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.