Mediated Activation-Induced Cell Death by Interfering with Fas Signaling1

[CANCER RESEARCH 63, 7475–7482, November 1, 2003] CD158 Receptor Controls Cytotoxic T-Lymphocyte Susceptibility to Tumor- Mediated Activation-Induced Cell Death by Interfering with Fas Signaling1

Asma Gati, Nadia Guerra, Catherine Gaudin, Sylvie Da Rocha, Bernard Escudier, Yann Le´cluse, Ali Bettaieb, Salem Chouaib, and Anne Caignard2 Institut National de la Sante´ et de la Recherche Me´dicale U487 from Institut Fe´de´ratif de Recherche 54 [A. G., N. G., C. G., S. D. R., S. C., A. C.], Unite´ des The´rapies Innovantes [B. E.], and Centre National de la Recherche Scientifique Unite´Mixte de Recherche 1598, Institut Gustave Roussy [Y. L.], 94805 Villejuif; Ecole Pratique des Hautes Etudes-Institut National de la Sante´et de la Recherche Me´dicale U517, Dijon, France [A. B.]

ABSTRACT molecules, KIRs are potent inhibitory receptors. They recognize specific polymorphisms on the classical HLA-A, -B, and C-molecules. Renal cell carcinoma-infiltrating lymphocytes express killer cell immu- All of these inhibitory receptors have in common one or more ITIMs -noglobulin-like receptors (KIRs) that inhibit antitumor CD8؉ T-cell func in their cytoplasmic tails. On tyrosine phosphorylation, ITIMs recruit tions and may contribute to local self-tolerance. In the present study, to better examine the functional consequences of KIR engagement on CTL– the tyrosine phosphatase SHP-1, which can dephosphorylate mole- tumor interactions, we investigated the influence of KIR2DL1/CD158a on cules involved in immunoreceptor tyrosine-based activation motif- CTL survival. We show that both KIR؉ and KIR؊ antigen-specific CTLs induced signaling pathways (6, 7). KIR2DL (CD158a and b) receptors express Fas and Fas ligand and were susceptible to activation-induced cell possessing two immunoglobulin domains are recognized by specific death (AICD) triggered by coated anti-CD3 monoclonal antibodies. In mAbs and distinguish HLA-C alleles (8). KIR؉ CTLs, anti-CD158a monoclonal antibodies partially inhibited anti- Inhibitory KIR receptors are also expressed by a small subset of CD3-induced AICD. Interestingly, T-cell receptor activation by cognate peripheral T cells in healthy individuals and counterbalance TCR- -tumor cells induced apoptosis in KIR؉ CTLs but not in KIR؊ CTLs. In mediated activation (9–11). These T cells express a memory pheno addition, co-engagement of T-cell receptors and KIRs by tumor cells type (CD28Ϫ, IL-2R␤), have a restricted TCR repertoire (9, 12), and decreased tumor-mediated CTL apoptosis. Blocking the interaction of lack CCR7. These KIRϩ T cells may expand in response to chronic KIR/HLA-Cw4 resulted in the restoration of tumor-induced AICD. Most importantly, our data indicate that KIR engagement affected two proxi- antigenic stimulation to self-antigen and most likely represent a pre- mal events of Fas signaling pathway, a sustained c-FLIP-L induction and cise subset of the memory T-cell pool (13, 14). Expression of inhib- a decrease in caspase 8 activity. These studies provide evidence that tumor itory KIRs appears late in T-cell maturation on antigen-experienced T cells selectively favor the local persistence of nonfunctional KIR؉ CTLs cells, and their expression probably involves modalities different from by promoting their survival. those on NK cells. The presence of tolerant antigen-specific T cells has been documented in experimental models and, more rarely, in human tumors INTRODUCTION (15, 16). In renal tumors, the possibility to select clonal cytotoxic effectors from TILs by means of KIR expression allowed us to study RCC3 represents a unique model of human immunogenic tumors. the mechanism of peripheral tolerance in these tumor-associated spe- The clinical responses in metastatic patients treated with cytokines