CD8+ tumor-infiltrating lymphocytes are primed for Fas-mediated activation-induced cell death but are not apoptotic in situ

Induction of Fas-mediated activation-induced cell death in antitumor T cells has been hypothesized to permit tumor escape from immune destruction. Several laboratories have proposed that expression of Fas ligand (L) by tumor is the basis for this form of T cell tolerance. In this study, we characterized murine tumor-infiltrating lymphocytes (TIL) for activation status, cell cycle status, level of apoptosis, cytokine secretion, and proliferative capacity. TILs express multiple activation markers (circa CD69, CD95L, CD122, and LFA-1) and contain IL-2 and IFN-gamma mRNAs, but are neither cycling nor apoptotic in situ. In addition, TIL are dramatically suppressed in proliferative response and do not secrete IL-2 and IFN-gamma. However, upon purification and activation in vitro, TIL secrete high levels of IL-2 and IFN-gamma, enter S phase, and then die by Fas-mediated apoptosis. Activation by injection of anti-TCR Ab or IL-2 into tumor-bearing mice induced TIL entrance into S phase preceding apoptosis, showing that TIL have functional TCR-mediated signal transduction in situ. Our data demonstrate that TIL, not tumor, express both Fas and FasL, are arrested in G(1), do not secrete cytokine in situ, and, upon activation in vitro and in vivo, rapidly die by activation-induced cell death.     

The role of the common cytokine receptor gamma-chain in regulating IL-2-dependent, activation-induced CD8+ T cell death.

IL-2-dependent, activation-induced T cell death (AICD) plays an important role in peripheral tolerance. Using CD8+ TCR-transgenic lymphocytes (2C), we investigated the mechanisms by which IL-2 prepares CD8+ T cells for AICD. We found that both Fas and TNFR death pathways mediate the AICD of 2C cells. Neutralizing IL-2, IL-2R alpha, or IL-2R beta inhibited AICD. In contrast, blocking the common cytokine receptor gamma-chain (gamma c) prevented Bcl-2 induction and augmented AICD. IL-2 up-regulated Fas ligand (FasL) and down-regulated gamma c expression on activated 2C cells in vitro and in vivo. Adult IL-2 gene-knockout mice displayed exaggerated gamma c expression on their CD8+, but not on their CD4+, T cells. IL-4, IL-7, and IL-15, which do not promote AICD, did not influence FasL or gamma c expression. These data provide evidence that IL-2 prepares CD8+ T lymphocytes for AICD by at least two mechanisms: 1) by up-regulating a pro-apoptotic molecule, FasL, and 2) by down-regulating a survival molecule, gamma c.     

T cell–tumor cell: a fatal interaction?

Fas (Apo-1/CD95) is a cell-surface protein that is responsible for initiating a cascade of proteases (caspases) culminating in apoptotic cell death in a variety of cell types. The function of the Fas/FasL system in the dampening of immune responses to infectious agents through the autocrine deletion of activated T cells has been well documented. More recently, it has been proposed that tumor cells express FasL, presumably to avoid immune detection. In this review, we focus on the role of the interaction of Fas and FasL in the modulation of antitumor responses. We critically examine the evidence that FasL is expressed by tumor cells and explore alternative explanations for the observed phenomena in vitro and in vivo. By reviewing data that we have generated in our laboratory as well as reports from the literature, we will argue that the Fas/FasL system is a generalized mechanism used in an autocrine fashion to regulate cell survival and expansion in response to environmental and cellular cues. We propose that FasL expression by tumor cells, when present, is indicative of a perturbed balance in the control of proliferation while “immune privilege” is established by “suicide” of activated antitumor T cells, a form of activation-induced cell death. Received: 5 May 1998 / Accepted: 20 May 1998     

Differential role of tyrosine phosphorylation in the induction of apoptosis in T cell clones via CD95 or the TCR/CD3-complex

