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Prepublished online June 21, 2002; doi:10.1182/blood-2002-02-0643

Engagement of the inhibitory receptor CD158a interrupts TCR signaling preventing dynamic membrane reorganization in CTL / tumor cell interaction Nadia Guerra, Frederique Michel, Asma Gati, Catherine Gaudin, Zohar Mishal, Bernard Escudier, Oreste Acuto, Salem Chouaib and Anne Caignard

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From bloodjournal.hematologylibrary.org by guestJune on June 2013. For personal use only. Blood First Edition Paper, prepublished online 21,5,2002; DOI 10.1182/blood-2002-02-0643

Engagement of the inhibitory receptor CD158a interrupts TCR signaling preventing dynamic membrane reorganization in CTL / tumor cell interaction

Nadia Guerra*, Frédérique Michel‡, Asma Gati*, Catherine Gaudin*, Zohar Mishal§, Bernard Escudier¶, Oreste Acuto‡, Salem Chouaib* and Anne Caignard*

*

Institut National de la Santé et de la Recherche Médicale (INSERM) U487, ¶ Unité des Thérapies

innovantes, Institut Gustave Roussy, 94805 Villejuif, Cedex 94, France, ‡Unité d’Immunologie Moléculaire, Institut Pasteur, 75724 Paris Cedex 15, France, § Institut Andre Lwoff - CNRS, Hôpital Paul Brousse, 94801, Villejuif, France

To whom reprint requests and pages proof should be addressed: Anne Caignard, INSERM 487, Institut Gustave Roussy, PR1, 39, rue Camille Desmoulins, 94805 Villejuif Cedex 94, France. Tel 33/1/42 11 50 36 ; Fax : 33/1/42 11 52 88. email : [email protected].

Short title: Inhibition of immunological synapse by NK receptor

Key words: Signal transduction, inhibitory NK receptor, lipid rafts, tumor specific CTL clone, renal tumors

1

Copyright 2002 American Society of Hematology


Acknowledgments : NG and AG are recipient of a grant from the Association pour la Recherche sur le Cancer. The study was supported by grants from INSERM, the Association pour la Recherche sur le Cancer (grants 2038 and 5253). We thank Dr Frédéric Triebel for providing CTL clones. We thank Prof Alessandro Moretta for helpful discussions and for kind gift of anti-KIR mAbs. We thank Yann Lécluse for flow cytometry analysis. We thank Dr Salvatore Valitutti for technical advises on confocal analysis and for helpful discussions.

2


Abstract:

Renal cell carcinoma (RCC) infiltrating lymphocytes (TIL) express Killer cell Ig-like receptors (KIR) that inhibit the antitumor CD8+T cell lysis. In the present study, to better examine the functional consequences of KIR engagement on CTL/Tumor interaction, we have investigated the influence of KIR CD158a on early steps of T cell activation. We show that coengagement of TCR and CD158a by tumor cells inhibited tyrosine phosphorylation of early signaling proteins ZAP-70 and LAT, lipid rafts coalescence and TCR/CD3 accumulation at the CTL/tumor cell interface. In addition, the guanine exchange factor Vav was not phosphorylated and no actin cytoskeleton rearrangement was observed. Our data indicate a role of KIR CD158a in the dynamic events induced by TCR triggering, preventing CTL membrane reorganization, and subsequent completion of CTL activation program. Accordingly, the expression of CD158 by TIL may favor tumor cell escape to the immune response.

3


Introduction

In human, inhibitory NK receptors for HLA-I molecules include killer cell Ig-like receptors (KIRs), the CD94/NKG2A heterodimer and Ig-like transcript 2 (ILT-2)/Leukocyte Ig-like receptor 1 (LIR-1). While KIRs recognize specific polymorphisms on the classical HLA-A, B and C molecules, CD94/NKG2A recognizes non-classical HLA-E molecules assembled with a peptide from the leader sequence of HLA-I molecules 1,2. ILT-2/LIR-1 expressed by NK, T cells and myeloid cells recognizes a broad range of cellular HLA-I molecules 3 as well as viral class I-like molecule, UL-18 4. All these inhibitory receptors have in common one or more immuno-receptor tyrosine-based inhibition motifs (ITIMs) in their cytoplasmic tails. Upon tyrosine phosphorylation, ITIMs recruit tyrosine phosphatases Src homology 2 domain-containing protein 1 (SHP-1) and /or SHP-2 which can dephosphorylate molecules involved in immuno-tyrosine-based activation motif (ITAM) induced signaling pathways 5,6

. KIR2D CD158 receptors possessing two Ig domains are recognized by specific mAbs and

distinguish HLA-C alleles7.

