lasting since rechallenge of galectin-2-treated mice after a 14-day interval still resulted in a decreased ear swelling. We were able to demonstrate that galectin-2 induced a reduction of MHC class I-restricted immune responses in the treated animals, which was mediated by the induction of apoptosis specifically in activated CD8(+) T cells. Additionally, we report that the galectin-2-binding protein CD29 is up-regulated on the surface of activated CD8(+) T cells compared with naive CD8(+) T cells or CD4(+) T cells, suggesting that increased galectin-2/CD29 signaling might be responsible for the proapoptotic effects of galectin-2 on activated CD8(+) T cells. Taken together, these data indicate that galectin-2 may represent a novel therapeutic alternative for the treatment of CD8-mediated inflammatory disorders such as contact allergy.

Dendritic cell activation by combined exposure to anti-CD40 plus interleukin (IL)-12 and IL-18 efficiently stimulates anti-tumor immunity.

Despite as yet limited clinical effectiveness, dendritic cell (DC)-based immunotherapy remains a promising approach for the treatment of cancer, but requires further improvement in its immunostimulatory effectiveness. Potent anti-tumor immunity often depends on the induction of type 1 (T(H)1) immune responses. Therefore, we combined different DC maturation stimuli that are known to induce T(H)1 immunity [anti-CD40, interleukin (IL)-12, IL-18], with the aim to trigger a T(H)1 driven anti-tumor CTL response. When compared with untreated DC or DC treated with anti-CD40 alone, DC matured with anti-CD40 plus IL-12 and IL-18 expressed significantly more IFN-gamma and IL-12, induced enhanced CD8(+) T-cell proliferation, prolonged synaptic interaction with T cells and increased CD8(+) T-cell-mediated cytotoxicity. To analyse if these DC are able to induce efficient anti-tumor immunity, mice carrying a B16-OVA tumor were treated with tumor antigen (TA)-loaded DC that had been exposed to anti-CD40 or to anti-CD40 plus IL-12 and IL-18. Our data show that anti-CD40 plus IL-12 and IL-18 matured DC are superior to controls in retarding tumor growth. These data indicate that maturation of DC with anti-CD40 plus IL-12 and IL-18 potently stimulates the generation of an anti-tumor immune response and may lead to improved immunotherapeutic capacity of DC vaccination.

SWAP-70 associates transiently with macropinosomes.

Cells accomplish the non-selective uptake of extracellular fluids, antigens and pathogens by the endocytic process of macropinocytosis. The protein SWAP-70 is a widely expressed, pleckstrin-homology (PH) domain-containing protein that marks a transitional subset of actin filaments in motile cells. Here we report that the protein SWAP-70 associates transiently with macropinosomes in dendritic cells and NIH/3T3 fibroblasts. The association of SWAP-70 with macropinosomes is preceded by the accumulation of Rac-GTP and followed by that of Rab5. Three regions of SWAP-70, the N-terminal region, the PH domain and the C-terminal region, contribute in a combinatorial manner to the transient association with newly formed macropinosomes in the cell periphery and occasionally with aged macropinosomes on their passage to the cell center. These data identify SWAP-70 as a transient component of early macropinosomes.

FK506 controls CD40L-induced systemic autoimmunity in mice.

