Treatment of melanoma with 5-fluorouracil or dacarbazine in vitro sensitizes cells to antigen-specific CTL lysis through perforin/granzyme- and Fas-mediated pathways

Several factors may influence sensitivity of melanoma cells to CTL lysis. One is the avidity of the CTL TCR. A second is that certain cytotoxic drugs have been reported to sensitize cancer cells to CTL lysis through Fas-mediated apoptosis. In this study, we examined whether antineoplastic agents 5-fluorouracil (5-FU) and dacarbazine (DTIC) sensitize melanoma cells to lysis of G209 peptide-specific CTL. Our results show that CTL generated from PBMC are HLA-A2 restricted and gp100 specific. Treatment with 5-FU or DTIC sensitized melanoma cells to lysis of G209-specific CTL. Most importantly, 5-FU- or DTIC-treated melanoma cells also became sensitive to low-avidity CTL, which per se are less cytolytic to melanomas. We sought to identify apoptotic pathways mediating this effect. The enhanced cytolysis was mediated through the perforin/granzyme pathway. Although 5-FU up-regulated FasR expression on melanoma cells, sensitization was not blocked by anti-Fas Ab, and the G209-specific CTL was Fas ligand (FasL) negative. However, when G209-specific CTL were stimulated to express FasL, FasL signaling also contributed to enhanced cytolysis. DTIC treatment, which did not increase FasR expression, also sensitized FasL-mediated killing induced by neutralizing anti-Fas Ab. For CD95L-positive G209-specific CTL, the sensitization was primarily mediated through the perforin/granzyme pathway regardless of up-regulation of FasR. The findings demonstrate that cytotoxic drug-mediated sensitization primes both perforin/granzyme and Fas-mediated killing by melanoma-specific CTL. Considering that most of autoreactive antitumor CTL are low avidity, the findings provide experimental basis for understanding cytotoxic and immunologic therapeutic synergy in melanoma.     

Tumor-specific CTL kill murine renal cancer cells using both perforin and Fas ligand-mediated lysis in vitro, but cause tumor regression in vivo in the absence of perforin

Kidney cancer is a devastating disease; however, biological therapies have achieved some limited success. The murine renal cancer Renca has been used as a model for developing new preclinical approaches to the treatment of renal cell carcinoma. Successful cytokine-based approaches require CD8(+) T cells, but the exact mechanisms by which T cells mediate therapeutic benefit have not been completely identified. After successful biological therapy of Renca in BALB/c mice, we generated CTLs in vitro using mixed lymphocyte tumor cultures. These CTL mediated tumor-specific H-2K(d)-restricted lysis and production of IFN-gamma, TNF-alpha, and Fas ligand (FasL) in response to Renca. CTL used both granule- and FasL-mediated mechanisms to lyse Renca, although granule-mediated killing was the predominant lytic mechanism in vitro. The cytokines IFN-gamma and TNF-alpha increased the sensitivity of Renca cells to CTL lysis by both granule- and FasL-mediated death pathways. Adoptive transfer of these anti-Renca CTL into tumor-bearing mice cured most mice of established experimental pulmonary metastases, and successfully treated mice were immune to tumor rechallenge. Interestingly, we were able to establish Renca-specific CTL from mice gene targeted for perforin (pfp(-/-)) mice. Although these pfp(-/-) CTL showed reduced cytotoxic activity against Renca, their IFN-gamma production in the presence of Renca targets was equivalent to that of wild-type CTL, and adoptive transfer of pfp(-/-) CTL was as efficient as wild-type CTL in causing regression of established Renca pulmonary metastases. Therefore, although granule-mediated killing is of paramount importance for CTL-mediated lysis in vitro, some major in vivo effector mechanisms clearly are independent of perforin.     

