*Purpose: Lymph nodes (LNs) have been regarded as primary activation sites for alloimmunity following transplantation, but our group has revealed an additional dimension to their function as critical locales for alloimmune regulation as well. Draining LNs (DLNs) must undergo massive structural changes for the proper generation of alloimmune responses. Although the importance of DLNs to transplant tolerance has been established, how their stromal compartment contributes to this process has never been studied in detail. Our hypothesis is that sustained post-transplant immune activation within the DLN results in fibrosis of the DLN and impairs its regulatory function.

*Methods: We used CCL19-Cre^+/- x iDTR conditional knockout mice to determine whether fibroblastic reticular cells (FRCs) are the primary source of extracellular matrix (ECM) accumulation. We employed CCL19-Cre^+/- x P16^fl/fl conditional knockout mice to investigate the role of senescence in FRCs post-transplantation. We also generated CCL19-Cre^+/- x herpes virus entry mediator (HVEM)^fl/fl conditional knockout mice to investigate the role of LIGHT (also known as TNFSF14 or CD258) and the HVEM pathway in inducing senescence in FRCs. Last, we isolated, characterized, and expanded FRCs ex vivo to investigate their effect on reprogramming the inflammatory milieu of the DLNs.

*Results: Serial analysis of the DLN at numerous timepoints post-organ transplantation revealed continuous expansion of extracellular matrix (ECM), and FRCs were identified as the primary source of these fibers. Following repetitive skin transplantation, FRCs adopted a senescence-associated secretory phenotype (SASP) marked by massive generation of ECM fibers and a pro-inflammatory milieu in the LN. LIGHT induced the SASP via binding to HVEM on the surface of FRCs. Notably, administration of FRCs via systemic injection ameliorated fibrosis and increased the regulatory T cell population in the DLNs following repetitive skin transplantation.

*Conclusions: These data highlight for the first time the critical importance of LN fibrosis and how key signaling pathways control the behavior of FRCs during transplantation. We have also introduced a novel strategy to support transplant tolerance by reprogramming the stroma of the DLN.

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