Autoimmunity results from loss of mechanisms controlling self-reactivity. Autoimmune disorders play an increasingly important role and CD40-CD40 ligand (CD40L) interaction on immunocompentent cells is able to break established immunotolerance. To study the effects of the calcineurin-inhibitor FK506 on CD40L-induced systemic autoimmunity, CD40L transgenic (tg) mice, which spontaneously develop a mixed connective tissue-like disease, were treated with FK506 after onset of overt autoimmunity. Interestingly, FK506-treated CD40L tg mice showed significantly reduced autoimmune dermatitis scores and markedly decreased numbers of lesional infiltrating leukocytes. This finding was associated with diminished lymphadenopathy induced by FK506 treatment. Furthermore, FK506 suppressed the development of cytotoxic/autoreactive CD8(+) T cells as evidenced by the reduced expression of T cell activation markers in treated CD40L tg mice. Importantly, FK506 induced a significant reduction in autoantibody titers in the serum of CD40L tg animals. As CD40L tg mice develop nephritis leading to loss of renal function proteinuria was determined after FK506 injections. Notably, FK506 treatment re-established renal function as indicated by significantly reduced uric protein concentrations of treated CD40L tg mice. Together, these findings show the beneficial therapeutic effects of FK506 for the treatment of CD40L-induced autoimmunity. Additionally, these results demonstrate that FK506 is able to suppress ongoing severe autoimmune responses.

In vitro and in vivo imaging of initial B-T-cell interactions in the setting of B-cell based cancer immunotherapy.

There has been a growing interest in the use of B cells for cancer vaccines, since they have yielded promising results in preclinical animal models. Contrary to dendritic cells (DCs), we know little about the migration behavior of B cells in vivo. Therefore, we investigated the interactions between CD40-activated B (CD40B) cells and cytotoxic T cells in vitro and the migration behavior of CD40B cells in vivo. Dynamic interactions of human antigen-presenting cells (APCs) and T cells were observed by time-lapse video microscopy. The migratory and chemoattractant potential of CD40B cells was analyzed in vitro and in vivo using flow cytometry, standard transwell migration assays, and imaging of fluorescently labeled murine CD40B cells. Murine CD40B cells show migratory features similar to human CD40B cells. They express important lymph node homing receptors which were functional and induced chemotaxis of T cells in vitro. Striking differences were observed with regard to interactions of human APCs with T cells. CD40B cells differ from DCs by displaying a rapid migratory pattern undergoing highly dynamic, short-lived and sequential interactions with T cells. In vivo, CD40B cells are home to the secondary lymphoid organs where they accumulate in the B cell zone before traveling to the B/T cell boundary. Moreover, intravenous (i.v.) administration of murine CD40B cells induced an antigen-specific cytotoxic T cell response. Taken together, this data show that CD40B cells home secondary lymphoid organs where they physically interact with T cells to induce antigen-specific T cell responses, thus underscoring their potential as cellular adjuvant for cancer immunotherapy.

Release of IL-12 by dendritic cells activated by TLR ligation is dependent on MyD88 signaling, whereas TRIF signaling is indispensable for TLR synergy.

Recently, it has been shown that certain combinations of TLR ligands act in synergy to induce the release of IL-12 by DCs. In this study, we sought to define the critical parameters underlying TLR synergy. Our data show that TLR ligands act synergistically if MyD88- and TRIF-dependent ligands are combined. TLR4 uses both of these adaptor molecules, thus activation via TLR4 proved to be a synergistic event on its own. TLR synergy did not affect all aspects of DC activation but enhanced primarily the release of certain cytokines, particularly IL-12, whereas the expression of costimulatory molecules remained unchanged. Consequently, synergistic activation of DC did not affect their ability to induce T cell proliferation but resulted in T(H)1-biased responses in vitro and in vivo. Furthermore, we examined the impact of TLR ligand combinations on primary DC in vitro but observed only modest effects with a combination of CpG + Poly (I:C). However, noticeable synergy in terms of IL-12 production by DCs was detectable in vivo after systemic administration of CpG + Poly (I:C). Finally, we show that synergy is partially dependent on IFNAR signaling but does not require the release of IFNs to the enviroment, suggesting an autocrine action of type I IFNs.

LFA-1 contributes to signal I of T-cell activation and to the production of T(h)1 cytokines.

