CCR7 Expression Distinguishes Functionally Distinct pTfh1 Subsets with Roles in Malaria-Specific Immunity
T-follicular CD4 T (Tfh) cells play essential roles in antibody induction during infection and following vaccination. In humans, peripheral Tfh (pTfh) cells are commonly analysed based on expression of CXCR3 and CCR6, with different subsets of pTfh (pTfh1, pTfh2, pTfh17) associated with antibody induction in a context-dependent manner. In malaria, the specific roles of pTfh subsets in antibody development is not clear. Several studies in human malaria infection and vaccination have identified an important role of pTfh2 cells, which associate with antibody development while pTfh1 cells do not. However, in vitro studies and animal models highlight that pTfh1 cells are key drivers of cytophilic antibody development, which are protective. To dissect these contradictions, we mapped the heterogeneity of pTfh cells in healthy individuals and individuals with controlled human malaria infection using scRNAseq. We identified two, previously unidentified, pTfh1-like subsets with functional relevance, which can be defined based on CCR7 expression. CCR7pos pTfh1 cells have increased capacity to produce IL-21, whereas CCR7neg pTfh1 cells express markers of cytotoxicity. In controlled human malaria infection, we show that both CCR7pos and CCR7neg pTfh1 cells, along with Tfh2 cells, clonally expand, are transcriptionally and phenotypically activated, and are malaria specific. However, only CCR7pos pTfh1 and pTfh2 cells associated with antibody responses to infection. Our data advance our knowledge of Tfh cell diversity, and may inform the development of vaccines and therapeutics that target these key CD4 T cell responses. Overall design: In brief, healthy malaria naive individuals underwent Controlled Human Malaria Infection with 2800 viable P. falciparum parasitized RBCs, and peripheral parasitaemia was measured at least daily by qPCR?. Participants were treated with antimalarial drugs at day 8 of infection when parasitaemia reaches approximately 20,000 parasites/ml. Blood samples (from 5 studies across 6 independent cohorts) were collected prior to infection (day 0), at peak infection (day 8) and 14 or 15 and 27-36 days (end of study, EOS) after inoculation (in analyses these time points are grouped as 0, 8, 14/15 and EOS). PBMCs were isolated by Ficoll-Paque (Sigma, USA) density gradient centrifugation, isolated PBMCs were cryopreserved in 10% DMSO/FBS. Participants were healthy malaria naïve adults with no prior exposure to malaria or residence in malaria-endemic regions.  Samples used here were from samples opportunistically collected from volunteers who consented to donate blood for immunological studies within the parent clinical trial. As such, no sample size estimation was performed for this immunology study.