Spatial transcriptomics maps molecular and cellular requirements for CD4+ T cell-dependent immunity to malaria.

CD4+ T cells orchestrate adaptive immunity to circulating malaria parasites; yet cellular interactions and molecular mechanisms controlling Th1 and Tfh differentiation in the spleen remain to be fully defined in vivo. Here, using a murine model of CD4-dependent immunity, we tested if Slide-seqV2, a spatial transcriptomic method with near single-cell resolution, could determine the locations of multiple CD4+ T cell subsets and potentially interacting cellular partners in the spleen during infection. Firstly, Slide-seqV2 readily mapped splenic cellular structure and microanatomical change during infection. Next, computational integration with scRNA-seq reference datasets of splenocytes, stromal cells, and specifically of polyclonal CD4+ T cells and B cells, mapped the relative locations of multiple cell-types within this dense tissue. scRNA-seq of B cells over time mapped emergence of germinal centre B cells, red pulp-located plasmablasts and atypical B cells, and uncovered a prolonged CD4+ T-cell-independent, follicular bystander B cell response marked by Sca-1 and Ly6C upregulation. scRNA-seq of activated, polyclonal CD4+ T cells revealed their similarity to our previous TCR transgenic models. Importantly, spatial analysis revealed polyclonal Th1 cells co-localised with CXCL9/10-producing monocytes in the red pulp, while polyclonal Tfh-like cells were located close to CXCL13-expressing B cell follicles, consistent with our previous CXCR3/CXCR5 competition model of Th1/Tfh bifurcation. CRISPR/Cas9 disruption of either or both CXCR3 and CXCR5 in naive Plasmodium-specific CD4+ T cells had unexpectedly minor effects on Th1 differentiation in vivo. Instead, CXCR5 was essential for maximising clonal expansion, suggesting a role for splenic CXCL13+ cells in supporting CD4+ T cell proliferation in malaria. Thus, spatial transcriptomics at near single-cell resolution was feasible in densely packed secondary lymphoid tissue, providing multiple insights into mechanisms controlling splenic polyclonal CD4+ T cell and B cell differentiation during infection. HighlightsO_LISlide-seqV2 maps splenic microanatomy, including stromal and immune cell location. C_LIO_LIBystander activation of all follicular B cells occurs in malaria, marked by Sca-1/Ly6C upregulation. C_LIO_LISingle naive polyclonal CD4+ T cells differentiate mostly into Th1 and Tfh cells in malaria. C_LIO_LICell-cell colocalization analysis positions Th1 cells with monocytes in red pulp, and Tfh cells with Cxcl13+ B cell follicles. C_LIO_LICXCR5, but not CXCR3, supports parasite-specific CD4+ T cell clonal expansion. C_LI