Defining the Immune Checkpoint Landscape in Human Colorectal Cancer Highlights the Relevance of the TIGIT/CD155 Axis for Optimizing Immunotherapy

International audienceWhile immune checkpoint (IC) therapies, particularly those targeting the PD-1/PD-L1 axis, have revolutionized the treatment of melanoma and several other cancers, their effect remains very limited in colorectal cancer (CRC). To define a comprehensive landscape of ICs in the human CRC tumor microenvironment (TME), we evaluated, using multiparametric flow cytometry, their ex vivo expression via tumor-infiltrating lymphocytes (TILs) (n = 40 CRCs) as well as that of their respective ligands on tumor and myeloid cells (n = 29). Supervised flow cytometry analyses showed that (i) most CD3+ TILs expressed PD-1 and TIGIT and, to a lesser extent, Tim-3, Lag3 and NKG2A, and (ii) EpCAM+ tumor cells and CD11b+ myeloid cells differed in their IC ligand expression profile, with a strikingly high expression of CD155 by tumor cells. An in situ analysis of IC and their ligands using immunohistochemistry on paraffin sections of CRC confirmed the overexpression of TIGIT and its ligand, CD155, in the TME. Most interestingly, an unsupervised clustering analysis of IC co-expression on CD4+ and CD8+ TILs identified two tumor subgroups, named IChigh and IClow. Altogether, our findings highlight the TIGIT/CD155 axis as a potential target that could be used in combination IC therapy in CRC

Involvement of the NLRC5 / MHC class I axis in human colorectal cancer immune surveillance

International audienceMost of patients with colorectal cancer (CRC) without microsatellite instability fail torespond to immune checkpoint blockade (ICB) through poorly understood changes in thetumor microenvironment. Recently, we provided evidence that the tonus of tumor infiltratingT lymphocytes (TILs) is associated to the intra-tumoral activity of Caspase-1 (doi:10.3390/cancers13020189). Bulk RNA-seq analysis of CRC revealed a greater expression level of NLRfamily CARD domain containing 5 (NLRC5) in tumors with a detectable Caspase-1 activity.Given that NLRC5 is a regulator of major histocompatibility complex class I (MHC-I) antigenpresentation, we postulated that NLRC5 may modulate cancer immune surveillance andresponsiveness to ICB. We thereby evaluated the significance of the intratumoral changesin NLRC5 expression in several cohorts of patients. We show that NLRC5 is a favorableprognostic factor of overall survival in 100 CRC and of responsiveness to ICB in 45 metastaticCRC. In agreement, NLRC5 expression in KRAS wild type CRC is of better prognosis andpositively correlates with PD1/PD-L1 axis. Conversely, a negative correlation is observedbetween PRMT5, an epigenetic modifier that represses NLRC5 expression, and PD-1/PD-L1axis. Finally, functional studies using co-cultures of TILs isolated from CRC fragments andcolonic cancer cell lines show that enforced expression of NLRC5 in tumor cells enhancesthe cytotoxic activity of CD8+ TILs (cytokine production, degranulation), via a MHC-I-dependent mechanism. Overall, our findings suggest that NLRC5 could be a valuablebiomarker in CRC and that increasing the NLRC5/MHC-I axis in tumor cells could enhanceanti-tumor immunity and immunotherapeutic responses

Involvement of the NLRC5 / MHC class I axis in human colorectal cancer immune surveillance

International audienceMost of patients with colorectal cancer (CRC) without microsatellite instability fail torespond to immune checkpoint blockade (ICB) through poorly understood changes in thetumor microenvironment. Recently, we provided evidence that the tonus of tumor infiltratingT lymphocytes (TILs) is associated to the intra-tumoral activity of Caspase-1 (doi:10.3390/cancers13020189). Bulk RNA-seq analysis of CRC revealed a greater expression level of NLRfamily CARD domain containing 5 (NLRC5) in tumors with a detectable Caspase-1 activity.Given that NLRC5 is a regulator of major histocompatibility complex class I (MHC-I) antigenpresentation, we postulated that NLRC5 may modulate cancer immune surveillance andresponsiveness to ICB. We thereby evaluated the significance of the intratumoral changesin NLRC5 expression in several cohorts of patients. We show that NLRC5 is a favorableprognostic factor of overall survival in 100 CRC and of responsiveness to ICB in 45 metastaticCRC. In agreement, NLRC5 expression in KRAS wild type CRC is of better prognosis andpositively correlates with PD1/PD-L1 axis. Conversely, a negative correlation is observedbetween PRMT5, an epigenetic modifier that represses NLRC5 expression, and PD-1/PD-L1axis. Finally, functional studies using co-cultures of TILs isolated from CRC fragments andcolonic cancer cell lines show that enforced expression of NLRC5 in tumor cells enhancesthe cytotoxic activity of CD8+ TILs (cytokine production, degranulation), via a MHC-I-dependent mechanism. Overall, our findings suggest that NLRC5 could be a valuablebiomarker in CRC and that increasing the NLRC5/MHC-I axis in tumor cells could enhanceanti-tumor immunity and immunotherapeutic responses