CTLs contain and use intracellular stores of FasL distinct from cytolytic granules

CTL lyse target cells through the release of cytolytic granule contents and cell surface expression of Fas ligand (FasL). Current models suggest that FasL is stored in cytolytic granules and that FasL cell surface expression would be subject to the same controls as degranulation. We demonstrate that murine CTLs undergo two waves of FasL cell surface expression after stimulation. The first wave is from a pre-existing pool of FasL, and the second wave requires new protein synthesis. Signaling for FasL expression appears to be finely tuned as a weak signal preferentially induced surface translocation of the stored FasL, whereas a strong signal preferentially triggered the expression of de novo synthesized FasL. The early FasL is differentially regulated from degranulation, as there were multiple circumstances whereby rapid FasL cell surface expression and FasL-dependent killing occurred in the absence of detectable degranulation. Furthermore, we found through confocal microscopy that stored FasL resides in vesicles distinct from cytolytic granules. Our data clearly show that CTL degranulation and FasL lytic mechanisms are fully independent with respect to stored component localization and regulation.     

Differential requirement for IFN-gamma in CTL maturation in acute murine graft-versus-host disease

Although IFN-gamma is the archetypal Th1 cytokine, its role in CTL maturation is uncertain. We used an in vivo mouse model of CTL development, parent-into-F(1) acute graft-vs-host disease (AGVHD), to evaluate this issue. In AGVHD, transfer of naive parental T cells into F(1) hosts stimulates the development of allospecific CTL effectors that eliminate host lymphocytes, particularly B cells. Complete elimination of IFN-gamma, using IFN-gamma-deficient donors and administering anti-IFN-gamma mAb, suppressed B cell elimination, down-regulated TNF-alpha production, and enhanced Th2 cytokine production, but did not allow the B cell expansion characteristic of chronic GVHD (CGVHD). Because complete CTL inhibition results in full-blown CGVHD that is IFN-gamma independent, these observations indicate that IFN-gamma elimination only partially blocks CTL development. IFN-gamma elimination did not inhibit donor T cell engraftment or activation in the AGVHD model, but almost completely blocked Fas/Fas ligand (FasL) gene expression, protein up-regulation, and Fas/FasL-mediated CTL killing. In contrast, IFN-gamma elimination only partially inhibited perforin gene expression and perforin-mediated CTL activity. The contributions of IFN-gamma to CTL development were indirect, because IFN-gamma receptor-deficient donor cells differentiated normally into allospecific CTLs. Consistent with the view that the Fas/FasL and perforin pathways each mediate CTL killing in AGVHD, the absence of both perforin and IFN-gamma (perforin knockout donor cells and anti-IFN-gamma mAb) converted AGVHD to CGVHD. Thus, both IFN-gamma-dependent induction of Fas/FasL and IFN-gamma-independent induction of perforin contribute to CTL-mediated elimination of host B cells in AGVHD. Suppression of both pathways is required for typical CGVHD development.     

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.     

Interleukin-4 Diminishes CD8+ Respiratory Syncytial Virus-Specific Cytotoxic T-Lymphocyte Activity In Vivo

Although interleukin-4 (IL-4) has been implicated in respiratory syncytial virus (RSV)-enhanced disease, the mechanism by which it modulates immune responses to primary RSV infection remains unclear. We have developed a system to investigate the effect of IL-4 on RSV epitope-specific cytotoxic T-lymphocyte (CTL) effector function in vivo, using an H-2K(d)-restricted RSV M2 epitope. BALB/c mice were infected with recombinant vaccinia virus (rVV) constructed to express RSV M2 protein (vvM2) alone or coexpress M2 and IL-4 (vvM2/IL-4). Splenocytes were assessed for M2-specific CTL activity in a direct (51)Cr release assay and intracellular gamma interferon (IFN-gamma) production by fluorescence-activated cell sorting analysis. Mice infected with vvM2/IL-4 had less M2-specific primary CTL activity than those infected with vvM2. M2-specific CTL frequency, as measured by M2 peptide-induced intracellular IFN-gamma production, was diminished in the vvM2/IL-4 group, partially accounting for the reduction of CTL activity. Mice immunized with either construct were challenged intravenously with RSV 4 weeks postimmunization, and direct CTL were measured. These results demonstrate that local expression of IL-4, at the time of antigen presentation, diminishes the cytolytic activity of primary and memory CD8(+) RSV-specific CTL responses in vivo.     

