Generation, expansion, gene delivery, and single-cell profiling in rhesus macaque plasma B cells

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. [Display omitted] •Expansion (10-fold) and differentiation (50% IgG) of NHP B cells into plasma cells•Engineered rhesus monkeys using AAV(D-J) serotype achieve 60% transduction rate•CD59 and CD79A can serve as surrogate markers for rhesus monkey plasma cells•NHP plasma cells exhibit increases in PD-L1 and MHC class I expression Protein and peptide drugs, including monoclonal antibodies, represent about 10% of the pharmaceutical market but face issues like poor solubility and short half-lives. Recent advances using engineered human plasma cells have shown promise in mice, but their safety and effectiveness need further testing in non-human primates, which provide better models for long-term evaluation. Cheng et al. develop efficient methods for generating and engineering rhesus monkey plasma cells ex vivo. Using single-cell RNA sequencing, they identify both less-recognized plasma cell markers conserved in humans and unique markers specific to rhesus monkeys.