B-Cells Derived EVs Shape Melanoma Response to Immune Checkpoint Therapy

In recent years, the understanding of the immune tumor microenvironment has evolved to include B cells as active participants rather than mere bystanders. Retrospective studies investigating the presence of B cells and B cells-associated signatures within tumors have revealed a significant correlation with therapeutic outcomes. Although conflicting data exist regarding whether B cells act as pro-tumor or anti-tumor agents, it is becoming increasingly clear that understanding B cells as a heterogeneous population, shaped by tumor microenvironment cues, is essential. This study explores the crucial roles played by B cells and their extracellular vesicles (EVs) in shaping responses to immune checkpoint blockade (ICB) therapy. We have independently verified the significant enrichment of B cells in ICB therapy responders in comparison to non-responder in tumors of melanoma patients. B cell depletion experiments in an in vitro tumor-killing assay demonstrate a clear impairment of T cell-mediated anti-tumor activity. To investigate the clinical relevance, EVs were isolated from melanoma patient tumors, and fractioned into subpopulations, including B cell-derived EVs. MiRNA profiling of CD19+ EVs identifies miR-99a-5p as a top candidate, significantly upregulated in responder EVs. In downstream assays, we observed a B cell-dependent phenotype of miR-99a-5p, where its silencing impaired T cell-mediated anti-tumor cytotoxicity in an in vitro tumor-killing assay. Mechanistically, miR-99a-5p appears to regulate B cell cell-cycle, favoring non-homologous end joining DNA repair pathway, and promoting class-switch recombination. Collectively, data reported herein emphasizes the indispensable role of B cells and their derived EVs in shaping cancer immunotherapy outcome. Overall design: To investigte the role of B cell-derived EVs in melanoma immunotherapy response

近年来，学界对肿瘤免疫微环境（immune tumor microenvironment）的认知持续深化，现已明确B细胞并非仅作为被动旁观者，而是主动参与肿瘤免疫过程的核心群体之一。针对肿瘤内B细胞及其相关特征的回顾性研究均证实，B细胞浸润与患者治疗结局存在显著相关性。尽管关于B细胞究竟发挥促瘤还是抗肿瘤功能的研究结论尚存分歧，但越来越多的证据表明，将B细胞视作受肿瘤微环境信号塑造的异质性群体进行研究，是解析其功能的关键前提。
本研究旨在揭示B细胞及其分泌的细胞外囊泡（extracellular vesicles, EVs）在调控免疫检查点阻断（immune checkpoint blockade, ICB）治疗应答过程中的关键作用。我们通过独立实验证实：在黑色素瘤患者的肿瘤组织中，接受ICB治疗的应答者体内B细胞富集程度显著高于无应答者。体外肿瘤杀伤实验中的B细胞耗竭实验显示，T细胞介导的抗肿瘤活性会因此受到明显抑制。
为探究该现象的临床相关性，我们从黑色素瘤患者的肿瘤组织中分离得到细胞外囊泡，并将其分选为多个亚群，其中涵盖B细胞来源的细胞外囊泡。对CD19阳性细胞外囊泡的miRNA谱分析结果显示，miR-99a-5p是排名最靠前的候选分子，在应答者来源的细胞外囊泡中表达显著上调。
后续功能实验证实，miR-99a-5p的生物学功能依赖于B细胞：在体外肿瘤杀伤实验中，沉默该miRNA会显著削弱T细胞介导的抗肿瘤细胞毒性。
机制层面的研究表明，miR-99a-5p可调控B细胞周期，偏好激活非同源末端连接DNA修复通路，并促进抗体类别转换重组。综上，本研究数据明确了B细胞及其衍生的细胞外囊泡在调控癌症免疫治疗结局中不可或缺的核心作用。
整体实验设计：探究B细胞来源的细胞外囊泡在黑色素瘤免疫治疗应答中的调控作用