The beta(2) integrins are important for both transendothelial migration of leukocytes and T-cell activation during antigen presentation. In T cells, triggering of leukocyte functional antigen-1 (LFA-1) is required for full activation and T-helper (Th)1/Th2 differentiation. We used CD18-deficient (CD18(-/-)) mice to examine the role of LFA-1 in the activation of T cells. Compared with wild-type controls, CD18(-/-) T cells proliferated normally when stimulated with antibodies against CD3 and CD28, but secreted significantly less IFN-gamma and IL-2 than their wild-type counterparts. However, when T cells were stimulated with dendritic cells (DCs) that provide additional LFA-1 ligation, the proliferation of CD18(-/-) T cells was significantly reduced, whereas cytokine production remained impaired. The diminished proliferative capacity of CD18(-/-) T cells could be fully compensated for by additional triggering of the T-cell receptor, but not by additional stimulation through the costimulatory molecule, CD28. Thus, ligation of LFA-1 on T cells participates in regulation of Th1 cytokines in vivo. In addition, LFA-1 primarily exerts an effect as an enhancer of TCR signalling and does not facilitate classical costimulation.

The level of friend retrovirus replication determines the cytolytic pathway of CD8+ T-cell-mediated pathogen control.

Cytotoxic T cells (CTL) play a central role in the control of viral infections. Their antiviral activity can be mediated by at least two cytotoxic pathways, namely, the granule exocytosis pathway, involving perforin and granzymes, and the Fas-FasL pathway. However, the viral factor(s) that influences the selection of one or the other pathway for pathogen control is elusive. Here we investigate the role of viral replication levels in the induction and activation of CTL, including their effector potential, during acute Friend murine leukemia virus (F-MuLV) infection. F-MuLV inoculation results in a low-level infection of adult C57BL/6 mice that is enhanced about 500-fold upon coinfection with the spleen focus-forming virus (SFFV). Both the low- and high-level F-MuLV infections generated CD8+ effector T cells that were essential for the control of viral replication. However, the low-level infection induced CD8+ T cells expressing solely FasL but not the cytotoxic molecules granzymes A and B, whereas the high-level infection resulted in induction of CD8+ effector T cells secreting molecules of the granule exocytosis pathway. By using knockout mouse strains deficient in one or the other cytotoxic pathway, we found that low-level viral replication was controlled by CTL that expressed FasL but control of high-level viral replication required perforin and granzymes. Additional studies, in which F-MuLV replication was enhanced experimentally in the absence of SFFV coinfection, supported the notion that only the replication level of F-MuLV was the critical factor that determined the differential expression of cytotoxic molecules by CD8+ T cells and the pathway of CTL cytotoxicity.

Friend retrovirus infection of myeloid dendritic cells impairs maturation, prolongs contact to naive T cells, and favors expansion of regulatory T cells.

Retroviruses have developed immunmodulatory mechanisms to avoid being attacked by the immune system. The mechanisms of this retrovirus-associated immune suppression are far from clarified. Dendritic cells (DCs) have been attributed a decisive role in these pathogenic processes. We have used the Friend retrovirus (FV) mouse model in order to acquire further knowledge about the role of infection of DCs in virus-induced immunosuppression. About 20% of the myeloid DCs that were generated from the bone marrow of FV-infected mice carried FV proteins. The infection was productive, and infected DCs transmitted the virus in cell culture and in vivo. FV infection of DCs led to a defect in DC maturation, as infected cells expressed very little costimulatory molecules. Live imaging analysis of the cell contact between DCs and T cells revealed prolonged contacts of T cells with infected DCs compared with uninfected DCs. Although naive T cells were still activated by FV-infected DCs, this activation did not result in antigen-specific T-cell proliferation. Interestingly, infected DCs expanded a population of Foxp3(+) regulatory T cells with immunosuppressive potential, suggesting that the contact between naive T cells and retrovirus-infected DCs results in tolerance rather than immunity. Thus, retroviral infection of DCs leads to an expansion of regulatory T cells, which might serve as an immune escape mechanism of the virus.