cific T cells. and the rare but documented spontaneous regressions indicate that an Apoptotic death of lymphocytes is an important homeostatic mech- immune response may at least partly control tumor growth (1). How- anism of peripheral tolerance to self-antigens (17, 18). The induction ever, these tumors are also characterized by several immune defects. of apoptosis in mature T cells after antigenic stimulation, referred as RCC infiltrating lymphocytes display poor proliferative and lytic AICD, is an important process for terminating and controlling the capacities, leading to a global functional anergy. In this regard, several expansion of activated T cells. The death of mature T cells involves mechanisms associated with alteration of TCR signaling and immu- either suicide or fratricide and in most cases depends on the Fas/FasL nosuppressive factors secretion by tumor cells have been reported (2, pathway. Ligation of Fas by homotrimeric FasL results in clustering 3). We recently provided evidence in support of a potential role of of Fas and recruitment of the adaptor protein FADD to the clustered inhibitory NK receptors (KIRs) in the alteration of TIL cytolytic Fas through their mutual death domain. FADD also contains two function (4, 5). death effector domains through which it can recruit pro-caspase 8, We have previously shown that inhibitory NK receptors belonging leading to activation of the caspase cascade (19). The cellular homo- to the Killer immunoglobulin-type family KIR, expressed by 5–40% logue of the virus-encoded molecule FLIP that can inhibit Fas-L of CD8ϩ TILs, contribute to the altered cytotoxic activity of tumor- induced-cell death in vitro is suggested to play a central role in the reactive CTLs (4, 5). Among inhibitory NK receptors for HLA-I regulation of T-cell homeostasis in vivo (20, 21). In the present studies, we investigated the role of the KIR engage- Received 3/21/03; revised 8/6/03; accepted 8/13/03. ϩ The costs of publication of this article were defrayed in part by the payment of page ment on survival of antigen-specific KIR CTLs in response to charges. This article must therefore be hereby marked advertisement in accordance with stimulation by autologous tumor cells. We demonstrate that KIR 18 U.S.C. Section 1734 solely to indicate this fact. engagement on tumor-specific CTLs favors their survival as a conse- 1 This work was supported by INSERM, the Association pour la Recherche sur le Cancer (Grants 2038 and 5253 to A. C.). A. G. and N. G. were recipients of grants from quence of inhibition of AICD. The present studies emphasize that Association pour la Recherche sur le Cancer and Ligue Nationale Contre le Cancer, KIR, in addition to its inhibition of CTL lytic function, also plays a respectively. role in the control of T-cell homeostasis by interfering with Fas 2 To whom requests for reprints should be addressed, at INSERM U487, Institut Gustave Roussy, PR1, 39 rue Camille Desmoulins, 94805 Villejuif, Cedex 94, France. signaling. Phone: 33-42-11-50-36; Fax: 33-42-11-52-88; E-mail: [email protected]. 3 The abbreviations used are: RCC, renal cell carcinoma; TCR, T-cell receptor; NK, natural killer; KIR, killer immunoglobulin-type receptor; TIL, tumor-infiltrating lympho- MATERIALS AND METHODS cyte; ITIM, immunoreceptor tyrosine-based inhibition motif; SHP-1, Src homology 2 domain-containing protein 1; mAb, monoclonal antibody; IL, interleukin; AICD, activa- Culture of Tumors Cells and CTLs. Tumor cells derived from RCC were tion-induced cell death; FasL, Fas ligand; FADD, Fas-associated death domain; FLIP, FLICE inhibitory protein; PE, phycoerythrin; PI, propidium iodide; DISC, death inducing maintained in DMEM/Ham F12 medium supplemented with 10% FCS and 1% signaling complex. Ultroser G (Life Technologies, Inc., Cergy Pontoise, France). Tumor cells 7475

