pre-GC and GC B cell scRNAseq

Following infection or vaccination, activated B cells at extrafollicular sites or within germinal centers (GCs) undergo vigorous clonal proliferation. Proliferating lymphocytes have been shown to undertake lactate dehydrogenase A (LDHA)-dependent aerobic glycolysis; however, the specific role of this metabolic pathway in a B cell transitioning from a naïve to a highly proliferative, activated state remains poorly defined. Here, we deleted LDHA in a stage- and cell-specific manner. We find that ablation of LDHA in a naïve B cell did not profoundly affect its ability to undergo a T cell-independent extrafollicular B cell response. On the other hand, LDHA-deleted naïve B cells had a severe defect in the capacities to form GCs and mount GC-dependent antibody responses. In addition, loss of LDHA in T cells severely compromised B cell-dependent immune responses. Strikingly, when LDHA was deleted in activated, as opposed to naïve, B cells, there were only minimal effects on the GC reaction and in the generation of high-affinity antibodies. These findings strongly suggest that naïve and activated B cells have distinct metabolic requirements that are further regulated by niche and cellular interactions. We performed scRNA sequencing to understand role of LDHA in the activation and proliferation of germinal center (GC) B cells. We combined this technology to HASH-sequencing in order to measure analyze LDHA-sufficient and -deficient GCs in triplicate and BCR sequencing to detect clonality and validate hypermutation in the early potential GC cells.

在感染或疫苗接种后，定位于滤泡外区域或生发中心（Germinal Centers, GCs）内的活化B细胞会发生旺盛的克隆增殖。已有研究表明，增殖中的淋巴细胞依赖乳酸脱氢酶A（Lactate Dehydrogenase A, LDHA）进行有氧糖酵解；然而，该代谢通路在B细胞从初始态向高度增殖的活化态转变过程中的具体作用仍未明确。本研究通过阶段特异性与细胞特异性的方式敲除LDHA。我们发现，在初始B细胞中敲除LDHA并不会显著影响其发生T细胞非依赖性滤泡外B细胞应答的能力。与之相反，缺失LDHA的初始B细胞在形成生发中心以及引发生发中心依赖性抗体应答方面存在严重缺陷。此外，在T细胞中敲除LDHA会严重削弱B细胞依赖性免疫应答。值得注意的是，相较于初始B细胞，在活化B细胞中敲除LDHA仅对生发中心反应以及高亲和力抗体的产生产生极轻微的影响。上述研究结果强烈表明，初始B细胞与活化B细胞具有截然不同的代谢需求，且该需求进一步由微环境与细胞间相互作用所调控。我们开展了单细胞RNA测序（single-cell RNA sequencing, scRNA-seq），以探究LDHA在生发中心B细胞的活化与增殖过程中的作用。我们将该技术与HASH测序（HASH-sequencing）相结合，用于对LDHA充足与LDHA缺失的生发中心进行三份重复样本的检测分析，并结合B细胞受体（B-cell receptor, BCR）测序以检测克隆性并验证早期潜在生发中心细胞的高突变情况。