Regulatory B cells (Bregs), a specialized subset of B cells that modulate immune responses and maintain immune tolerance in malignant tumors, have not been extensively investigated in the context of bladder cancer (BLCA). This study aims to elucidate the roles of Bregs and Breg-related genes in BLCA. We assessed Breg infiltration levels in 34 pairs of BLCA and corresponding paracancerous tissues using immunohistochemical staining. We conducted transwell and wound healing assays to evaluate the impact of Bregs on the malignant phenotype of SW780 and T24 cells. Breg-related genes were identified through gene sets and transcriptional analysis. The TCGA-BLCA cohort served as the training set, while the IMvigor210 and 5 GEO cohorts were used as external validation sets. We employed LASSO regression and random forest for feature selection and developed a risk signature using Cox regression. Primary validation of the risk signature was performed through immunohistochemical staining and RT-qPCR experiments using the 34 local BLCA samples. Additionally, we employed transfection assays and flow cytometry to investigate Breg expansion ability and immunosuppressive functions. Breg levels in BLCA tissues were significantly elevated compared to paracancerous tissues (P < 0.05) and positively correlated with tumor malignancy (P < 0.05). Co-incubation of SW780 and T24 cells with Bregs resulted in enhanced invasion and migration abilities (all P < 0.05). We identified 27 Breg-related genes, including CD96, OAS1, and CSH1, which were integrated into the risk signature. This signature demonstrated robust prognostic classification across the 6 cohorts (pooled HR = 2.25, 95% CI = 1.52–3.33). Moreover, the signature exhibited positive associations with advanced tumor stage (P < 0.001) and Breg infiltration ratios (P < 0.05) in the local samples. Furthermore, the signature successfully predicted immunotherapeutic sensitivity in three cohorts (all P < 0.05). Knockdown of CSH1 in B cells increased Breg phenotype and enhanced suppressive ability against CD8 + T cells (all P < 0.05). Bregs play a pro-tumor role in the development of BLCA. The Breg-related gene signature established in this study holds great potential as a valuable tool for evaluating prognosis and predicting immunotherapeutic response in BLCA patients.

中文翻译：

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研究调节性 B 细胞和调节性 B 细胞相关基因对膀胱癌进展和免疫治疗敏感性的影响

调节性 B 细胞 (Bregs) 是一种专门的 B 细胞亚群，可调节恶性肿瘤的免疫反应并维持免疫耐受，但尚未在膀胱癌 (BLCA) 中得到广泛研究。本研究旨在阐明 Breg 和 Breg 相关基因在 BLCA 中的作用。我们使用免疫组织化学染色评估了 34 对 BLCA 和相应癌旁组织中的 Breg 浸润水平。我们进行了 Transwell 和伤口愈合测定，以评估 Bregs 对 SW780 和 T24 细胞恶性表型的影响。通过基因集和转录分析鉴定了 Breg 相关基因。 TCGA-BLCA 队列作为训练集，而 IMvigor210 和 5 GEO 队列作为外部验证集。我们采用 LASSO 回归和随机森林进行特征选择，并使用 Cox 回归开发风险签名。使用 34 个本地 BLCA 样本通过免疫组织化学染色和 RT-qPCR 实验对风险特征进行初步验证。此外，我们采用转染测定和流式细胞术来研究 Breg 扩增能力和免疫抑制功能。 BLCA组织中Breg水平较癌旁组织显着升高（P < 0.05），且与肿瘤恶性程度呈正相关（P < 0.05）。 SW780和T24细胞与Bregs共孵育导致侵袭和迁移能力增强（均P < 0.05）。我们鉴定了 27 个 Breg 相关基因，包括 CD96、OAS1 和 CSH1，它们已整合到风险特征中。该特征证明了 6 个队列的稳健预后分类（汇总 HR = 2.25，95% CI = 1.52-3.33）。此外，该特征与局部样本中的晚期肿瘤分期（P < 0.001）和 Breg 浸润率（P < 0.05）呈正相关。此外，该特征成功预测了三个队列的免疫治疗敏感性（所有 P < 0.05）。 B细胞中CSH1的敲低增加了Breg表型并增强了对CD8 + T细胞的抑制能力（所有P < 0.05）。 Bregs 在 BLCA 的发展中发挥促肿瘤作用。本研究中建立的 Breg 相关基因特征具有作为评估 BLCA 患者预后和预测免疫治疗反应的有价值工具的巨大潜力。