Downloaded from cancerres.aacrjournals.org on October 5, 2021. © 2003 American Association for Cancer Research. KIR RECEPTOR ENHANCES CTL SURVIVAL were HLA genotyped RCC7 [HLA-A2, A29, B51, B44, Cw14, and Cw16 gation-independent apoptosis, for 16 h. In some experiments, CTLs were (Cw4 supertype)], RCC6 [HLA-A1, A2, B51, B8, Cw7 and Cw14 (Cw3 incubated with anti-CD158a (1 ␮g/ml) for 10 min before the beginning of the supertype)], and RCC5 [HLA-A1, A32, B7, B49, and Cw7]. apoptotic treatment or with the selective caspase 8 inhibitor Z-IETD-FMK (25 CTLs were obtained after in vitro stimulation of TILs derived from RCC7 mM; Apotech, Coger, France), added 20 min before the anti-CD3 or anti-Fas for 2–6 weeks and subsequent cloning by limiting dilution. CTL clones were mAbs. expanded [3 ϫ 103 cells/well on irradiated feeder cells (7 ϫ 104 cells/well of Western Blot Analysis. Total cellular extracts were prepared by lysing allogeneic peripheral blood mononucleated cells) and 104 cells/well of an cells in ice-cold buffer [50 mM Tris (pH 7.5), 150 mM NaCl, 1% Triton X-100, allogeneic EBV-transformed cell line (LAZ509) in the presence of IL-2 (100 1mM phenylmethylsulfonyl fluoride, 10 ␮g/ml aprotinin, 10 ␮g/ml leupeptin]. IU/ml)] and allowed to proliferate for 10 days. At the end of expansion, CTLs Equivalent protein extracts (10 ␮g) were denatured by boiling in SDS and were frozen and used for the different experiments. ␤-mercaptoethanol, separated by SDS-PAGE and transferred onto Hybond Cytotoxicity Assay and CTL Survival Rescue Assay. The cytolytic ac- membranes (Amersham, Saclay, France). The efficiency of the electrotransfer tivity of T-cell clones against tumor cells was measured in a 4-h 51Cr-release was assessed by Ponceau Red staining of the membranes. Blots were blocked assay. Tumor cells were used in amounts of 2 ϫ 103 cells/well, and the E:T overnight with Tris-buffered saline containing 5% nonfat dry milk and probed ratio ranged from 10:1 to 2:1. In some experiments, mAbs EB6 (anti-CD158a, with the anti-FLIP mAb NF6 (IgG1; Apotech, Coger SA, France) for 1 h. After IgG1), GL183 (anti-CD158b, IgG1), B1.23.2 (anti-HLA-B or -C, IgG2a), or washing, blots were incubated with the appropriate secondary horseradish W6.32 (anti-HLA-I, IgG1) or control murine IgG were added at saturating peroxidase-conjugated antibodies. The complexes were detected by use of the concentrations at the beginning of the cytolytic assay. ECL detection kit (Amersham Corp., Buckinghamshire, United Kingdom). The cytolytic activity of T-cell clones was also assessed in a CD3-redirected Densitometric analysis, including correction for background, was performed lysis assay using P815 mastocytoma mouse cells. Briefly, 51Cr-labeled P815 with NIH Image software. (3 ϫ 103) cells coated with decreasing doses of anti-CD3⑀ (10 ␮g/ml–1 ng/ml) Detection of Caspase 8 Activation. Caspase 8 activity was measured by were incubated with T cells at a 5:1 ratio. Data were expressed as the flow cytometry using the CaspaseTag Caspase 8 (LETD) Activity kit (Inter- percentages of specific lysis at the indicated E:T ratio. The percentage of gen, Burlington, MA). CTLs were stimulated for 16 h by RCC targets at a 1:1 specific 51Cr release was calculated as: (experimental release Ϫ spontaneous tumor:CTL ratio and then treated for 1 h with the caspase tag fluorescent release)/(total release Ϫ spontaneous release) ϫ 100. reagent. Apoptosis was measured by use of annexin V and PI on the gated Flow Cytometric Detection of Cell Surface Molecules. The phenotypes CD8APC-labeled T cells. of CTLs and tumor cells were determined by direct fluorescence. Briefly, 2 ϫ 105 cells were incubated for 30 min at 4°C with conjugated mAbs: RESULTS EB6-PE, CD3-FITC, CD56-PECy5, CD8-FITC, and UB2-PE (anti-Fas, IgG1), purchased from Immunotech (Marseille, France), and B-R17-PE (anti-FasL, Down-Modulation of the Lytic Activity of RCC-Specific CTLs IgG1), purchased from Diaclone (Besanc¸on, France). by KIRs. The present studies were based on the use of two antigen- CTL Stimulation and Cell Death Analysis. Early apoptotic events were specific CTLs isolated from a renal tumor (RCC7 A2Cw4): KIRϩ evaluated by an annexin V labeling method using the Vybrant apoptosis assay Ϫ kit (Molecular Probes, Interchim, Montluc¸on, France). AICD was evaluated 4D4 CTLs, which express a KIR2DL1 (CD158a) receptor, and KIR after triggering of TCRs on IL-2-starved CTLs by coated anti-CD3 (UCHT1) 22G7 CTLs. The lytic activity of both CTLs is restricted by HLA-A2 mAb (1 ␮g/ml), alone or in combination with control mouse IgG (1 ␮g/ml), or molecules. In addition, KIRϩ 4D4 displays faint membrane expres- with the anti-KIR mAb anti-CD158a (EB6; 1 ␮g/ml) for 6 h. Cells (2 ϫ 105 sion of CD158e, but CD158k is absent. Reverse transcription-PCR cells) from each sample were then washed twice with cold PBS and resus- analysis of KIR transcripts revealed that KIR2DL2, KIR2DL4, pended in a binding buffer [50 mM HEPES, 700 mM NaCl, 12.5 mM CaCl2 (pH KIR2DS1, and KIR2DS2 transcripts were present in 4D4 KIRϩ CTLs 7.4)] at a concentration of 1 ϫ 106 cells/ml. One hundred ␮l of this suspension (Table 1). Both CTLs express ILT2, but CD94/NKG2A was not were reacted with 5 ␮l of annexin V-FITC and 1 ␮lof100␮g/ml PI. The expressed. Whereas KIRϩ 4D4 CTLs recognize a self-antigen, KIRϪ mixture was gently vortexed and then incubated for 15 min at room temper- 22G7 CTLs recognize a private tumor antigen expressed by RCC7 ature. After incubation, 400 ␮lof1ϫ binding buffer was added to each tube. (22). As shown in Fig. 1A, lysis of autologous RCC7 tumor cells by Analysis by flow cytometry was conducted within1hofassay completion for optimal results. PI-stained cells, CD3-untreated cells, and annexin V-stained 4D4 was efficiently increased by anti-HLA-B/C mAbs that block cells were run first to optimize the settings. Viable apoptotic cells were KIR/HLA-Cw4 interaction, resulting in a lytic potential similar to that differentiated from necrotic cells by flow cytometry after PI staining of of 22G7. In addition, 22G7 lytic activity was decreased by anti-HLA-I nonpermeabilized cells. Apoptotic cells were defined as annexin Vϩ/PIϪ. mAb (Fig. 1B). KIR engagement by tumor cells also reduced IFN␥ Results were plotted as the percentage of annexin Vϩ cells and PIϪ cells. In production by KIRϩ 4D4 CTLs (Fig. 1C). In the two clones, TCR some experiments, anti-FasL (4H9, IgG1) purchased from Immunotech was triggering induced efficient lysis as measured by anti-CD3 redirected used to block Fas/FasL interaction. lysis of Fc-receptor-positive P815 cells. In KIRϪ 22G7 CTLs, a low Alternatively, CTLs were stimulated by tumor cells for 24 h in the absence concentration of anti-CD3 was sufficient to trigger lysis, whereas a of IL-2. After coculture, CTLs were removed gently and stained with Allo- higher concentration of anti-CD3 was required to trigger lysis by phycocyanin-conjugated anti-CD8 mAb, and apoptosis was measured in the ϩ ϩ Ϫ ϩ KIR 4D4 CTLs (Fig. 1D). annexin V /PI /CD8 population. In some experiments, anti-HLA-B/C ؉ ؊ mAbs were added in saturating concentrations to block KIR/HLA-C interac- Anti-CD 3-Induced AICD in KIR and KIR CTLs. As de- ϩ Ϫ tions. picted in Fig. 2A, both KIR and KIR CTLs express Fas receptor T-cell apoptosis was induced by incubation with 1–0.5 ␮M staurosporine and FasL. Although the mean fluorescence intensities of Fas were (Sigma, Saint Quentin Fallavier, France) for2horwith anti-Fas mAb 7C11 higher on KIRϪ CTLs, both CTLs displayed similar susceptibility to (IgG1; purchased from Immunotech, Marseille, France), which triggers aggre- agonistic anti-Fas mAb (data not shown). Both CTLs were susceptible

