Nadine Gervois

Inhibition of antigen-induced T cell response and antibody-induced NK cell cytotoxicity by NKG2A : association of NKG2A with SHP-1 and SHP-2 protein-tyrosine phosphatases

Subsets of T and natural killer (NK) lymphocytes express the CD94‐NKG2A heterodimer, a receptor for major histocompatibility complex class I molecules. We show here that engagement of the CD94‐NKG2A heterodimer inhibits both antigen‐driven tumor necrosis factor (TNF) release and cytotoxicity on melanoma‐specific human T cell clones. Similarly, CD16‐mediated NK cell cytotoxicity is extinguished by cross‐linking of the CD94‐NKG2A heterodimer. Combining in vivo and in vitro analysis, we report that both I/VxYxxL immunoreceptor tyrosine‐based inhibition motifs (ITIM) present in the NKG2A intracytoplasmic domain associate upon tyrosine phosphorylation with the protein tyrosine phosphatases SHP‐1 and SHP‐2, but not with the polyinositol phosphatase SHIP. Determination of the dissociation constant, using surface plasmon resonance analysis, indicates that NKG2A phospho‐ITIM interact directly with the SH2 domains of SHP‐1 and SHP‐2 with a high affinity. Engagement of the CD94‐NKG2A heterodimer therefore appears as a protein‐tyrosine phosphatase‐based strategy that negatively regulates both antigen‐induced T cell response and antibody‐induced NK cell cytotoxicity. Our results suggest that this inhibitory pathway sets the threshold of T and NK cell activation.

Expression and Release of HLA-E by Melanoma Cells and Melanocytes: Potential Impact on the Response of Cytotoxic Effector Cells

HLA-E are nonclassical MHC molecules with poorly characterized tissue distribution and functions. Because of their capacity to bind the inhibitory receptor, CD94/NKG2A, expressed by NK cells and CTL, HLA-E molecules might play an important role in immunomodulation. In particular, expression of HLA-E might favor tumor cell escape from CTL and NK immunosurveillance. To address the potential role of HLA-E in melanoma immunobiology, we assessed the expression of these molecules ex vivo in human melanoma biopsies and in melanoma and melanocyte cell lines. Melanoma cell lines expressed no or low surface, but significant intracellular levels of HLA-E. We also report for the first time that some of them produced a soluble form of this molecule. IFN-γ significantly increased the surface expression of HLA-E and the shedding of soluble HLA-E by these cells, in a metalloproteinase-dependent fashion. In contrast, melanocyte cell lines constitutively expressed HLA-E molecules that were detectable both at the cell surface and in the soluble form, at levels that were poorly affected by IFN-γ treatment. On tumor sections, a majority of tumor cells of primary, but a low proportion of metastatic melanomas (30–70 and 10–20%, respectively), expressed HLA-E. Finally, HLA-E expression at the cell surface of melanoma cells decreased their susceptibility to CTL lysis. These data demonstrate that HLA-E expression and shedding are normal features of melanocytes, which are conserved in melanoma cells of primary tumors, but become dependent on IFN-γ induction after metastasis. The biological significance of these findings warrants further investigation.

Therapeutic efficacy of melanoma-reactive TIL injected in stage III melanoma patients

Abstract. Adoptive therapy for cancer using tumor-infiltrating lymphocytes (TIL) has mainly been investigated in cancer patients with advanced stage disease. The limited clinical success has not been encouraging, although this might be explained by poor TIL specificity and/or high tumor burden. To re-evaluate the effectiveness of adoptive therapy, we analyzed the capacity of tumor-reactive TIL injection in preventing the further development of disease in stage III melanoma patients after complete tumor resection. A phase II/III randomized trial was performed on 88 melanoma patients, who received autologous TIL plus interleukin-2 (IL-2) or IL-2 only. The duration of relapse-free survival was analyzed, taking into account the immunological specificity of injected TIL and the number of metastatic lymph nodes removed before treatment. Kaplan-Meyer analysis revealed that the injection of tumor-reactive TIL was statistically correlated with prolonged relapse-free survival in patients with only one metastatic lymph node. Therefore, improved clinical outcome could be obtained after adoptive therapy by selecting appropriate groups of patients and monitoring the specificity of the injected TIL populations.

