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This work sought to confirm the human-like expression of exhaustion and senescence markers in a mouse model with a humanized immune system (HIS): the Balb/c Rag2KO IL2rgcKO SirpαNOD Flk2KO HLA-A2HHD (BRGSF-A2) mouse reconstituted with human CD34+ cord blood cells. With regard to senescence markers, the percentage of CD57+ T cells was higher in the bone marrow (BM) than in the spleen or blood. The same was true for KLRG1+ hCD8+ T cells. With regard to exhaustion markers, the percentage of programmed death 1 (PD-1+) T cells was higher in the BM than in the spleen or blood; the same was true for TIGIT+ hCD4+ cells. These tissue-specific differences were related to both higher proportions of memory T cells in BM and intrinsic differences in expression within the memory fraction. In blood samples from HIS mice and healthy human donors (HDs), we found that the percentage of KLRG1+ cells among hCD8+ T cells was lower in HIS compared to HDs. The opposite was true for CD4+ T cells. Unexpectedly, a high frequency of KLRG1+ cells was observed among naive T cells in HIS mice. CD57 expression on T cells was similar in blood samples from HIS mice and HDs. Likewise, PD-1 expression was similar in the two systems, although a relatively low proportion of HIS hCD4+ T cells expressed TIGIT. The BRGSF-A2 HIS mouse's exhaustion and senescence profile was tissue specific and relatively human like; hence, this mouse might be a valuable tool for determining the preclinical efficacy of immunotherapies.

The high expression of inducible nitric oxide synthase (NOS2) by myeloid-derived suppressor cells (MDSCs) is a key mechanism of immune evasion in cancer. Recently we reported that NOS2 is also expressed by γδ T cells in melanoma, contributing to their polarization towards a pro-tumor phenotype. The molecular mechanisms underlying regulation of NOS2 expression in tumor-induced γδ T cells remain unexplored. By using the model of mice transgenic for the ret oncogene (Ret mice) that develops a spontaneous metastatic melanoma, we evidence that interleukin (IL)-1β and IL-6 drive NOS2 expression in γδ T cells. Indeed, their in vivo neutralization lessens the γδ T cell capacity to produce not only NOS2, but also IL-17 involved in the recruitment of MDSCs at the primary tumor site. The treatment also delayed tumor cell dissemination and induced vitiligo in a significant proportion of Ret mice. Interestingly, Ret mice developing a less aggressive melanoma, characterized by the spontaneous development of a concomitant autoimmune vitiligo, exhibit a weaker concentration of inflammatory cytokines and a reduction of tumor infiltrating γδ T cells expressing NOS2, when compared to Ret mice without any signs of vitiligo. Overall our results support that the level of inflammation at the tumor site regulates NOS2 expression by γδ T cells and the development of vitiligo associated melanoma.

γδ T lymphocytes may exert either protective or tumor-promoting functions in cancer, mostly based on their polarization toward interferon (IFN)-γ or interleukin (IL)-17 productions, respectively. Here, we demonstrate that γδ T cells accelerate the spontaneous metastatic melanoma development in a model of transgenic mice for the human RET oncogene (Ret mice). We identify unanticipated roles of inducible nitric oxide synthase (NOS2) in favoring the recruitment of pro-tumor γδ T cells within the primary tumor. γδ T cells isolated from Ret mice deficient for NOS2 produced more IFNγ and less IL-17 than their counterparts from Ret mice. By supporting IL-17 production by γδ T cells, NOS2 leads to the recruitment of polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs) and metastasis formation. NOS2 also reduces the cytotoxicity of γδ T cells toward melanoma cells. Finally, we detected NOS2 expressing γδ T cells in the primary tumor and tumor-draining lymph nodes in Ret mice, but also in human melanoma. Overall our results support that this NOS2 autocrine expression is responsible for the polarization of γδ T cells toward a pro-tumor profile.