Nathalie Labarrière

CD4CD8αα Lymphocytes, A Novel Human Regulatory T Cell Subset Induced by Colonic Bacteria and Deficient in Patients with Inflammatory Bowel Disease

Gut bacterium Faecalibacterium prausnitzii activates a newly identified set of human IL-10-producing Treg cells (CD4CD8αα lymphocytes), revealing a mechanism by which commensal microbes contribute to host immunity.

PD-1 expression conditions T cell avidity within an antigen-specific repertoire

ABSTRACT Despite its negative regulatory role on tumor-specific T cells, Programmed cell death 1 (PD-1) is also a marker of activated tumor-infiltrating T cells. In cancer, PD-1 blockade partially reverses T cell dysfunction allowing the amplification of tumor reactive T cells. Here, we investigated the role of PD-1 signaling on effector/memory human T cells specific for shared melanoma antigens, derived from blood. We documented for the first time the existence of melanoma-specific T cell clones unable to express PD-1. This stable feature was due to the persistent methylation of the PDCD1 promoter. These PD-1neg clones were of lower avidity than their PD-1pos counterparts, suggesting that high-affinity-specific T cell clones unable to express PD-1 are not or rarely present in peripheral blood, as they are probably eliminated by negative selection, due to their high reactivity. We also documented the existence of such PD-1neg T cell clones in melanoma tumor-infiltrating lymphocytes (TIL), which also exhibited a lower functional avidity than PD-1pos TIL clones. This clearly shows that PD-1 expression identifies antigen-specific T cell clonotypes of high functional avidity. Finally, we demonstrated that PD-1 blockade during the in vitro selection process of Melan-A-specific T cells favored the amplification of higher avidity T cell clonotypes. This preferential amplification of high-avidity memory T cells upon PD-1 blockade resonates with the expansion of reactive T cells, including neo-antigen-specific T cells observed in anti-PD-1-treated patients. This feature should also be a useful biomarker of clinical efficiency, while providing new insights for adoptive transfer treatments.

A Full GMP Process to Select and Amplify Epitope-Specific T Lymphocytes for Adoptive Immunotherapy of Metastatic Melanoma

A number of trials of adoptive transfer of tumor-specific T lymphocytes have been performed in the last 20 years in metastatic melanoma, with increasingly encouraging results as the relevant melanoma antigens were identified and the purity/specificity of injected T cells improved. We have previously described a sorting method of epitope-specific T lymphocytes that uses magnetic beads coated with HLA/peptide complexes and we suggested that this method could be applied to a clinical setting. In the present work, we provide a detailed description of the whole GMP process of sorting and amplification of clinical grade T cells specific for the melanoma antigens Melan-A and MELOE-1. All the reagents used in this process including the sorting reagent were produced in GMP conditions and we document the optimization of the different steps of the process such as peptide stimulation, sorting, and amplification. The optimized procedure, validated in 3 blank runs in a clinical setting, allowed the production of at least 108 pure (>90%) Melan-A- and MELOE-1-specific T cells within 28 days starting with 100 mL of blood from metastatic melanoma patients. This GMP process is thus ready to be used in an upcoming phase I/II clinical trial on metastatic melanoma patients.

PD-1 expression on tumor-specific T cells: Friend or foe for immunotherapy?

ABSTRACT Inhibitory properties of PD-1 receptor engagement on activated T cells are well established in physiologic and pathological contexts. In cancer, the use of checkpoint blockade, such as anti-PD-1 antibodies, becomes progressively a reference treatment of a growing number of tumors. Nonetheless, it is also established that PD-1 expression on antigen-specific T cells reflects the functional avidity and anti-tumor reactivity of these T cells. We will discuss this dual significance of PD-1 expression on tumor-specific T cells, due to a complex regulation and the opportunity to exploit this expression to define, monitor and exploit tumor-reactive T cells for immunotherapy purposes.

An additional ORF on meloe cDNA encodes a new melanoma antigen, MELOE-2, recognized by melanoma-specific T cells in the HLA-A2 context.

