HLA-G-mediated inhibition of antigen- specific cytotoxic T lymphocytes

International Immunology, Vol. 11, No. 8, pp. 1351–1356

© 1999 The Japanese Society for Immunology

HLA-G-mediated inhibition of antigenspecific cytotoxic T lymphocytes Fre´de´rique-Anne Le Gal, Be´atrice Riteau1, Christine Sedlik1, Iman Khalil-Daher1, Catherine Menier1, Jean Dausset2, Jean-Ge´rard Guillet, Edgardo D. Carosella1 and Nathalie Rouas-Freiss1 Laboratoire d’Immunologie des Pathologies Infectieuses et Tumorales, INSERM U445, Institut Cochin de Ge´ne´tique Mole´culaire, 27, rue du Faubourg St Jacques, Universite´ Rene´ Descartes, 75014 Paris, France 1Service de Recherches en He ´ mato-Immunologie, CEA-DSV-DRM, Hoˆpital Saint-Louis, 1, avenue Claude-Vellefaux, 75475 Paris Cedex 10, France 2CEPH-Fondation Jean Dausset, 27, rue Juliette-Dodu, 75010 Paris Cedex, France

Keywords: immunotolerance, killer inhibitory receptor

Abstract In the present study, we demonstrate that the non-classical MHC class I molecule HLA-G impairs specific cytolytic T cell functions in addition to its well-established inhibition of NK lysis. The antigen-specific cytotoxic T lymphocyte (CTL) response analyzed was mediated by CD8F T cells specific for the influenza virus matrix epitope, M58–66, presented by HLA-A2. The transfection of HLA-G1 cDNA in target cells carrying the M58–66 epitope reduced their lysis by these virusspecific CTL. This HLA-G-mediated inhibition of antigen-specific CTL lysis was (i) peptide dose dependent, (ii) reversed by blocking HLA-G with a specific mAb and (iii) still observed despite the blockade of HLA-E/CD94/NKG2A interaction. By inhibiting both CTL and NK functions, HLA-G appears to have an extensive role in immune tolerance. Introduction Unlike classical HLA class I molecules, the non-classical HLA-G molecule is only found in certain tissues, presents a limited polymorphism, and is produced by alternative transcription of spliced mRNAs that encode at least six different membrane-bound and soluble HLA-G isoforms (1,2). The HLA-G1 isoform has a classical HLA class I structure, consisting of α1, α2 and α3 extracellular domains non-covalently associated with β2-microglobulin, and has been detected as a membrane-bound protein at the cell surface (3,4). Studies on the immunological functions of HLA-G over the past few years have identified it as a key mediator in immune tolerance (5) by protecting HLA-G1 target cells from NK cytolysis through interaction with killer inhibitory receptors (KIR) (4–11). In line with this, we have demonstrated that HLA-G molecules protect (i) trophoblast cells from the lytic activity of maternal uterine NK cells ex vivo, making it important in materno-fetal tolerance (12) and (ii) HLA-G1 melanomas cells from NK lysis, which may be one way in which tumors escape immunosurveillance (13). However, HLA-G modulation of T cell-mediated immunity remains to be addressed. The presence of KIR on T lymphocytes (14,15), their interaction with HLA class I molecules (16–19) and recent data on HLA-

G expression in the thymus (20) suggest that HLA-G molecules act on T lymphocyte responses. In the present study, we have investigated whether HLAG1, in addition to its well-established NK inhibitory properties, inhibits T cell function by analyzing a well-characterized cytotoxic T lymphocyte (CTL) response specific for the influenza virus matrix epitope M58–66 presented by HLA-A2 (21). We used an HLA-A21 target cell line transfected with HLAG1 cDNA to show that the antigen-specific CTL lysis was significantly reduced. These results provide the first direct evidence that HLA-G1 molecules play an important role in the recognition of MHC-restricted, antigen peptide-specific T cells. Methods

Cell lines and peptide The M8 (HLA-A1, -A2 and -B12, and B-40) human melanoma cell line was kindly provided by F. Jotereau (INSERM U211, Nantes, France). M8 transfectants were obtained as previously described (4) using vectors containing pRc-RSV-HLA-G1 and

