The therapeutic applications of regulatory T cells (T_regs) include treating autoimmune diseases, graft‐versus‐host disease and induction of transplantation tolerance. For ex‐vivo expanded T_regs to be used in deceased donor transplantation, they must be able to suppress T cell responses to a broad range of human leukocyte antigen (HLA). Here, we present a novel approach for the expansion of polyspecific T_regs in cynomolgus macaques that was adapted from a good manufacturing practice‐compliant protocol. T_regs were isolated by fluorescence‐activated cell sorting (FACS) and expanded in the presence of a panel of CD40L‐stimulated B cells (CD40L‐sBc). Prior to T_reg culture, CD40L‐sBc were expanded in vitro from multiple major histocompatibility complex (MHC)‐disparate macaques. Expanded T_regs expressed high levels of forkhead box protein 3 (FoxP3) and Helios, a high percentage of T_reg‐specific demethylated region (TSDR) demethylation and strong suppression of naïve T cell responses in vitro. In addition, these T_regs produced low levels of inflammatory cytokines and were able to expand post‐cryopreservation. Specificity assays confirmed that these T_regs were suppressive upon activation by any antigen‐presenting cells (APCs) whose MHC was shared by CD40L‐sBc used during expansion, proving that they are polyspecific. We developed an approach for the expansion of highly suppressive cynomolgus macaque polyspecific T_regs through the use of a combination of CD40L‐engineered B cells with the potential to be translated to clinical studies. To our knowledge, this is the first report that uses a pool of MHC‐mismatched CD40L‐sBc to create polyspecific T_regs suitable for use in deceased‐donor transplants.

中文翻译：

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CD40L 刺激的 B 细胞用于多特异性非人灵长类动物调节性 T 细胞的离体扩增，用于转化研究

调节性 T 细胞 (T _regs ) 的治疗应用包括治疗自身免疫性疾病、移植物抗宿主病和诱导移植耐受。为了使体外扩增的 T _regs用于已故供体移植，它们必须能够抑制 T 细胞对广泛的人类白细胞抗原 (HLA) 的反应。_{在这里，我们提出了一种在食蟹猴中扩展多特异性 T regs}的新方法，该方法改编自符合良好生产实践的协议。通过荧光激活细胞分选 (FACS) 分离T _regs，并在一组 CD40L 刺激的 B 细胞 (CD40L-sBc) 存在下扩增。在 T _reg之前培养，CD40L-sBc从多个主要组织相容性复合体 (MHC) 不同的猕猴体外扩增。扩展的 T _reg表达高水平的叉头盒蛋白 3 (FoxP3) 和 Helios，高百分比的 T _{reg特异性去甲基化区域 (TSDR) 去甲基化和}体外幼稚 T 细胞反应的强烈抑制。此外，这些 T _regs产生低水平的炎性细胞因子，并且能够扩大冷冻保存后的范围。特异性测定证实这些 T _regs任何抗原呈递细胞 (APC) 的 MHC 与扩增期间使用的 CD40L-sBc 共享的任何抗原呈递细胞 (APC) 激活后均具有抑制性，证明它们是多特异性的。我们开发了一种方法，通过使用 CD40L 工程 B 细胞的组合来扩展高度抑制的食蟹猴多特异性 T _regs，并有可能转化为临床研究。据我们所知，这是第一份使用 MHC 不匹配的 CD40L-sBc 池来创建适用于已故供体移植的多特异性 T _{regs的报告。}