Urotensin II is a New Chemotactic Factor for UT Receptor-Expressing Monocytes

Urotensin II (U-II), a vasoactive cyclic neuropeptide which activates the G protein-coupled receptor UT receptor, exerts various cardiovascular effects and may play a role in the pathophysiology of atherosclerosis. In this study, we report that the UT receptor is expressed and functional on human PBMC and rat splenocytes. PBMC surface expression of the UT receptor was mainly found in monocytes and NK cells, also in a minority of B cells, but not in T cells. Stimulation of monocytes with LPS increased UT receptor mRNA and protein expression. Cloning and functional characterization of the human UT receptor gene promoter revealed the presence of NF-κB-binding sites involved in the stimulation of UT receptor gene expression by LPS. Activation of the UT receptor by U-II induced chemotaxis with maximal activity at 10 and 100 nM. This U-II effect was restricted to monocytes. Analysis of the signaling pathway involved indicated that U-II-mediated chemotaxis was related to RhoA and Rho kinase activation and actin cytoskeleton reorganization. The present results thus identify U-II as a chemoattractant for UT receptor-expressing monocytes and indicate a pivotal role of the RhoA-Rho kinase signaling cascade in the chemotaxis induced by U-II.

Expression of CD94/NKG2-A on Human T Lymphocytes Is Induced by IL-12: Implications for Adoptive Immunotherapy

NK cell receptors (NKRs) are expressed on a subset of human T cells, predominantly CD8+, within which they can modulate TCR-mediated functions. In an attempt to identify the mechanisms leading to NKR expression, we analyzed the capacity of IL-12 to modulate the expression by T cells of the components of the CD94/NKG2-A inhibitory receptor, a member of the C-type lectin-like family of NKR. We show that IL-12 induces the expression of NKG2-A and/or CD94 by CD8+ T cells in culture, and that this induction was mediated neither by IFN-γ nor by IL-15. We also show, using the redirected killing assay, that IL-12-induced expression of both CD94 and NKG2-A led to the acquisition by T cells of a functional inhibitory receptor. Expression of the CD94/NKG2-A inhibitory receptor was also induced by IL-12 during T cell Ag stimulation so that in the presence of this cytokine a high proportion of melanoma-reactive CTL induced from PBL by melanoma peptide stimulation expressed this receptor. This study emphasizes the implication of IL-12 in the modulation of immune responses through NKR induction.

Dominant TCR Vα usage by virus and tumor‐reactive T cells with wide affinity ranges for their specific antigens

We have studied the TCR features and functional responses of three sets of human cytolytic T cell (CTL) clones, recognizing antigenic peptides presented by HLA‐A2 and derived from the Epstein‐Barr virus proteins BMLF1 and BRLF1 and from the melanoma protein Melan‐A/MART‐1. Within each set, a majority of clones used a recurrent Vα region, even though they expressed highly diverse TCR β chains and V(D)J junctional sequences. Functional assays and peptide/MHC multimer binding studies indicated that this restricted Vα usage was not associated with the affinity/avidity of the CTL clones. The Vα dominance, which may be a frequent feature of antigen‐specific T cells, likely reflects a restricted geometry of TCR/peptide/MHC complexes, primarily determined by Vα CDR.

Apoptotic body–loaded dendritic cells efficiently cross-prime cytotoxic T lymphocytes specific for NA17-A antigen but not for Melan-A/MART-1 antigen

DCs hold promise for cancer immunotherapy due to their functional ambivalence: iDCs internalize antigens, then mDCs trigger naive T‐cell activation. However, no consensus has been reached concerning the optimal mode of antigen acquisition for efficient cross‐priming of TAA‐specific CTLs, and this remains a field of investigation. Here, we used highly purified apobodies derived from an HLA‐A*0201‐negative melanoma line as a source of tumor antigens for HLA‐A*0201 DCs. We compared in vitro mDCs loaded with apobodies to DCs loaded with antigenic peptides, NA17‐A1–9 and Melan‐A/MART‐126–35 A27L analogue, for their capacity to stimulate melanoma antigen‐specific T cells from autologous PBLs. Apobody phagocytosis did not induce spontaneous DC maturation, but phagocytic DCs were still responsive to maturation signals, resulting in a functional ability to activate antigen‐specific lymphocytes. NA17‐A‐specific T lymphocytes were activated by both types of stimulation, whereas only peptide‐pulsed DCs stimulated the growth of Melan‐A/MART‐1‐specific lymphocytes. We also observed a lack of staining of melanoma‐derived apobodies with a Melan‐A‐specific MAb, suggesting protein alteration during apoptosis induction. After HLA‐A*0201/NA17‐A multimer sorting, antigen‐specific lymphocytes induced by mature DCs loaded with either peptide or apobodies displayed similar functional capacity against peptide‐pulsed T2 cells and melanoma cells. Therefore, apobody‐loaded DCs can achieve T‐cell priming similar to that induced by peptide‐pulsed DCs, provided that the apoptotic process allows the preservation of antigen expression.

Human monocyte-derived macrophages and dendritic cells are comparably effective in vitro in presenting HLA class I-restricted exogenous peptides.