Correspondence to: N. Rouas-Freiss Transmitting editor: J.-F. Bach

Received 8 February 1999, accepted 6 May 1999

1352 HLA-G inhibits antigen-specific CTL response the pRc-RSV vector alone as a control. Cells were maintained in RPMI medium supplemented with 10% inactivated FCS, 2 mM L-glutamine, 1 µg/ml gentamycin and fungizone (Sigma, St Louis, MO). HLA-G transfectants were selected by growth in medium containing 1 mg/ml geneticin (Sigma). The human choriocarcinoma HLA-G1 cell line JEG-3 (ATCC, Rockville, MD) was cultured in DMEM (Sigma) supplemented with 10% heat-inactivated FCS, antibiotics and 2 mM L-glutamine. All the cell lines used in this study were free from mycoplasma. The peptide M58–66, GILGFVFTL, derived from the matrix of the influenza virus, was synthesized by Neosystem (Strasbourg, France).

harvested after 1 week in culture. To determine the KIR phenotype of this CTL line, cells were stained with the indicated anti-KIR mAb revealed with a F(ab9)2 goat antimouse IgG antibody conjugated to fluorescein, and with phycoerythrin-conjugated anti-Vβ17 mAb and Quantum Redconjugated anti-CD8 mAb followed by three-color immunofluorescence analysis. Control aliquots were stained with the corresponding isotype-matched antibodies to evaluate nonspecific binding to cells. The parameters were collected in Listmode files: linear forward scatter, linear side scatter, log FITC, log phycoerythrin and log Quantum Red fluorescence.

mAb and flow cytometry analysis

Cytolysis was measured with a standard 4 h 31Cr-release test. CTL effectors and targets labeled with 51Cr (100 µCi of 51Crlabeled sodium chromate; Amersham), and pulsed (or not) with M58–66 peptide for 1 h 30 min, were incubated together at different E:T ratios in U-bottomed microtiter plates. The radioactivity released into supernatants was counted in a Cobra γ-counter. The percentage of specific lysis was calculated as follows: percent specific lysis 5 [(c.p.m. experimental well – c.p.m. spontaneous release)/(c.p.m. maximum release – c.p.m. spontaneous release)]3100. Results are presented as the means of triplicate samples. In experiments in which mAb were used to block KIR interaction, target cells or effector cells were incubated with the corresponding mAb, washed and incubated with a F(ab9)2 goat anti-mouse IgG antibody (Jackson ImmunoResearch, West Grove, PA) in order to prevent antibody-dependent cell cytotoxicity.

The following mAb were used: 87G, IgG2b anti-HLA-G1 kindly provided by D. Geraghty (Fred Hutchinson Cancer Research, Seattle); BB7.2, IgG2b anti-HLA-A2 (ATCC); 4H84, IgG1 anti-HLA-G kindly provided by S. Fisher and M. McMaster (University of California, San Francisco); XA.185, IgG1 antiCD94 mAb and Z 270, IgG1 anti-NKG2A mAb were kindly provided by A. Moretta (University of Genova); anti-CD8 conjugated to Quantum Red (Sigma); and anti-Vβ17 conjugated to phycoerythrin (Immunotech, Marseille, France). For flow cytometry assays, cells were washed in PBS and stained with the corresponding mAb in PBS/2% FCS for 30 min at 4°C. The cells were washed twice and analyzed directly in a flow cytometer (FACS Vantage; Becton Dickinson, Le Pont-de-Claix, France) if the mAb used was conjugated to fluorochrome or stained with an F(ab9)2 goat anti-mouse IgG antibody conjugated to phycoerythrin (Immunotech) for FACS analysis. Control aliquots were stained with an isotypematched antibody to evaluate non-specific binding to target cells. The cytometer was calibrated using Fluoresbrite Calibration grade 2 µYG-microspheres (Polysciences, Fischer Scientific, Osi, France) and the calibration points were set using the Lysys II program software, ‘by eye’. The parameters were collected in Listmode files: linear forward scatter, log side scatter and log phycoerythrin fluorescence. Off-line analysis was conducted using Lysys II software as supplied by Becton Dickinson.

