Dynamic Regulation of Tfh Clonal Selection During the Germinal Center Reaction. Dynamic Regulation of Tfh Clonal Selection During the Germinal Center Reaction

The Germinal center is a dynamic microenvironment wherein B cells expressing high affinity antibody variants produced by hypermutation are selected for clonal expansion by limiting numbers of T follicular helper cells. Although a great deal is known about the mechanisms that control B cell selection in the germinal center, far less is understood about the clonal behavior of the T follicular helper cells that regulate this process. Here we report on the dynamic behavior of clones of T follicular helper cells during the germinal center reaction. We find that like germinal center B cells, T follicular helper cells undergo antigen dependent selection during the germinal center reaction resulting in differential proliferative expansion and contraction. Increasing the amount of antigen presented in the germinal center leads to increased T follicular cell division. Competition between T follicular helper cell clones is mediated by T cell receptor affinity for peptide-MHC ligand. Higher affinity T cells expanding preferentially in the germinal center show increased expression of genes downstream of the T cell receptor, genes required for metabolic reprogramming, cell division and cytokine production. These dynamic changes lead to dramatic remodeling of the functional T follicular cell repertoire during the germinal center reaction. Overall design: OTII-Fucci cells were adoptively transferred into C57Bl/6 mice that were immunized with NP-OVA and then boosted on day 7 with high and low affinity aDEC-APLs or nothing and analyzed after 18 hours. To examine the transcriptional programs associated with increased TCR affinity driven Tfh division we performed bulk mRNA sequencing. To determine whether there is dynamic redistribution of Tfh clones during a polyclonal immune response we followed clonotypes in the same mouse longitudinally by performing hemi-splenectomy on days 7 and 21 after immunization with NP-OVA. To ensure that we assayed T cells entering the spleen during the initial immune response we used mice that carry a tamoxifen inducible Cre in the CD62L locus and a tdTomato indicator (SellCreERT2 ROSAtdTmice). Tamoxifen injection into SellCreERT2 ROSAtdT reporter mice labels naïve T cells, but not Tfh cells and immunization recruits labeled naïve cells into the Tfh compartment. Naïve cells were labeled in reporter mice before immunization and following immunization tdTomato expressing Tfh were purified from hemisplectomized mice on days 7 and 21 after NP-OVA injection. Single cell RNA sequencing was carried out using Cellular Indexing of Transcriptomes and Epitopes by Sequencing (CITE-Seq) method.

生发中心（germinal center）是一类动态微环境，其中表达经体细胞高频突变产生的高亲和力抗体变体的B细胞，会通过数量受限的滤泡辅助性T细胞（T follicular helper cell，Tfh）被筛选以进行克隆扩增。尽管学界对生发中心内调控B细胞筛选的机制已有较为充分的认知，但对调控这一过程的滤泡辅助性T细胞的克隆行为却所知甚少。本研究报道了生发中心反应过程中滤泡辅助性T细胞克隆的动态行为。我们发现，与生发中心B细胞类似，滤泡辅助性T细胞在生发中心反应过程中会经历抗原依赖性选择，进而产生差异性的增殖扩增与收缩。增加生发中心内呈递的抗原量，可促进滤泡辅助性T细胞的分裂增殖。滤泡辅助性T细胞克隆间的竞争由T细胞受体（T cell receptor，TCR）对肽-MHC配体的亲和力所介导。在生发中心中优先扩增的高亲和力T细胞，其TCR下游基因、代谢重编程所需基因、细胞分裂相关基因以及细胞因子产生相关基因的表达水平均显著上调。这些动态变化会在生发中心反应过程中，对滤泡辅助性T细胞的功能库造成显著重塑。实验整体设计：将OTII-Fucci细胞过继转移至经NP-OVA免疫的C57BL/6小鼠中，并于免疫后第7天分别以高亲和力、低亲和力aDEC-APLs进行加强免疫，或不进行额外处理，于18小时后对小鼠进行分析。为探究与TCR亲和力驱动的Tfh增殖增强相关的转录程序，我们开展了批量mRNA测序。为明确多克隆免疫应答过程中Tfh克隆是否存在动态重分布，我们通过在NP-OVA免疫后第7天和第21天对小鼠实施半脾切除术，纵向追踪同一只小鼠体内的克隆型。为确保我们检测的是初始免疫应答过程中进入脾脏的T细胞，我们使用了在CD62L基因座携带他莫昔芬诱导型Cre及tdTomato标记物的小鼠（SellCreERT2 ROSAtdT小鼠）。向SellCreERT2 ROSAtdT报告基因小鼠注射他莫昔芬，可特异性标记初始T细胞而非Tfh细胞，免疫应答会将标记的初始细胞招募至Tfh细胞群中。我们在免疫前对报告基因小鼠的初始T细胞进行标记，于NP-OVA注射后第7天和第21天，从半脾切除小鼠体内纯化表达tdTomato的Tfh细胞。本研究采用基于测序的转录组与表位细胞索引法（Cellular Indexing of Transcriptomes and Epitopes by Sequencing，CITE-Seq）开展单细胞RNA测序。