Stage of primary infection with lymphocytic choriomeningitis virus determines predisposition or resistance of mice to secondary bacterial infections.

We investigated the effect of a primary non-lethal infection with lymphocytic choriomeningitis virus (LCMV) on the course and outcome of a secondary infection with the Gram-negative Salmonella enterica serovar Typhimurium or the Gram-positive Listeria monocytogenes in mice. We found that at each stage of the viral infection the susceptibility of mice to bacterial super-infections changes dramatically and depends also on whether the secondary infection is a Gram-positive or Gram-negative one. The study shows that the outcome of the secondary infection is determined by a delicate balance between the overproduction of and the hypersensitivity to inflammatory cytokines (TNF-alpha and IFN-gamma), as well as by the changes in blood leukocytes occurring in mice in the course of viral infection.

IL-10 controls ultraviolet-induced carcinogenesis in mice.

UV radiation-induced immunosuppression contributes significantly to the development of UV-induced skin cancer by inhibiting protective immune responses. IL-10 has been shown to be a key mediator of UV-induced immunosuppression. To investigate the role of IL-10 during photocarcinogenesis, groups of IL-10(+/+), IL-10(+/-), and IL-10(-/-) mice were chronically irradiated with UV. IL-10(+/+) and IL-10(+/-) mice developed skin cancer to similar extents, whereas IL-10(-/-) mice were protected against the induction of skin malignancies by UV. Because UV is able to induce regulatory T cells, which play a role in the suppression of protective immunity, UV-induced regulatory T cell function was analyzed. Splenic regulatory T cells from UV-irradiated IL-10(-/-) mice were unable to confer immunosuppression upon transfer into naive recipients. UV-induced CD4+CD25+ T cells from IL-10(-/-) mice showed impaired suppressor function when cocultured with conventional CD4+CD25- T cells. CD4+CD25- T cells from IL-10(-/-) mice produced increased amounts of IFN-gamma and enhanced numbers of CD4+TIM-3+ T cells were detectable within UV-induced tumors in IL-10(-/-) mice, suggesting strong Th1-driven immunity. Mice treated with CD8+ T cells from UV-irradiated IL-10(-/-) mice rejected a UV tumor challenge significantly faster, and augmented numbers of granzyme A+ cells were detected within injected UV tumors in IL-10(-/-) animals, suggesting marked antitumoral CTL responses. Together, these findings indicate that IL-10 is critically involved in antitumoral immunity during photocarcinogenesis. Moreover, these results point out the crucial role of Th1 responses and UV-induced regulatory T cell function in the protection against UV-induced tumor development.

Active MAC-1 (CD11b/CD18) on DCs inhibits full T-cell activation.

The beta2 integrins are important for transendothelial migration of leukocytes as well as for T-cell activation during antigen presentation. Despite abundant expression of beta2 integrins on antigen-presenting cells (APCs), their functional relevance for antigen presentation is completely unclear. We show here that dendritic cells (DCs) from CD18-deficient mice, which lack all functional beta2 integrins, have no defect in antigen presentation. Moreover, DCs from normal mice express inactive beta2 integrins that do not become activated on contact with T cells, at least in vitro. Pharmacologic activation of beta2 integrins on DCs results in a significant reduction of their T cell-activating capacity. This effect is mediated by Mac-1 (CD11b/CD18) on DCs because it could be reversed via blocking antibodies against CD18 and CD11b. Furthermore, the antigen-presenting capacity of macrophages, which express constitutively active beta2 integrins, is significantly enhanced on Mac-1 blockade. We therefore conclude that active CD11b/CD18 (Mac-1) on APCs directly inhibits T-cell activation.

Agonists of proteinase-activated receptor-2 affect transendothelial migration and apoptosis of human neutrophils.

