Tfh2 and a subset of Tfh1 cells associate with antibody-mediated immunity to malaria

High-affinity antibody production depends on CD4+ T-follicular helper (Tfh) cells. In humans, peripheral blood Tfh cells are heterogenous, as evidenced by differential expression of the chemokine receptors, CXCR3 and CCR6, which to date have served to classify three subsets, pTfh1, pTfh2 and pTfh17. Although pTfh1 responses dominate during blood-stage Plasmodium infections, a clear association with protective antibody responses remains to be described. We hypothesise that pTfh cells exhibit greater phenotypic and functional heterogeneity than that described by CXCR3/CCR6 alone, and that these more nuanced pTfh subsets play distinct roles during Plasmodium infection. We map pTfh cell heterogeneity in healthy individuals prior to and during controlled human malaria infection (CHMI) using parallel scRNA-seq and VDJ-seq. We uncover two pTfh1 subsets or differential phenotypic states, distinguishable by CCR7 expression. Prior to infection, Tfh1-CCR7neg cells exhibit higher baseline expression of inflammatory cytokines and genes associated with cytotoxicity. While Tfh1-CCR7pos cells have higher GC signatures. Indeed, during CHMI, Tfh1-CCR7pos, Tfh1-CCR7neg, and Tfh2 cells, all clonally expand and become activated. However, only Tfh1-CCR7pos and Tfh2 cells positively associate with protective antibody production. Hence, our data reveal further complexity amongst human Tfh cells, and highlight two distinct subsets associated with antibody-mediated immunity to malaria.高亲和力抗体的产生依赖于CD4+滤泡辅助性T（T follicular helper, Tfh）细胞。在人体中，外周血滤泡辅助性T（peripheral blood Tfh, pTfh）细胞具有异质性，这一点可通过趋化因子受体CXCR3与CCR6的差异表达得以佐证；截至目前，学界正是基于这两个受体的表达模式将pTfh细胞划分为pTfh1、pTfh2及pTfh17三个亚型。尽管在红内期疟原虫感染过程中pTfh1型应答占据主导地位，但目前仍尚未明确其与保护性抗体应答之间的明确关联。本研究提出假说：相较于仅通过CXCR3/CCR6划分的亚型，pTfh细胞的表型与功能异质性更为丰富，且这些更为精细的pTfh亚型在疟原虫感染过程中发挥着各不相同的作用。本研究借助并行单细胞RNA测序（single-cell RNA sequencing, scRNA-seq）与VDJ测序（VDJ sequencing, VDJ-seq）技术，绘制了健康个体在受控人体疟疾感染（controlled human malaria infection, CHMI）前后的pTfh细胞异质性图谱。研究发现了两种基于CCR7表达可区分的pTfh1亚型或差异化表型状态：感染前，Tfh1-CCR7阴性细胞的炎症细胞因子及细胞毒性相关基因的基础表达水平更高，而Tfh1-CCR7阳性细胞则表现出更高的生发中心（germinal center, GC）特征基因表达。确实，在CHMI过程中，Tfh1-CCR7阳性、Tfh1-CCR7阴性及Tfh2细胞均会发生克隆扩增并被激活，但仅有Tfh1-CCR7阳性细胞与Tfh2细胞与保护性抗体的产生呈正相关。综上，本研究的数据揭示了人类Tfh细胞更为复杂的异质性，并明确了两种与疟疾抗体介导免疫相关的独特亚型。