Localization of Fas ligand in cytoplasmic granules of CD8+ cytotoxic T lymphocytes and natural killer cells: participation of Fas ligand in granule exocytosis model of cytotoxicity

Fas ligand (FasL) has been implicated in cytotoxic T lymphocyte (CTL)- and natural killer (NK) cell-mediated cytotoxicity. In the present study, we investigated the localization of FasL in murine CTL and NK cells. Immunocytochemical staining showed that FasL was stored in cytoplasmic granules of CD8+ CTL clones and in vivo activated CTL and NK cells, where perforin and granzyme A also resided. Immunoelectron microscopy revealed that FasL was localized on outer membrane of the cytoplasmic granules, while perforin was localized in internal vesicles. Western blot analysis showed that the membrane-type FasL of 40 kDa was stored in CD8+ CTL clones but not in CD4+ CTL clones. By utilizing a granule exocytosis inhibitor (TN16), we demonstrated that FasL translocated onto cell surface upon degranulation of anti-CD3-stimulated CD8+ CTL clones. Moreover, TN16 markedly inhibited the FasL-mediated cytotoxicity by CD8+ T cell clones and NK cells. These results suggested a substantial contribution of FasL to granule exocytosis-mediated target cell lysis by CD8+ CTL and NK cells.     

RKTS-33, an epoxycyclohexenone derivative that specifically inhibits Fas ligand-dependent apoptosis in CTL-mediated cytotoxicity

Cytotoxic T lymphocytes (CTLs) eliminate virus-infected cells and tumor cells by two distinct killing pathways, mediated by lytic granules containing perforin and by Fas ligand (FasL). ECH [(2R,3R,4S)-2,3-epoxy-4-hydroxy-5-hydroxymethyl-6-(1E)-propenyl-cyclohex-5-en-1-one] has been shown to inhibit FasL-dependent apoptosis or the killing pathway in short-term culture. However, since ECH exhibited cell toxicity in long-term culture, we attempted the synthesis of less toxic epoxycyclohexenone derivatives. In the present study, we found that RKTS-33 [(2R,3R,4S)-2,3-epoxy-4-hydroxy-5-hydroxymethyl-cyclohex-5-en-1-one] has cell toxicity lower than ECH in long-term culture, and further investigated the inhibitory effect of RKTS-33 on CTL-mediated killing pathways. RKTS-33 did not affect cell-surface expression of FasL upon CD3 stimulation, but profoundly inhibited the FasL-dependent killing pathway mediated by CD4+ and CD8+ CTLs, indicating that RKTS-33 specifically blocks target cell apoptosis but not CTL function. By contrast, RKTS-33 did not affect the perforin-dependent killing pathway in CD8+ CTLs. These results indicate that RKTS-33 is a specific inhibitor of the FasL-dependent killing pathway in CTL-mediated cytotoxicity.     

ECH, an epoxycyclohexenone derivative that specifically inhibits Fas ligand-dependent apoptosis in CTL-mediated cytotoxicity

CTL eliminate cells infected with intracellular pathogens and tumor cells by two distinct mechanisms mediated by Fas ligand (FasL) and lytic granules that contain perforin and granzymes. In this study we show that an epoxycyclohexenone derivative,(2R,3R,4S)-2,3-epoxy-4-hydroxy-5-hydroxymethyl-6-(1E)-propenyl-cyclohex-5-en-1-one (ECH) specifically inhibits the FasL-dependent killing pathway in CTL-mediated cytotoxicity. Recently, we have reported that ECH blocks activation of procaspase-8 in the death-inducing signaling complex and thereby prevents apoptosis induced by anti-Fas Ab or soluble FasL. Consistent with this finding, ECH profoundly inhibited Fas-mediated DNA fragmentation and cytolysis of target cells induced by perforin-negative mouse CD4+ CTL and alloantigen-specific mouse CD8+ CTL pretreated with an inhibitor of vacuolar type H+-ATPase concanamycin A that selectively induces inactivation and proteolytic degradation of perforin in lytic granules. However, ECH barely influenced perforin/granzyme-dependent DNA fragmentation and cytolysis of target cells mediated by alloantigen-specific mouse CD8+ CTL. The components of lytic granules and the granule exocytosis pathway upon CD3 stimulation were also insensitive to ECH. In conclusion, our present results demonstrate that ECH is a specific nonpeptide inhibitor of FasL-dependent apoptosis in CTL-mediated cytotoxicity. Therefore, ECH can be used as a bioprobe to evaluate the contributions of two distinct killing pathways in various CTL-target settings.     

