Laurent Derré

BTLA mediates inhibition of human tumor-specific CD8+ T cells that can be partially reversed by vaccination

The function of antigen-specific CD8+ T cells, which may protect against both infectious and malignant diseases, can be impaired by ligation of their inhibitory receptors, which include CTL-associated protein 4 (CTLA-4) and programmed cell death 1 (PD-1). Recently, B and T lymphocyte attenuator (BTLA) was identified as a novel inhibitory receptor with structural and functional similarities to CTLA-4 and PD-1. BTLA triggering leads to decreased antimicrobial and autoimmune T cell responses in mice, but its functions in humans are largely unknown. Here we have demonstrated that as human viral antigen-specific CD8+ T cells differentiated from naive to effector cells, their surface expression of BTLA was gradually downregulated. In marked contrast, human melanoma tumor antigen-specific effector CD8+ T cells persistently expressed high levels of BTLA in vivo and remained susceptible to functional inhibition by its ligand herpes virus entry mediator (HVEM). Such persistence of BTLA expression was also found in tumor antigen-specific CD8+ T cells from melanoma patients with spontaneous antitumor immune responses and after conventional peptide vaccination. Remarkably, addition of CpG oligodeoxynucleotides to the vaccine formulation led to progressive downregulation of BTLA in vivo and consequent resistance to BTLA-HVEM-mediated inhibition. Thus, BTLA activation inhibits the function of human CD8+ cancer-specific T cells, and appropriate immunotherapy may partially overcome this inhibition.

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.

Comprehensive analysis of the frequency of recognition of melanoma-associated antigen (MAA) by CD8 melanoma infiltrating lymphocytes (TIL): implications for immunotherapy

Fifty‐nine tumor‐infiltrating lymphocyte (TIL) cultures established from melanoma‐invaded lymph nodes were screened for recognition of 28 melanoma‐associated antigens (MAA) in association with31 HLA molecules. Twenty‐three (39%) TIL lines reacted to at least one melanoma antigen. Melanosomal proteins were recognized by 19 TIL populations and the most prominent responses against these proteins were directed against Melan‐A/MART‐1 (mainly in association with HLA‐A*0201) and gp100 (in association with diverse HLA contexts). Ten TIL populations reacted against 10 tumor‐specific antigens, in association with 8 different HLA molecules. HLA‐A*0201 and B*3501‐restricted responses were the most frequent with, respectively, 17 and 7 responses directed against 5 distinct antigens. Unexpectedly, the recognition by TIL of different MAA was frequently restricted by a single HLA in individual tumors, and there was no evidence for the existence of dominant MAA epitopes between tumors,except for Melan‐A/MART‐1 antigen. This analysis also led to the detection of 21 new HLA‐peptide complexes recognized by melanoma TIL. This study, which is to our knowledge the most comprehensive analysis of TIL specificity to tumor antigens, has several implications for the design of immunotherapeutic strategies based on immunization against selected tumor epitopes.

Inducible Hsp70 as target of anticancer immunotherapy: Identification of HLA-A*0201-restricted epitopes

The design of a broad application tumor vaccine requires the identification of tumor antigens expressed in a majority of tumors of various origins. We questioned whether the major stress‐inducible heat shock protein Hsp70 (also known as Hsp72), a protein frequently overexpressed in human tumors of various histological origins, but not in most physiological normal tissues, constitutes a tumor antigen. We selected the p391 and p393 peptides from the sequence of the human inducible Hsp70 that had a high affinity for HLA‐A*0201. These peptides were able to trigger a CTL response in vivo in HLA‐A*0201‐transgenic HHD mice and in vitro in HLA‐A*0201+ healthy donors. p391‐ and p393‐specific human and murine CTL recognized human tumor cells overexpressing Hsp70 in a HLA‐A*0201‐restricted manner. Tetramer analysis of TILs showed that these Hsp70 epitopes are targets of an immune response in many HLA‐A*0201+ breast cancer patients. Hsp70 is a tumor antigen and the Hsp70‐derived peptides p391 and p393 could be used to raise a cytotoxic response against tumors of various origins.

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.

Identification of Five New HLA-B*3501-Restricted Epitopes Derived from Common Melanoma-Associated Antigens, Spontaneously Recognized by Tumor-Infiltrating Lymphocytes

We previously described HLA-B35-restricted melanoma tumor-infiltrating lymphocyte responses to frequently expressed melanoma-associated Ags: tyrosinase, Melan-A/MART-1, gp100, MAGE-A3/MAGE-A6, and NY-ESO-1. Using clones derived from these TIL, we identified in this study the corresponding epitopes. We show that five of these epitopes are new and that melanoma cells naturally present all the six epitopes. Interestingly, five of these epitopes correspond to or encompass melanoma-associated Ag epitopes presented in other HLA contexts, such as A2, A1, B51, and Cw3. In particular, the HLA-B35-restricted Melan-A epitope is mimicked by the peptide 26–35, already known as the most immunodominant melanoma epitope in the HLA-A*0201 context. Because this peptide lacked adequate anchor amino acid residues for efficient binding to HLA-B35, modified peptides were designed. Two of these analogues were found to induce higher PBL- and tumor-infiltrating lymphocyte-specific responses than the parental peptide, suggesting that they could be more immunogenic in HLA-B*3501 melanoma patients. These data have important implications for the formulation of polypeptide-based vaccines as well as for the monitoring of melanoma-specific CTL response in HLA-B*3501 melanoma patients.