In the peripheral blood of healthy individuals, low percentages of KIR+T cells express a memory CD28-, CD45RA-, CD45RO+ phenotype and a restricted TCR repertoire

8,9

. These cells may expand

in response to chronic antigenic stimulation to self-antigen and most likely represent a precise subset of the memory T cell pool 10,11. It has been reported that inhibitory NK receptors expressed by CD8+ cells were able to counterbalance TCR-mediated activation by inhibitory signals that are transduced upon specific binding to HLA-I molecules 8,12,13.

A few studies have reported that CD94 and KIR are also expressed by CD8+ T cells infiltrating tumors

14-16

. We previously reported that renal tumors contained particular CD8+ infiltrating T cells

that express an inhibitory NK receptor from the KIR family (CD158), specific for HLA-C molecules. In vitro, these KIR+ CTL correspond to potential highly lytic effectors but their functional activity towards tumor cells is strongly inhibited by the NK receptor17. In the present study, a tumor derived CD158a+ CTL was stimulated by tumor cells expressing or not the KIR ligand to investigate the 4


influence of the KIR engagement in the early biochemical and morphological events leading to cell activation. This system constitutes an accurate model to analyze the interplay between TCR/MHC and KIR/MHC interactions during physiological CTL/target activation.

The recognition of MHC/peptide complex by TCR results in the activation of protein tyrosine kinases (PTK) 18. Among them, ZAP-70 phosphorylates the adaptor molecule linker for activation of T cells (LAT) that plays a key role in linking TCR to phospholipase C 1 and Ras pathways 19. LAT is also implicated in actin cytoskeleton remodeling required for the TCR induced membrane spreading

20

.

TCR mediated-signaling and reorganization of the actin cytoskeleton are further connected through intracellular signaling proteins such as Vav, Rho family GTP-ases, SLP-76 and Arp2/3

21-23

.

Consequently, the accumulated actin at the contact between T and APC was found to stabilize a continuous contact which allows the clustering of TCR, co-stimulatory and signaling molecules into lipid rafts. Several groups have documented the importance of these membrane rafts in TCR mediated signal transduction

24-26

. Besides its inhibitory effect on cytotoxicity and cytokine production 27, the

engagement of inhibitory receptors at the T cell/APC interface is also likely to influence the outcome of the CTL/tumor interaction by modulating the cellular and morphological events linked to T cell activation and cytotoxicity. We took advantage of confocal microscopy to visualize the membrane interactions between a CD158a+CTL and tumor cells. In this physiological CTL/tumor cell interaction, we evidence that the KIR triggering interfered with early TCR signaling and membrane reorganization, preventing completion of the T cell activation program.

5


Results:

Inhibition of the cytotoxic activity and Ca2+ response of CTL by CD158a NK receptor

4D4 CTL clone expressing a VB13.1 TCR and a unique CD158a receptor (specific for HLA-Cw4 supertype alleles) was derived from tumor infiltrating lymphocytes of a renal tumor. This tumor contained 2% of CD158a+ CD8+ T cells and VB13.1+T cells corresponded to 16% of the CD158a+ T cells (data not shown). This CTL is HLA-A2 restricted and recognized an antigen expressed by tumor cells of different origins (renal, melanoma, breast) as well as normal renal cells and EBV-transformed B cells. In order to analyze the respective role of TCR/MHC and KIR/MHC engagement on CTL activation, 4D4 CTL clone was stimulated by autologous (HP) renal tumor cells or allogeneic renal tumor cell lines (DM and GE) displaying different combinations of HLA-A and C molecules. 4D4 CTL efficiently lyzed HLA-A2+ HLA-Cw3 supertype tumor cell line (RCC-DM). In contrast, autologous HLA-A2+ HLA-Cw4 cell line (RCC-HP) was lysed by 4D4 CTL only when the CD158a/HLA-Cw4 interaction was blocked with anti-HLA-B/C mAb. The HLA-A2- cell line (RCCGE) was not recognized even in the presence of blocking mAb (Fig. 1A, left panel). EBV-transformed B cells (LCL) derived from the same RCC patients were lysed with a similar pattern as tumor targets indicating that 4D4 CTL recognizes a self antigen (Fig. 1A, right panel). In addition, the lysis of DM cell lines was markedly inhibited in presence of anti-CD3 or anti-HLA-A2 mAbs evidencing that the lytic activity of 4D4 CTL is TCR-mediated and restricted by HLA-A2 molecules (data not shown).

