The B Cell Subtypes and VDJ Repertoire of Young Adult Rhesus Macaques

Rhesus macaques (RMs) are widely employed as a pre-clinical model in vaccination and infectious disease studies, yet their B cell immunobiology and immunogenetics remain ill-characterized. In this study, single-cell RNA/VDJ-seq (scRNA/VDJ-seq) was conducted on peripheral blood mononuclear cell (PBMC) samples from six RMs to describe the transcriptomic and V(D)JC repertoires of B cells and subtypes. 12 RM B cell clusters of distinct transcriptional states were identified, including IgM+ memory B cells (MBC), class-switched MBC, CD11c+ MBC and activated B cells. Novel gene signatures were also characterized for each B cell subtype, such as FCRL2 and CD24 for circulating marginal-zone-like B cells. In addition, VDJ repertoire properties of the global B cell population and each B cell subtype were elucidated, including IGH/K/L-V(D)JC gene family and subtype usage, class-switch recombination (CSR) status, somatic hypermutation (SHM) rate and levels, complementarity-determining region heavy chain (CDRH3) amino acid (AA) length and CDRH3 AA hydrophobicity scores. Interesting insights included the 1:1 ratio of kappa and lambda light chain usage and a preferential decreased IGHV3 but increased IGHV1 and 5 gene family usage in IGHG1 than IGHM-bearing B cells. Altogether, this study through comprehensive transcriptomic analyses identifies 12 distinct RM B cell subtypes paired with their respective V(D)JC repertoire, unraveling the complexity of B cell heterogeneity and improving future pre-clinical studies that can translate insights from this important non-human primate model to the understanding of human immunobiology. Overall design: 6 peripheral blood mononuclear cells (PBMCs) samples from healthy juvenile Indian-origin rhesus macaques (RMs), 2 male and 4 female, 2-3 years old, Macaca mulatta, were collected for single-cell RNA and VDJ sequencing.

恒河猴（Rhesus macaques, RMs）已被广泛用作疫苗研发与传染病研究中的临床前模型，但其B细胞免疫生物学与免疫遗传学特征仍有待深入阐明。本研究对6只恒河猴的外周血单个核细胞（peripheral blood mononuclear cell, PBMC）样本开展了单细胞RNA/VDJ测序（single-cell RNA/VDJ-seq, scRNA/VDJ-seq），以解析B细胞及其亚型的转录组与V(D)JC抗原受体库特征。研究共鉴定出12个具有独特转录状态的恒河猴B细胞簇，包括IgM+记忆B细胞（IgM+ memory B cells, MBC）、类别转换记忆B细胞、CD11c+记忆B细胞与活化B细胞。同时为各B细胞亚型鉴定了全新的基因特征，例如循环边缘区样B细胞的特征基因为FCRL2与CD24。此外，本研究还阐明了整体B细胞群及各B细胞亚型的VDJ抗原受体库特性，包括IGH/K/L-V(D)JC基因家族与亚型使用偏好、类别转换重组（class-switch recombination, CSR）状态、体细胞超突变（somatic hypermutation, SHM）频率与水平、重链互补决定区3（complementarity-determining region heavy chain, CDRH3）的氨基酸（amino acid, AA）长度及CDRH3氨基酸疏水性评分。有趣的研究发现包括：κ与λ轻链使用比例为1:1；相较于表达IGHM的B细胞，表达IGHG1的B细胞中IGHV3基因家族使用偏好显著降低，而IGHV1与IGHV5基因家族使用比例则显著升高。综上，本研究通过全面的转录组分析，鉴定出12种独特的恒河猴B细胞亚型，并匹配了各自的V(D)JC抗原受体库，揭示了B细胞异质性的复杂机制，为未来将这一重要非人灵长类模型的研究成果转化至人类免疫生物学研究提供了支撑。总体实验设计：采集6只健康幼年印度来源恒河猴（Macaca mulatta）的外周血单个核细胞样本，其中雄性2只、雌性4只，年龄为2~3岁，随后开展单细胞RNA测序与VDJ测序。