Ex vivo Detectable Human CD8 T-Cell Responses to Cancer-Testis Antigens

Clinical trials have shown that strong tumor antigen-specific CD8 T-cell responses are difficult to induce but can be achieved for T-cells specific for melanoma differentiation antigens, upon repetitive vaccination with stable emulsions prepared with synthetic peptides and incomplete Freunds adjuvant. Here, we show in four melanoma patients that ex vivo detectable T-cells and thus strong T-cell responses can also be induced against the more universal cancer-testis antigens NY-ESO-1 and Mage-A10. Interestingly, all patients had ex vivo detectable T-cell responses against multiple antigens after serial vaccinations with three peptides emulsified in incomplete Freunds adjuvant. Antigen-specific T-cells displayed an activated phenotype and secreted IFNgamma. The robust immune responses provide a solid basis for further development of human T-cell vaccination.

Intravaginal TLR agonists increase local vaccine-specific CD8 T cells and human papillomavirus-associated genital-tumor regression in mice

Human papillomaviruses (HPV)-related cervical cancer is the second leading cause of cancer death in women worldwide. Despite active development, HPV E6/E7 oncogene-specific therapeutic vaccines have had limited clinical efficacy to date. Here, we report that intravaginal (IVAG) instillation of CpG-ODN (TLR9 agonist) or poly-(I:C) (TLR3 agonist) after subcutaneous E7 vaccination increased ∼fivefold the number of vaccine-specific interferon-γ-secreting CD8 T cells in the genital mucosa (GM) of mice, without affecting the E7-specific systemic response. The IVAG treatment locally increased both E7-specific and total CD8 T cells, but not CD4 T cells. This previously unreported selective recruitment of CD8 T cells from the periphery by IVAG CpG-ODN or poly-(I:C) was mediated by TLR9 and TLR3/melanoma differentiation-associated gene 5 signaling pathways, respectively. For CpG, this recruitment was associated with a higher proportion of GM-localized CD8 T cells expressing both CCR5 and CXCR3 chemokine receptors and E-selectin ligands. Most interestingly, IVAG CpG-ODN following vaccination led to complete regression of large genital HPV tumors in 75% of mice, instead of 20% with vaccination alone. These findings suggest that mucosal application of immunostimulatory molecules might substantially increase the effectiveness of parenterally administered vaccines.

Tumor Antigen–Specific FOXP3+ CD4 T Cells Identified in Human Metastatic Melanoma: Peptide Vaccination Results in Selective Expansion of Th1-like Counterparts

We have previously shown that vaccination of HLA-A2 metastatic melanoma patients with the analogue Melan-A(26-35(A27L)) peptide emulsified in a mineral oil induces ex vivo detectable specific CD8 T cells. These are further enhanced when a TLR9 agonist is codelivered in the same vaccine formulation. Interestingly, the same peptide can be efficiently recognized by HLA-DQ6-restricted CD4 T cells. We used HLA-DQ6 multimers to assess the specific CD4 T-cell response in both healthy individuals and melanoma patients. We report that the majority of melanoma patients carry high frequencies of naturally circulating HLA-DQ6-restricted Melan-A-specific CD4 T cells, a high proportion of which express FOXP3 and proliferate poorly in response to the cognate peptide. Upon vaccination, the relative frequency of multimer+ CD4 T cells did not change significantly. In contrast, we found a marked shift to FOXP3-negative CD4 T cells, accompanied by robust CD4 T-cell proliferation upon in vitro stimulation with cognate peptide. A concomitant reduction in TCR diversity was also observed. This is the first report on direct ex vivo identification of antigen-specific FOXP3+ T cells by multimer labeling in cancer patients and on the direct assessment of the impact of peptide vaccination on immunoregulatory T cells.

Vaccination-induced functional competence of circulating human tumor-specific CD8 T-cells.

T‐cells specific for foreign (e.g., viral) antigens can give rise to strong protective immune responses, whereas self/tumor antigen‐specific T‐cells are thought to be less powerful. However, synthetic T‐cell vaccines composed of Melan‐A/MART‐1 peptide, CpG and IFA can induce high frequencies of tumor‐specific CD8 T‐cells in PBMC of melanoma patients. Here we analyzed the functionality of these T‐cells directly ex vivo, by multiparameter flow cytometry. The production of multiple cytokines (IFNγ, TNFα, IL‐2) and upregulation of LAMP‐1 (CD107a) by tumor (Melan‐A/MART‐1) specific T‐cells was comparable to virus (EBV‐BMLF1) specific CD8 T‐cells. Furthermore, phosphorylation of STAT1, STAT5 and ERK1/2, and expression of CD3 zeta chain were similar in tumor‐ and virus‐specific T‐cells, demonstrating functional signaling pathways. Interestingly, high frequencies of functionally competent T‐cells were induced irrespective of patients age or gender. Finally, CD8 T‐cell function correlated with disease‐free survival. However, this result is preliminary since the study was a Phase I clinical trial. We conclude that human tumor‐specific CD8 T‐cells can reach functional competence in vivo, encouraging further development and Phase III trials assessing the clinical efficacy of robust vaccination strategies.