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.     

Immunosensitization of melanoma tumor cells to non-MHC Fas-mediated killing by MART-1-specific CTL cultures

The discovery of human melanoma rejection Ags has allowed the rational design of immunotherapeutic strategies. One such Ag, MART-1, is expressed on >90% of human melanomas, and CTL generated against MART-1(27-35) kill most HLA A2.1(+) melanoma cells. However, variant tumor cells, which do not express MART-1, down-regulate MHC, or become resistant to apoptosis, will escape killing. Cytotoxic lymphocytes kill by two main mechanisms, the perforin/granzyme degranulation pathway and the TNF/Fas/TNF-related apoptosis-inducing ligand superfamily of apoptosis-inducing ligands. In this study, we examined whether cis-diaminedichloroplatinum (II) cisplatin (CDDP) sensitizes MART-1/HLA A2.1(+) melanoma and melanoma variant tumor cells to non-MHC-restricted, Fas ligand (FasL)-mediated killing by CTL. MART-1(27-35)-specific bulk CTL cultures were generated by pulsing normal PBL with MART-1(27-35) peptide. These CTL cultures specifically kill M202 melanoma cells (MART-1(+), HLA A2.1(+), FasR(-)), and MART-1(27-35) peptide-pulsed T2 cells (FasR(+)), but not M207 melanoma cells (MART-1(+), HLA A2.1(-), FasR(-)), FLU(58-66) peptide-pulsed T2 cells, or DU145 and PC-3 prostate cells (MART-1(-), HLA A2.1(-), FasR(+)). CDDP (0.1-10 microg/ml) sensitized non-MART-1(27-35) peptide-pulsed T2 to the CD8(+) subset of bulk MART-1-specific CTL, and killing was abolished by neutralizing anti-Fas Ab. Furthermore, CDDP up-regulated FasR expression and FasL-mediated killing of M202, and sensitized PC-3 and DU145 to killing by bulk MART-1-specific CTL cultures. These findings demonstrate that drug-mediated sensitization can potentiate FasL-mediated killing by MHC-restricted CTL cell lines, independent of MHC and MART-1 expression on tumor cells. This represents a novel approach for potentially controlling tumor cell variants found in primary heterogeneous melanoma tumor cell populations that would normally escape killing by MART-1-specific immunotherapy.     

Gammadelta T cells promote a Th1 response during coxsackievirus B3 infection in vivo: role of Fas and Fas ligand

Fas/Fas ligand (FasL) interactions regulate disease outcome in coxsackievirus B3 (CVB3)-induced myocarditis. MRL(+/+) mice infected with CVB3 develop severe myocarditis, a dominant CD4(+) Th1 (gamma interferon [IFN-gamma(+)]) response to the virus, and a predominance of gammadelta T cells in the myocardial infiltrates. MRL lpr/lpr and MRL gld/gld mice, which lack normal expression of Fas and express a mutated FasL, respectively, have minimal myocarditis and show a dominant CD4(+) Th2 (interleukin-4 [IL-4(+)]) phenotype to CVB3. Spleen cells from virus-infected wild-type, lpr, and gld animals proliferate equally to virus in vitro. Adoptive transfer of gammadelta T cells from hearts of CVB3-infected MRL(+/+) mice (FasL(+)) into infected MRL gld/gld recipients (FasL(-)/Fas(+)) restores both disease susceptibility and Th1 cell phenotype. However, transfer of these cells into MRL lpr/lpr recipients (FasL(+)/Fas(-)) did not promote myocarditis and the viral response remained Th2 biased. This paralleled the expression of very high surface levels of FasL by myocardial gammadelta T cells, as well as their propensity to selectively lyse Th2 virus-specific CD4(+) T cells. These results demonstrate that Fas/FasL interactions conferred by gammadelta T cells on lymphocyte subpopulations may regulate the cytokine response to CVB3 infection and pathogenicity.     

