Clonal evolution and specificity of the human T follicular helper cell response to Plasmodium falciparum circumsporozoite protein

T follicular helper (TFH) cells play a crucial role in the development of long-lived, quality-improved B cell responses after infection and vaccination. However, little is known about their clonal evolution. Here we assessed the cell phenotype, clonal dynamics, and TCR specificity of human circulating TFH (cTFH) cells at monoclonal level during successive malaria immunizations with radiation-attenuated Plasmodium falciparum (Pf) sporozoites. Repeated parasite exposures induced a dynamic, polyclonal cTFH response with high frequency of cells specific to the Pf circumsporozoite protein (PfCSP), the main surface protein of sporozoites and a validated vaccine target. Repeated immunizations were required to induce detectable PfCSP-reactive cTFH cell responses to a small number of epitopes. HLA-restrictions and differences in TCR generation probability explain the high targeting frequency of the polymorphic Th2R/T* region over the conserved T1 epitope. The vast majority of anti-Th2R/T* TCRs failed to tolerate natural polymorphisms in their target peptide sequence suggesting that parasite diversity limits natural boosting of the cTFH cell response in endemic areas and protection from non-vaccine strains. Among convergent anti-Th2R/T* TCRs with high sequence similarity, subtle differences in CDR3 composition discriminated cross-reactive from non-cross-reactive cTFH cells. Thus, our study provides deep molecular and cellular insights into the kinetics, fine specificity and HLA-restrictions of the anti-cTFH cell response that are of direct relevance for the design of PfCSP-based malaria vaccines by guiding the selection of PfCSP peptides that induce optimal B cell help.