Tumor-induced apoptosis of human IL-2-activated NK cells: role of natural cytotoxicity receptors

We provide evidence that tumor cells can induce apoptosis of NK cells by engaging the natural cytotoxicity receptors (NCR) NKp30, NKp44, and NKp46. Indeed, the binding between NCR on NK cells and their putative ligands on tumor target cells led to NK cell apoptosis, and this event was abolished by blocking NCR/NCR-ligand interaction by anti-NCR-specific mAbs. The engagement of NCR induced up-regulation of Fas ligand (FasL) mRNA, FasL protein synthesis, and release. In turn, FasL interacting with Fas at NK cell surface causes NK cell suicide, as apoptosis of NK cells was inhibited by blocking FasL/Fas interaction with specific mAbs. Interestingly, NK cell apoptosis, but not killing of tumor target cells, is inhibited by cyclosporin A, suggesting that apoptosis and cytolysis are regulated by different biochemical pathways. These findings indicate that NCR are not only triggering molecules essential for antitumor activity, but also surface receptors involved in NK cell suicide.     

Aberrant CD3- and CD28-mediated signaling events in cord blood T cells are associated with dysfunctional regulation of Fas ligand-mediated cytotoxicity

There have been numerous reports of decreased acute and chronic graft-vs-host disease (GVHD) in patients receiving HLA-matched or HLA-disparate umbilical cord transplants. However, little is known about the mechanisms underlying the low incidence of GVHD in umbilical cord blood transplantation (CBT). In this study, we examined CD3- and CD28-mediated functional properties and signaling events in CB T cells (CBTCs). Dual stimulation of peripheral blood TCs (PBTCs) and bone marrow TCs (BMTCs) with mAbs to CD3- and CD28-induced expressions of Fas ligand (FasL), as well as CD25 and CD154 (CD40L), whereas defective induction of these activation-associated cell surface molecules were observed in CBTCs. Engagement of both CD3 and CD28 induced FasL-mediated cytotoxicity in peripheral blood TCs (PBTCs) but not CBTCs; however, both of these tissue sources possess intrinsically similar proliferative responsiveness. Analysis of CD3- and CD28-induced signal transduction revealed a deficiency in signaling events that involved repressed tyrosine phosphorylation and enzymatic activities of a family of mitogen-activated protein kinases, extracellular signal-regulated kinase 2, stress-activated protein kinase/c-jun N-terminal kinase (SAPK/JNK), and p38mapk, as well as p56lck and ZAP-70 in CBTCs compared with those in PBTCs. These results suggest that CD3- and CD28-mediated signaling events blockage in CBTCs may be responsible for dysfunction of FasL-mediated cytotoxicity and lead to the low incidence of severe GVHD in CBT.     

Disparate cytotoxic activity of nickel-specific CD8+ and CD4+ T cell subsets against keratinocytes

Allergic contact dermatitis (ACD) is the result of an exaggerated immune reaction to haptens mediated by skin-homing T cells, but the effector mechanisms responsible for the tissue damage are poorly understood. Here we studied the capacity of distinct subsets of hapten-specific T cells to induce apoptosis in autologous keratinocytes. Skin- and blood-derived nickel-specific CD8+ T cytotoxic 1 (Tc1) and Tc2 clones as well as CD4+ Th1 and Th2 expressed the cutaneous lymphocyte-associated Ag and exhibited strong MHC-restricted cytotoxicity against nickel-coupled B lymphoblasts, as detected by the [3H]TdR release assay. Both Tc1 and Tc2 clones, but not CD4+ T cells, displayed a significant cytotoxic activity against resting nickel-modified keratinocytes. Following IFN-gamma treatment, keratinocytes expressed MHC class II and ICAM-1 and became susceptible to Th1-mediated, but not Th2-mediated, cytotoxicity. The molecules of the two major cytotoxic pathways, Fas ligand (FasL) and perforin, were expressed by Tc1, Tc2, and Th1 cells, whereas Th2 cells expressed only FasL. Experiments performed in the presence of specific inhibitors of the perforin (concanamycin A) and FasL (brefeldin A) pathway indicated that perforin-mediated killing dominated in Tc1 and Tc2, and FasL-mediated cytotoxicity prevailed in Th2 clones, with a more heterogeneous behavior in the case of Th1 cells. Finally, perforin mRNA was expressed in ACD lesional skin, as assessed by RT-PCR analysis. In aggregate, our results indicate that keratinocytes can be target of multiple hapten-specific CTL responses, that may have distinct roles in the epidermal injury during ACD.     