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.     

Alternative mechanisms of respiratory syncytial virus clearance in perforin knockout mice lead to enhanced disease

Virus-specific cytotoxic T lymphocytes are key effectors for the clearance of virus-infected cells and are required for the normal clearance of respiratory syncytial virus (RSV) in mice. Although perforin/granzyme-mediated lysis of infected cells is thought to be the major molecular mechanism used by CD8(+) cytotoxic T lymphocytes for elimination of virus, its role in RSV has not been reported. Here, we show that viral clearance in perforin knockout (PKO) mice is slightly delayed but that both PKO and wild-type mice clear virus by day 10, suggesting an alternative mechanism of RSV clearance. Effector T cells from the lungs of both groups of mice were shown to lyse Fas (CD95)-overexpressing target cells in greater numbers than target cells expressing low levels of Fas, suggesting that Fas ligand (CD95L)-mediated target cell lysis was occurring in vivo. This cell lysis was associated with a delay in RSV-induced disease in PKO mice compared to the time of disease onset for wild-type controls, which correlated with increased and prolonged production of gamma interferon and tumor necrosis factor alpha levels in PKO mice. We conclude that while perforin is not necessary for the clearance of primary RSV infection, the use of alternative CTL target cell killing mechanisms is less efficient and can lead to enhanced disease.     

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.     

Evaluation of human immunodeficiency virus type 1 (HIV-1)-specific cytotoxic T-lymphocyte responses utilizing B-lymphoblastoid cell lines transduced with the CD4 gene and infected with HIV-1.

Analysis of major histocompatibility complex-restricted cytotoxic T lymphocytes (CTL) capable of killing human immunodeficiency virus type 1 (HIV-1)-infected targets is essential for elucidating the basis for HIV-1 disease progression and the potential efficacy of candidate vaccines. The use of primary CD4+ T cells with variable infectivity as targets for such studies has significant limitations, and immortal autologous cells with high levels of CD4 expression that can be consistently infected with HIV-1 would be of much greater utility. Therefore, we transduced Epstein-Barr-virus-transformed B-lymphoblastoid cell lines (LCL) with a retroviral vector, LT4SN, containing the human CD4 gene. Stable LCL in which more than 95% of cells expressed membrane CD4 were obtained. Aliquots were infected with HIV-1, and, after 4 to 7 days, nearly all of the cells contained cytoplasmic gag and produced high levels of p24 antigen. The ability of major histocompatibility complex-restricted CD8+ CTL to lyse such HIV-1-infected CD4-transduced LCL (LCL-CD4HIV-1) was evaluated. These autologous targets were lysed by CTL generated from an HIV-1-uninfected vaccinee over a broad range of effector-to-target ratios. Similarly, the LCL-CD4HIV-1 were efficiently lysed by fresh circulating CTL from HIV-1-infected individuals, as well as by CTL activated by in vitro stimulation. Both HIV-1 env- and gag-specific CTL effectors lysed LCL-CD4HIV-1, consistent with the cellular expression of both HIV-1 genes. The LCL-CD4HIV also functioned as stimulator cells, and thus are capable of amplifying CTL against multiple HIV-1 gene products in HIV-1-infected individuals. The ability to produce HIV-1-susceptible autologous immortalized cell lines that can be employed as target cells should enable a more detailed evaluation of vaccine-induced CTL against both homologous and disparate HIV-1 strains. Furthermore, the use of LCL-CD4HIV-1 should facilitate the analysis of the range of HIV-1 gene products recognized by CTL in seropositive persons.     

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.     

