*Purpose: The lymph node (LN) is the primary site of alloimmunity activation and regulation during transplantation. Fibroblastic reticular cells (FRCs), the prominent cells of the LN stroma, are master arbiters of the LN microenvironment, and they regulate T cell trafficking across high endothelial venules (HEVs) into the LN as well as their positioning alongside dendritic cells. Meca79 mAb recognizes peripheral node addressin (PNAd), a glycoprotein family expressed exclusively by LN-restricted HEVs. We have demonstrated previously that Meca79-conjugated polymeric NPs (Meca79-NPs) enter LNs through HEVs following systemic administration in mice, forming the basis of a LN-targeted drug delivery platform. Here, we hypothesize that LN-targeted nanodelivery of the costimulatory blockade agent anti-CD40L will enhance cardiac allograft tolerance in mice following transplantation, through potentiation of immunoregulatory FRC activity.

*Methods: We transplanted cardiac allografts into CCL19^Cre x iDTR mice, in which FRCs can be conditionally ablated, to examine the importance of FRCs to allograft survival. Through intravital microscopy, we tested the interaction between CD11c⁺ dendritic cells and T cells in the LNs of these mice. Meanwhile, we tested how FRCs alter CD4⁺ T cell differentiation in vitro. Then, we encapsulated the immune therapeutic anti-CD40L inside Meca79-NP (Meca79-anti-CD40L-NP) and tested its delivery to draining lymph nodes (DLNs) following skin transplantation and its efficacy in prolonging heart allograft survival in mice through intravenous injection.

*Results: FRC depletion in CCL19^Cre x iDTR mice abrogated the tolerogenic effect of anti-CD40L treatment following heart transplantation. Intravital microscopy showed that FRCs facilitated the interaction between CD11c⁺ dendritic cells and T cells in the LN. FRCs also induced the differentiation of CD4⁺ T cells to Tregs but suppressed their differentiation to effector T cells in vitro. DLNs of cardiac transplant recipients that received Meca79-anti-CD40L-NP contained more regulatory T cells and lower rejection, as signified by smaller immune cell infiltrates, than the soluble anti-CD40L-treated group at 7 days post-transplantation. Finally, combined treatment with Meca79-anti-CD40L-NP and rapamycin resulted in a markedly longer allograft survival than soluble anti-CD40L and rapamycin (mean of 80 days vs. 24 days, respectively, p<0.001).

*Conclusions: These data demonstrate that FRC are the master regulators of the LN microenvironment following transplantation andestablish the efficacy and feasibility of our first-in-class therapeutic approach to promote transplant survival through delivery of costimulation blockade to the LN.

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