The influence of the KIR receptor was assessed on the intracellular calcium [Ca2+]i release, one of the earliest event of CTL activation. Figure 1B shows the percentages of T cells releasing intracellular Ca2+ following stimulation by LCL cell lines. Quantitatively, tumor cells RCC-DM triggered high [Ca2+]i release by CTL (>600 mM) when compared to a stimulation by RCC-HP that engaged KIR and by a non-relevant target (RCC-GE) (Fig. 1C, left panel). In addition, preincubation of LCL-HP with blocking anti-HLA-B/C mAb restored a significant [Ca2+]i release by CTL comparable to the

6


one detected in response to LCL-DM (Fig. 1C, right panel). Thus, the difference of susceptibility of HP and DM cell lines to specific CTL was exclusively accounted for by CD158a receptor engagement.

Lack of lipid rafts polarization on the CD3-activated CTL by CD158a NK receptor

Data depicted in Fig. 2A indicate that triggering of KIR by specific mAbs dramatically abrogated the CD3-redirected lysis of murine FcR+ P815 cells. Thus, TCR and KIR were triggered on CTL by microbeads coated with anti-CD3 or anti-CD3 + anti-CD158a mAbs and membrane reorganization was analysed using confocal microscopy (Fig 2B). Stimulation by anti-CD3 coated beads (beads exhibit light red autofluorescence) induced reorganization of membrane lipid rafts visualized on FITCCT-B labeled CTL by formation of fluorescent patches and protusion of the CTL at the contact site between cells and beads when compared to unstimulated CTL (Fig. 2B, a and e). In addition, labeling with anti-Ptyr mAb revealed a bright polarized red and green overlapping staining indicating an increased accumulation of phosphorylated proteins at the cell/bead contact compared to light diffuse staining in control cells (Fig. 2B, b and f). Co-triggering of the TCR and KIR on CTL did not induce redistribution and clustering of FITC-CT-B labeled rafts and staining with anti-Ptyr mAb revealed a weak and diffuse staining (Fig. 2B, c and d), suggesting that KIR engagement affects TCR-mediated tyrosine phosphorylation. When CTL were stimulated by beads coated with anti-CD3 + anti-HLADR, similar results as those observed with anti-CD3 alone were obtained whereas anti-CD158 coated beads had no effect on CTL (data not shown). In three independent experiments the numbers of CTL that formed fluorescent patches at the cell/bead contact were counted and revealed that the triggering of KIR significantly decreased the percentages of CTL aggregating lipid rafts (5% versus 32%). This strongly indicates that KIR play a role in the prevention of lipid rafts polarization.

Prevention of lipid rafts polarization and CD3 clustering on CTL by tumor cells co-triggering TCR and KIR

7


Using fluorescence imaging, we investigated the effect of simultaneous co-triggering of TCR and KIR receptors on the membrane redistribution events at the contact zone between CTL and RCC targets. FITC-CT-B labeled CTL were stimulated with HP or DM tumor targets and stained with anti-Ptyr mAbs (Ptyr-Cy5, red) after heteroconjugates fixation. Major differences between CTL/RCC-HP and CTL/RCC-DM conjugates were observed at this level of resolution. Fig. 3A shows a typical image of interface between CTL and RCC-DM tumor cell with strong lipid rafts concentration at the contact zone with the tumor cell, that colocalizes in the overlay picture with the dense red anti-Ptyr staining (Fig. 3A, upper panels). The lower panels correspond to the contact zone of CTL/RCC-HP conjugates characterized by the absence of accumulation of fluorescent lipid rafts (green) and undetectable level of phosphorylation (Fig. 3A, lower panels). Quantitative analysis revealed that only 10% of the CTL/RCC-HP conjugates showed a tight polarization of the fluorescence at contact site whereas fluorescent patches were evident in 32% of CTL/RCC-DM conjugates. In addition, in Fig. 3B double staining with anti-CD3 mAb and anti-Ptyr revealed a zone of dense CD3 staining at the contact with RCC-DM tumor cell that overlapped with regions of plasma membrane containing the highest Ptyr accumulation. TCR/CD3 accumulation was observed in aggregated lipid rafts (data not shown). In CTL/RCC-HP conjugates (Fig. 3B, lower panel), anti-CD3 staining displayed a ring like pattern and was not associated with PTK activation. These results demonstrate that CD158a engagement by its specific ligand HLA-Cw4 on RCC-HP significantly impaired lipid rafts coalescence, TCR/CD3 capping and early TCR signaling through PTK activation during CTL/tumor cell contact.

