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Recently, the inhibitory CD94/NKG2A receptor has joined the group of immune checkpoints (ICs) and its expression has been documented in NK cells and CD8 + T lymphocytes in several cancers and some infectious diseases. In colorectal cancer (CRC), we previously reported that NKG2A + tumor-infiltrating lymphocytes (TILs) are predominantly CD8 + αβ T cells and that CD94 overexpression and/or its ligand HLA-E were associated with a poor prognosis. This study aimed to thoroughly characterize the NKG2A + CD8 + TIL subpopulation and document the impact of NKG2A on anti-tumor responses in CRC. Our findings highlight new features of this subpopulation: (i) enrichment in colorectal tumors compared to paired normal colonic mucosa, (ii) their character as tissue-resident T cells and their majority terminal exhaustion status, (iii) co-expression of other ICs delineating two subgroups differing mainly in the level of NKG2A expression and the presence of PD-1, (iv) high functional avidity despite reduced proliferative capacity and finally (v) inhibition of anti-tumor reactivity that is overcome by blocking NKG2A. From a clinical point of view, these results open a promising alternative for immunotherapies based on NKG2A blockade in CRC, which could be performed alone or in combination with other IC inhibitors, adoptive cell transfer or therapeutic vaccination.

BackgroundGenome editing offers unique perspectives for optimizing the functional properties of T cells for adoptive cell transfer purposes. So far,PDCD1editing has been successfully tested mainly in chimeric antigen receptor T (CAR-T) cells and human primary T cells. Nonetheless, for patients with solid tumors, the adoptive transfer of effector memory T cells specific for tumor antigens remains a relevant option, and the use of high avidity T cells deficient for programmed cell death-1 (PD-1) expression is susceptible to improve the therapeutic benefit of these treatments.MethodsHere we used the transfection of CAS9/sgRNA ribonucleoproteic complexes to editPDCD1gene in human effector memory CD8+T cells specific for the melanoma antigen Melan-A. We cloned edited T cell populations and validatedPDCD1editing through sequencing and cytometry in each T cell clone, together with T-cell receptor (TCR) chain’s sequencing. We also performed whole transcriptomic analyses on wild-type (WT) and edited T cell clones. Finally, we documented in vitro and in vivo through adoptive transfer in NOD scid gamma (NSG) mice, the antitumor properties of WT and PD-1KO T cell clones, expressing the same TCR.ResultsHere we demonstrated the feasibility to editPDCD1gene in human effector memory melanoma-specific T lymphocytes. We showed that PD-1 expression was dramatically reduced or totally absent onPDCD1-edited T cell clones. Extensive characterization of a panel of T cell clones expressing the same TCR and exhibiting similar functional avidity demonstrated superior antitumor reactivity against a PD-L1 expressing melanoma cell line. Transcriptomic analysis revealed a downregulation of genes involved in proliferation and DNA replication in PD-1-deficient T cell clones, whereas genes involved in metabolism and cell signaling were upregulated. Finally, we documented the superior ability of PD-1-deficient T cells to significantly delay the growth of a PD-L1 expressing human melanoma tumor in an NSG mouse model.ConclusionThe use of such lymphocytes for adoptive cell transfer purposes, associated with other approaches modulating the tumor microenvironment, would be a promising alternative to improve immunotherapy efficacy in solid tumors.

Purpose: The recent success of anti-PD1 antibody in metastatic colorectal cancer (CRC) patients with microsatellite instability (MSI), known to be associated with an upregulated Th1/Tc1 gene signature, provides new promising therapeutic strategies. However, the partial objective response highlights a crucial need for relevant, easily evaluable, predictive biomarkers. Here we explore whether in situ assessment of Tbet+ tumor infiltrating lymphocytes (TILs) reflects a pre-existing functional antitumor Th1/Tc1/IFNγ response, in relation with clinicopathological features, microsatellite status and expression of immunoregulatory molecules (PD1, PDL1, IDO-1). Methodology: In two independent cohorts of CRC (retrospective n = 80; prospective n = 27) we assessed TILs density (CD3, Tbet, PD1) and expression profile of PDL1 and IDO-1 by immunohistochemistry/image analysis. Furthermore, the prospective cohort allowed to perform ex vivo CRC explant cultures and measure by Elisa the IFNγ response, at baseline and upon anti-PD1 treatment. Results: The density of Tbet+ TILs was significantly higher in MSI CRC, especially in the medullary subtype but also in a subgroup of MSS (microsatellite stable), and positively correlated with PD1 and PDL1 expression, but not with IDO-1. Finally, a high number of Tbet+ TILs was associated with a favorable overall survival. These Tbet+ TILs were functional as their density positively correlated with basal IFNγ levels. In addition, the combined score of Tbet+ PD1+ TILs coupled with IDO-1 expression predicted the magnitude of the IFNγ response upon anti-PD1. Conclusion: Altogether, immunohistochemical quantification of Tbet+ TILs is a reliable and accurate tool to recapitulate a preexisting Th1/Tc1/IFNγ antitumor response that can be reinvigorated by anti-PD1 treatment.