Skin is the first barrier preventing microorganism invasion in host. Wounds destroy this defense barrier and, without an appropriate care, may lead to sepsis. Neutrophil activation and immigration plays an important role at the inflammatory stage of wound healing. Neutrophils are known to express proteinase-activated receptors (PARs), which can be activated by serine proteases, also by enzymes involved in wound healing. We previously reported that PAR(2) agonists up-regulate cell adhesion molecule expression and cytokine production by human neutrophils. Here, we demonstrate that PAR(2) agonists (serine proteases as well as synthetic peptides) reduce transendothelial migration of neutrophils and prolong their life in vitro. Synthetic PAR(2) agonist also enhanced protective interferon (IFN)gamma-induced FcgammaRI expression at neutrophil cell surface. Of note, IFNgamma is a cytokine, which was used in clinical trials to reactivate human neutrophil functions during sepsis. Moreover, we observed a significant increase of PAR(2) expression on cell surface of neutrophils from septic patients as compared with healthy volunteers. Together, our results indicate that PAR(2) may be involved in the pathophysiology of neutrophil-endothelial interactions during wound healing or later during sepsis in humans, potentially by affecting neutrophil apoptosis, transendothelial migration and Fcgamma receptor-mediated phagocytosis.

Structure and duration of contact between dendritic cells and T cells are controlled by T cell activation state.

The adaptive immune response is initiated when naive T cells interact with dendritic cells (DC). However, the physicodynamics as well as the molecules that constitute the contact plane (immunological synapse) between DC and T cells are not well understood. We show here that for the formation of stable conjugates, T cells need to be preactivated by DC in a CD80/86- and antigen dose-dependent manner. When activated, T cells induce cytoskeletal reorganization within DC via CD40-CD40L signaling. Polarization of the actin and fascin cytoskeleton in DC is associated with sustained DC-T cell contacts, strong T cell proliferation and a Th1 response. Organized contact planes with clearly separated patches containing TCR or CD11a are also formed. Thus, DC-T cell interactions take place in a sequential, interdependent fashion: first, DC "license" naive T cells to engage DC in an antigen dose- and CD80/86-dependent fashion. Then, these preactivated T cells induce cytoskeletal reorientation in DC, resulting in sustained DC-T cell contacts and subsequent T cell activation. These results demonstrate that T cells control the mode of interaction based on information gathered from DC.

Systemic administration of a TLR7 ligand leads to transient immune incompetence due to peripheral-blood leukocyte depletion.

Toll-like receptor (TLR) ligands lead to the induction of proinflammatory cytokines and are potent enhancers of specific immune responses. We show here that a single systemic dose of R-848, a ligand for TLR7, potently enhanced hapten sensitization during the induction of contact hypersensitivity (CHS). However, R-848 administration also resulted in a rapid and almost complete depletion of leukocytes from the blood. This effect was transient and was associated with general induction of endothelial adhesiveness. In response to R-848, endothelial cells up-regulated adhesion molecules in vitro and in vivo and leukocytes exhibited increased rolling on endothelia in R-848-treated animals. Adhesion molecule induction appeared to be a direct effect, because endothelial cells expressed TLR7 in vitro and in vivo. After R-848 treatment, the tissue residence time of leukocytes was markedly prolonged in all major peripheral organs. The resulting transiently reduced availability of peripheral-blood leukocytes (PBLs) (TRAP) significantly inhibited otherwise potent CHS responses until the effector cells returned. Thus, although TLR7 ligands are effective adjuvants for the induction of cell-mediated immunity, they can transiently inhibit the elicitation of localized immune responses, possibly due to a systemic endothelial activation throughout the vasculature.

The differential contribution of granzyme A and granzyme B in cytotoxic T lymphocyte-mediated apoptosis is determined by the quality of target cells.