Decreased phosphorylation of TCR-induced tyrosine kinase ZAP-70 and LAT upon KIR engagement

KIR receptors are rapidly phosphorylated on tyrosine residue of ITIM and recruits phosphatase SHP-1 that is responsible for the KIR inhibitory effect on TCR-mediated signaling 28,29. In the present model, immunoprecipitation of the PTK ZAP-70 showed that a low amount of tyrosine phosphorylation of this PTK was observed following CTL stimulation by RCC and LCL-HP target cells that engage the KIR when compared to stimulation with RCC and LCL-DM cells (Fig. 4A, upper panel). Although

8


less obvious, immunoprecipitation of LAT, the ZAP-70 substrate also showed that KIR engagement by HP cell lines reduced LAT phosphorylation. As indicated by densitometric values, there was a 2.48 fold lower LAT phosphorylation in response to RCC HP stimulation (Fig. 4A, lower panel). In addition, preventing triggering of KIR during CTL stimulation by masking HLA-C molecules with anti- HLA-B/C mAb (Fig. 4B, lane 3) on RCC-HP targets cells resulted in a significantly restored phosphorylation on tyrosine residues of ZAP-70 and LAT proteins. These results evidence that KIR triggering decreases early TCR mediated signaling in tumor specific 4D4 CTL clone.

Failure of TCR-induced Vav phosphorylation and actin cytoskeleton redistribution upon KIR ligation

Protein tyrosine phosphorylation patterns of 4D4 CTL clone after stimulation by the previous RCC targets revealed marked differences in CTL stimulated with targets expressing the KIR ligand (RCCHP, Fig 5, lane2) compared to the stimulation with targets engaging TCR only (RCC-DM, lane 3). Compared to unstimulated CTL (lane1) and to background tyrosine phosphorylated proteins in HP and DM tumor cells (lanes 4 and 5), stimulation of CTL with the susceptible DM targets (lane 3) resulted in phosphorylation of proteins around 95kD and 76kD that were not detected in CTL stimulated by RCC-HP (lane 2). Immunoblotting with specific Abs indicated that these proteins presumably corresponded to Vav and SLP-76 proteins, respectively (Fig 5). Indeed, immunoprecipitation of Vav shows that triggering of KIR by RCC-HP targets totally prevents Vav phosphorylation compared to RCC-DM targets that do not engage the KIR (Fig 5B, lane 2 and 5). Interestingly, preventing KIR triggering by specific mAbs on HP tumor cells restored the tyrosine phosphorylation of Vav (Fig 5 B, lane 3). In addition, confocal microscopy analyses further indicate that actin cytoskeleton rearrangement in CTL was greatly affected by KIR engagement. Fig 5C shows that in CTL/RCC-DM conjugates, polymerized F-actin (red staining) was accumulated at the same zone than polarized lipid rafts (green) (Fig 5C, upper panels). In contrast, an homogeneous distribution of membrane rafts and no marked actin polymerization were observed in CTL stimulated by RCC-HP targets (Fig 5C, lower panels).

9


Discussion :

Renal cell carcinoma are considered as potentially immunogeneic tumors on the basis of the rare but documented spontaneous regressions reported and in light of the clinical responses obtained in some patients treated with cytokines. Although RCC are infiltrated by numerous T cells confirming that the immune system may control the growth of these tumors, this tumor model is also characterized by the existence of various immune defects that result in the global functional alteration of the cytotoxic T effector cells

30-32

. It is known that human solid tumors mostly express tissue specific or self-antigens

that may induce local tolerance and cytotoxic T cells infiltrating RCC may correspond to antigen experienced CTL. In accordance with this hypothesis, we have previously shown that clonal KIR+CTL, bearing a memory activated phenotype, infiltrated RCC 17. These KIR+CTL that recognize tissue specific antigens and/or self-antigens represent potentially lytic T cells but their activity against tumor targets is inhibited by the KIR 17,33. In the present study, we took advantage of a tumor derived 4D4 CTL clone, expressing a unique CD158a receptor (specific for HLA-Cw4 supertype) that recognizes a HLA-A2-restricted self antigen, to determine the influence of such receptor on the early and late steps of CTL activation. 4D4 CTL clone efficiently lysed allogeneic HLA-A2+, Cw3+ DM targets whereas lysis and intracellular Ca2+ mobilization towards autologous HLA-A2+, Cw4+ HP targets were abrogated following specific CD158a triggering.