Western blot analysis Cells were lysed in buffer containing 1% NP 40 and the protein concentration was estimated. Aliquots (20 µg) of total protein were separated in 10% SDS–PAGE. The gels were blotted onto nitrocellulose membranes (Hybond; Amersham, Little Chalfont, UK), and the membranes were blocked by incubation with PBS containing 0.2% Tween 20 and 5% BSA. The membrane was then probed with the 4H84 mAb (overnight at 4°C) and washed in PBS containing 0.2% Tween 20. The membrane was subsequently incubated for 40 min at room temperature with peroxidase conjugated sheep anti-mouse IgG antibody, washed thoroughly and stained with ECL Western blot detection reagent (Amersham). Finally the membrane was exposed to Kodak film at room temperature.

Generation of influenza peptide M58–66-specific CTL The CTL line specific for the matrix peptide of the influenza A virus was generated from PBMC of donor HC12 (HLA-A2, A3, -B7, -B60 and -C7) as previously described (22) and

Cytotoxicity assays

Results and discussion The inhibition of CTL response by HLA-G molecules was assessed using target cells bearing HLA-G in a antigenspecific CTL model (21). The CTL effectors were induced in vitro from the PBMC of a healthy donor by incubation with the HLA-A2.1-restricted influenza virus peptide M58–66 for 1 week giving rise to the HC12-J7 cell line. The HLA-A21 M8 cells were used as target cells, since no HLA-G transcript has been detected in this cell line (13). HLA-G1 cDNA, coding for the full-length HLA-G isoform, was transfected into M8 cells to obtain a target cell line expressing both HLA-G1 and the HLA-A2 restriction molecule. We first looked for the presence of the HLA-G1 molecule on the cell-surface by FACS analysis using the anti-HLA-G1 87G mAb. The M8 control cell line transfected with the vector alone (M8-RSV) did not express HLA-G1 molecules, whereas the HLA-G1transfected M8 cells (M8-HLA-G1) had a high concentration of HLA-G1 molecules on their surface (Fig. 1A). Western blot analysis using the anti-HLA-G denatured heavy chain 4H84 mAb detected a 39 kDa protein on M8-HLA-G1, as well as on JEG-3, the HLA-G1 control cell line. In contrast, no HLAG1 protein was detected on M8-RSV (Fig. 1B). Both M8-RSV and M8-HLA-G1 cells were then sensitized with the M58–66 influenza virus peptide and used as target cells for the M58–66-specific CTL effectors among the HC12J7 cell line. A CTL cytotoxicity assay performed at different E:T ratios showed that lysis of M8-HLA-G1 cells was significantly reduced compared to that of M8-RSV cells (Fig. 2A). Interestingly, this inhibition did not occur at a high peptide concentra-

HLA-G inhibits antigen-specific CTL response 1353 tion (10–1 µg/ml), suggesting that the off-signal triggered by HLA-G can be overcome by excess antigen (Fig. 2B). In order to confirm that the decreased lysis of M8-HLA-G1 was not due to decreased expression of HLA-A2 molecules (and therefore diminished peptide presentation), we checked by FACS analysis that both M8-RSV and M8-HLA-G1 had similar levels of expression of HLA-A2 molecules (Fig. 1A). To further demonstrate that inhibition of CTL lysis was due to HLA-G1 cell-surface molecules, cytotoxicity assays were performed with target cells that had been incubated with the anti-HLAG1 87G mAb or an irrelevant isotypic control Ab. In this experiment, we carefully blocked the Fc portion of the antiHLA-G1 mAb used to prevent antibody-dependent cell cytotoxicity occurring. Results showed that HLA-G1-mediated inhibition of antigen-specific CTL lysis could be reversed by masking HLA-G1 with 87G mAb (Fig. 3). This clearly

Fig. 1. The 39 kDa HLA-G1 protein is present at the cell surface of the M8-HLA-G1 transfectants. HLA-G1 expression on the transfectants of the M8 cell line was detected by (A) cytofluorometry using the antiHLA-G1 87 G mAb (bold profiles) and an isotype-matched control antibody (light profiles), and (B) Western blot analysis using the 4H84 mAb specific for the denatured HLA-G heavy chain. The HLAA2 expression level on both transfectants was determined by (A) cytofluorometry using the anti-HLA-A2 BB7.2 mAb (bold profiles).

