<b>Radical hemithorax radiotherapy induces an increase in circulating PD-1</b>^{<strong>+ </strong>}<b>T lymphocytes and in the soluble levels of PD-L1 in Malignant Pleural Mesothelioma patients: a possible synergy with PD-1/PD-L1 targeting treatment?</b>

Malignant Pleural Mesothelioma (MPM) is an aggressive tumor associated with asbestos exposure, characterized by a poor prognosis, managed with surgery, chemotherapy and radiotherapy. Recently, immunotherapy gives a survival advantage compared to chemotherapy, but limited to the non-epithelioid histotype, the rarest type. Radical hemithorax radiotherapy (RHRT) improves the Overall Survival (OS) of MPM patients, irrespective of histotype, and is able to induce immunomodulatory effects. In this study we aime to investigate changes in circulating T lymphocytes phenotype and activity, in MPM patients undergoing RHRT, to evaluate a possible therapeutic space for immunotherapy in this setting.To assess immunomodulatory effects of RHRT we evaluate peripheral blood samples of 35 MPM patients collected before treatment, at the end of RT, and 1 month later. We first notice that higher Lymphocyte-to-Monocyte Ratio (LMR) levels, before RT, are associated with an improved OS. The immune monitoring performed by ELISA assays reveals a significant increase in the serum levels of sPD‐L1 and IFN‐γ at the end of RHRT. Furthermore, the percentage of PD‐1+ cells, evaluated by flow cytometry, significantly raise after RHRT in T cells, both CD4+ and CD8+. Also the proportion of proliferative cells is significantly expanded after RHRT in all T cell subtypes. After treatment we observe a significant increase in the number of patients showing WT-1 specific CD4+ T cells, measured by intracellular staining. The TCR repertoire analysis, investigated by Next Generation Sequencing, reveals an increased number of expanded T-cell clones after RHRT, and an association between TCR clonality and the percentage of proliferating cytotoxic T lymphocytes. The comparison of TCR sequences obtained in our cohort with those described in a literature cohort of MPM patients, reveals common entries, specific for MPM-associated antigens including WT-1.In this setting, pre-treatment levels of LMR index seem to have a positive prognostic role, and RHRT would appear to induce immunomodulating effects, potential biomarkers for immunotherapy eligibility: i.e. increased PD-1+ T lymphocytes, proliferating T cells, expanded T cell clones and augmented levels of sPD-L1. These data suggest the design of a prospective study evaluating a maintenance immunotherapy after RHRT in MPM, even in the epithelioid histotype.恶性胸膜间皮瘤（Malignant Pleural Mesothelioma, MPM）是一种与石棉暴露相关的侵袭性肿瘤，预后不良，临床治疗手段包括手术、化疗与放疗。近年来，免疫治疗相较化疗可为患者带来生存获益，但仅适用于最为罕见的非上皮样组织学亚型。根治性半胸放疗（Radical hemithorax radiotherapy, RHRT）可改善MPM患者的总生存期（Overall Survival, OS），且不受组织学亚型影响，同时能够诱导免疫调节效应。本研究旨在探究接受RHRT的MPM患者循环T淋巴细胞的表型与活性变化，以评估该临床场景下免疫治疗的潜在应用价值。为评估RHRT的免疫调节效应，本研究收集了35例MPM患者的外周血样本，分别于治疗前、放疗结束时及治疗后1个月进行检测。本研究首先发现，放疗前较高的淋巴细胞-单核细胞比值（Lymphocyte-to-Monocyte Ratio, LMR）与更优的总生存期相关。通过酶联免疫吸附试验（ELISA）开展的免疫监测结果显示，RHRT结束时患者血清中可溶性PD-L1（sPD-L1）与干扰素γ（IFN-γ）水平显著升高。此外，经流式细胞术（flow cytometry）检测的PD-1阳性细胞占比，在RHRT后的CD4阳性与CD8阳性T细胞中均显著提升；所有T细胞亚群的增殖细胞比例亦在RHRT后显著扩增。治疗后，通过胞内染色检测到的WT-1特异性CD4阳性T细胞阳性患者数量显著增加。通过下一代测序（Next Generation Sequencing, NGS）开展的T细胞受体（TCR）库分析显示，RHRT后扩增的T细胞克隆数量增多，且T细胞受体克隆性与增殖性细胞毒性T淋巴细胞占比存在相关性。将本研究队列获取的T细胞受体序列与文献报道的MPM患者队列序列进行比对，可发现二者存在针对MPM相关抗原（包括WT-1）的共有特异性序列。在此研究背景下，治疗前的LMR水平似乎具有正向预后价值，而RHRT可诱导免疫调节效应，包括PD-1阳性T淋巴细胞增多、T细胞增殖活跃、T细胞克隆扩增及血清sPD-L1水平升高等潜在的免疫治疗适应证生物标志物。上述数据提示，可开展前瞻性研究以评估MPM患者在接受RHRT后接受维持免疫治疗的效果，即使针对上皮样组织学亚型患者亦可行此探索。