IFN-gamma-dependent delay of in vivo tumor progression by Fas overexpression on murine renal cancer cells

The role of Fas in the regulation of solid tumor growth was investigated. Murine renal carcinoma (Renca) cells were constitutively resistant to Fas-mediated killing in vitro, but exhibited increased expression of Fas and sensitivity to Fas-mediated killing after exposure to IFN-gamma and TNF. Transfected Renca cells overexpressing Fas were efficiently killed in vitro upon exposure to anti-Fas Ab (Jo2). When Fas-overexpressing Renca cells were injected into syngenic BALB/c mice, there was a consistent and significant delay in tumor progression, reduced metastasis, and prolonged survival that was not observed for Renca cells that overexpressed a truncated nonfunctional Fas receptor. The delay of in vivo tumor growth induced by Fas overexpression was not observed in IFN-gamma-/- mice, indicating that IFN-gamma is required for the delay of in vivo tumor growth. However, there was a significant increase of infiltrated T cells and in vivo apoptosis in Fas-overexpressing Renca tumors, and Fas-overexpressing Renca cells were also efficiently killed in vitro by T cells. In addition, a strong therapeutic effect was observed on Fas-overexpressing tumor cells by in vivo administration of anti-Fas Ab, confirming that overexpressed Fas provides a functional target in vivo for Fas-specific ligands. Therefore, our findings demonstrate that Fas overexpression on solid tumor cells can delay tumor growth and provides a rationale for therapeutic manipulation of Fas expression as a means of inducing tumor regression in vivo.     

Interleukin-4-mediated downregulation of cytotoxic T lymphocyte activity is associated with reduced proliferation of antigen-specific CD8+ T cells

During virus infection, exogenous IL-4 strongly downregulates expression of antiviral cytokines and cytotoxic T lymphocyte (CTL) responses. In this study, we have employed a T cell receptor (TCR) transgenic system to more closely investigate the effect of IL-4 on CTL activity. This system involves mice transgenic for an H2-Kb restricted TCR recognising an ovalbumin (OVA)-specific peptide (OT-I mice), and recombinant vaccinia viruses expressing the gene for OVA (VV-OVA), or OVA together with IL-4 (VV-OVA-IL-4). Spleen cells from OT-I mice were adoptively transferred to irradiated C57BL/6 mice infected with VV-OVA or VV-OVA-IL-4. Five days following transfer, markedly stronger CTL activity was detected in VV-OVA- than in VV-OVA-IL-4-infected recipients. The reduction in CTL activity was associated with a reduction in the number of OVA-specific CD8+ T cells. Proliferation of cells from VV-OVA-IL-4-infected recipients was dramatically reduced, and this is a likely explanation for the IL-4-mediated reduction in the total number of OVA-specific cells and the reduced cytotoxic activity. On a per cell basis, the production of IFNgamma and cytotoxic activity of OVA-specific CD8+ cells was not influenced by IL-4. Taken together, our results indicate that the reduction in CTL activity by exogenous IL-4 is due to a reduced number of antigen-specific effectors, and does not involve a downregulation of effector function of these cells.     

Cytotoxic T cells specifically induce Fas on target cells, thereby facilitating exocytosis-independent induction of apoptosis

Cytotoxic T (Tc) cells deficient in perforin lyse Fas-negative targets after lengthy incubation periods. This process is independent of granzymes, and killing occurs via the Fas pathway for the following reasons. Interaction of perforin-deficient Tc cells with Fas-negative targets leads to an up-regulation of Fas that is dependent on Ag recognition, de novo synthesis, and transport of proteins to the target cell surface. Treatment of effectors with brefeldin A, but not with the exocytosis inhibitor concanamycin, inhibited this process. Lysis of targets is inhibited by anti-Fas Abs, soluble mouse Fas-Fc, and the caspase-cascade inhibitor, crm-A. Targets from Fas-mutant lpr mice are refractory to lysis, and Tc cells from mice deficient in Fas- and perforin-mediated lysis do not lyse Fas-negative targets. The possible relevance of this exocytosis-independent cytolytic process in the regulation of T cell activity and control of pathogens is discussed.