Fig. 3. HLA-G1-mediated inhibition is blocked by the anti-HLA-G1 87 G mAb. The M8 transfectants were incubated with the M58–66 peptide at a final concentration of 0.005 µg/ml, then pretreated with 87 G or a control antibody at 20 µg/ml and used in a cytotoxicity assay with the HC12-J7 cell line. The results are expressed as the percentage specific lysis recorded in a 4 h 51Cr-release assay. Similar results were obtained with M8-RSV cells treated with either the control Ab or the 87G mAb. The SD of the mean of the triplicates was ,8% and the spontaneous release never exceeded 10% of the maximum release. This experiment was repeated at least 3 times, giving the same pattern of inhibition.

Fig. 2. HLA-G1 expression on target cells inhibits antigen-specific CTL lysis in a peptide concentration-dependent manner. M8 transfectants were incubated (A) with or without the M58–66 peptide at a final concentration of 0.01 µg/ml and used in a cytotoxicity assay with the HC12J7 cell line at various E:T ratios or (B) with various M58–66 peptide concentrations and used in a cytotoxicity assay with the HC12-J7 cells at the E:T ratio of 30:1. The results are expressed as the percentage specific lysis recorded in a 4 h 51Cr-release assay. The SD of the mean of the triplicates was ,8% and the spontaneous release never exceeded 10% of the maximum release. This experiment was repeated at least 3 times, giving the same pattern of inhibition.

1354 HLA-G inhibits antigen-specific CTL response demonstrates that this CTL inhibition results from a direct interaction between HLA-G1 and inhibitory receptors carried by the M58–66-specific effector cells. We next addressed the question of the inhibitory receptors involved in the HLA-G1-mediated protection. Two KIR that interact directly with HLA-G1 have been recently identified on NK and T lymphocytes. One is p49, which is composed of two extracellular Ig-like domains and a single ITIM in its cytoplasmic tail (23); the other is ILT-2, which belongs to a new family of Ig-SF receptors (24,25). Since biased usage of the TCR Vβ17 chain in response to the influenza matrix epitope M58–66 has been demonstrated in HLA-A2.1 individuals (26) and in the HC12 donor in particular (21), we determined the KIR expression on the Vβ171 CD81 cells, corresponding to the viral M58–66-specific CTL effectors. Only 10% of the Vβ171 CD81 cell population was ILT-21 by three-color FACS analysis (data not shown). Thus, the inhibitory action of HLAG1 is probably not mediated by this KIR in our experiments. We could not determine whether p49 was present on the Vβ171 CD81 cell population and implicated in the HLA-G1mediated inhibition we observed because no anti-p49 mAb is currently available. However, such inhibition may be the result of interaction with an as-yet unknown KIR. We are currently examining this point. Recent studies showed that HLA-G can play an indirect inhibitory role by stabilizing HLA-E with its signal peptide sequence and allowing HLA-E interaction with the CD94/ NKG2A inhibitory receptor (27,28). The CD94/NKG2A is a lectin-like inhibitory receptor present on NK and T cells that interacts specifically with HLA-E (29). To confirm the direct inhibitory role of HLA-G in our model, we investigated the involvement of the HLA-E/CD94/NKG2A interaction in the lysis inhibition we observed. For this purpose, we first checked the expression of HLA-E in both M8-RSV and M8-HLAG1 cell lines. Carrying out metabolic labelling followed by immunoprecipitation of HLA class I by W6.32 mAb, we observed in both cell lines a weak 42 kDa band corresponding to the expected mol. wt of HLA-E (data not shown). The presence of HLA-E was expected, since both M8 cell lines bear HLA class I molecules, such as HLA-A1 and HLA-A2, which contain within their leader sequences nonapeptides that induce the cell-surface expression of HLA-E and its interaction with the CD94/NKG2A receptor. We then determined whether the Vβ171 CD81 cells corresponding to the M58–66-specific CTL effectors expressed CD94/NKG2A. A three-color FACS analysis (Fig. 4) showed that the Vβ171 CD81 cells accounted for 58% of the total HC12-J7 cells after 1 week of in vitro stimulation by the influenza peptide and that 93% of these Vβ171 CD81 cells were CD941 and 84% were NKG2A1. Since HLA-E was expressed on our targets and most of the Vβ171 CD81 cells were CD941 NKG2A1, we looked at the effect of their interaction on the negative regulation of M58–66-specific CTL lysis. For this purpose, we used anti-CD94/NKG2A mAb because no anti-HLA-E mAb is currently available. Blocking the CD94/NKG2A receptors increased the lysis of the M58–66-pulsed M8-RSV target cells (Fig. 5). Thus the HLA-E/CD94/NKG2A interaction occurs in our system, leading to inhibition of lysis. These data are in good agreement with those of previous studies showing that the presence of CD94/

