Mature but not immature Fas ligand (CD95L)-transduced human monocyte-derived dendritic cells are protected from Fas-mediated apoptosis and can be used as killer APC

Several in vitro and animal studies have been performed to modulate the interaction of APCs and T cells by Fas (CD95/Apo-1) signaling to delete activated T cells in an Ag-specific manner. However, due to the difficulties in vector generation and low transduction frequencies, similar studies with primary human APC are still lacking. To evaluate whether Fas ligand (FasL/CD95L) expressing killer APC could be generated from primary human APC, monocyte-derived dendritic cells (DC) were transduced using the inducible Cre/Loxp adenovirus vector system. Combined transduction of DC by AdLoxpFasL and AxCANCre, but not single transduction with these vectors, resulted in dose- and time-dependent expression of FasL in >70% of mature DC (mDC), whereas <20% of immature DC (iDC) expressed FasL. In addition, transduction by AdLoxpFasL and AxCANCre induced apoptosis in >80% of iDC, whereas FasL-expressing mDC were protected from FasL/Fas (CD95/Apo-1)-mediated apoptosis despite coexpression of Fas. FasL-expressing mDC eliminated Fas(+) Jurkat T cells as well as activated primary T cells by apoptosis, whereas nonactivated primary T cells were not deleted. Induction of apoptosis in Fas(+) target cells required expression of FasL in DC and cell-to-cell contact between effector and target cell, and was not dependent on soluble FasL. Induction of apoptosis in Fas(+) target cells required expression of FasL in DC, cell-to-cell contact between effector and target cell, and was not dependent on soluble FasL. The present results demonstrate that FasL-expressing killer APC can be generated from human monocyte-derived mDC using adenoviral gene transfer. Our results support the strategy to use killer APCs as immunomodulatory cells for the treatment of autoimmune disease and allograft rejection.     

Peripheral deletion of antigen-specific T cells leads to long-term tolerance mediated by CD8+ cytotoxic cells

Peripheral deletion is one mechanism by which potentially self-reactive clones are removed whether they escape thymic deletion. We have examined the consequences of deleting Ag-specific T cells by i.v. injection of soluble Ag. Deletion of DO11.10 T cells by peptide was mediated predominately via a Fas/FasL mechanism. Animals that underwent deletion were tolerant to subsequent immunization with Ag, even when tolerant mice were given fresh Ag-specific DO11.10 T cells before immunization. Tolerance was mediated by CD8(+) T cells that killed the DO11.10-transgenic T cells in vivo. These data demonstrate that the programmed cell death of large numbers of T cells leads to peripheral tolerance mediated by CD8(+) CTLs.     

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.     

Mutation in the Fas pathway impairs CD8+ T cell memory

Fas death pathway is important for lymphocyte homeostasis, but the role of Fas pathway in T cell memory development is not clear. We show that whereas the expansion and contraction of CD8+ T cell response against Listeria monocytogenes were similar for wild-type (WT) and Fas ligand (FasL) mutant mice, the majority of memory CD8+ T cells in FasL mutant mice displayed an effector memory phenotype in the long-term in comparison with the mainly central memory phenotype displayed by memory CD8+ T cells in WT mice. Memory CD8+ T cells in FasL mutant mice expressed reduced levels of IFN-gamma and displayed poor homeostatic and Ag-induced proliferation. Impairment in CD8+ T cell memory in FasL mutant hosts was not due to defective programming or the expression of mutant FasL on CD8+ T cells, but was caused by perturbed cytokine environment in FasL mutant mice. Although adoptively transferred WT memory CD8+ T cells mediated protection against L. monocytogenes in either the WT or FasL mutant hosts, FasL mutant memory CD8+ T cells failed to mediate protection even in WT hosts. Thus, in individuals with mutation in Fas pathway, impairment in the function of the memory CD8+ T cells may increase their susceptibility to recurrent/latent infections.     

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.     