Recent experimental data have shown that mice could be immunized efficiently, in particular against cancer, by the injection of antigen‐loaded dendritic cells (DC) or macrophages (MPH). In the present work, these two antigen‐presenting cells (APC) were prepared in humans from circulating mononuclear cells (MNC). MPH were obtained from MNC that were cultured in hydrophobic plastic bags and purified by elutriation. DC were from the culture of adherent elutriation‐purified monocytes in the presence of granulocyte–macrophage colony‐stimulating factor (GM‐CSF) and interleukin‐4 (IL‐4). The two APC were prepared in parallel from the same donors and their phenotype and antigen‐presenting capacity were compared. DC differed from MPH by a higher expression of HLA‐DR and CD23 and a lower expression of CD14, CD64 and of adhesion molecules. DC and MPH were comparably effective in (a) enhancing the mitotic response of autologous lymphocytes to immobilized anti‐CD3 (accessory function); (b) presenting melanoma peptides to specific cytotoxic T lymphocyte (CTL) clones; and (c) stimulating the generation of CTL directed against a myxovirus influenza peptide. However, DC were more effective than MPH in inducing the mitotic response of allogeneic peripheral blood leucocytes (PBL), possibly because of their higher expression of HLA class II molecules. In conclusion, DC and MPH prepared from blood MNC did not differ substantially in their ability to activate HLA class I‐restricted T‐cell responses by exogenous peptide presentation.

Serum Soluble HLA-E in Melanoma: A New Potential Immune-Related Marker in Cancer

Background Tumor-derived soluble factors, including soluble HLA molecules, can contribute to cancer immune escape and therefore impact on clinical course of malignant diseases. We previously reported that melanoma cells produce, in vitro, soluble forms of the non-classical MHC class I molecule HLA-E (sHLA-E). In order to investigate sHLA-E production by various tumors and to address its potential value as a tumor-associated marker, we developed a specific ELISA for the quantification of sHLA-E in biological fluids. Methodology/Principal Findings We developed a sHLA-E specific and sensitive ELISA and we showed that serum sHLA-E levels were significantly elevated (P<0.01) in melanoma patients (n = 127), compared with healthy donors (n = 94). sHLA-E was also detected in the culture supernatants of a wide variety of tumor cell lines (n = 98) including melanomas, kidney, colorectal and breast cancers. Cytokines regulation of sHLA-E production by tumor cells was also carried out. IFN-γ, IFN-α and TNF-α were found to upregulate sHLA-E production by tumor cells. Conclusions/Significance In view of the broad tumor tissue release of HLA-E and its up-regulation by inflammatory cytokines, sHLA-E should be studied for its involvement in immune responses against tumors. Interestingly, our results demonstrated a positive association between the presence of serum sHLA-E and melanoma. Therefore, the determination of sHLA-E levels, using ELISA approach, may be investigated as a clinical marker in cancer patients.

Unique Functional Status of Natural Killer Cells in Metastatic Stage IV Melanoma Patients and Its Modulation by Chemotherapy

Purpose: Immunotherapy is an alternative for metastatic melanoma patients resistant to chemotherapy. Natural killer (NK) cells are powerful antileukemia effectors and their role in solid tumors is suspected. NK cell activation is regulated by a balance between activating receptors, which detect stress molecules on tumor cells, and HLA-I specific inhibitory receptors. Here, we studied the phenotype and function of NK cells in stage IV metastatic melanoma patients. Experimental Design: Circulating NK cells from 35 healthy donors and 51 patients were studied: 24 patients before chemotherapy (prechemotherapy), 17 patients 1 month after 1 to 4 lines of chemotherapy (postchemotherapy), and 10 patients analyzed pre- and postchemotherapy. NK functionality was carried out toward 2 primary metastatic melanoma cell lines, analyzed for the expression of NK receptor ligands. Results: NK cells from prechemotherapy patients exhibit an NKp46dim/NKG2Adim phenotype. In contrast, NK cells from postchemotherapy patients display high expression of NKp46 and NKG2A receptors. Purified NK cells from patients are efficiently activated in response to melanoma cells. Melanoma cells express different level of NKG2D ligands and HLA-I molecules. In agreements with their phenotype, NK cells from pre- and postchemotherapy patients present distinct functional status toward these primary melanoma cells. A dynamic label free assay was used to determine the pathways involved in the lysis of melanoma cells by IL-2–activated NK cells. NKG2D, NCR (natural cytotoxicity receptor), and DNAM-1 are involved in the NK-mediated lysis of melanoma cells. Conclusions: These results provide new arguments and clues to design NK cell–based immunotherapeutic strategies for melanoma patients. Clin Cancer Res; 17(9); 2628–37. ©2011 AACR.