Fig. 4. The CD81Vβ171 cells present among the HC12-J7 cell population expressed KIR. Cells were analyzed by cytofluorometry and were gated on (A) dot-plot with respect to light scatter properties and (B) expression of double-labeled CD81 Vβ171 cells. Subsequent analyses were sorted on the window selected in (B) to show correlated expression of CD81Vβ171 with either (C) CD94 and (D) NKG2A receptors. The number indicated at the right of each quadrant corresponds to the percentage of (B) CD81Vβ171 cells among the HC12-J7 cell population and (C and D) KIR1 cells among the CD81Vβ171 HC12-J7 cells. Controls were the same cells stained with the corresponding isotype-matched control antibodies. Data from one representative experiment out of four are shown.

Fig. 5. HLA-G inhibition is not mediated through interactions with CD94/NKG2A. The M8 transfectants were incubated with the M58– 66 peptide at a final concentration of 0.002 µg/ml, then used in a cytotoxicity assay with the HC12-J7 cells preincubated with either (i) anti-CD94 and anti-NKG2A mAb as culture supernatants or (ii) control antibodies at 20 µg/ml. The results are expressed as the percentage specific lysis recorded in a 4 h 51Cr-release assay. The SD of the mean of the triplicates was ,8% and the spontaneous release never exceeded 10% of the maximum release. Data from one representative experiment out of three are shown.

NKG2A on antigen-specific CTL impairs their specific cytolytic activity (19,30). Although resulting in an increased lysis, treatment of the HC12-J7 with anti-CD94/NKG2A mAbs

HLA-G inhibits antigen-specific CTL response 1355 towards M8-HLA-G1 target cells did not fully restore lysis to that of the M8-RSV (Fig. 5). These results confirm recent studies showing that HLA-G1-mediated NK inhibition does not act via CD94/NKG2A (31). Considered together, our data show for the first time that HLA-G1 can directly block CTL function and that inhibition occurs even so HLA-E/CD94/ NKG2A interaction was impaired. The inhibition of the antigen-specific CTL response by HLAG provides new insight into the role of HLA-G in preventing allograft rejection. Hence, HLA-G could protect the semiallogeneic fetus by preventing maternal CTL allo-responses, in addition to inhibiting decidua NK cells during pregnancy (12). These findings may open new possibilities for treating tissue-graft rejection, since transplant rejection is primarily mediated by T lymphocytes. They may also be relevant to tumor immunology. As recently suggested, the interactions of KIR with their ligands on tumor cells in vivo may affect antitumor responses mediated by both innate and acquired immune effector cells (16,17). We have recently described the ectopic expression of HLA-G on melanoma cells leading to inhibition of tumor cell lysis by NK cells (13). We believe that HLA-G may also constitute a way for tumors to escape from CTL immunosurveillance. Acknowledgements We thank Owen Parkes for reading and correcting the manuscript.

Abbreviations CTL HLA KIR

cytotoxic T lymphocyte human leukocyte antigen killer inhibitory receptor

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