CD4+ T cells display a spectrum of recall dynamics during re-infection with malaria parasites.
Children in malaria-endemic regions can experience multiple Plasmodium infections over a short period of time, with in vitro CD4+ T cell recall responses becoming more regulatory with increasing age and exposure. This suggests that repeated infection qualitatively changes CD4+ T cells, although the heterogeneity and dynamics of these responses await systematic analysis in vivo. Here, we examined TCR transgenic PbTII and polyclonal CD4+ T cells during Plasmodium re-infection in mice, in conjunction with scRNA-seq/TCR-seq and spatial transcriptomics at near single-cell resolution. PbTII cells gave rise to multiple antigen-experienced states in different areas of the spleen after primary infection and antimalarial treatment, including ongoing GC responses and T-cell zone memory. Upon re-infection, Th1-memory PbTII cells initiated a rapid effector response prior to proliferating, while GC Tfh cells of the same antigen specificity were entirely refractory within the same organ. Transcriptome dynamic modelling and network analysis of Th1 recall revealed a biphasic wave of RNA processing that firstly preceded immune effector transcription, and later accompanied cellular proliferation. Importantly, Th1 recall constituted a partial facsimile of primary Th1 responses, with no unique genes amongst the small subset of those upregulated upon re-infection. Finally, we noted a similar spectrum of antigen-experienced states and recall dynamics by polyclonal CD4+ T cells with diverse TCRs. Therefore, during re-infection with Plasmodium, persisting GC Tfh cells remained unaltered transcriptionally, Tcm/Tfh-like cells exhibited minimal proliferation, and Th1-memory cells displayed a rapid, proliferating IL-10-producing Tr1 response consistent with a shift towards immune-regulation. These data highlight a broad spectrum of simultaneous CD4+ T cell responses that occur in the spleen during re-infection with malaria parasites. HighlightsO_LISplenic TCR transgenic CD4+ T cells are highly heterogeneous prior to re-infection. C_LIO_LIPersisting GC Tfh cells are refractory to re-activation during re-infection. C_LIO_LITh1-memory cells rapidly upregulate RNA processing prior to effector function and proliferation. C_LIO_LITh1-recall is an imperfect but faithful facsimile of primary Th1 responses. C_LIO_LIA spectrum of recall states is observed in polyclonal CD4+ T cells with diverse TCRs. C_LI