Activated T cells undergo apoptosis when the Fas-antigen (APO-1, CD95) is ligated by Fas Ligand (FasL) or agonistic anti-Fas antibodies. Repeated stimulation of T lymphocytes via the TCR/CD3-complex induces activation-induced cell death (AICD) associated with FasL surface expression. FasL binding to Fas molecules triggers the Fas-dependent death signaling cascade. Since it is still controversial whether Fas-induced cell death is associated with tyrosine kinase activity, we investigated the tyrosine kinase activation requirements in anti-Fas antibody-induced cell death and AICD in human T cell clones. We report that cell death triggered by anti-Fas antibody is not accompanied by an increase in tyrosine phosphorylation and cannot be blocked by inhibitors of protein tyrosine kinases (PTK). Under the same conditions, AICD of T cell clones is clearly associated with tyrosine kinase activation. In fact, semiquantitative RT-PCR analysis of FasL mRNA expression triggered in T cell clones via the TCR/CD3-complex revealed that tyrosine phosphorylation is required for functional FasL mRNA and surface expression.     

Chronic morphine treatment promotes specific Th2 cytokine production by murine T cells in vitro via a Fas/Fas ligand-dependent mechanism

Improper homeostasis of Th1 and Th2 cell differentiation can promote pathological immune responses such as autoimmunity and asthma. A number of factors govern the development of these cells including TCR ligation, costimulation, death effector expression, and activation-induced cell death (AICD). Although chronic morphine administration has been shown to selectively promote Th2 development in unpurified T cell populations, the direct effects of chronic morphine on Th cell skewing and cytokine production by CD4(+) T cells have not been elucidated. We previously showed that morphine enhances Fas death receptor expression in a T cell hybridoma and human PBL. In addition, we have demonstrated a role for Fas, Fas ligand (FasL), and TRAIL in promoting Th2 development via killing of Th1 cells. Therefore, we analyzed whether the ability of morphine to affect Th2 cytokine production was mediated by regulation of Fas, FasL, and TRAIL expression and AICD directly in purified Th cells. We found that morphine significantly promoted IL-4 and IL-13 production but did not alter IL-5 or IFN-gamma. Furthermore, morphine enhanced the mRNA expression of Fas, FasL and TRAIL and promoted Fas-mediated AICD of CD4(+) T cells. Additionally, blockade of Fas/FasL interaction by anti-FasL inhibited the morphine-induced production of IL-4 and IL-13 and AICD of CD4(+) T cells. These results suggest that morphine preferentially enhances Th2 cell differentiation via killing of Th1 cells in a Fas/FasL-dependent manner.     

Th17 cells undergo Fas-mediated activation-induced cell death independent of IFN-gamma

IL-17-secreting CD4+ T cells (Th17 cells) play a critical role in immune responses to certain infections and in the development of many autoimmune disorders. The mechanisms controlling homeostasis in this cell population are largely unknown. In this study, we show that murine Th17 cells undergo rapid apoptosis in vitro upon restimulation through the TCR. This activation-induced cell death (AICD), a common mechanism for elimination of activated T cells, required the Fas and FasL interaction: Fas was stably expressed, while FasL was up-regulated upon TCR reactivation of Th17 cells; Ab ligation of Fas induced Th17 cell death; and AICD was completely absent in Th17 cells differentiated from gld/gld CD4+ T cells. Thus, the Fas/FasL pathway is essential in regulating the AICD of Th17 cells. Interestingly, IFN-gamma, a cytokine previously found to be important for the AICD of T cells, did not affect Th17 cell apoptosis. Furthermore, Th17 cells derived from mice deficient in IFN-gamma receptor 1 (IFN-gammaR1-/-) underwent AICD similar to wild-type cells. Thus, AICD of Th17 cells occurs via the Fas pathway, but is independent of IFN-gamma.     

Fas-dependent, activation-induced cell death of gammadelta cells.

Activated gammadelta T cells undergo apoptosis upon restimulation of their T cell receptor (TCR)/CD3 complex. We demonstrate that in these cells, the activation-induced cell death (AICD) is mediated by Fas and Fas ligand (FasL) interaction. The activated gammadelta T cells are prone to AICD initiated by exposure to mitogens, anti-TCR/CD3 antibodies, as well as specific antigens such as Daudi cells or ethylpyrophosphate (Etpp). Cells that have been activated twice, and consequently more susceptible to AICD than primary cells, display augmented tyrosine phosphorylation in comparison with control cells. These studies outline a mechanism that may regulate gammadelta T cell activities in immune responses and limit the expansion of activated T cells repeatedly exposed to antigens.     