The inflammasome of tumor cells modulates the biology of tumor-infiltrating T lymphocytes in colorectal cancer

International audienceIn colorectal cancer (CRC), little is known about mechanisms by which tumor cells can influence the biology of Tumor Infiltrating T lymphocytes (TILs) present in the tumor microenvironment. One of these mechanisms could be modulation of the inflammasome of tumor cells. The inflammasome is a molecular platform present in normal intestinal epithelial cells, whose effector protein, caspase-1, can rapidly mature IL-18 and generate a mucosal Th1 (IFNγ) response. However, the inflammasome status of tumor cells in CRC and its potential role in the biology of TILs are unknown yet. This prospective study aimed to determine in CRC patients (n = 50) : i) the status of the inflammasome (caspase-1 / IL-18 axis) in tumor cells according to their microsatellite status [unstable (MSI) or stable (MSS)], in relation with the density of Th1/Tc1 TILs (T-bet+) and with the levels of the prototype Th1 cytokine IFNγ, secreted in an ex vivo explant culture model of CRC we developed and ii) the impact of the inflammasome-dependent cytokines potentially secreted by tumor cells on the biology of isolated TILs. Our study delineates two major groups of patients. The first group (33% of cases, mostly MSS with a low immunoscore) featured no active caspase-1 in tumor cells, no or low levels of mature IL-18 and IFNγ and only few T-bet+ TILs in the tumor microenvironment. This profile could correspond to an immune escape mechanism facilitating tumor progression. The second group (66% of cases, both MSI and MSS with high immunoscore) featured active caspase-1 in tumor cells associated with mature IL-18 secretion, high density of T-bet+ TILs expressing IL-18Rα and IFNγ release in most cases. In addition, isolated IL-18Rα+ TILs cultured with recombinant IL-18 were able to secrete IFNγ, either left unstimulated or stimulated with anti-CD3. In these CRC, the inflammasome of tumor cells, maintained and active, can contribute to a Th1/Tc1 antitumor response elicited by TILs present in the microenvironment that can modulate tumor growth. Interestingly, in a few cases of this second group (MSI CRC), the numerous T-bet+ TILs were unable to generate a Th1 response. Noticeably, these TILs express numerous immune checkpoints including PD1 and TIGIT, potentially responsible for their exhaustion. This study is the first to delineate functional interactions between tumor cells and TILs in CRC, using ex vivo explant cultures. Altogether, our findings support the inflammasome of tumor cells as a potential new therapeutic target to strengthen the Th1/Tc1 immune response in CRC, in association with immune checkpoint blockers. This work is supported by the “Ligue contre le Cancer Grand Ouest”Citation Format: Linda Bilonda, Delphine Dansette, Cecile Deleine, Romain Oger, Nicolas Jouand, Juliette Podevin, Pierre Fourquier, Emilie Thibaudeau, Jerome Chetritt, Jean-François Mosnier, Celine Bossard, Nadine Gervois, Anne Jarry. The inflammasome of tumor cells modulates the biology of tumor-infiltrating T lymphocytes in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4061

Targeting NKG2A to boost anti-tumor CD8 T-cell responses in human colorectal cancer

International audienceRecently, 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

The inflammasome of tumor cells in colorectal cancer : a potential target to strengthen the Th1/Tc1 response of Tumor Infiltrating T lymphocytes (TILs)

International audienc

The inflammasome of tumor cells in colorectal cancer : a potential target to strengthen the Th1/Tc1 response of Tumor Infiltrating T lymphocytes (TILs)

International audienc

The Caspase-1/IL-18 Axis of the Inflammasome in Tumor Cells: A Modulator of the Th1/Tc1 Response of Tumor-Infiltrating T Lymphocytes in Colorectal Cancer

International audienceIn 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

The density of Tbet+ tumor-infiltrating T lymphocytes reflects an effective and druggable preexisting adaptive antitumor immune response in colorectal cancer, irrespective of the microsatellite status

International audiencePurpose: 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

Increased antitumor efficacy of PD-1- deficient melanoma-specific human lymphocytes

International audienceBACKGROUND:Genome editing offers unique perspectives for optimizing the functional properties of T cells for adoptive cell transfer purposes. So far, PDCD1 editing 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.METHODS:Here we used the transfection of CAS9/sgRNA ribonucleoproteic complexes to edit PDCD1 gene in human effector memory CD8+ T cells specific for the melanoma antigen Melan-A. We cloned edited T cell populations and validated PDCD1 editing 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.RESULTS:Here we demonstrated the feasibility to edit PDCD1 gene in human effector memory melanoma-specific T lymphocytes. We showed that PD-1 expression was dramatically reduced or totally absent on PDCD1-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.CONCLUSION:The 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