We characterized a new melanoma antigen derived from one of the multiple open reading frames (ORFs) of the meloe transcript. The meloe gene is overexpressed in melanomas as compared to other cancer cell lines and normal tissues. The corresponding transcript is rather unusual, in that it does not contain a long unique ORF but multiple short ORFs. We recently characterized a tumor epitope derived from a polypeptide (MELOE-1) encoded by the ORF1230–1370 and involved in relapse prevention of melanoma patients treated with autologous tumor infiltrating lymphocytes (TIL). Here we show that the ORF285–404 encodes a polypeptide called MELOE-2 that also generated a HLA-A2 epitope recognized by a melanoma-specific T cell clone derived from the same TIL population from which we derived the MELOE-1-specific T cell clone. We also showed that HLA-A2 melanoma cells were spontaneously recognized by the MELOE-2-specific T cell clone, and we detected the presence of MELOE-2 reactive T cells in another TIL population infused to a patient who remained relapse-free after TIL treatment. These results demonstrate that translation of meloe transcript in melanoma cells can produce at least two immunogenic polypeptides, MELOE-1 and MELOE-2, from two distinct ORFs that could be relevant target for melanoma immunotherapy.

Oncolytic viruses sensitize human tumor cells for NY-ESO-1 tumor antigen recognition by CD4+ effector T cells.

ABSTRACT Oncolytic immunotherapy using oncolytic viruses (OV) has been shown to stimulate the antitumor immune response by inducing the release of tumor-associated antigens (TAA) and danger signals from the dying infected tumor cells. In this study, we sought to determine if the lysis of tumor cells induced by different OV: measles virus, vaccinia virus, vesicular stomatitis virus, herpes simplex type I virus, adenovirus or enterovirus, has consequences on the capacity of tumor cells to present TAA, such as NY-ESO-1. We show that the co-culture of NY-ESO-1neg/HLA-DP4pos melanoma cells with NY-ESO-1pos/HLA-DP4neg melanoma cells infected and killed by different OV induces an intercellular transfer of NY-ESO-1 that allows the recognition of NY-ESO-1neg/HLA-DP4pos tumor cells by an HLA-DP4/NY-ESO-1(157–170)-specific CD4+ cytotoxic T cell clone, NY67. We then confirmed this result in a second model with an HLA-DP4+ melanoma cell line that expresses a low amount of NY-ESO-1. Recognition of this cell line by the NY67 clone is largely increased in the presence of OV productive infection. Altogether, our results show for the first time another mechanism of stimulation of the anti-tumor immune response by OV, via the loading of tumor cells with TAA that sensitizes them for direct recognition by specific effector CD4+ T cells, supporting the use of OV for cancer immunotherapy.

CD40L confers helper functions to human intra-melanoma class-I-restricted CD4+CD8+ double positive T cells

ABSTRACT Although CD4+CD8+ double positive (DP) T cells represent a small fraction of peripheral T lymphocytes in healthy human donors, their frequency is often increased under pathological conditions (in blood and targeted tissues). In solid cancers such as melanoma, we previously demonstrated an enrichment of tumor reactive CD4lowCD8highαβ DP T cells among tumor-infiltrating lymphocytes of unknown function. Similarly to their single positive (SP) CD8+ counterparts, intra-melanoma DP T cells recognized melanoma cell lines in an HLA-class-I restricted context. However, they presented a poor cytotoxic activity but a strong production of diverse Th1 and Th2 cytokines. The aim of this study was to clearly define the role of intra-melanoma CD4lowCD8highαβ DP T cells in the antitumor immune response. Based on a comparative transcriptome analysis between intra-melanoma SP CD4+, SP CD8+ and DP autologous melanoma-infiltrating T-cell compartments, we evidenced an overexpression of the CD40L co-stimulatory molecule on activated DP T cells. We showed that, like SP CD4+ T cells, and through CD40L involvement, DP T cells are able to induce both proliferation and differentiation of B lymphocytes and maturation of functional DCs able to efficiently prime cytotoxic melanoma-specific CD8 T-cell responses. Taken together, these results highlight the helper potential of atypical DP T cells and their role in potentiating antitumor response.