CD95 stimulation results in the formation of a novel death effector domain protein-containing complex

Stimulation of CD95 (APO-1/Fas) by its natural ligand CD95L (APO-1L/FasL) leads to the formation of the death-inducing signaling complex. Here we report that upon CD95 stimulation in several T and B cell lines, a novel signaling complex is formed, which we term complex II. Complex II is composed of the death effector domain proteins as follows: procaspase-8a/b, three isoforms of c-FLIP (c-FLIP(L), c-FLIP(S), c-FLIP(R)), and FADD. Notably, complex II does not contain CD95. Based on our findings we suggest that CD95 signaling includes two steps. The first step involves formation of the death-inducing signaling complex at the cell membrane. The second step involves formation of the cytosolic death effector domain protein-containing complex that may play an important role in amplification of caspase activation.     

Fas ligand is required for resolution of granulomatous experimental autoimmune thyroiditis

We previously suggested that CD8(+) T cells promoted resolution of granulomatous experimental autoimmune thyroiditis (G-EAT) at least in part through regulation of Fas ligand (FasL) expression on thyroid epithelial cells. To directly evaluate the role of the Fas pathway in G-EAT resolution, Fas- and FasL-deficient mice on the NOD.H-2h4 background were used as recipients of activated G-EAT effector cells. When MTg-primed wild-type (WT) donor splenocytes were activated and transferred to WT recipients, thyroid lesions reached maximal severity on day 20 and resolved on day 50. Fas, FasL, and FLIP were up-regulated, and many apoptotic inflammatory cells were detected in recipient thyroids on day 20. Fas was predominantly expressed by inflammatory cells, and FasL and FLIP were mainly expressed by thyroid epithelial cells. After depletion of CD8(+) T cells, G-EAT resolution was delayed, FLIP and FasL were predominantly expressed by inflammatory cells, and few inflammatory cells were apoptotic. When WT donor splenocytes were transferred to gld recipients, disease severity on day 20 was similar to that in WT recipients, but resolution was delayed. As in CD8-depleted WT recipients, there were few apoptotic inflammatory cells, and FLIP and FasL were expressed primarily by inflammatory cells. These results indicated that the expression of functional FasL in recipient mice was critical for G-EAT resolution. WT cells induced minimal disease in lpr recipients. This was presumably because donor cells were eliminated by the increased FasL on lpr recipient cells, because donor cells were not eliminated, and the mice developed G-EAT if lpr recipients were given anti-FasL mAb.     

Cellular and molecular requirements for rejection of B16 melanoma in the setting of regulatory T cell depletion and homeostatic proliferation

We have recently demonstrated that adoptive transfer of regulatory T cell-depleted polyclonal T cells into lymphopenic mice leads to rejection of B16 melanoma, which generated an opportunity to study host requirements for tumor rejection when it effectively occurred. CD8(+) T cell priming and tumor rejection required tumor Ag cross-presentation, as evidenced by tumor outgrowth in Kb(-/-) bone marrow chimeric or B71/2(-/-) mice. CD4(+) T cells were additionally required for optimal tumor control, although not through classical CD4 "help," as the frequency of primed CD8(+) T cells was similar in the absence of CD4(+) T cells, and tumor rejection did not depend upon CD40-CD40L interactions or on IL-2 production by CD4(+) T cells. Rather, CD4(+) T cells appeared to act at the effector phase of tumor rejection and responded to B16-derived Ags in vitro. At the effector phase, IFN-γ production by transferred T cells, but not host cells, was necessary. IFN-γ acted either on host or tumor cells and was associated with reduced tumor vascularity. Finally, tumor rejection occurred after transfer of TNF-α, perforin, or FasL-deficient T cells. However, perforin/FasL double-knockout T cells failed to reject, arguing that the killing of B16 melanoma cells could occur either via the cytotoxic granule or Fas pathways. Collectively, these results support a model in which host tumor Ag cross-presentation primes adoptively transferred T cells, which remain functional in the setting of homeostatic proliferation and regulatory T cell depletion, and which promote tumor rejection via IFN-γ and lysis via cytotoxic granules and/or FasL.     