BackgroundThe immune system gradually deteriorates with age and nutritional status is a major factor in immunosenescence. Of the many nutritional factors implicated in age-related immune dysfunction, vitamin A may be a good candidate, since vitamin A concentrations classically decrease during aging whereas it may possess important immunomodulatory properties via its active metabolites, the retinoic acids. This prompted us to investigate the immune response induced by retinoids in adults and elderly healthy subjects. Before and after oral supplementation with 13cis retinoic acid (0.5 mg/kg/day during 28 days), whole blood cells were phenotyped, and functions of peripheral blood mononuclear cells (PBMC) and polymorphonuclear cells (PMN) were investigated by flow cytometry and ELISA tests.ResultsIn both young adults (n = 20, 25 ± 4 years) and older subjects (n = 20, 65 ± 4 years), retinoic acid supplementation had no effect on the distribution of leukocyte subpopulations or on the functions of PBMC (Il-2 and sIl-2R production, membrane expression of CD25). Concerning PMN, retinoic acid induced an increase in both spontaneous migration and cell surface expression of CD11b in the two different age populations, whereas bactericidal activity and phagocytosis remained unchanged.ConclusionsWe demonstrated that retinoic acid induces the same intensity of immune response between adult and older subjects, and more specifically affects PMN functions, i.e. adhesion and migration, than PBMC functions.

Background: Natural Killer (NK) cells are key actors of innate immunity that supervise the organism's cells, and fight against viral infections and cancer development through their cytotoxic activity. This cytotoxic activity is modulated by cytokines and hormones and could be influenced by physiological or pathological conditions. New techniques for measuring NK cytotoxic activity by flow-cytometry have recently been developed, and they correlated strongly with the standard chromium ( 51 Cr) release assay. Our aim was to implement a previously published enhanced green fluorescent protein (EGFP)-K562 flow cytometric method and use it to evaluate NK cytotoxic activity under different nutritional conditions. Methods: NK effector cells were isolated from peripheral blood mononuclear cells, and a K562 cell line stably transfected by EGFP was used as target cells. Different ana-

The optimization of adoptive transfer approaches of anti-tumor T cells requires both the functional improvement of the injected T cells and the modulation of the tumor microenvironment, favoring the recruitment of these T cells and their activation. We have recently shown the therapeutic benefit of two approaches tested individually in a melanoma model wich were on one hand the adoptive transfer of specific T cells deficient for the expression of the inhibitory receptor PD-1, and on the other hand PD-L1 targeted alpha therapy (TAT). In this study, we sought to investigate the efficacy of these two therapies combined, compared to each monotherapy, in order to evaluate the synergy between these two approaches, in the same melanoma model. Here we used melanoma-specific T-cell clones, previously validated for the edition of PDCD1 gene and with previously demonstrated superior anti-tumor activity than their wild-type counterparts, after adoptive transfer in NSG mice engrafted with PD-L1 expressing human melanoma tumors. We also used a previously validated TAT approach, using a 213 Bi-anti-human-PD-L1 mAb, alone or in combination with adoptive cell transfer, in the same mouse model. We confirmed previous results obtained with each monotherapy and documented the safety and the superior ability of a combination between the adoptive transfer of PD-1 deficient T cells and TAT targeting PD-L1 to control the growth of melanoma tumors in NSG mice. This study provides the first proof-of-concept of the efficacy of a combination therapy using TAT, adoptive cell transfer and genomic editing of IC-coding genes.

In colorectal cancer (CRC), a high density of T lymphocytes represents a strong prognostic marker in subtypes of CRC. Optimized immunotherapy strategies to boost this T-cell response are still needed. A good candidate is the inflammasome pathway, an emerging player in cancer immunology that bridges innate and adaptive immunity. Its effector protein caspase-1 matures IL-18 that can promote a T-helper/cytotoxic (Th1/Tc1) response. It is still unknown whether tumor cells from CRC possess a functional caspase-1/IL-18 axis that could modulate the Th1/Tc1 response. We used two independent cohorts of CRC patients to assess IL-18 and caspase-1 expression by tumor cells in relation to the density of TILs and the microsatellite status of CRC. Functional and multiparametric approaches at the protein and mRNA levels were performed on an ex vivo CRC explant culture model. We show that, in the majority of CRCs, tumor cells display an activated and functional caspase-1/IL-18 axis that contributes to drive a Th1/Tc1 response elicited by TILs expressing IL-18Rα. Furthermore, unsupervised clustering identified three clusters of CRCs according to the caspase-1/IL-18/TIL density/interferon gamma (IFNγ) axis and microsatellite status. Together, our results strongly suggest that targeting the caspase-1/IL-18 axis can improve the anti-tumor immune response in subgroups of CRC.