Complementary approaches with purified molecules or transfected cytolytic effector cells have suggested that both, granzyme A (gzmA) and granzyme B (gzmB), similarly contribute to CTL-mediatedand perforin (perf)-dependent apoptotic nuclear damage (DNA fragmentation) in target cells. Studies employing gzmA or gzmB single-knockout mice on the other hand indicated that gzmB is the prominent CTL effector molecule for the rapid induction of DNA fragmentation, with gzmA playing only a minor part. We have now taken ex vivo-derived virus-specific or in vitro generated alloreactive CTL from mice deficient in either gzmA or gzmB and a panel of three target cells to reinvestigate this unresolved issue. We show that rapid CTL-mediated DNA fragmentation of L1210.3 target cells is solely dependent on gzmB, whereas the DNA fragmentation of EL4.F15 target cells by the same CTL population is mainly induced by gzmA and only marginally by gzmB. Moreover, CTL-induced apoptosis of a third target cell, MC57G, was partially dependent on both gzmA and gzmB activities. The differential contribution of the two gzms to apoptosis was further verified by their distinct sensitivity tocaspase inhibitors. The data suggest that both, gzmA and gzmB, have a similar potential to induce rapid perf-mediated apoptosis but that their individual contribution to the underlying intracellular processes is dictated by the quality of the target cell.

Granzymes are essential for natural killer cell-mediated and perf-facilitated tumor control.

Studies with perforin-deficient mice firmly established perforin as a key element in cytotoxic T cell (CTL) / natural killer (NK) cell-mediated tumor control but did not reveal the role of granzyme (gzm) A and B. A contribution of gzm in these processes was indicated by earlier in vitro experiments employing purified effector molecules demonstrated that tumor cell apoptosis and death only occurred in the presence of both, perf and gzm. However, recent work using mice deficient in either gzmA, gzmB or both gzm suggested that only perf but neither of the two gzm are critical for tumor surveillance by CTL or NK cells. In light of the conflicting results we have re-investigated this issue by analyzing the potential of mice deficient in one or more component(s) of the exocytosis pathway to control NK-sensitive syngeneic MHC class I-defective RMA-S tumor cells in vivo. Our results show that in contrast to wild-type mice, mice deficient for both gzm exhibit an uncontrolled tumor growth with a time kinetic similar to that of perforin-deficient mice. Together with the finding that a defect of mice in either gzmA or gzmB alone also leads to an increased susceptibility to tumor growth, at least to a certain extent when compared to wild-type mice, the data clearly indicate that a concerted action of perforin and the two gzm is mandatory for optimal NK cell-mediated tumor control in vivo. Most notably, the in vivo potential of the respective NK cell populations was only reflected by their nucleolytic, but not their cytolytic activities in vitro.

Antigen-induced cell death of T effector cells in vitro proceeds via the Fas pathway, requires endogenous interferon-gamma and is independent of perforin and granzymes.

Activation of resting T cells usually leads to their proliferation and differentiation into effector cells and a subsequent decline following elimination of the antigen. A situation of excessive antigen density may result in T cell receptor (TCR)-induced deletion of T effector cells, a process termed antigen-induced cell death (AgICD). Previous studies indicate that AgICD of cytotoxic T cells may be induced by either of the two key cytotoxic processes, granule exocytosis, including perforin and granzymes, or the Fas ligand (FasL)/Fas pathway. By using in vitro-polyclonally activated or ex vivo-derived virus-induced T cell populations from mice with mutations or targeted gene defects in one or more components of the two key cytolytic pathways we now show that TCR-induced apoptosis is only impaired in the absence of FasL and/or Fas, but not in the absence of perforin and/or granzymes. Furthermore, antibody-blockage of FasL alone is sufficient to inhibit early T cell death. Inhibition of both, FasL and tumor necrosis factor (TNF-alpha) is required to abrogate late apoptosis by AgICD. The fact that antibodies to IFN-gamma also inhibit AgICD suggests that the perforin plus granzyme-independent and FaSL and/or TNF-alpha facilitated process of AgICD of T effector cells is tightly regulated by endogenous IFN-gamma.