Positive and negative consequences of soluble Fas ligand produced by an antigen-specific CD4(+) T cell response in human carcinoma immune interactions.

The influence of a human CD4(+) T cell response in anti-carcinoma immune reactions remains largely uncharacterized. Here, we made use of a major histocompatibility complex (MHC) class-II-restricted, anti-ras oncogene-specific CD4(+) T cell line produced previously in vivo from a patient with metastatic carcinoma in a peptide-based phase I trial. Using this patient-derived T cell line as a potentially relevant cell type, we examined the consequences of the anti-carcinoma CD4(+) T cell response, with emphasis on specific lymphokines potentially important for the regulation of Fas/Fas ligand (FasL) interactions. Antigen (Ag)-specific CD4(+) T cells produced substantial amounts of IFN-gamma following recognition of MHC class-II-matched Ag-presenting cells expressing the cognate peptide. The IFN-gamma promoted significant upregulation of Fas on the surface of colon carcinoma cells and sensitized these targets to Fas-mediated apoptosis and Ag-specific CD8(+) cytotoxic T lymphocyte (CTL)-mediated lysis involving a Fas-based effector mechanism. Moreover, Ag-stimulated CD4(+) T cells secreted soluble FasL (sFasL), which induced the death of TNF-resistant/refractory colon, breast, and ovarian carcinoma cells. Interestingly, although CD4(+)-derived sFasL expressed cytotoxic activity, the recovery of carcinoma cells which resisted Fas-mediated lysis displayed enhanced metastatic ability in vivo, compared with the unselected parental population, in an athymic mouse model. Thus, a tumor-specific CD4(+) T cell response may have both positive and negative consequences in human carcinoma via the production of proinflammatory cytokines such as IFN-gamma and/or sFasL that may (1) improve or facilitate CTL-target engagement, contact-independent effector mechanisms, and the overall lytic outcome and (2) potentially select for Fas-resistant tumor cells that escape immune destruction, which may thus impact the metastatic process.     

Cell death induced by the Fas/Fas ligand pathway and its role in pathology.

Engagement of the cell death surface receptor Fas by Fas ligand (FasL) results in apoptotic cell death, mediated by caspase activation. Cell death mediated via Fas/FasL interaction is important for homeostasis of cells in the immune system and for maintaining immune-privileged sites in the body. Killing via the Fas/FasL pathway also constitutes an important pathway of killing for cytotoxic T cells. Fas ligand is induced in activated T cells, resulting in activation-induced cell death by the Fas/FasL pathway. Recently it has been shown that the Fas receptor can also be up-regulated following a lesion to the cell, particularly that induced by DNA-damaging agents. This can then result in killing of the cell by a Fas/FasL-dependent pathway. Up-regulation of Fas receptor following DNA damage appears to be p53 dependent.     

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.     

T cell receptor-induced activation and apoptosis in cycling human T cells occur throughout the cell cycle

Previous studies have found conflicting associations between susceptibility to activation-induced cell death and the cell cycle in T cells. However, most of the studies used potentially toxic pharmacological agents for cell cycle synchronization. A panel of human melanoma tumor-reactive T cell lines, a CD8+ HER-2/neu-reactive T cell clone, and the leukemic T cell line Jurkat were separated by centrifugal elutriation. Fractions enriched for the G0-G1, S, and G2-M phases of the cell cycle were assayed for T cell receptor-mediated activation as measured by intracellular Ca(2+) flux, cytolytic recognition of tumor targets, and induction of Fas ligand mRNA. Susceptibility to apoptosis induced by recombinant Fas ligand and activation-induced cell death were also studied. None of the parameters studied was specific to a certain phase of the cell cycle, leading us to conclude that in nontransformed human T cells, both activation and apoptosis through T cell receptor activation can occur in all phases of the cell cycle.     

