DataSheet_1_B Cells Are Required to Generate Optimal Anti-Melanoma Immunity in Response to Checkpoint Blockade.pdf

Immunotherapies such as checkpoint blockade therapies are known to enhance anti-melanoma CD8+ T cell immunity, but only a fraction of patients treated with these therapies achieve durable immune response and disease control. It may be that CD8+ T cells need help from other immune cells to generate effective and long-lasting anti-tumor immunity or that CD8+ T cells alone are insufficient for complete tumor regression and cure. Melanoma contains significant numbers of B cells; however, the role of B cells in anti-melanoma immunity is controversial. In this study, B16 melanoma mouse models were used to determine the role of B cells in anti-melanoma immunity. C57BL/6 mice, B cell knockout (KO) C57BL/6 mice, anti-CD19, and anti-CXCL13 antibody-treated C57BL/6 mice were used to determine treatment efficacy and generation of tumor-specific CD8+ T cells in response to PD-L1 blockade alone or combination with TLR-7/8 activation. Whole transcriptome analysis was performed on the tumors from B cell depleted and WT mice, untreated or treated with anti-PD-L1. Both CD40-positive and CD40-negative B cells were isolated from tumors of TLR-7/8 agonist-treated wild-type mice and adoptively transferred into tumor-bearing B cell KO mice, which were treated with anti-PD-L1 and TLR-7/8 agonist. Therapeutic efficacy was determined in the presence of activated or inactivated B cells. Microarray analysis was performed on TLR-7/8-treated tumors to look for the B cell signatures. We found B cells were required to enhance the therapeutic efficacy of monotherapy with anti-PD-L1 antibody and combination therapy with anti-PD-L1 antibody plus TLR-7/8 agonist. However, B cells were not essential for anti-CTLA-4 antibody activity. Interestingly, CD40-positive but not CD40-negative B cells contributed to anti-melanoma immunity. In addition, melanoma patients’ TCGA data showed that the presence of B cell chemokine CXCL13 and B cells together with CD8+ T cells in tumors were strongly associated with improved overall survival. Our transcriptome data suggest that the absence of B cells enhances immune checkpoints expression in the tumors microenvironment. These results revealed the importance of B cells in the generation of effective anti-melanoma immunity in response to PD-1-PD-L1 blockade immunotherapy. Our findings may facilitate the design of more effective anti-melanoma immunotherapy.

免疫治疗，如检查点阻断疗法，已知能增强抗黑色素瘤CD8+ T细胞的免疫反应，然而，仅有一部分接受这些治疗的患者能够获得持久性的免疫反应和疾病控制。或许，CD8+ T细胞需要其他免疫细胞的协助以产生有效且持久的抗肿瘤免疫，或者CD8+ T细胞本身对于肿瘤的完全消退和治愈可能是不够的。黑色素瘤含有大量的B细胞，然而B细胞在抗黑色素瘤免疫中的作用尚存在争议。在本研究中，我们利用B16黑色素瘤小鼠模型来探究B细胞在抗黑色素瘤免疫中的作用。我们使用了C57BL/6小鼠、B细胞敲除（KO）的C57BL/6小鼠、抗CD19和抗CXCL13抗体处理的C57BL/6小鼠，以确定单独使用PD-L1阻断或与TLR-7/8激活联合使用时的治疗有效性和肿瘤特异性CD8+ T细胞的生成。对B细胞耗竭和野生型小鼠的肿瘤进行了全转录组分析，分析了未经处理或接受抗PD-L1治疗的小鼠。从接受TLR-7/8激动剂治疗的野生型小鼠的肿瘤中分离出CD40阳性和B细胞以及CD40阴性B细胞，并将其输注到接受抗PD-L1和TLR-7/8激动剂治疗的B细胞KO小鼠体内。在存在激活或非激活B细胞的情况下评估了治疗效果。对TLR-7/8处理的肿瘤进行了微阵列分析，以寻找B细胞的特征。我们发现，B细胞对于增强抗PD-L1单克隆抗体和抗PD-L1与TLR-7/8激动剂联合疗法的治疗效果是必需的。然而，B细胞对于抗CTLA-4抗体的活性并非必需。有趣的是，CD40阳性的B细胞而非CD40阴性的B细胞对抗黑色素瘤免疫做出了贡献。此外，黑色素瘤患者的TCGA数据表明，肿瘤中B细胞趋化因子CXCL13以及与CD8+ T细胞共存与患者总生存率的改善密切相关。我们的转录组数据分析表明，B细胞的缺失增强了肿瘤微环境中免疫检查点的表达。这些结果揭示了B细胞在应对PD-1-PD-L1阻断免疫治疗时产生有效抗黑色素瘤免疫的重要性。我们的发现可能有助于设计更有效的抗黑色素瘤免疫治疗方案。