It is established that KIR receptors counteract the signaling initiated by the TCR/CD3 receptor in CTL and the underlining mechanism involves at least in part, the recruitment of SHP-1 following phosphorylation of the KIR ITIMs by Src family kinases. SHP-1 extinguishes signaling on neighboring ITAMs and efficiently inhibits CD3 phosphorylation 28, suggesting a sequential aspect in the response to TCR and KIR co-engagement. Our data show that when 4D4 CTL clone was stimulated by antigen positive targets lacking the KIR ligand (DM targets), ZAP-70 and its substrate LAT, two proteins known to be essential for the propagation of TCR-mediated signal were tyrosine phosphorylated

19

. In contrast, after 4D4 CTL stimulation by HP cells that trigger the CD158a

10


receptor, we observed a reduced level of ZAP-70 and LAT phosphorylation. As shown for cytotoxicity and calcium response, preventing triggering of KIR by masking of HLA-C molecules on HP targets restored the phosphorylation of these two essential signaling proteins indicating that KIR efficiently downregulated initial TCR-mediated signaling. Analysis of morphological events by confocal microscopy studies indicate that TCR induced signaling takes place in the membrane rafts and further illustrate that TCR and CD158 co-engagement abrogates TCR/CD3 and Ptyr accumulation at the CTL/target contact zone. Following CTL/target specific conjugate, phosphorylation of KIR on CTL 4D4 recruits SHP-1 (data not shown) may inhibit the signaling by the few TCR engaged. Based on these results, we may assume that TCR and KIR act concomitantly with early signaling of TCR involving the rapid phosphorylation of ITIM on KIR and further downregulation of TCR signaling. Recent data on early clustering of the TCR complex position of KIR

35

34

as well as on the binding kinetics and spatial

, emphasize the inhibitory role of KIR on the early consequences of TCR

engagement with its MHC/peptide ligand interrupting the activation program.

It is clearly established that the linker for activation of T cell LAT, predominantly localized into lipid rafts, plays a central role in signal transduction upon TCR engagement. Indeed, TCR-mediated tyrosine phosphorylation of LAT results in the formation of a structural scaffold of downstream signaling proteins such as Vav, PLC and SLP-76, critical for the propagation of the signal 19,36,37 and for the dynamic actin polymerization 20. Furthermore, it was shown that Vav, the guanine nucleotide exchange factor (GEF) for Rac/Cdc42 small GTP-ases regulates actin cytoskeleton reorganization, the association of actin to CD3 chain and TCR/CD3 capping

21,22,38

. In our model, TCR and KIR co-

engagement by tumor cells decreased LAT activation, and completely abrogated the tyrosine phosphorylation of Vav that was however restored when KIR triggering was prevented by specific mAbs. Tyrosine phosphorylation of Vav has been correlated with its GEF activity for RhoGTPases39. Thus, the inhibitory effect of KIR triggering on Vav phosphorylation correlated with the absence of actin cytoskeleton redistribution towards HP targets. Our data suggest that Vav activation is a crucial event in the control of CTL activation by KIR since it links TCR signaling dependent-cytoskeleton rearrangement to lipid rafts clustering 40.

11


The dynamic reorganization of membrane proteins, also termed immunological synapse, is initiated by coalescence of lipid rafts and correlates with the sustained activation of T cell 41,42. In cytotoxic T cell effectors, the immunological synapse brings the T cell secretory apparatus into close contact with the target for a polarized release of cytokines and cytotoxic mediators

43,44

. Our data show that KIR

engagement by targets was effective in inhibiting TCR-mediated lipid rafts coalescence, TCR/CD3 and phosphotyrosine protein accumulation at the contact site, as the underlining mechanism for abrogation of CTL mediated killing. Our findings are supported by a recent report of Colonna et al outlining the existence of a cross talk between ILT-2 and TCR with a reduction of both TCR signaling and TCR-induced reorganization of the actin cytoskeleton at the interface of the APC-T cell after ILT2 ligation 45.

Taken together, the present studies emphasize that although KIR+CTL effectively recognized renal tumor cells, KIR engagement induced an alteration of early TCR/CD3 signaling and prevented the membrane rafts reorganization resulting in target protection from lysis. KIR has been proposed as a regulator of homeostasis of memory T cell subsets, involved in their in vivo survival 11. In the context of an immune response to tumor, the KIR receptor may be involved in the local functional alteration of self specific CTL. In renal tumors, KIR+CTL reflect the presence of an ongoing anti-tumor immune response of memory T cells induced in response to chronic antigen stimulation by tumor cells. Although the mechanisms controlling the induction of such inhibitory receptors on CTL are not known, their influence on tumor specific CTL functions is remarkable and the expression of KIR by TIL represent a powerful mechanism that contribute to antigen specific CD8+ dysfunction. In that context, an in vivo up-regulation of CD94-NKG2A by antiviral CTL responsible for their functional alteration and virus induced oncogenesis was recently evidenced 46. In renal tumors, the presence of KIR+CTL highlights a particular regulation of cytotoxic effectors and may partly explain the beneficial effect currently, not clearly understood of the allogeneic hematopoïetic stem cell transplantation in RCC patients

47,48

. The existence of such CTL should be taken into account in any

immunotherapeutic protocols in RCC patients.

