Nathalie Labarrière

Colorectal cancer (CRC) progression is driven by dynamic interactions among tumor cells, immune infiltrates, and the gut microbiota. While regulatory T cells (Tregs) may contribute to immune suppression in CRC, the role of non-conventional Tregs remains poorly defined. We identified a non-conventional population of microbiota-induced Tregs in the human colonic mucosa that co-expressed CD4, CD8α, CXCR6, and CCR6, termed DP8α Tregs, that exert potent immunomodulatory properties in different inflammatory settings. Their status and role in CRC, however, have not been investigated. Here, using multiparametric flow cytometry in a prospective cohort of CRC patients, we showed that DP8α Tregs are significantly enriched in tumors compared to paired non-tumoral colonic mucosa. Tumor-infiltrating DP8α Tregs displayed elevated expression of the CD39/CD73 ectonucleotidases, as well as CCR5, consistent with a suppressive phenotype within the tumor microenvironment. Functional co-culture assays further demonstrated that sorting DP8α Tregs from CRC tumors inhibited both CD4 and CD8 T-cell proliferation, an effect largely reversed by pharmacological inhibition of CD39 and CD73, which was associated with reduced IL-2 levels. Together, these findings show that DP8α Tregs enriched in the CRC tumor microenvironment, are able to suppress effector T-cell responses through the purinergic pathway and support further investigation of their contribution to immune regulation in CRC.

Although anti-PD-1 antibodies are approved as first-line treatment for patients with metastatic melanoma (MM), many patients remain resistant. Adoptive cel

Inflammatory responses can be of critical importance in determining both the course and outcome of infectious diseases. Buruli ulcer, a neglected tropical disease caused by Mycobacterium ulcerans ( M. ulcerans ), has been reported to induce both immunosuppressive and proinflammatory responses in the skin. Here, we found that genetic inactivation of the orphan receptor GPR84 in mice leads to spontaneous healing of ulcerative lesions, recapitulating the rare spontaneous healing observed in human Buruli ulcer. Mechanistically, M. ulcerans infection induced Gpr84 expression through Toll-like receptor 2 engagement, promoting proinflammatory cytokine release through a self-amplifying loop that sustained receptor expression and inflammatory signaling. Moreover, pharmacological inhibition of GPR84 with the antagonist PBI-4050, in combination with antibiotics, accelerated tissue repair in infected GPR84-competent mice, which was associated with attenuation of inflammatory responses. Together, these findings establish GPR84 as a promising target for host-directed therapeutic interventions in Buruli ulcer and its expression as a potential biomarker of lesion activity.

Cancer cells rely on alternative modes of translation for protein synthesis, promoting internal ribosome entry site (IRES)-dependent translation of mRNA subsets encoding pro-oncogenic factors. Furthermore, ribosomes translate mRNA with lower fidelity in tumor cells. Our bioinformatics analyses highlighted large numbers of non-canonical peptides specific to cancer cells derived from aberrant translation of IRES-containing oncogene mRNAs. Interestingly, these peptides contain multiple shared neoepitope candidates. To provide a complete proof-of-concept, we selected one HLA-A2 epitope candidate (LLL) derived from c-MYC , which was confirmed to be immunogenic and expressed selectively on tumor cells by targeted immunopeptidomics. This peptide is generated by a (+ 1) ribosomal frameshift of c-MYC mRNA occurring during the oncogenic transformation. TCR-engineered T cells specific to LLL epitope selectively recognized and killed tumor cells, confirming the therapeutic potential of targeting this neoepitope. Altogether, these results validate a new class of actionable shared neoepitopes derived from non-canonical translation of oncogenes.

The search for reliable shared tumor-specific antigens (TSAs) to improve cancer immunotherapy is on-going. The so-called non-coding regions of the genome have recently been shown to give rise to immunogenic peptides, including the melanoma-specific antigen MELOE-1 which is translated from the long intergenic non-coding RNA (lincRNA) meloe in an IRES-dependent manner. Here, we present a strategy to systematically identify tumor-specific antigens produced by ORFs within lincRNAs with IRES-like upstream structures. We provide evidence suggesting that in the melanocytic lineage a significant proportion of the selected lincRNAs can produce immunogenic peptides. T cell repertoires against some of these peptides were found in peripheral blood mononuclear cells (PBMCs) from healthy donors and melanoma patients, and in tumor-infiltrating lymphocytes (TILs) from metastatic melanoma patients. Finally, CD8+ T cell lines from melanoma patients specific for three of the characterized HLA-A *0201 epitopes could recognize melanoma cell lines, which were enhanced by reticular stress. Thus, these peptides may represent a new class of shared TSAs in melanoma and are attractive candidates for evaluation as targets for immunotherapy in preclinical studies. In addition, our selection strategy has the potential to identify new lincRNA-derived antigens in other cancers.

