Key Points Circulating plasmablasts are a major B cell subpopulation that expresses GZMB. GZMB+ B cells can be efficiently expanded ex vivo from total B cells. GZMB+ B cells strongly suppress both autologous and allogenic T cell proliferation. Granzyme B–expressing B cells have been shown to be an important regulatory B cell subset in humans. However, it is unclear which subpopulations of B cells express GZMB under normal conditions and which protocols effectively induce ex vivo expansion of GZMB+ B cells. We found that in the peripheral blood of normal individuals, plasmablasts were the major B cell subpopulation that expressed GZMB. However, when using an in vitro plasmablast differentiation protocol, we obtained only 2% GZMB+ B cells. Nevertheless, using an expansion mixture containing IL-21, anti-BCR, CpG oligodeoxynucleotide, CD40L, and IL-2, we were able to obtain more than 90% GZMB+ B cells after 3 d culture. GZMB+ B cells obtained through this protocol suppressed the proliferation of autologous and allogenic CD4+CD25− effector T cells. The suppressive effect of GZMB+ B cells was partially GZMB dependent and totally contact dependent but was not associated with an increase in effector T cell apoptosis or uptake of GZMB by effector T cells. Interestingly, we showed that GZMB produced by B cells promoted GZMB+ B cell proliferation in ERK1/2-dependent manner, facilitating GZMB+ B cell expansion. However, GZMB+ B cells tended to undergo apoptosis after prolonged stimulation, which may be considered a negative feedback mechanism to limit their uncontrolled expansion. Finally, we found that expanded GZMB+ B cells exhibited a regulatory phenotype and were enriched in CD307bhi, CD258hiCD72hi, and CD21loPD-1hi B cell subpopulations. Our study, to our knowledge, provides new insight into biology of GZMB+ B cells and an efficient method to expand GZMB+ B cells for future cell therapy applications.

中文翻译：

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有效扩增具有强调节特性的表达人颗粒酶 B 的 B 细胞

要点 循环浆母细胞是表达 GZMB 的主要 B 细胞亚群。GZMB+ B 细胞可以从总 B 细胞中有效地体外扩增。GZMB+ B 细胞强烈抑制自体和同种异体 T 细胞增殖。表达颗粒酶 B 的 B 细胞已被证明是人类重要的调节性 B 细胞亚群。然而，尚不清楚哪些 B 细胞亚群在正常条件下表达 GZMB，以及哪些方案能有效诱导 GZMB+ B 细胞的体外扩增。我们发现在正常个体的外周血中，浆母细胞是表达 GZMB 的主要 B 细胞亚群。然而，当使用体外浆母细胞分化方案时，我们只获得了 2% 的 GZMB+ B 细胞。然而，使用含有 IL-21、抗 BCR、CpG 寡脱氧核苷酸、CD40L 和 IL-2 的扩增混合物，我们能够在培养3天后获得超过90%的GZMB+ B细胞。通过该方案获得的 GZMB+ B 细胞抑制了自体和同种异体 CD4+CD25-效应 T 细胞的增殖。GZMB+ B 细胞的抑制作用部分依赖于 GZMB，完全依赖于接触，但与效应 T 细胞凋亡或效应 T 细胞摄取 GZMB 的增加无关。有趣的是，我们发现 B 细胞产生的 GZMB 以 ERK1/2 依赖性方式促进 GZMB+ B 细胞增殖，促进 GZMB+ B 细胞扩增。然而，GZMB+ B 细胞在长时间刺激后倾向于发生凋亡，这可能被认为是限制其不受控制的扩张的负反馈机制。最后，我们发现扩增的 GZMB+ B 细胞表现出调节表型并富含 CD307bhi，CD258hiCD72hi 和 CD21loPD-1hi B 细胞亚群。据我们所知，我们的研究提供了对 GZMB+ B 细胞生物学的新见解，并提供了一种有效的方法来扩展 GZMB+ B 细胞以用于未来的细胞治疗应用。