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

CD4 memory T cells play an important role in protective immunity and are a key target in vaccine development. Many studies have focused on T central memory (TCM) cells, while the existence and functional significance of T follicular helper (TFH) memory cells is controversial. Here we show that TFH memory cells are highly susceptible to NAD induced cell death (NICD) during isolation from tissues, leading to their under-representation in prior studies. NICD blockade reveals the persistence of abundant TFH memory cells, with high expression of hallmark TFH markers, that persist to at least 400 days after infection, by which time TCM cells are no longer found. Using single cell RNA-seq we demonstrate that TFH memory cells are transcriptionally distinct from TCM cells, maintain stemness and self-renewal gene expression, and, in contrast to TCM cells, are multipotent following recall. Surprisingly, TFH memory cells concurrently express a distinct glycolytic signature similar to trained immune cells, including elevated expression of mTOR, HIF-1 and cAMP regulated genes. Late disruption of glycolysis/ICOS signaling leads to TFH memory cell depletion concomitant with decreased splenic plasma cells and circulating antibody titers, demonstrating both unique homeostatic regulation of memory TFH and their sustained function during the memory phase of the immune response. These results highlight the metabolic heterogeneity underlying distinct memory T cell subsets and establish TFH memory cells as an attractive target for the induction of long-lived adaptive immunity. HIGHLIGHTS