Human peripheral blood T regulatory cells (Tregs), functionally primed CCR4+ Tregs and unprimed CCR4- Tregs, regulate effector T cells using FasL

Regulatory CD25(+)CD4(+) T cells (Tregs) play an important role in the control of peripheral tolerance. In this study we demonstrate that human peripheral blood Tregs can be divided into two distinct populations based on the expression of CCR4. The majority ( approximately 75%) of freshly isolated Tregs express CCR4 and presumably represent memory-type Tregs. Interestingly, CCR4(-) Tregs require anti-CD3 Ab-mediated activation to acquire a regulatory activity, while CCR4(+) Tregs appear to be already primed to suppress the proliferation of CD8(+) T cells. CCR4 is also expressed on CD25(low)CD4(+) T cells (CCR4(+) non-Tregs) that mostly suppress Th1-type polarization without affecting T cell proliferation, presumably via the production of immunomodulatory cytokines like IL-10. In contrast, CCR4(+) Tregs express FasL to primarily regulate T cell proliferation via a contact-mediated process involving FasL/Fas signaling, a major regulatory pathway of T cell homeostasis. Finally, we also demonstrate that the depletion of CCR4(+) T cells leads to Th1-type polarization of CD4(+) T cells and augmentation of CD8(+) T cell responses to tumor Ags.     

Fas-mediated suicide of tumor-reactive T cells following activation by specific tumor: selective rescue by caspase inhibition

CD8+ T lymphocytes that specifically recognize tumor cells can be isolated and expanded ex vivo. While the lytic properties of these cells have been well described, their fate upon encounter with cognate tumor is not known. We performed reverse 51Cr release assays in which the lymphocyte effectors rather than the tumor cell targets were radioactively labeled. We found that melanoma tumor cells caused the apoptotic death of tumor-specific T cells only upon specific MHC class I-restricted recognition. This death was entirely blockable by the addition of an Ab directed against the Fas death receptor (APO-1, CD95). Contrary to the prevailing view that tumor cells cause the death of anti-tumor T cells by expressing Fas ligand (FasL), our data suggested that FasL was instead expressed by T lymphocytes upon activation. While the tumor cells did not express FasL by any measure (including RT-PCR), functional FasL (as well as FasL mRNA) was consistently found on activated anti-tumor T cells. We could successfully block the activation-induced cell death with z-VAD-fmk, a tripeptide inhibitor of IL-1 beta-converting enzyme homologues, or with anti-Fas mAbs. Most importantly, these interventions did not inhibit T cell recognition as measured by IFN-gamma release, nor did they adversely affect the specific lysis of tumor cell targets. These results imply that Fas-mediated activation-induced cell death could be a limiting factor in the in vivo efficacy of adoptive transfer of class I-restricted CD8+ T cells and provide a means of potentially enhancing their growth in vitro as well as their function in vivo.     

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.     

Loss of Fas (CD95/APO-1) expression by antigen-specific cytotoxic T cells is reversed by inhibiting DNA methylation

Elimination of clonally expanded peripheral CD8 T cells was thought to involve apoptosis induction mediated principally by TNF, but recently Fas (CD95/APO-1) has been shown to play a role in certain responses. Here we study Fas expression and sensitivity to its ligation on murine CD8 cells specific for the CW3 antigen expressed by transfected P815 cells. Fas was progressively downregulated after successive in vitro restimulations of antigen-specific CD8 cells, until clones became Fas negative and totally resistant to the effects of recombinant Fas ligand. In contrast, Fas expression by in vivo restimulated antigen-specific cells did not diminish. Loss of Fas expression in vitro was not totally irreversible, since it could be reinduced by inhibition of DNA methylation. Understanding how Fas may be differentially regulated in vivo and in vitro is an important issue for the optimal manipulation of T cells for adoptive immunotherapy protocols.     

Absence of weight loss during Cryptosporidium infection in susceptible mice deficient in Fas-mediated apoptosis

Apoptosis plays a major role in the development of pathogenesis due to a number of microbial infections. Epithelial cells have been previously shown to die through apoptosis during in vitro infection by the Apicomplexan parasite Cryptosporidium parvum. We now test the possibility that Fas (APO-1/CD95)-dependent apoptosis of uninfected cells, due to enhanced expression of the Fas ligand (FasL) on infected cells, may contribute to the pathology of cryptosporidiosis. Expression of the FasL increased by a large amount on the surface of intestinal epithelial cells infected with C. parvum, and the increase was limited exclusively to infected cells. In addition, a significant increase in FasL expression was observed in epithelial cells from the small intestine of mice infected with C. parvum. Finally, whereas wild-type mice depleted of CD4(+) lymphocytes lost weight during C. parvum infection, CD4(+) cell-depleted lpr mice (deficient in Fas function) infected with C. parvum gained weight at the same rate as undepleted wild-type or lpr mice. These results suggest that bystander Fas-dependent apoptosis of uninfected epithelial cells may exacerbate the weight loss associated with cryptosporidiosis.     

