Innate-like T cell subset commitment in the murine thymus is independent of TCR characteristics and occurs during proliferation.

How T-cell receptor (TCR) characteristics determine subset commitment during T-cell development is still unclear. Here, we addressed this question for innate-like T cells, mucosal-associated invariant T (MAIT) cells, and invariant natural killer T (iNKT) cells. MAIT and iNKT cells have similar developmental paths, leading in mice to two effector subsets, cytotoxic (MAIT1/iNKT1) and IL17-secreting (MAIT17/iNKT17). For iNKT1 vs iNKT17 fate choice, an instructive role for TCR affinity was proposed but recent data argue against this model. Herein, we examined TCR role in MAIT and iNKT subset commitment through scRNAseq and TCR repertoire analysis. In our dataset of thymic MAIT cells, we found pairs of T-cell clones with identical amino acid TCR sequences originating from distinct precursors, one of which committed to MAIT1 and the other to MAIT17 fates. Quantitative in silico simulations indicated that the number of such cases is best explained by lineage choice being independent of TCR characteristics. Comparison of TCR features of MAIT1 and MAIT17 clonotypes demonstrated that the subsets cannot be distinguished based on TCR sequence. To pinpoint the developmental stage associated with MAIT sublineage choice, we demonstrated that proliferation takes place both before and after MAIT fate commitment. Altogether, we propose a model of MAIT cell development in which noncommitted, intermediate-stage MAIT cells undergo a first round of proliferation, followed by TCR characteristics-independent commitment to MAIT1 or MAIT17 lineage, followed by an additional round of proliferation. Reanalyzing a published iNKT TCR dataset, we showed that this model is also relevant for iNKT cell development.

A conserved transcriptional program for MAIT cells across mammalian evolution.

Mucosal-associated invariant T (MAIT) cells harbor evolutionarily conserved TCRs, suggesting important functions. As human and mouse MAIT functional programs appear distinct, the evolutionarily conserved MAIT functional features remain unidentified. Using species-specific tetramers coupled to single-cell RNA sequencing, we characterized MAIT cell development in six species spanning 110 million years of evolution. Cross-species analyses revealed conserved transcriptional events underlying MAIT cell maturation, marked by ZBTB16 induction in all species. MAIT cells in human, sheep, cattle, and opossum acquired a shared type-1/17 transcriptional program, reflecting ancestral features. This program was also acquired by human iNKT cells, indicating common differentiation for innate-like T cells. Distinct type-1 and type-17 MAIT subsets developed in rodents, including pet mice and genetically diverse mouse strains. However, MAIT cells further matured in mouse intestines to acquire a remarkably conserved program characterized by concomitant expression of type-1, type-17, cytotoxicity, and tissue-repair genes. Altogether, the study provides a unifying view of the transcriptional features of innate-like T cells across evolution.

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

While 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.

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

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+ αß 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.