Analysis of infiltrating γδ T cells in cutaneous melanoma: a hypothetic crosstalk for new immunotherapy

Introduction: Melanoma is one of the most frequently occurring skin cancer and remains an important cause of mortality mainly in Caucasian populations. In clinical practice, the standard treatment for localized melanoma is surgical resection but for metastatic melanoma, few treatments are available, where chemo and radio-therapies are rarely indicated. Immunotherapy has revolutionized the management of metastatic melanoma that, as in others tumors, uses your body's own immune system to help fight cancer. Therapies targeting the immune checkpoint molecules have achieved objective responses in melanoma. Several receptors and/or ligands, such as PDL1, PD-1 and CTLA-4, have been identified as important regulatory molecules but these therapies are not effective for a large number of patients. The importance of the immune response in the clinical outcome was correlated with increased number of tumor-infiltrating lymphocytes (TILs) in melanoma and in several other tumors. Many studies underline the role of γδ T lymphocytes in the anti-tumor surveillance with a marked cytotoxic effect towards tumor cells. We investigated the expression of some novel immune checkpoint molecules, such as TIM3 (T-cell immunoglobulin and mucin-domain containing-3), TIGIT (Tcell Ig and ITIM domain) and LAG3 (lymphocyte activation gene-3) on T lymphocytes. Material and Methods: From 2018 to 2021, 54 adult patients were enrolled in our study with clinical suspicion of melanoma, at the Plastic Surgery Department of the University Hospital in Palermo. A blood drawing (in a dry test tube) and a fragment (about 1 mm) of perilesional skin was taken before the tumoral excision. All bioptical samples were processed immediately through mechanical and enzymatic digestion. After the immunological cells were analysed by flow cytometry. We conducted also the immunofluorescence staining on tumoral samples and bioinformatics analysis through dataset online. Results: The analysis of the histological examination reports showed that in 75% of cases the diagnosis of suspected melanoma was confirmed. The infiltrating and circulating T lymphocytes analysis showed that the cell percentage was higher in peripheral blood than in peritumoral tissues in particular 5 on melanoma’s metastases. The study of the immunocheckpoint molecules expression (PD1, TIGIT, LAG3 and TIM3) showed higher values in the infiltrating compared to circulating T cells. In addition, a detailed analysis of these data in correlation with pathological stage of melanoma, showed that, on perilesional infiltrating T lymphocytes, PD-1 is more expressed in melanomas in situ, then decreasing from pt1a as pathological stage increases. TIM3, TIGIT and LAG3, were expressed in other stage. The circulating T lymphocytes investigation showed variable values for pathological stage, except TIGIT, whose values were higher in the ptis and pt1a. In the mestastases, all immunocheckpoints were more expressed, expecially LAG3. Furthermore, the bioinformatic analysis showed that immunotherapy with checkpoint inhibitors (anti-PD1, anti-CTL4 and anti- PD1/CTL4) caused an increase in memory T lymphocytes, with a marked migratory phenotype and an increase in depleted γδ lymphocytes. Conclusions: In the literature, there are few data on the LAG3, TIM3 and TIGIT expression in melanoma, therefore, this study may be able to contribute in this regard. LAG 3 is the highest of all the immunocheckpoints analyzed. Further studies will be needed to fully understand the function of these molecules in order to develop new immunotherapy protocols. In particular, the impact of neutralizing LAG3 with certain inhibitors should be further studied to understand the functional role of LAG3 in anti-PD1 resistant patients.