Fas expression on antigen-specific T cells has costimulatory, helper, and down-regulatory functions in vivo for cytotoxic T cell responses but not for T cell-dependent B cell responses

Fas-mediated apoptosis is an important contributor to contraction of Ag-driven T cell responses acting only on activated Ag-specific T cells. The effects of targeted Fas deletion on selected cell populations are well described however little is known regarding the consequences of Fas deletion on only activated Ag-specific T cells. We addressed this question using the parent-into-F(1) (P-->F(1)) model of acute or chronic (lupus-like) graft-vs-host disease (GVHD) as a model of either a CTL-mediated or T-dependent B cell-mediated response, respectively. By transferring Fas-deficient lpr donor T cells into Fas-intact F(1) hosts, the in vivo role of Ag-specific T cell Fas can be determined. Our results demonstrate a novel dichotomy of Ag-specific T cell Fas function in that: 1) Fas expression on Ag-activated T cells has costimulatory, helper, and down-regulatory roles in vivo and 2) these roles were observed only in a CTL response (acute GVHD) and not in a T-dependent B cell response (chronic GVHD). Specifically, CD4 T cell Fas expression is important for optimal CD4 initial expansion and absolutely required for help for CD8 effector CTL. Donor CD8 T cell Fas expression played an important but not exclusive role in apoptosis and down-regulation. By contrast, CD4 Fas expression played no detectable role in modulating chronic GVHD induction or disease expression. These results demonstrate a novel role for Ag-specific T cell Fas expression in in vivo CTL responses and support a review of the paradigm by which Fas deficiency accelerates lupus in MRL/lpr lupus-prone mice.     

A novel role of CD30L/CD30 signaling by T-T cell interaction in Th1 response against mycobacterial infection

A CD30 ligand (CD30L, CD153) is a type II membrane-associated glycoprotein belonging to the TNF family. To illustrate the potential role of CD30L in CD4(+) Th1 cell responses, we investigated the fate of Ag-specific CD4(+) T cells in CD30L-deficient (CD30L(-/-)) mice after Mycobacterium bovis bacillus Calmette-Guérin (BCG) infection. The number of bacteria was significantly higher in organs of CD30L(-/-) mice than in wild-type (WT) mice 4 wk postinfection. The numbers of purified protein derivative- or Ag85B-specific-IFN-gamma-producing-CD4(+) T cells in spleen, lung, or peritoneal exudate cells were significantly fewer in CD30L(-/-) mice than in WT mice. During the infection, CD30L was expressed mainly by CD44(+)CD3(+)CD4(+) T cells but not by CD3(+)CD8(+) T cells, B cells, dendritic cells, or macrophages. Costimulation with agonistic anti-CD30 mAb or coculturing with CD30L-transfected P815 cells restored IFN-gamma production by CD4(+) T cells from BCG-infected CD30L(-/-) mice. Coculturing with CD30L(+/+)CD4(+) T cells from BCG-infected WT mice also restored the number of IFN-gamma(+)CD30L(-/-)CD4(+) T cells. When transferred into the CD30L(+/+) mice, Ag-specific donor CD30L(-/-) CD4(+) T cells capable of producing IFN-gamma were restored to the compared level seen in CD30L(+/+) CD4(+) T cells on day 10 after BCG infection. When naive CD30L(+/+) T cells were transferred into CD30L(-/-) mice, IFN-gamma-producing-CD4(+) Th1 cells of donor origin were normally generated following BCG infection, and IFN-gamma-producing-CD30L(-/-)CD4(+) Th1 cells of host origin were partly restored. These results suggest that CD30L/CD30 signaling executed by CD30(+) T-CD30L(+) T cell interaction partly play a critical role in augmentation of Th1 response capable of producing IFN-gamma against BCG infection.