Loss of B7-H1 expression by recipient parenchymal cells leads to expansion of infiltrating donor CD8+ T cells and persistence of graft-versus-host disease

Previous experimental studies have shown that acute graft-versus-host disease (GVHD) is associated with two waves of donor CD8(+) T cell expansion. In the current studies, we used in vivo bioluminescent imaging, in vivo BrdU labeling, and three different experimental GVHD systems to show that B7-H1 expression by recipient parenchymal cells controls the second wave of alloreactive donor CD8(+) T cell expansion and the associated second phase of GVHD. Loss of B7-H1 expression by parenchymal cells during the course of GVHD was associated with persistent proliferation of donor CD8(+) T cells in GVHD target tissues and continued tissue injury, whereas persistent expression of B7-H1 expression by parenchymal cells led to reduced proliferation of donor CD8(+) T cells in GVHD target tissues and resolution of GVHD. These studies demonstrate that parenchymal cell expression of B7-H1 is required for tolerizing infiltrating T cells and preventing the persistence of GVHD. Our results suggest that therapies designed to preserve or restore expression of B7-H1 expression by parenchymal tissues in the recipient could prevent or ameliorate GVHD in humans.     

Effect of graft-versus-host disease (GVHD) on host hematopoietic progenitor cells is mediated by Fas-Fas ligand interactions but this does not explain the effect of GVHD on donor cells.

The acute graft-versus-host disease (GVHD) generated in BDF1 mice by the injection of spleen cells from the C57BL/6 parental strain induces a direct cell-mediated attack on host lymphohematopoietic populations, resulting in the reconstitution of the host with donor hematopoietic stem cells. We examined the effect of GVHD on the donor and host hematopoiesis in parental-induced acute GVHD. The bone marrow was hypoplastic and the number of hematopoietic progenitor cells significantly decreased at 4 weeks after GVHD induction. However, extramedullary splenic hematopoiesis was present and the number of hematopoietic progenitor cells in the spleen significantly increased at this time. Fas expression on the host spleen cells and bone marrow cells significantly increased during weeks 2 to 8 of GVHD. Host cell incubation with anti-Fas Ab induced apoptosis, and the number of hematopoietic progenitor cells decreased during these weeks. A significant correlation between the augmented Fas expression on host bone marrow cells and the decreased number of host bone marrow cells by acute GVHD was observed. Furthermore, the injection of Fas ligand (FasL)-deficient B6/gld spleen cells failed to affect host bone marrow cells. Although Fas expression on repopulating donor cells also increased, Fas-induced apoptosis by the repopulating donor cells was not remarkable until 12 weeks, when more than 90% of the cells were donor cells. The number of hematopoietic progenitor cells in the bone marrow and the spleen by the repopulating donor cells, however, decreased over an extended time during acute GVHD. This suggests that Fas-FasL interactions may regulate suppression of host hematopoietic cells but not of donor hematopoietic cells. Hematopoietic dysfunctions caused by the reconstituted donor cells are independent to Fas-FasL interactions and persisted for a long time during parental-induced acute GVHD.     

IFN-gamma and Fas ligand are required for graft-versus-tumor activity against renal cell carcinoma in the absence of lethal graft-versus-host disease

To determine the mechanisms of graft-versus-tumor (GVT) activity in the absence of graft-versus-host disease (GVHD) against a solid tumor, we established two allogeneic bone marrow transplantation models with a murine renal cell carcinoma (RENCA). The addition of 0.3 x 10(6) donor CD8(+) T cells to the allograft increased the survival of tumor-bearing mice without causing GVHD. The analysis of CD8(+) T cells deficient in cytotoxic molecules demonstrated that anti-RENCA activity is dependent on IFN-gamma and Fas ligand (FasL), but does not require soluble or membrane-bound TNF-alpha, perforin, or TRAIL. Recipients of IFN-gamma(-/-) CD8(+) T cells are unable to reject RENCA compared with recipients of wild-type CD8(+) T cells and, importantly, neither group develops severe GVHD. IFN-gamma(-/-) CD8(+) T cells derived from transplanted mice are less able to kill RENCA cells in vitro, while pretreatment of RENCA cells with IFN-gamma enhances class I and FasL expression and rescues the lytic capacity of IFN-gamma(-/-) CD8(+) T cells. These results demonstrate that the addition of low numbers of selected donor CD8(+) T cells to the allograft can mediate GVT activity without lethal GVHD against murine renal cell carcinoma, and this GVT activity is dependent on IFN-gamma and FasL.     

