Center of Immunotherapy and Immunity, Seattle Children Research Institute, Seattle, WA 98101, USA; Molecular Engineering and Science Institute, University of Washington, Seattle, WA 98195, USA.
Center of Immunotherapy and Immunity, Seattle Children Research Institute, Seattle, WA 98101, USA.
Cell Rep Methods. 2024 Oct 21;4(10):100878. doi: 10.1016/j.crmeth.2024.100878. Epub 2024 Oct 14.
A key step in developing engineered B cells for therapeutic purposes is evaluation in immunocompetent, large-animal models. Therefore, we developed methods to purify, expand, and differentiate non-human primate (NHP; rhesus macaque) B cells. After 7 days in culture, B cells expanded 10-fold, differentiated into a plasma cell phenotype (CD38, CD138), and secreted immunoglobulin G. Using single-cell sequencing and flow cytometry, we verified the presence of plasma cell genes in differentiated NHP B cells and unearthed less-recognized markers, such as CD59 and CD79A. In contrast with human cells, we found that the immune checkpoint molecule CD274 (PD-L1) and major histocompatibility complex (MHC) class I molecules were upregulated in NHP plasma cells in the transcriptional data. Lastly, we established the conditions for efficient transduction of NHP B cells with adeno-associated virus (AAV) vectors, achieving a delivery rate of approximately 60%. We envision that this work will accelerate proof-of-concept studies using engineered B cells in NHPs.
为了治疗目的而构建工程化 B 细胞的关键步骤是在免疫功能正常的大型动物模型中进行评估。因此，我们开发了从非人灵长类动物（NHP；恒河猴）中纯化、扩增和分化 B 细胞的方法。在培养 7 天后，B 细胞扩增了 10 倍，分化为浆细胞表型（CD38、CD138）并分泌免疫球蛋白 G。通过单细胞测序和流式细胞术，我们验证了分化的 NHP B 细胞中存在浆细胞基因，并发现了一些不太被认可的标志物，如 CD59 和 CD79A。与人类细胞不同，我们发现转录数据中，免疫检查点分子 CD274（PD-L1）和主要组织相容性复合体（MHC）I 类分子在 NHP 浆细胞中上调。最后，我们建立了用腺相关病毒（AAV）载体有效转导 NHP B 细胞的条件，转导效率约为 60%。我们设想这项工作将加速在 NHP 中使用工程化 B 细胞进行概念验证研究。