Table 1 Membrane and mRNA expression of KIRs in 4D4 KIRϩ CTLs Values are the percentage of positive cells (mean fluorescence intensity values in parentheses). KIR2D transcripts KIR3D transcripts

DL1 DS1 DL2 DS2 DL3 DS3 DL4 DS4 DS5 DL1 DS1 DL2 ϩϩ ϪϪ ϩϩ ϪϪ ϪϪ ϪϪ ϩϩ ϪϪ ϪϪ ϪϪ ϩ ϩ ILT2 CD158a CD158b CD158e CD158k 100% (60) 100% (1700) Ͻ5% 80% (44) Ͻ5% 7476

Fig. 1. KIR engagement modulates the lytic activity of KIRϩ CTLs. A and B, lysis of autologous RCC7 tumor cells by KIRϩ 4D4 CTLs (A) and KIRϪ 22G7 CTLs (B) in the presence of saturating concentrations of anti-HLA mAbs or isotype-matched control immunoglobulin (cIgG). C, production of IFN␥ by KIRϩ 4D4 CTLs in response to RCC7 A2Cw4 is increased when KIR/ HLA-C interaction is blocked. D, dose–response curves for CD3-redirected lysis of P815 cells showing CD3-redirected lysis of the KIRϩ and KIRϪ CTL clones.

to anti-CD3-induced AICD measured by annexin V/PI staining, and targets for 24 h, T-cell death was measured by triple staining with low concentrations of anti-CD3 mAbs (up to 0.25 ␮g/ml) produced anti-CD8 and annexin V/PI. As clearly shown in Fig. 4A, RCC6 50–60% apoptosis in the two CTLs. However, in response to higher A2Cw3 cells, which optimally activate TCRs on 4D4 KIRϩ CTLs concentrations of coated anti-CD3 mAbs (Ͼ0.5 ␮g/ml), 85–90% of induce significant AICD, whereas RCC5 A1Cw3 cells did not, con- 22G7 KIRϪ CTLs became apoptotic, whereas the percentages of firming that cognate tumor cell-induced AICD was antigen dependent. apoptosis remained unchanged in 4D4 KIRϩ CTLs (Fig. 2B). As Interestingly, apoptosis of 4D4 KIRϩ CTLs was clearly reduced in evidence of the involvement of Fas/FasL in AICD in our experimental response to autologous RCC7 A2Cw4 cells, indicating that engage- model, we showed that anti-FasL mAb completely abrogates AICD ment of the KIRs by tumor cells decreased T-cell death and favored induced by anti-CD3 in both 4D4 KIRϩ CTLs (3% versus 49%) and KIRϩ CTL survival. In addition, the presence of blocking anti-HLA- 22G7 KIRϪ CTLs (14% versus 72%), as shown in Fig. 2C. B/C mAbs during the stimulation with RCC7 restored 4D4 apoptosis Triggering of KIRs by Specific mAbs Reduced Anti-CD3- to the level obtained in response to RCC6 A2Cw3 tumor cells (Fig. -Induced Apoptosis in KIR؉ CTLs. The data depicted in Fig. 3A 4B). Furthermore, increasing the tumor:CTL ratio to 2:1 led to apo show that simultaneous triggering of TCRs and KIRs on 4D4 KIRϩ ptosis in up to 90% of 4D4 after TCR stimulation by cognate RCC6 CTLs with coated anti-CD3 and anti-CD158a mAbs resulted in a tumor cells (Fig. 4C). Tumor cells did not stimulate nonmemory significant decrease of apoptosis compared with anti-CD3 and CD28ϩ/22G7KIRϪ CTLs to AICD (data not shown). isotype-matched control (cIgG). Triggering of KIR receptor alone did KIR Engagement Induced Down-Regulation of AICD by Inter- not induce CTL apoptosis. The decrease in apoptosis varied from 35% fering with Fas Signaling. Because the above experiments revealed to 60% in four independent experiments. In addition, TCR activation- that KIR affects the Fas/FasL-mediated AICD pathway, we therefore independent apoptosis induced by staurosporine, an inhibitor of the asked whether this receptor interferes with Fas signaling. For this protein kinase C pathway, led to death of the two CTLs, and the purpose, 4D4 KIRϩ CTLs were simultaneously stimulated with anti- triggering of KIRs had no effect on 4D4 KIRϩ CTLs (Fig. 3). These Fas (7C11) and anti-CD158a mAbs, and T-cell death was evaluated results indicate that the influence of KIRs on AICD is presumably by annexin V/PI. The data shown in Fig. 5A revealed that KIR2DL1 mediated through a modulation of TCR-driven signaling events. engagement resulted in a significant inhibition (Ͼ50%) of anti-Fas- KIR Engagement by Tumor Cells Reduced Tumor-Induced- induced apoptosis. Dose–response experiments revealed that supra- AICD in KIR؉ CTLs. The above experiments indicate that TCR optimal Fas stimulation (Ͼ0.75 ␮g/ml) was not inhibited by KIR activation resulted in AICD in tumor-specific 4D4 KIRϩ and 22G7 triggering (Fig. 5A). It is well established that Fas/FasL can be KIRϪ CTLs and that KIRs partially control this response in KIRϩ proximally blocked by the expression of c-FLIP, a caspase 8 homo- CTLs. We then asked whether TCR activation by tumors controls logue that binds FADD without transducing a death signal (23). The AICD in our model and whether KIR engagement influences AICD. long form of c-FLIP-L shares extensive homology with procaspase 8 For this purpose, CTLs were incubated with tumor cells that engaged and contains two death effector domains that interact with FADD, but either TCRs or KIRs or both. After CTL stimulation with tumor has a mutation in the caspase-like domain that renders it inactive. In 7477

