Long-lived T follicular helper cells retain plasticity and help sustain humoral immunity.

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 (T-cm) cells, whereas the existence and functional significance of long-lived T follicular helper (T-fh) cells are controversial. Here, we show that T-fh 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 T-fh cells with high expression of hallmark T-fh markers to at least 400 days after infection, by which time T-cm cells are no longer found. Using single-cell RNA-seq, we demonstrate that long-lived T-fh cells are transcriptionally distinct from T-cm cells, maintain stemness and self-renewal gene expression, and, in contrast to T-cm cells, are multipotent after recall. At the protein level, we show that folate receptor 4 (FR4) robustly discriminates long-lived T-fh cells from T-cm cells. Unexpectedly, long-lived T-fh 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 T-fh cell depletion concomitant with decreased splenic plasma cells and circulating antibody titers, demonstrating both unique homeostatic regulation of T-fh and their sustained function during the memory phase of the immune response. These results highlight the metabolic heterogeneity underlying distinct long-lived T cell subsets and establish T-fh cells as an attractive target for the induction of durable adaptive immunity.