Immune profile associated to vitiligo onset in melanoma patients undergoing treatment with checkpoint inhibitors gives insight into the raising of the adverse event and of the effective anti-tumor response

Immunotherapy with checkpoint inhibitors is an efficient treatment for metastatic melanoma. Development of vitiligo upon immunotherapy represents a specific immune-related adverse event (irAE) diagnosed in 15% of patients and associated with a positive clinical response. Therefore, a detailed characterization of immune cells during vitiligo onset in melanoma patients would give insight into the immune mechanisms mediating both this irAE and the anti-tumor response. To better understand these aspects, we analyzed T cell subsets from peripheral blood of metastatic melanoma patients undergoing treatment with anti-programmed cell death protein (PD)-1 antibodies. Stratification of patients for developing or not developing vitiligo during therapy revealed an association between blood reduction of mucosal associated invariant T (MAIT), T helper (h) 17, natural killer (NK) CD56bright, and T regulatory (T-reg) cells and vitiligo onset. To deeply characterize the tumoral T cell response concomitant to vitiligo onset, we analyzed T cell content in skin biopsies collected from melanoma patients who developed vitiligo. Consistently with the observed blood reduction of Th17 cells in melanoma patients developing vitiligo during immunotherapy, we found an enrichment of Th17 cells in the vitiligo skin biopsy, suggesting a migration of Th17 cells from the blood into the autoimmune lesion. To further characterize T cells in vitiligo skin lesion of melanoma patients, we sequenced T cell receptor (TCR) of cells derived from biopsies of vitiligo and primary melanoma of the same patient. Interestingly, we found different TCR sequences between vitiligo and primary melanoma lesions, except for a few cases showing the same TCR sequences. In contrast, shared TCR sequences were identified between vitiligo and metastatic tissues of the same patient. These data indicate that T cell response against normal melanocytes, which is involved in vitiligo onset, is not mainly mediated by the reactivation of specific T cell clones infiltrating primary melanoma but may be elicited by T cell clones targeting metastatic tissues. Altogether, our data indicate that anti-PD-1 therapy induces a de novo immune response, composed of different T cell subtypes, whose role may be related to the development of vitiligo and the response against metastatic tumor. In this study we performed TCR-Seq of PBMC (10 biological replicateas), primary melanoma (15 biological replicates), metastasis (5 biological replicates) and vitiligo (10 biological replicates) samples deriving from patients affected by metastatic melanoma, who were treated with anti-PD1 immunotherapy at IDI-IRCCS. ***Submitter states that Adaptive Biotechnologies does not release raw data***

免疫检查点抑制剂（checkpoint inhibitors）疗法是治疗转移性黑色素瘤的有效手段。接受该免疫治疗后出现白癜风（vitiligo），是一类在15%患者中确诊的特异性免疫相关不良事件（immune-related adverse event, irAE），且与良好的临床应答相关。因此，对黑色素瘤患者白癜风发作期间的免疫细胞进行详细表征，将有助于阐明介导该免疫相关不良事件以及抗肿瘤应答的免疫机制。

为进一步明晰上述问题，本研究对接受抗程序性死亡蛋白1（PD-1）抗体治疗的转移性黑色素瘤患者的外周血T细胞亚群进行了分析。按治疗期间是否出现白癜风对患者进行分层后发现，外周血中黏膜相关恒定T细胞（mucosal associated invariant T, MAIT）、辅助性T细胞17（Th17）、自然杀伤（NK）CD56bright细胞以及调节性T细胞（T-reg）的减少与白癜风发作存在关联。

为深入表征白癜风发作时伴随的肿瘤T细胞应答，本研究对出现白癜风的黑色素瘤患者的皮肤活检样本中的T细胞含量进行了分析。与免疫治疗期间出现白癜风的黑色素瘤患者外周血中Th17细胞减少的结果一致，我们在白癜风皮肤活检样本中检测到Th17细胞的富集，提示Th17细胞从外周血迁移至自身免疫损伤部位。

为进一步表征黑色素瘤患者白癜风皮损中的T细胞特征，我们对同一患者的白癜风皮损与原发性黑色素瘤组织来源的T细胞受体（T cell receptor, TCR）进行了测序。有趣的是，除少数病例存在相同的TCR序列外，白癜风皮损与原发性黑色素瘤病灶的TCR序列存在显著差异。与之相反，同一患者的白癜风皮损与转移灶组织中可检测到共享的TCR序列。

上述数据表明，参与白癜风发作的针对正常黑素细胞的T细胞应答，并非主要由浸润原发性黑色素瘤的特异性T细胞克隆活化所介导，而可能由靶向转移灶组织的T细胞克隆所触发。

综上，本研究数据显示，抗PD-1疗法可诱导由不同T细胞亚型组成的全新免疫应答，其功能可能与白癜风的发生以及转移性肿瘤的应答相关。本研究对来自接受抗PD-1免疫治疗的转移性黑色素瘤患者的样本进行了TCR测序，样本类型包括外周血单个核细胞（PBMC，10例生物学重复）、原发性黑色素瘤组织（15例生物学重复）、转移灶组织（5例生物学重复）以及白癜风皮损组织（10例生物学重复），所有患者均在IDI-IRCCS接受治疗。***提交者声明：自适应生物技术公司（Adaptive Biotechnologies）不发布原始数据***