Emergence of High-Avidity Melan-A–Specific Clonotypes as a Reflection of Anti–PD-1 Clinical Efficacy

Therapeutic strategies using anti-PD-1-blocking antibodies reported unparalleled effectiveness for melanoma immunotherapy, but deciphering immune responses modulated by anti-PD-1 treatment remains a crucial issue. Here, we analyzed the composition and functions of the large Melan-A-specific T-cell repertoire in the peripheral blood of 9 melanoma patients before and after 2 months of treatment with anti-PD-1. We observed amplification of Melan-A-specific Vß subfamilies undetectable before therapy (thereafter called emerging Vß subfamilies) in responding patients, with a predominant expansion in patients with a complete response. These emerging Vß subfamilies displayed a higher functional avidity for their cognate antigen than Vß subfamilies not amplified upon anti-PD-1 therapy and could be identified by a sustained coexpression of PD-1 and TIGIT receptors. Thus, in addition to the emergence of neoantigen-specific T cells previously documented upon anti-PD-1 therapy, our work describes the emergence of high-avidity Melan-A-specific clonotypes as a surrogate marker of treatment efficacy. Cancer Res; 77(24); 7083-93. ©2017 AACR.

Thoracic and cutaneous sarcoid-like reaction associated with anti-PD-1 therapy: longitudinal monitoring of PD-1 and PD-L1 expression after stopping treatment

BackgroundImmune checkpoint inhibitors (ICI) target T cell inhibitory pathways that are responsible for cancer tolerance by down-modulating immune functions. ICI have revolutionized patients care with lung cancer. Nevertheless, restoring endogenous antitumor T-cell responses can induce immune related adverse events, such as sarcoidosis.Case presentationWe report here the first case of a thoracic and cutaneous sarcoid-like reaction in a patient with a relapsing unresectable non-small cell lung cancer (NSCLC) treated with nivolumab, an anti-PD-1 mAb. The expression of PD-1 and its ligands, PD-L1 and PD-L2, was assessed by flow cytometry on peripheral blood mononuclear cells (PBMC) and compared to patients who had discontinued nivolumab therapy without having developed any immune related adverse events. PD-L1 expression was transiently increased on B cells, T cells and monocytes, whereas PD-L2 expression was not modulated. PD-1 was transiently undetectable when PD-L1 was maximal, before returning to basal level. Sarcoidosis spontaneously resolved, without corticotherapy.ConclusionThis case sheds the light on a complex regulation of PD-L1 expression in vivo on PBMC after nivolumab arrest and triggers the question of monitoring the expression of immune checkpoint on immune cells during and after treatment with ICI.

Merkel cell carcinoma and cellular cytotoxicity: sensitivity to cellular lysis and screening for potential target antigens suitable for antibody-dependent cellular cytotoxicity

The recent success of checkpoint inhibitors in the treatment of Merkel cell carcinoma (MCC) confirms that MCC tumors can be immunogenic. However, no treatment directly targeting the tumor is available for use in combination with these checkpoint inhibitors to enhance their efficacity. This study was carried out to characterize MCC line sensitivity to cellular lysis and to identify cell surface antigens that could be used for direct targeting of this tumor. For five representative MCC lines, the absence or low expression of MICA, MICB, HLA-I, and ICAM-1 was associated with low level of recognition by NK cells and T lymphocytes. However, expression of HLA-I and ICAM-1 and sensitivity to cellular lysis could be restored or increased after exposure to INFγ. We tested 41 antibodies specific for 41 different antigens using a novel antibody-dependent cellular cytotoxicity (ADCC) screening system for target antigens. Anti-CD326 (EpCAM) was the only antibody capable of inducing ADCC on the five MCC lines tested. Because MCC tumors are often directly accessible, local pharmacologic manipulation to restore HLA class-I and ICAM-1 cell surface expression (and thus sensitivity to cell lysis) can potentially benefit immune therapeutic intervention. In line with this, our observation that ADCC against EpCAM can induce lysis of MCC lines and suggests that therapeutic targeting of this antigen deserves to be explored further.