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Objectives: Lymphoepithelioma‐like carcinoma (LELC) is an uncommon lung cancer, typically observed in young, non‐smoking Asian populations. LELC is associated with Epstein–Barr virus (EBV) infection of lung tumor cells of epithelial origin, suggesting a carcinogenic role of EBV as observed in nasopharyngeal carcinoma (NPC). Here, we studied the antigen specificity and phenotype of EBV‐specific CD8+ T cells in blood and tumor of one LELC patient positive for EBV infection in lung tumor cells. Methods: Using multiplex MHC class I tetramers, mass cytometry and mRNA sequencing, we studied EBV‐specific CD8+ T cells at the transcriptomic and phenotypic levels in blood and tumor tissues of the LELC patient. Results: Lymphoepithelioma‐like carcinoma lung tumor cells were positive for EBV infection. In both blood and tumor tissues, we detected two populations of EBV‐specific CD8+ T cells targeting the EBV lytic cycle proteins: BRLF1 and BMLF1. Transcriptomic analyses of these two populations in the tumor, which can be considered as tumor‐specific, revealed their distinct exhausted profile and polyclonal TCR repertoire. High‐dimensional phenotypical analysis revealed the distinct phenotype of these cells between blood and tumor tissues. In tumor tissue, EBV‐specific CD8+ TILs were phenotypically heterogeneous, but consistently expressed CD39. Unexpectedly, although the LELC tumor cells expressed abundant PD‐L1, these tumor‐specific CD8+ tumor‐infiltrating lymphocytes (TILs) mostly did not express PD‐1. Conclusion: Epstein–Barr virus‐specific CD8+ TILs in EBV‐driven tumor are heterogeneous and partially lack PD‐1 expression, suggesting that anti‐PD1/PD‐L1 immunotherapy may not be an appropriate strategy for disinhibiting EBV‐specific cells in the treatment of LELC patients. [ABSTRACT FROM AUTHOR]

Invariant natural killer T (iNKT) cells expressing the retinoic acid receptor-related orphan receptor γt (RORγt) and producing IL-17 represent a minor subset of CD1d-restricted iNKT cells (iNKT17) in C57BL/6J (B6) mice. We aimed in this study to define the reasons for their low distribution and the sequence of events accompanying their normal thymic development. We found that RORγt+ iNKT cells have higher proliferation potential and a greater propensity to apoptosis than RORγt− iNKT cells. These cells do not likely reside in the thymus indicating that thymus emigration, and higher apoptosis potential, could contribute to RORγt+ iNKT cell reduced thymic distribution. Ontogeny studies suggest that mature HSAlow RORγt+ iNKT cells might develop through developmental stages defined by a differential expression of CCR6 and CD138 during which RORγt expression and IL-17 production capabilities are progressively acquired. Finally, we found that RORγt+ iNKT cells perceive a strong TCR signal that could contribute to their entry into a specific 'Th17 like' developmental program influencing their survival and migration. Overall, our study proposes a hypothetical thymic developmental sequence for iNKT17 cells, which could be of great use to study molecular mechanisms regulating this developmental program. [ABSTRACT FROM AUTHOR]

The success of cancer immunotherapies such as immune checkpoint inhibitors, CAR T-cells and immune cell engagers have provided clinicians with tools to bypass some of the limitations of cancer immunity. However, numerous tumour factors curtail the immune response against cancer and limit the efficiency of immuno-oncology (IO) therapies. Acidification of the extra-cellular tumour environment consecutive to aberrant cancer cell metabolism is a well-known promoter of oncogenic processes that also acts as an immune regulator. Yet, the suppressive mechanisms of low extra-cellular pH on anti-cancer immunity remain poorly understood. Recent reports have suggested that GPR65, a Gαs-coupled proton-sensing GPCR broadly expressed in the immune system, may act as an immune suppressant detrimental to anti-tumour immunity. So far, the immuno-regulatory properties of GPR65 in acidic milieux have mostly been documented in macrophages and myeloid cells. Our computational evaluation of GPR65's transcriptomic expression profile and potential as an IO target using public datasets prompted us to further investigate its functions in human T-cells. To this end, we identified and validated GPR65 small molecule inhibitors active in in vitro cellular assays and we showed that GPR65 inhibition promoted the killing capacity of antigen-specific human T-cells. Our results broaden the scope of GPR65 as an IO target by suggesting that its inhibition may enhance T-cell anti-tumour activity and provide useful pharmacological tools to further investigate the therapeutic potential of GPR65 inhibition., Competing Interests: All authors are or were employees of Laboratoires Servier, who sponsored the research. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Li, Melchiore, Kantari-Mimoun, Mouton, Knockaert, Philippon, Chanrion, Bourgeois, Lefebvre, Elhmouzi-Younes, Blanc, Ramon Olayo and Laugel.)

Tumor-specific T cells likely underpin effective immune checkpoint-blockade therapies. Yet, most studies focus on Treg cells and CD8 + tumor-infiltrating lymphocytes (TILs). Here, we study CD4 + TILs in human lung and colorectal cancers and observe that non-Treg CD4 + TILs average more than 70% of total CD4 + TILs in both cancer types. Leveraging high dimensional analyses including mass cytometry, we reveal that CD4 + TILs are phenotypically heterogeneous, within each tumor and across patients. Consistently, we find different subsets of CD4 + TILs showing characteristics of effectors, tissue resident memory (Trm) or exhausted cells (expressing PD-1, CTLA-4 and CD39). In both cancer types, the frequencies of CD39 - non-Treg CD4 + TILs strongly correlate with frequencies of CD39 - CD8 + TILs, which we and others have previously shown to be enriched for cells specific for cancer-unrelated antigens (bystanders). Ex-vivo , we demonstrate that CD39 - CD4 + TILs can be specific for cancer-unrelated antigens, such as HCMV epitopes. Overall, our findings highlight that CD4 + TILs can also recognize cancer-unrelated antigens and suggest measuring CD39 expression as a straightforward way to quantify or isolate bystander CD4 + T cells., Competing Interests: E.W.N is a co-founder, advisor and shareholder of ImmunoScape Pte. Ltd. E.W.N. is an advisor for Neogene Therapeutics and Nanostring Technologies. All other authors declare no potential conflicts of interest., (© 2022 The Author(s). Published with license by Taylor & Francis Group, LLC.)

Kupffer cells are the liver-resident macrophages lining the sinusoids and are mostly known for their role of scavengers, as crucial keepers of organ integrity. But due to the many fundamental functions of the liver notably linked to detoxication, metabolism, protein synthesis, or immunology, Kupffer cells are exposed to a dynamic environment and constantly adapt themselves by modulating their gene and protein expressions. In this context, the characterization of these cells at steady-state and upon challenges may be limited by the classical microscopy or flow cytometry which allow for the use of only few selected markers. On the other end, transcriptomic approach, although being very powerful, can be costly and time-consuming. So mass cytometry offers a good compromise, allowing for the monitoring of a representative set of markers (up to 40) in a simple experiment. Herein, we describe a straightforward experimental and analysis workflow for Kupffer cell characterization by mass cytometry.