Vasoactive intestinal peptide and pituitary adenylate cyclase-activating polypeptide inhibit T cell-mediated cytotoxicity by inhibiting Fas ligand expression

We reported recently that the neuropeptides vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP) protect CD4+ T cells against Ag-induced apoptosis by down-regulating the expression of Fas ligand (FasL). Because the cytotoxic activity of CD8+ CTLs is mediated through two mechanisms, which involve the perforin/granzyme and the FasL/Fas pathways, in this study we investigated the effects of VIP/PACAP on the generation and activity of allogeneic CTLs, of CD8+ T1 and T2 effector cells and of alloreactive peritoneal exudate cytotoxic T cells (PEL) generated in vivo. VIP/PACAP did not affect perforin/granzyme-mediated cytotoxicity, perforin gene expression, or granzyme B enzymatic activity, but drastically inhibited FasL/Fas-mediated cytotoxicity against allogeneic or syngeneic Fas-bearing targets. VIP/PACAP inhibit CTL generation, but not the activity of competent CTLs. The inhibition is associated with a profound down-regulation of FasL expression, and these effects are mediated through both VPAC1 and VPAC2 receptors. VIP/PACAP inhibit the FasL/Fas-mediated cytotoxicity of T1 effectors and do not affect T2 cytotoxicity, which is entirely perforin/granzyme mediated. Similar effects were observed in vivo. Both the FasL/Fas-mediated cytotoxicity and FasL expression of cytotoxic allogeneic PELs generated in vivo in the presence of VIP or PACAP were significantly reduced. We conclude that, similar to their effect on CD4+ T cells, the two structurally related neuropeptides inhibit FasL expression in CD8+ cytotoxic T cells and the subsequent lysis of Fas-bearing target cells.     

Cutting edge: RANTES regulates Fas ligand expression and killing by HIV-specific CD8 cytotoxic T cells.

Based on the previous observation that RANTES mediates the cytotoxic activity of human HIV-specific CD8+ T cells via the chemokine receptor CCR3, we studied the effect of this chemokine on different effector CD8+ cytolytic cells requiring Fas/Fas ligand (FasL) or perforin-dependent pathway. In CTLs derived from PBMCs of HIV-infected patients, both the spontaneous and the RANTES-induced cytotoxicity were inhibited by anti-FasL neutralizing Abs. In contrast, allogeneic CTLs or NK cells killing through perforin were not affected by RANTES and anti-FasL Ab. Accordingly, RANTES enhanced the expression of FasL in a concentration- and time-dependent manner in HIV-specific CTLs, whereas anti-RANTES Ab decreased markedly FasL expression. Finally, cell surface expression of FasL protein in HIV-specific CTLs was also up-regulated by eotaxin, a selective ligand for CCR3. Our observations show that the action of RANTES via CCR3 is necessary to regulate FasL expression on HIV-specific CD8+ T cells that kill through the Fas/FasL pathway.     

Crosstalk between keratinocytes and T lymphocytes via Fas/Fas ligand interaction: modulation by cytokines.

Apoptosis mediated by Fas/FasL interaction plays an important role during many inflammatory skin disorders. To estimate whether the expression of FasL, the ligand for Fas, might be regulated by cytokines we stimulated primary human keratinocytes with several pro- and anti-inflammatory cytokines. Keratinocytes cultured to subconfluence expressed FasL constitutively. Cells stimulated with the proinflammatory cytokines IL-1beta, TNF-alpha, IFN-gamma, and IL-15, respectively, increased significantly their intracellular as well as cell surface-bound FasL expression in a time- and dose-dependent manner. This cytokine-induced FasL expression was dependent on new protein synthesis. Despite enhanced expression of cell surface-bound FasL, no release of soluble FasL was measured in the cell supernatants determined by ELISA. Stimulation of the cells with IL-6, IL-10, IL-12, TGF-beta1, and GM-CSF did not modulate the constitutive FasL expression, but IFN-gamma-mediated FasL up-regulation was significantly diminished by IL-10 and TGF-beta1, respectively. Up-regulation of FasL on IFN-gamma-stimulated keratinocytes led to increased apoptosis within monolayers cultured for 48 h. Moreover, coculture experiments performed with Fas+ Jurkat T cells revealed that enhanced FasL expression on IFN-gamma-stimulated keratinocytes induced apoptosis in cocultured T cells, demonstrating that up-regulated FasL was functionally active. In summary, our data suggest the important regulatory role of cytokine-controlled Fas/FasL interaction in the cross-talk between keratinocytes and skin-infiltrating T cells for maintenance of homeostasis in inflammatory skin processes.     