activation, which was inhibited by KIR engagement (data not shown). Co-engagement of TCRs and KIRs on 4D4 by KIR ligand-positive RCC7 for 16 h also resulted in low caspase 8 activity compared with RCC6, which engages only TCRs (Fig. 5C, upper panels). When KIR/HLA-Cw4 interaction was blocked during stimulation of RCC7 cells, the caspase 8 activity in 4D4 was comparable to that of RCC6 cells (Fig. 5C, lower panels). Finally, specific caspase 8 inhibitor clearly reduced anti-CD3-triggered AICD as well as anti-Fas-induced apoptosis, indicating that AICD is caspase dependent (Fig. 5D). However, it exerted no clear effect in experiments using tumor- stimulated 4D4 KIRϩ CTLs. Stimulation by tumor cells required 16 h, and the inhibitor, by acting on tumor cells, may increase their resistance to CTL lysis. Taken together, our data indicate an essential role of KIRs in the control of AICD by a mechanism involving, at least in part, positive modulation of c-FLIP-L and a subsequent alteration of pro-caspase 8 activation. KIR Engagement by Tumor Cells Maintains T-Cell Survival. 4D4 KIRϩ CTLs were incubated for 3 days in cytokine-free medium in the absence or presence of different RCC targets. CTL survival was measured by triple staining as described above, and T cells were counted every 24 h during the time of culturing (Fig. 6). In accordance with the influence of KIRs on AICD, 4D4 KIRϩ CTL survival was higher in response to RCC7 A2Cw4 targets compared with RCC6 A2Cw3 targets, which do not engage the KIRs. In addition, a CTL rescue assay indicated that by controlling the CTL activation level, KIRs may protect them from functional exhaustion and preserve their lytic potential toward KIR ligand-lacking tumor cells (data not shown).

DISCUSSION It is well established that the immune system maintains an optimal number of lymphocytes and prevents autoimmune responses by eliminating any excess of nonfunctional and autoreactive T cells. It also supports the survival of antigen-specific T cells, thus promoting protective immune responses and immunological memory (24, 25). This balance is achieved by a complex network of lymphocyte survival and death signals, in which the Fas/FasL system plays a significant role to maintain T-cell homeostasis (26, 27). In response to repeated antigen challenges, the elimination of high-affinity CD8ϩ T cells by clonal downsizing occurs during viral

Fig. 2. Anti-CD3-induced AICD in KIRϩ and KIRϪ CTLs involved in the Fas/FasL pathway. A, expression of Fas antigen and FasL on KIRϩ 4D4 and KIRϪ 22G7 CTLs. Numbers represent mean fluorescence intensity values. B, dose–response curves showing AICD in 4D4 and 22G7 CTLs in response to coated anti-CD3 mAbs. AICD was induced in KIRϩ and KIRϪ CTLs by incubation with coated anti-CD3 mAbs for 6 h, and T-cell apoptosis was measured by annexin V/PI staining. C, CTLs were incubated for 10 min with anti-FasL or control IgM (IgMc) before the treatment with coated anti-CD3 mAbs (0.5 ␮g/ml) for 6 h. Numbers correspond to percentages of apoptotic cells. One representative of three experiments. four independent experiments, after triggering of TCRs and KIRs by anti-CD3 ϩ anti-CD158a mAbs, the cellular c-FLIP-L level was significantly increased 3 min poststimulation compared with the level of FLIP detected in anti-CD3 ϩ cIgG-stimulated CTLs. KIR engagement alone induced c-FLIP-L, but cotriggering of TCRs led to higher induction. Interestingly, in the presence of anti-CD158a mAbs, the Fig. 3. KIR triggering by mAbs (anti-CD158a) inhibits anti-CD3-induced AICD in c-FLIP-L level was sustained after 3 h, rendering the cells resistant to KIRϩ 4D4 CTLs. 4D4 CTLS were incubated with anti-CD158a mAbs or control IgG (IgGc) for 5 min at room temperature before treatment with coated anti-CD3 mAbs (1 AICD (Fig. 5B). ␮g/ml) for6h(top) or with staurosporine (0.5 ␮M; bottom) for 2 h. T-cell apoptosis was In addition, anti-CD3-induced AICD correlated with pro-caspase 8 measured by annexin V/PI labeling. Numbers correspond to percentages of apoptotic cells. 7478