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Methods

Culture of tumor cells and CTL: Tumor cells derived from RCC were maintained in Dulbecco modified Eagle medium/Ham F12 medium supplemented with 10% fetal calf serum (FCS) and Ultroser G (Gibco BRL, Scotland, UK). EBV-transformed B cell lines (LCL) from the same donors were maintained in RPMI 1640 medium supplemented with 10% FCS. Tumor cells were HLA genotyped HP : HLA-A2, A29, B51, B44, Cw15 Cw16 (Cw4 supertype), DM : HLA-A1, A2, B51, B8, Cw7, Cw14 (Cw3 supertype) and GE : HLA-A9, A32, B7, B49, Cw7. 4D4 CD158+ CTL clone obtained from direct cloning of tumor infiltrating lymphocytes from RCC (patient HP) was expanded on allogeneic feeder cells in RPMI1640 medium supplemented with 10% human serum and 30 U/ml IL-2 (Roussel Uclaff, Romainville, France). Briefly, 3x103 T cells/well platted with 7x104 irradiated allogeneic PBMC and 104 irradiated allogeneic LCL (LAZ509) were grown for 14 days. Expression of CD158a by 4D4 CTL was stable over time in culture.

Cytotoxicity assay : The cytolytic activity of 4D4 CD158a+ T cell clone against tumor cells was measured in a 4 h-chromium 51 (51Cr)- release assay as previously described 17. In some experiments, mAbs EB6 (anti-CD158a, IgG1), B1.23.2 (anti-HLA-B or C, IgG2a), L243 (anti-HLA-DR, IgG2a) or UCHT1 (anti-CD3, IgG1) were added at saturating concentrations at the beginning of the cytolytic assay.

Measurement of intracellular calcium concentration by flow cytometry: Target cells were labeled with PKH26 fluorescent cell linker (5.10-6 M dye Sigma), washed three times and rested for 1 h at 37°C. 4D4 CTL clone (5.106/ml) were labeled with 4µM Fluo 3-AM (Sigma) for 45 min at 37°C, washed twice with serum rich medium and resuspended in hepes-buffered medium. Mixed cells (5x104 CTL and 105 target cells /400µl) were centrifuged 1 min at 1.500 rpm, incubated 1 min at 37°C, gently resuspended and immediately analyzed on Flow cytofluorimeter (FACS-Calibur, Becton Dickinson). In blocking conditions, target cells were incubated with saturating concentration of anti-

13


HLA-B/C or irrelevant anti-HLA-DR mAbs, before stimulation. Ca2+ release was evaluated by measuring green fluorescence of Fluo3 emission at 526 nm. The concentration of intracellular calcium was calculated using the formula [Ca2+]i nM = KD (F-Fmin) / (Fmax-F) as previously reported

49

,

where KD represents the dissociation constant for Ca2+ bound-Fluo-3 and is 400 nM. Fmax and Fmin were evaluated by adding a of calcium ionophore (ionomycin 10 µg/ml, Sigma) followed by MnCl2 (2 mM, Sigma) to Fluo-3 loaded-CTL.

Receptor clustering and raft redistribution by confocal microscopy: Lipid raft aggregation on CTL stimulated by mAb coated-beads have been described