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.     

Differential T cell response in central and peripheral nerve injury: connection with immune privilege.

The central nervous system (CNS), unlike the peripheral nervous system (PNS), is an immune-privileged site in which local immune responses are restricted. Whereas immune privilege in the intact CNS has been studied intensively, little is known about its effects after trauma. In this study, we examined the influence of CNS immune privilege on T cell response to central nerve injury. Immunocytochemistry revealed a significantly greater accumulation of endogenous T cells in the injured rat sciatic nerve than in the injured rat optic nerve (representing PNS and CNS white matter trauma, respectively). Use of the in situ terminal deoxytransferase-catalyzed DNA nick end labeling (TUNEL) procedure revealed extensive death of accumulating T cells in injured CNS nerves as well as in CNS nerves of rats with acute experimental autoimmune encephalomyelitis, but not in injured PNS nerves. Although Fas ligand (FasL) protein was expressed in white matter tissue of both systems, it was more pronounced in the CNS. Expression of major histocompatibility complex (MHC) class II antigens was found to be constitutive in the PNS, but in the CNS was induced only after injury. Our findings suggest that the T cell response to central nerve injury is restricted by the reduced expression of MHC class II antigens, the pronounced FasL expression, and the elimination of infiltrating lymphocytes through cell death.     

The critical role of protein kinase C-theta in Fas/Fas ligand-mediated apoptosis

A functional immune system not only requires rapid expansion of antigenic specific T cells, but also requires efficient deletion of clonally expanded T cells to avoid accumulation of T cells. Fas/Fas ligand (FasL)-mediated apoptosis plays a critical role in the deletion of activated peripheral T cells, which is clearly demonstrated by superantigen-induced expansion and subsequent deletion of T cells. In this study, we show that in the absence of protein kinase C-theta (PKC-theta), superantigen (staphylococcal enterotoxin B)-induced deletion of Vbeta8(+) CD4(+) T cells was defective in PKC-theta(-/-) mice. In response to staphylococcal enterotoxin B challenge, up-regulation of FasL, but not Fas, was significantly reduced in PKC-theta(-/-) mice. PKC-theta is thus required for maximum up-regulation of FasL in vivo. We further show that stimulation of FasL expression depends on PKC-theta-mediated activation of NF-AT pathway. In addition, PKC-theta(-/-) T cells displayed resistance to Fas-mediated apoptosis as well as activation-induced cell death (AICD). In the absence of PKC-theta, Fas-induced activation of apoptotic molecules such as caspase-8, caspase-3, and Bid was not efficient. However, AICD as well as Fas-mediated apoptosis of PKC-theta(-/-) T cells were restored in the presence of high concentration of IL-2, a critical factor required for potentiating T cells for AICD. PKC-theta is thus required for promoting FasL expression and for potentiating Fas-mediated apoptosis.     

Apoptosis-inducing membrane vesicles. A novel agent with unique properties

The CD95 ligand (FasL) transmembrane protein is found on activated T cells and cells outside the immune system. A well-known turnover process of membrane FasL is mediated by matrix metalloproteinase, which generates soluble FasL (sFasL). Here, we demonstrate that membrane FasL turnover occurs effectively through the release of membrane vesicles. Quantitative analysis indicates that this process is as effective as sFasL release for FasL-3T3 cells but somewhat less effective for FasL-expressing T cells. The apoptosis-inducing membrane vesicles display unique properties not found in FasL-expressing cells and sFasL. Unlike sFasL, vesicle-associated FasL remained bioactive, killing the same panel of targets that are susceptible to FasL-expressing cells. In contrast to FasL-expressing T cells, FasL-mediated killing by vesicles do not involve LFA-1/ICAM interaction and do not depend on de novo protein synthesis. These observations indicate that the release of FasL-bearing vesicles contributes to the turnover of cell-associated FasL, but the impact of the bioactive FasL-expressing vesicles on the function of cell-associated FasL is different from that of sFasL.     

Fas ligand is enriched in the caveolae membrane domains of thymic epithelial cells

Both Fas and Fas ligand (FasL) are expressed in the thymus. Although reports suggest that they are important throughout the thymocyte maturation process their precise role remains elusive. The present paper characterizes the expression of FasL in the thymus and in the TEA3A1 and BT1B functional thymic epithelial cell (TEC) lines. FasL expression by thymus fractions, TEA3A1, and BT1B cells was detected by Northern blot analysis. In TEA3A1 cells, we discovered that FasL protein expression was localized to caveolae membrane domains. This restricted subcellular localization of FasL, together with reports describing the localization of the major histocompatibility complex proteins, the T cell receptor and Fas to caveolae membrane domains, may provide a mechanism for the deletion of thymocytes during negative selection. Finally, using semi-quantitative RT-PCR we found that FasL expression by TECs is regulated by glucocorticoids.     