Fig. 4. Tumor-induced AICD in KIRϩ CTLs is inhibited by KIR triggering. A, tumor-induced AICD in 4D4 KIRϩ CTLs by cognate but not noncognate tumor cells. CTLs were stimulated for 24 h with RCC targets, and CTL cell death was measured by annexin V/PI staining. Numbers correspond to percentages of apoptotic T cells (one representative experiments of four). B, tumor-induced AICD in 4D4 KIRϩ CTLs is restored by blocking HLA-C/KIR interactions. RCC7 targets were incubated with anti-HLA-B/C mAb for 10 min before the addition of CTL. C, tumor-induced AICD of KIRϩ CTLs is dependent on CTL:tumor ratios.

infections. It has been proposed that AICD serves a role in limiting KIR3DL on T-cell apoptosis was reported previously in transfected expansion of an immune response, in that few effector T cells may Jurkat T cells. In this model, KIR inhibited AICD by blocking FasL survive as memory T cells. It may be assumed that in response to induction in a KIR ligation-independent process involving inhibition overexpressed self-antigens (28), tumor-specific CTLs may behave of protein kinase C recruitment (36). In our model, triggering of KIR similarly. Although numerous reports support the existence of a tumor further decreased anti-CD3-induced AICD by affecting Fas signaling. attack phenomenon, tumor-induced AICD of CTLs has rarely been The dependence on KIR ligation may likely reflect the nature of the investigated (29, 30). We therefore took advantage of our experimen- T cells studied, i.e., antigen-specific CTLs versus KIR3DL-trans- tal model to further investigate whether the engagement of TCRs by fected Jurkat cells, and may be related to the levels of KIRs expressed tumor targets results in CTL AICD and whether KIRs are involved by these cells (36). in the regulation of CTL survival. Our studies demonstrate that AICD Recently we obtained evidence indicating that KIR engagement in response to tumor cells was high in KIRϩ CTLs and minimal in resulted in the attenuation of early signaling and the subsequent T-cell KIRϪ CTLs. The different behaviors of the two CTL clones may activation (22). In the present studies, we showed that the down- reflect the nature of the recognized antigen or the level of its expres- regulation of T-cell activation by KIRs interferes with the Fas path- sion. KIRϪ 22G7 CTLs recognize a private tumor-specific antigen, way, decreasing AICD. Exploring the mechanisms by which KIR and the cognate HLA-A2 restricted peptide may be sparse on targets engagement interferes with Fas-induced cell death, we showed by cells compared with self-antigens recognized by KIRϩ CTLs. In this confocal microscopy that the formation of CD95 clusters in response regard, whereas only a few TCR molecules are necessary to induce to anti-CD3 was not decreased by engagement of KIRs (data not cytolysis in activated CTLs (31), the triggering of AICD may require shown). We next investigated the proximal events of the Fas signaling higher numbers of TCR-MHC-peptide complexes. Both CTLs derived pathway, focusing on the c-FLIP protein, which acts as a dominant from blood (22G7 KIRϪ) and tumor (4D4 KIRϩ) may display negative of caspase 8. It is known that the affinity of FLIP/caspase 8 different TCR avidities (32–34). The importance of high-avidity CTLs heterodimer for FADD is much higher than that of the caspase 8 as potent cytotoxic effectors and their regulation by AICD were homodimer and is critical in determining the fate of Fas ligation (21). recently demonstrated in patients with chronic myeloid leukemia, KIR engagement was accompanied by an increase in c-FLIP-L levels indicating the likelihood of immune tolerance in patients with persis- in CTLs that was sustained after TCR and KIR co-engagement. The tent disease by elimination of high-avidity specific T cells (30). role of c-FLIP in the regulation of TCR-induced Fas-mediated apo- A role for KIRs in the survival of memory T cells and inhibition of ptosis in T cells has been documented in vitro (37, 38) and in vivo AICD was suggested recently (13, 14, 35). In our experimental model, (39). Fas-resistant T cells express high levels of c-FLIP compared sensitivity to AICD of KIRϩ CTLs in response to tumor-induced with sensitive T cells (40, 41). In addition, it has been demonstrated reactivation is in accordance with their memory effector phenotype that antigen concentration and costimulation signals are critical pa- and suggests that they correspond to preactivated, potentially autore- rameters in regulating AICD in memory T cells and that TCR medi- active T cells. Furthermore, the present studies indicate, for the first ated higher neosynthesis of c-FLIP mRNA levels in these T cells than time, a role of KIRs in the control of AICD in a tumor-specific CTL. in naive T cells (32). Furthermore, our data indicate that in addition to Triggering experiments using anti-CD158 mAbs indicated that KIR higher c-FLIP-L levels, KIR engagement correlated with a decrease in engagement indeed resulted in a significant enhancement of CTL pro-caspase 8 activation in KIRϩ CTLs. The critical role of pro- survival by inhibiting AICD. Nevertheless, KIR triggering had no caspase 8 in Fas signaling was demonstrated in mice with targeted effect on staurosporine-induced apoptosis, further confirming that caspase 8 or FADD mutations restricted to T-cell lineage that resulted triggering of KIRs results in the modulation of TCR signaling events. in complete blockage of Fas-induced apoptosis (42, 43). In humans, a With respect to the mechanisms involved in the inhibition of AICD by homozygous caspase 8 deficiency results in defective T-cell apoptosis KIRs, few data are available at present. The inhibitory effect of and activation (44). In peripheral T cells resistant to Fas, pro-caspase 7479