26

. Beads (polystyrene latex microspheres,

Polysciences) were coated with 10µg/ml of anti-CD3 (UCHT1, IgG1, Becton Dickinson, Le Pont de Claix, France); anti-CD3 + anti-CD158a (10 µg/ml) or anti-HLA-DR (10 µg/ml) and used at 2/1 ratio over T cells. 4D4 CTL clone labeled with fluorescent B subunit of cholera toxin (Sigma, 8µg/ml), FITC-CT-B (which binds to GM1 glycosphingolipids in the lipid rafts) was incubated with mAbcoated-beads for 15 min at 37°C. T cell-bead complexes were settled onto poly-l-lysine-coated slides. After fixation, CTL/beads were saturated with mouse IgG1 Abs and permeabilized for 10 min (Hepesbuffered PBS containing 3% BSA and 0.1% saponin) before staining with anti-Ptyr mAb (4G10, IgG2b, Euromedex, Souffelweyersheim, France) followed by goat anti-mouse IgG2b-Cy5 (Caltag laboratories, Burlingame, CA, USA). For CTL / tumor cell interaction analysis, CTL were mixed to PKH26 labeled tumor cells, centrifuged 2 min at 1500 rpm, settled onto slides after gentle resuspension and incubated at 37°C for 10 min. After fixation, CTL were labeled with anti-Ptyr or anti-CD3 mAb (UCHT1, IgG1) followed by the labeled-isotypic secondary Abs. Alternaltively, CTL/RCC conjugates were stained with Phalloidin Texas-red (Molecular Probes, Eugene OR) to detect polymerized F-actin. The samples were conserved in mounting medium (Vectashield, Biovalley) at +4°C and analyzed by laser scanning confocal microscopy, a Leica TCS Confocal System (Wetzler, Germany). Cells were considered to have clustered receptors if the staining pattern was crescent shaped and receptors polarized to one side of the cell 50. A total of 100 conjugates per slide were analysed to evaluate percentages.

14


Immunoblot and immunoprecipitation analysis: 4D4 CTL clone (5x105-106 per sample for immunoprecipitation and 2x105 per sample for whole cell extracts) were stimulated with target cells for 3 min at 37°C. CTL stimulations were performed at 7/1 E/T ratio for tumor cells and 5/1 ratio for LCL. Stimulated cells were rapidly pelleted and lysed for 20 min on ice in 1% NP-40 lysis buffer containing 20 mM tris-HCl (pH 7.5), 150 mM NaCl, 1mM EGTA and 1% of n-Dodecyl beta-Dmaltoside in presence of protease and phosphatase inhibitors: 10 µg/ml leupeptin, 10 mg/ml aprotinin, 1 mM Pefabloc-sc, 50 mM NaF, 10 mM Na4P2O7 and 1 mM NaVO4. Insoluble material was removed by centrifugation 10 min at 14,000 rpm at +4°C. Postnuclear lysate was kept to analyse the protein tyrosine phosphorylated profile and the remaining material was immunoprecipitated. Cell lysates were subjected to immunoprecipitation for 2 h with anti-LAT (rabbit anti-human p36 serum directed against 219-233 residues), anti-ZAP-70 (IB60, rabbit polyclonal Ab directed against 266-344 residues) preadsorbed to protein A-Sepharose, (O. Acuto, Paris, France) or anti- Vav (mixed H211 and C14 rabbit polyclonal Abs) (Santa Cruz Biotechnology, CA). Immunocomplexes were washed twice in 20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% NP-40, twice in 20 mM Tris-HCl, pH 7.5, 150 mM NaCl and boiled in SDS sample buffer before gel electrophoresis. Whole cell lysates and immunoprecipitated proteins were separated on SDS-polyacrylamide gels and blotted onto nitrocellulose membranes. Separated proteins were probed initially with mAb 4G10 (antiPtyr) and after stripping (0,1M glycine HCL pH 2,3) with specific antibodies : anti-LAT (L.Samelson, NIH, Bethesda); anti-ZAP-70, (IB60); anti-Vav-30 mAb (J. Griffin, Boston, MA) and M14, rabbit anti- human SLP-76 (O. Acuto, Paris, France). Proteins were revealed by peroxidase-conjugated goat anti- mouse or peroxidase-conjugated protein A and developed with the enhanced chemiluminescence detection system (Amersham Pharmacia Biotech, Orsay, France). Densitometric analysis was performed using Bioprofil Bio 1D Windows application V99.04. Values indicate the ratio of tyrosine phosphorylation to protein signal.

15


Figure legends:

Figure 1. Cytotoxicity and intracellular Ca2+ release of 4D4 CTL clone are inhibited by the CD158a receptor. (A) Cytotoxicity of CD158a+CTL clone 4D4 was assessed at 3/1:E/T ratio against RCC and EBV-B transformed cell lines (LCL) derived from three patients: autologous RCC-HP (HLA-A2, Cw4), allogeneic RCC-DM (HLA-A2, Cw3) and RCC-GE (HLA-A9, Cw3). Cytotoxicity was determined in absence (medium) or presence of anti-HLA-B/C or control anti-HLA-DR mAbs (one out of five experiments is shown). (B) Flow cytometry analysis of [Ca2+]i release by Fluo3-AM loaded CTL stimulated for 1 min at 37°C with PKH26 labeled target cells. Percentages correspond to CTL conjugates with calcium release out of total CTL. Target cells profile of LCL-HP alone is represented (left panel). (C) [Ca2+]i measurements were calculated from mean fluorescence values (see material and methods). Determination of [Ca2+]i release by CTL stimulated by RCC cells (left panel), LCL-DM or LCL-HP, coated or not with mAbs (right panel).

