抗血管生成治疗通过募集OSP+SEMA3A+B淋巴细胞逆转三阴乳腺癌免疫治疗耐受的机制研究

三阴乳腺癌（TNBC）预后差，免疫治疗极具前景，但单用疗效不佳。预实验发现：免疫联合抗血管生成治疗的肿瘤中OSP+SEMA3A+B和CD8+T细胞亚群等增多，OSP+SEMA3A+B细胞分泌的SEMA3A等增加；当阻断富集的B细胞后，其抑制肿瘤生长的效应降低，CD8+T及M1型巨噬细胞也减少。分析数据库发现，敲除SEMA3A的受体NRP1后调控巨噬细胞极化的cGKII基因表达下调，而cGKII的表达受FOXO转录因子家族调控，提示OSP+SEMA3A+B细胞可能通过SEMA3A/NRP1/FOXO/cGKII信号级联调控M1巨噬细胞的极化，从而激活CD8+T细胞以增敏抗PD-1治疗的机制。本研究拟通过体内外分子细胞实验、B细胞特异性基因敲除小鼠模型及患者临床标本分析，阐明抗血管生成治疗通过募集OSP+SEMA3A+B细胞逆转TNBC免疫治疗耐受的分子机制，并发现预测治疗敏感性的分子标志。

The prognosis of triple-negative breast cancer (TNBC) is poor. Immunotherapy including checkpoint blockade has great application prospect in the treatment of TNBC, however, PD-1/PD-L1 blockade monotherapy only showed marginal effect. Our preliminary experiments found that the tumor-infiltrating OSP+SEMA3A+B cells, CD8+ T cells and M1 macrophages increased significantly after the combinational treatment of anti-PD-1 and anti-angiogenic therapies, and the OSP+SEMA3A+B cells secreted much more cytokines including SEMA3A compared with the OSP-SEMA3A-B cells. Depletion of B cells significantly abrogated the antitumoral effects of anti-PD-1 and anti-angiogenic combination therapy, and reversed the increase of CD8+ T cells and M1 in tumor microenvironment. We next analyzed the GEO database and found that knockout of NRP1 decreased the expression level of cGKII, which is a gene known as a regulator of macrophage polarization; and the JASPAR software analysis showed that the promoter region of cGKII existed many FOXO transcription factor family binding sites. These results suggest that OSP+SEMA3A+B cell might secrete SEMA3A to induce polarization of M1 macrophages via SEMA3A/NRP1/FOXO/cGKII signaling pathway, and accordingly to stimulate recruitment and activation of cytotoxic CD8+ T cells in TNBC, which makes tumor sensitive to anti-PD-1 therapy. Based on this, by using in vitro and in vivo molecular and cellular experiments, B cell-specific gene knockout mice, and patient tumor specimens, we will investigate the mechanism of anti-angiogenic therapy sensitizing anti-PD-1 therapy via recruiting tumor-infiltrating OSP+SEMA3A+B lymphocytes. This study will reveal the molecular mechanism of tumor-infiltrating OSP+SEMA3A+B cells reversing immunotherapy tolerance, and detect the biomarker of predicting response to anti-PD-1 and anti-angiogenic combination therapy in TNBC.