Protection of bona fide T follicular memory cells during tissue isolation reveals their persistence, plasticity and functional impact

Abstract

Running title: TFH memory cell persistence, identity and function 2 SUMMARY T follicular helper (TFH) cells regulate antibody production. Following infection or vaccination, effector TFH cells give rise to long-lived memory TFH cells. The survival requirements, lineage flexibility and impact of TFH memory cells have not been fully elucidated. In this study we determined that TFH memory cells persist to at least 400 days after infection while T cells with a central memory phenotype are largely absent.Single cell RNA sequencing reveals that TFH memory cells express many genes associated with stemness and self-renewal; reconstruction of a developmental trajectory suggests that TFH memory cells occur earlier in pseudotime, giving rise to other Th cell subsets in a linear fashion. Surprisingly, TFH memory cells concurrently express a distinct metabolic signature similar to trained immune cells, including elevated expression of mTOR, HIF-1 and cAMP regulated genes. TFH memory cell survival is mediated by ICOS signaling which acts as an integrator of glycolytic and self-renewal programming. Inhibition of ICOS at late time points leads to a reduction in TFH memory cells concomitant with decreased splenic plasma cells and circulating antibody titers.These results highlight the metabolic heterogeneity underlying distinct CD4 memory T cell subsets, linking TFH memory cell survival to sustained humoral immunity. 3 HIGHLIGHTS• TFH memory cells are long-lived and transcriptionally distinct from T central memory cells • TFH memory cells are multipotent following recall• TFH memory cells can be maintained in the absence of but requires ICOS signaling and glycolysis• TFH memory cells support late phase antibody production by splenic plasma cells 8 et al., 2014). TCM and Th1 clusters (4-7) exhibited higher expression of IL-7r, S100a4, S100a6, Selplg and various integrins ( Figures 2B and 2D), while clusters 6 and 7 were enriched for genes strongly associated with Th1 differentiation including Cxcr6, Ccl5, Nkg7 and Id2 (Figures 2B and 2E). Among Th1 clusters, cluster 7 represented the subset highest in Selplg, Ly6c2 and Il7r, while cluster 6 expressed higher levels of Cxcr6 and Id2 and exhibited signatures of dysfunction and exhaustion ( Figures S2D-S2F) (Ciucci et al., 2019;Martinez et al., 2015). Cluster cell-type identities were further confirmed by scoring each cell for signature genes obtained from publicly available datasets and examining the distributions of these scores across clusters ( Figures 2F and S2C). TFH and Th1 signatures matched well with clusters 1-3 and 6-7 respectively, while clusters 4, 5 and 7 were enriched for the TCM precursor signature reported by Ciucci et. al. (Figure 2F) (Ciucci et al., 2019). Clusters 4 and 5 had the highest Ccr7 transcription, which negatively correlated with Cxcr5 across all cells ( Figure 2G). To facilitate visualization of the relationship between Ccr7 and Cxcr5, we plotted expression levels imputed using the Seurat R package ( Figure 2H) (Satija et al., 2015).Within TFH clusters...