Fig. 5. KIR engagement interferes with the Fas signaling pathway. A, KIRs decrease Fas-induced apoptosis. 4D4 KIRϩ CTLs, incubated with anti-CD158a (1 ␮g/ml) or cIgG for 10 min, were treated with increasing doses of anti-Fas mAbs (7C11) for 16 h, and apoptosis was measured by annexin V/PI staining. Numbers are percentages of apoptotic cells (percentages of annexin V/PI were Ͻ10%). B, analysis of c-FLIP by Western blotting in 4D4 KIRϩ CTLs stimulated by anti-CD158a, cIgG, anti-CD3ϩcIgG, or anti-CD3ϩanti- CD158a. Membrane was reprobed with antibody for actin to control for protein amounts. C, detection of pro-caspase 8 activity by flow cytometry in 4D4 CTLs stimulated for 16 h by RCC7, RCC6, and RCC5 tumor cells with use of the caspase Tag kit. 4D4 KIRϩ CTLs were stimulated by RCC7 in the presence of anti-HLA-BC or cIgG. Results are expressed as percentages of cells with caspase 8 activity. D, specific caspase inhibitor Z-IETD-FMK (25 ␮M) inhibits anti-CD3- and anti-Fas-triggered T-cell apoptosis. 7480

tion threshold, resulting in decreased lytic activity and improved survival. Intratumoral rescued KIRϩ CTLs with maintained lytic capacities do not represent a threat for tumor cells because the KIRs interrupt their activation, preventing the completion of the lytic process. Because there is growing knowledge of the antitumoral immune response, it is becoming evident that the challenge to improve cancer immunotherapy will be to maintain T-cell activation while preventing T-cell apoptosis and eliminating the effect of negative regulatory factors.

ACKNOWLEDGMENTS

We thank Dr. Guido Kroemer for helpful discussions. Fig. 6. CTLs escaping AICD lytic activity. CTLs were stimulated for 72 h with RCC7 A2Cw4, RCC6 A2Cw3, and RCC5 A1Cw3 targets, and T-cell survival was evaluated every 24 h. REFERENCES 1. Nathan, P., and Eisen, T. The biological treatment of renal-cell carcinoma and melanoma. Lancet Oncol., 3: 89–96, 2002. 8 recruitment to the DISC was decreased (45). In Jurkat T cells, AICD 2. Uzzo, R., Clark, P., Rayman, P., Bloom, T., Rybicki, L., Novick, A., Bukowski, R., and Finke, J. Alterations in NF␬B activation in T lymphocytes of patients with renal control by an immunosuppressive drug, LF 15-0195, involved caspase cell carcinoma. J. Natl. Cancer Inst. (Bethesda), 91: 718–721, 1999. 8 and caspase 10 activation at the DISC level (46). 3. Uzzo, R., Kolenko, V., Froelich, C., Tannenbaum, C., Molto, L., Novick, A., Bander, N., Bukowski, R., and Finke, J. 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