Immune checkpoint (IC) blockade and adoptive transfer of tumor-specific T-cells (ACT) are two major strategies to treat metastatic melanoma. Their combination can potentiate T-cell activation in the suppressive tumor microenvironment, but the autoimmune adverse effects associated with systemic injection of IC blockers persist with this strategy. ACT of tumor-reactive T-cells defective for IC expression would overcome this issue. For this purpose, PD-1 and TIGIT appear to be relevant candidates, because their co-expression on highly tumor-reactive lymphocytes limits their therapeutic efficacy within the tumor microenvironme,nt. Our study compares the consequences of PDCD1 or TIGIT genetic deletion on anti-tumor properties and T-cell fitness of melanoma-specific T lymphocytes. Transcriptomic analyses revealed down-regulation of cell cycle-related genes in PD-1^KO T-cells, consistent with biological observations, whereas proliferative pathways were preserved in TIGIT^KO T-cells. Functional analyses showed that PD-1^KO and TIGIT^KO T-cells displayed superior antitumor reactivity than their wild-type counterpart in vitro and in a preclinical melanoma model using immunodeficient mice. Interestingly, it appears that TIGIT^KO T-cells were more effective at inhibiting tumor cell proliferation in vivo, and persist longer within tumors than PD-1^KO T-cells, consistent with the absence of impact of TIGIT deletion on T-cell fitness. Taken together, these results suggest that TIGIT deletion, over PD-1 deletion, in melanoma-specific T-cells is a compelling option for future immunotherapeutic strategies.

Natural killer (NK) cells play a crucial role in the innate immune response to bacterial infections, including those due to Pseudomonas aeruginosa ( P. aeruginosa ) and Staphylococcus aureus ( S. aureus ). In vivo , it has been shown that NK cells are activated by innate accessory cells that detect the presence of bacteria and activate NK cells via a cytokine network. In vitro , several studies have shown that NK cells can also be activated without the help of accessory cells by direct contact with some bacteria species such as extracellular P. aeruginosa . Whether this phenomenon of direct activation is restricted to certain bacterial species, or whether it can be generalized, is still debated, as for example in the case of NK cell activation by S. aureus , which seems to require the intervention of accessory immune cells. Here, we show with co-incubation experiments between NK cells and two bacterial species, that, in the absence of accessory cells, NK cells are able to impede bacterial growth. This has been demonstrated for the P. aeruginosa PA14 strain, which is hypervirulent and known for its deleterious effects on NK cells, as well as for the S. aureus Newman strain. The monitoring of CD107a by flow cytometry suggests that NK cells degranulate after contact with S. aureus bacteria. Our study contributes to the idea that NK cells can be activated in the absence of any accessory cells by various species of bacteria, even an hypervirulent one, and therefore that NKs can directly have an antibacterial effect. This important insight may pave the way for new therapeutic approaches using antibacterial NK-cell engagers.

Antibiotics (ABX) compromise the efficacy of programmed cell death protein 1 (PD-1) blockade in cancer patients, but the mechanisms underlying their immunosuppressive effects remain unknown. By inducing the down-regulation of mucosal addressin cell adhesion molecule 1 (MAdCAM-1) in the ileum, post-ABX gut recolonization by Enterocloster species drove the emigration of enterotropic α4β7 ⁺ CD4 ⁺ regulatory T 17 cells into the tumor. These deleterious ABX effects were mimicked by oral gavage of Enterocloster species, by genetic deficiency, or by antibody-mediated neutralization of MAdCAM-1 and its receptor, α4β7 integrin. By contrast, fecal microbiota transplantation or interleukin-17A neutralization prevented ABX-induced immunosuppression. In independent lung, kidney, and bladder cancer patient cohorts, low serum levels of soluble MAdCAM-1 had a negative prognostic impact. Thus, the MAdCAM-1–α4β7 axis constitutes an actionable gut immune checkpoint in cancer immunosurveillance.

MicroRNAs (miRNA), small noncoding RNAs that regulate gene expression, exist not only in cells but also in a variety of body fluids. These circulating miRNAs could enable intercellular communication. miRNAs are packaged in membrane-encapsulated vesicles, such as exosomes, or protected by RNA-binding proteins. Here, we report that miRNAs included in human melanoma exosomes regulate the tumor immune response. Using microscopy and flow cytometry, we demonstrate that CD8+ T cells internalize exosomes from different tumor types even if these cells do not internalize vesicles as readily as other immune cells. We explored the function of melanoma-derived exosomes in CD8+ T cells and showed that these exosomes downregulate T-cell responses through decreased T-cell receptor (TCR) signaling and diminished cytokine and granzyme B secretions. The result reduces the cells' cytotoxic activity. Using mimics, we found that miRNAs enriched in exosomes—such as Homo sapiens (hsa)-miR-3187-3p, hsa-miR-498, hsa-miR-122, hsa-miR149, and hsa-miR-181a/b—regulate TCR signaling and TNFα secretion. Our observations suggest that miRNAs in melanoma-derived exosomes aid tumor immune evasion and could be a therapeutic target.