**= 0.0023 (two-tailed paired = 5 mice per Rabbit Polyclonal to RIN1 group). turned on CD8+ T cells is not known. Here we report that encounters with FRCs enhanced cytokine production and remodeled chromatin accessibility in newly activated CD8+ T cells via interleukin-6. These epigenetic changes facilitated metabolic reprogramming and amplified the activity of pro-survival pathways through differential transcription factor activity. Accordingly, FRC conditioning significantly enhanced the persistence of virus-specific CD8+ T cells in vivo and augmented their differentiation into tissue-resident memory T cells. Our study demonstrates that FRCs play a role beyond restricting T cell expansionthey can also shape the fate and function of CD8+ T cells. Lymph nodes (LNs) contain hematopoietic cell lineages and several specialized stromal cells, including blood endothelial cells, lymphatic endothelial cells, follicular dendritic cells, marginal reticular cells, integrin 7+ pericytes and fibroblastic reticular cells (FRCs)1C5. LN stromal cells have primarily been viewed as structural determinants, uninvolved in immune cell homeostasis or ongoing immune responses. However, a series of recent publications has uncovered several immunoregulatory properties of LN stromal cells. In particular, T cell zone FRCs are concentrated in the paracortical region (T cell zone) of the LN and are endowed with functions that regulate the activity of T lymphocytes6C11. FRCs express the lymphocyte chemoattractants CCL19 and CCL21, which function to support na?ve T cell trafficking across high endothelial venules and retain T cells in the LN paracortex through their ligation to CCR7 (refs. 4,12C16). In addition to migration, FRCs support na?ve T cell homeostasis by expressing CNQX disodium salt interleukin-7 (IL-7) and aid T cell priming by facilitating interactions between T cells and antigen-presenting dendritic cells13,17C19. During T cell priming, the FRC network elongates in response to signals from antigenbearing dendritic cellswhich allows space for T cell influx and clonal expansion20,21. Although the FRC network relaxes to facilitate an area for T cell expansion, direct signals from FRCs actively restrict and curtail proliferation of the expanding T cell pool. Multiple studies have shown that FRCs and activated T cells engage in a molecular cross-talk whereby T cell-derived interferon- (IFN-) and tumor necrosis factor (TNF) act synergistically to enable FRCs with suppressive capabilities that are mediated through the activity of inducible nitric oxide synthase (NOS2)22C24. However, only a portion of FRCs expressed NOS2 in vivo during T cell priming22,23. These data indicate that FRCs may have a functional role beyond restricting T cell expansion. Indeed, recent publications have described the functional diversity of LN FRCs given their ability to regulate B and plasma cell homeostasis25C27. These studies highlighted the pleiotropic nature of FRCs, thus raising the possibility of their functional heterogeneity also within the T cell zone. Therefore, whether FRCs can enhance the function or support the differentiation of newly activated CD8+ CNQX disodium salt T cells remains an open question. Here we report a previously unrecognized function of LN FRCs during T cell activation. In response to signals from activated T cells, FRCs upregulated molecules with immunostimulatory functionnamely ICOS ligand (ICOSL), CD40 and interleukin-6 (IL-6). Once CNQX disodium salt released from FRCs, IL-6 enhanced IL-2 and TNF production by activated CD8+ T cells. FRC-derived signals, including IL-6, led to chromatin remodeling in activated CD8+ T cells, which promoted the expression of genes involved in bioenergetic and pro-survival pathways. In vivo, FRC-conditioned T cells persisted significantly longer following adoptive transfer into virally infected animals and preferentially differentiated into tissue-resident memory T (TRM) cells. These data demonstrate that LN FRCs, which are closely positioned near T cells during priming, can transmit long-lasting signals into activated CD8+ T cells to support their function, survival and differentiation. Results FRCs can express functionally diverse molecules To investigate whether FRCs express molecules capable of promoting the functions of activated T cells, we expanded FRCs from primary LN stromal cell cultures as previously described22, and then cultured them alone or with splenocytes activated with soluble antibody against CD3 and CD28 (hereafter anti-CD3/CD28) for 16 h. We generated transcriptional profiles of FRCs from each condition and found that FRCs co-cultured with activated splenocytes significantly upregulated 2,390 genes compared to their resting counterparts ( 0.01) (Fig. 1a). Expected gene signatures induced by T cell-derived cytokines, such as IFN-, were significantly enriched in FRCs following exposure to activated T cells ( 0.1) (Fig. 1b). Expression analysis of individual genes confirmed previous findings but also uncovered some unanticipated results. Consistent with published reports22C24, FRCs upregulated expression of genes encoding molecules known to dampen T cell function, such as and (Fig. 1a,?,c).c). However, FRCs also upregulated molecules with immunostimulatory capabilities in response to activated T cells. We detected increased expression of and in FRCs co-cultured with activated T cells (Fig. 1a). We validated these findings by comparing ICOSL, CD40 and IL-6 protein expression in resting FRCs versus FRCs co-cultured with activated T cells. Flow cytometric analyses revealed a robust increase in ICOSL, CD40.