Non-apoptotic Fas signaling

Fas (Apo-1, CD95) and Fas-Ligand (FasL, CD95L) are typical members of the TNF receptor and TNF ligand family, respectively, with a pivotal role in the regulation of apoptotic processes, including activation-induced cell death, T-cell-induced cytotoxicity, immune privilege and tumor surveillance. Impairment of the FasL/Fas system has been implicated in liver failure, autoimmune diseases and immune deficiency. Thus, the FasL/Fas system was mainly appreciated with respect to its death-inducing capabilities. However, there is increasing evidence that activation of Fas can also result in non-apoptotic responses like cell proliferation or NF-kappaB activation. While the apoptotic features of the FasL/Fas system and the pathways involved are comparably well investigated, the pathways that are utilized by Fas to transduce proliferative and activating signals are poorly understood. This review is focused on the non-apoptotic functions of the FasL/Fas system. In particular, the similarities and differences of the molecular mechanisms of apoptotic and non-apoptotic Fas signaling are addressed.     

T cell receptor ligation triggers novel nonapoptotic cell death pathways that are Fas-independent or Fas-dependent

Short-term culture of activated T cells with IL-2 renders them highly susceptible to apoptotic death triggered by TCR cross-linking. Activation-induced apoptosis is contingent upon caspase activation and this is mediated primarily by Fas/Fas ligand (FasL) interactions that, in turn, are optimized by p38 mitogen-activated protein kinase (MAPK)-regulated signals. Although T cells from mice bearing mutations in Fas (lpr) or FasL (gld) are more resistant to activation-induced cell death (AICD) than normal T cells, a significant proportion of CD8(+) T cells and to a lesser extent CD4(+) T cells from mutant mice die after TCR religation. Little is known about this Fas-independent death process. In this study, we demonstrate that AICD in lpr and gld CD4(+) and CD8(+) T cells occurs predominantly by a novel mechanism that is TNF-alpha-, caspase-, and p38 MAPK-independent and has morphologic features more consistent with oncosis/primary necrosis than apoptosis. A related Fas- and caspase-independent, nonapoptotic death process is revealed in wild-type (WT) CD8(+) T cell blasts following TCR ligation and treatment with caspase inhibitors, the p38 MAPK inhibitor, SB203580, or neutralizing anti-FasL mAb. In parallel studies with WT CD4(+) T cells, two minor pathways leading to nonapoptotic, caspase-independent AICD were identified, one contingent upon Fas ligation and p38 MAPK activation and the other Fas- and p38 MAPK-independent. These data indicate that TCR ligation can activate nonapoptotic death programs in WT CD8(+) and CD8(+) T blasts that normally are masked by Fas-mediated caspase activation. Selective use of potentially proinflammatory oncotic death programs by activated lpr and gld T cells may be an etiologic factor in autosensitization.     

Fas and Fas ligand in embryos and adult mice: ligand expression in several immune-privileged tissues and coexpression in adult tissues characterized by apoptotic cell turnover

The cell surface receptor Fas (FasR, Apo-1, CD95) and its ligand (FasL) are mediators of apoptosis that have been shown to be implicated in the peripheral deletion of autoimmune cells, activation-induced T cell death, and one of the two major cytolytic pathways mediated by CD8+ cytolytic T cells. To gain further understanding of the Fas system., we have analyzed Fas and FasL expression during mouse development and in adult tissues. In developing mouse embryos, from 16.5 d onwards, Fas mRNA is detectable in distinct cell types of the developing sinus, thymus, lung, and liver, whereas FasL expression is restricted to submaxillary gland epithelial cells and the developing nervous system. Significant Fas and FasL expression were observed in several nonlymphoid cell types during embryogenesis, and generally Fas and FasL expression were not localized to characteristic sites of programmed cell death. In the adult mouse, RNase protection analysis revealed very wide expression of both Fas and FasL. Several tissues, including the thymus, lung, spleen, small intestine, large intestine, seminal vesicle, prostate, and uterus, clearly coexpress the two genes. Most tissues constitutively coexpressing Fas and FasL in the adult mouse are characterized by apoptotic cell turnover, and many of those expressing FasL are known to be immune privileged. It may be, therefore, that the Fas system is implicated in both the regulation of physiological cell turnover and the protection of particular tissues against potential lymphocyte-mediated damage.