16


Figure 2. KIR triggering prevents CD3-redirected lysis and CD3 induced membrane rafts reorganization. (A) Cytotoxic activity of 4D4 clone was assessed against anti-CD3-coated P815 murine cells. CTL 4D4 was added at 5/1 to target in absence (anti-CD3) or in presence of 10µg/ml of anti- CD158a (+anti-CD158) or anti-HLA-DR mAbs (+anti-HLA-DR). (B) Membrane reorganization of CTL clone 4D4 was analysed by confocal microscopy after stimulation for 15 min at 37°C with mAbs coated beads. CTL membrane was labeled with fluorescent B subunit of Cholera Toxin (FITC-

17


CT-B, green) and after cell permeabilization, CTL were labeled with anti-Ptyr mAb revealed by Cy5 labeled goat anti-mouse IgG (red). An overlay compacted image of serial optical sections is shown on left side panels (a, c and e), the green color has been removed on right panel (b, d and f) (magnification x 63).

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Figure 3. KIR engagement prevents lipid rafts polarization and signaling protein phosphorylation: Confocal analysis of serial optical sections collected at 0.3µm intervals. (A) 4D4 CTL clone labeled by FITC-CT-B (green) was stimulated with PKH26-labeled tumor cells (blue). 4D4 CTL clone was conjugated either with RCC DM (upper panels) or RCC HP (lower panels) and incubated 5 min at 37°C. After fixation, cells were stained with anti-Ptyr mAb (red). This picture represents an overlay and the side-side picture of the overlay showing labeling lipid rafts (CT-B-FITC) or tyrosine-phosphorylated proteins (Ptyr-Cy5) as indicated. (B) Stimulated 4D4 CTL clone was stimulated as in (A) and then stained with anti-CD3 mAb (green) and anti-Ptyr mAb (red).

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Figure 4: Inhibitory effect of KIR on TCR mediated ZAP-70 and LAT tyrosine phophorylation. (A) Post nuclear lysates of non stimulated CTL (lane 1) or stimulated by LCL or RCC targets were immunoprecipitated with polyclonal anti-ZAP-70 or anti-LAT Ab and analysed for tyrosine phosphorylation (left panels). (B) Same conditions of immunoprecipitation as in (A) of CTL (lane1) stimulated by RCC-HP (lane 2), masking of KIR ligand with anti-HLA-B/C (lane 3) or control antiHLA-DR mAbs (lane 4) before stimulation (right panels). Lower panels show membrane reprobing with corresponding Abs specific for precipitated proteins.

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Figure 5: KIR engagement prevents Vav tyrosine phosphorylation and the reorganization of actin cytoskeleton. (A) Tyrosine phosphorylated protein patterns were analyzed by immunoblot from total cell lysates of non stimulated 4D4 CTL (lane 1) or incubated 3 min at 37°C with RCC-HP (lane 2) or RCC-DM cell lines (lane 3). Ptyr-protein profil of RCC-HP (lane 4) and RCC-DM (lane 5) alone are shown. The lower panel shows membrane reprobing with mAb against SLP-76, the upper band is non specific detection of tumor cell proteins. Densitometric values are indicated in italic. (B) Post nuclear lysates of non stimulated CTL (lane 1) or stimulated by RCC-HP targets were immunoprecipitated with polyclonal anti-Vav Abs and analysed for tyrosine phosphorylation. CTL was stimulated by RCC-HP alone (lane 2), or in presence of anti-HLA-B/C (lane 3) or control antiHLA-DR mAbs (lane 4); alternatively CTL was stimulated by RCC-DM (lane 5). Lower panels show

21


membrane reprobing with corresponding Abs specific for precipitated proteins. (C) Confocal analysis was performed as indicated above. 4D4 CTL clone labeled by FITC-CT-B (green) was stimulated with RCC-DM (upper panels) or RCC-HP (lower panels) for 10 min at 37°C. After permeabilization, fixed cells were stained with Phalloidin/Texas red (red). This picture represents an overlay and the side-side picture of the overlay showing labeled lipid rafts (CT-B-FITC) or Phalloïdin/Texas red as indicated.

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