Preferential blockade of CD8(+) T cell responses by administration of anti-CD137 ligand monoclonal antibody results in differential effect on development of murine acute and chronic graft-versus-host diseases.

We investigated the effect of CD137 costimulatory blockade in the development of murine acute and chronic graft-vs-host diseases (GVHD). The administration of anti-CD137 ligand (anti-CD137L) mAb at the time of GVHD induction ameliorated the lethality of acute GVHD, but enhanced IgE and anti-dsDNA IgG autoantibody production in chronic GVHD. The anti-CD137L mAb treatment efficiently inhibited donor CD8(+) T cell expansion and IFN-gamma expression by CD8(+) T cells in both GVHD models and CD8(+) T cell-mediated cytotoxicity against host-alloantigen in acute GVHD. However, a clear inhibition of donor CD4(+) T cell expansion and activation has not been observed. On the contrary, in chronic GVHD, the number of CD4(+) T cells producing IL-4 was enhanced by anti-CD137L mAb treatment. This suggests that the reduction of CD8(+) T cells producing IFN-gamma promotes Th2 cell differentiation and may result in exacerbation of chronic GVHD. Our results highlight the effective inactivation of CD8(+) T cells and the lesser effect on CD4(+) T cell inactivation by CD137 blockade. Intervention of the CD137 costimulatory pathway may be beneficial for some selected diseases in which CD8(+) T cells are major effector or pathogenic cells. Otherwise, a combinatorial approach will be required for intervention of CD4(+) T cell function.     

ADAM10 regulates FasL cell surface expression and modulates FasL-induced cytotoxicity and activation-induced cell death

The apoptosis-inducing Fas ligand (FasL) is a type II transmembrane protein that is involved in the downregulation of immune reactions by activation-induced cell death (AICD) as well as in T cell-mediated cytotoxicity. Proteolytic cleavage leads to the generation of membrane-bound N-terminal fragments and a soluble FasL (sFasL) ectodomain. sFasL can be detected in the serum of patients with dysregulated inflammatory diseases and is discussed to affect Fas-FasL-mediated apoptosis. Using pharmacological approaches in 293T cells, in vitro cleavage assays as well as loss and gain of function studies in murine embryonic fibroblasts (MEFs), we demonstrate that the disintegrin and metalloprotease ADAM10 is critically involved in the shedding of FasL. In primary human T cells, FasL shedding is significantly reduced after inhibition of ADAM10. The resulting elevated FasL surface expression is associated with increased killing capacity and an increase of T cells undergoing AICD. Overall, our findings suggest that ADAM10 represents an important molecular modulator of FasL-mediated cell death.     

Involvement of TNF-related apoptosis-inducing ligand in human CD4+ T cell-mediated cytotoxicity

TNF-related apoptosis-inducing ligand (TRAIL) has been identified as a member of the TNF family that induces apoptosis in a variety of tumor cells, but its physiological functions are largely unknown. In the present study, we examined the expression and function of TRAIL in human CD4+ T cell clones by utilizing newly established anti-human TRAIL mAbs. Human CD4+ T cell clones, HK12 and 4HM1, exhibited perforin-independent and Fas ligand (FasL)-independent cytotoxicity against certain target cells, including T lymphoma (Jurkat) and keratinocyte (HaCaT) cell lines, which are susceptible to TRAIL-mediated cytotoxicity. In contrast to FasL, the expression of which was inducible upon anti-CD3 stimulation, TRAIL was constitutively expressed on HK12 and 4HM1 cells, and no further increase was observed after anti-CD3 stimulation. Spontaneous cytotoxic activities of resting HK12 and 4HM1 cells against Jurkat and HaCaT cells were blocked by anti-TRAIL mAb but not by anti-FasL mAb, and bystander cytotoxic activities of anti-CD3-stimulated HK12 and 4HM1 cells were abolished by the combination of anti-TRAIL and anti-FasL mAbs. These results indicate a differential regulation of TRAIL and FasL expression on human CD4+ T cell clones and that TRAIL constitutes an additional pathway of T cell-mediated cytotoxicity.