*Purpose: Expression of stromal laminin α5β1γ1 (511) in lymph nodes (LN) is associated with immunity, while laminin α4β1γ1 (411) is associated with tolerance. The laminins determine how alloreactive T cells enter high endothelial venules and traffic within LN. Using a conditional knock out mouse model, we investigated the hypothesis that laminin α5 depletion in stromal cells will alter LN structure to enhance allograft survival.

*Methods: Laminin α5-flox and Pdgfrb-Cre DNA sequences were confirmed through genotyping Pdgfrb-Cre^+/-x laminin α5^flox/flox(KO) mice. Mice and LN stromal cells were analyzed for genomic DNA and laminin transcripts. Lymphocytes and stromal cells were analyzed with immunohistochemistry and flow cytometry. C57BL/6 (WT) and KO mice received cardiac transplants from BALB/c donors.

*Results: Laminin α5 mRNA transcripts were depleted completely in fibroblastic reticular cells (FRC) and partially in blood endothelial cells (BEC) and lymphatic endothelial cells (LEC) in Pdgfrb-Cre^+/- x laminin α5^flox/floxmice. Consequently, the ratio of laminin α4:α5protein was increased in LNs, but not spleen of KO mice, as determined byimmunohistochemistry. Compared with WT controls, no differences in CD4+, CD8+, or CD3+ T cells, B cells (B220+), or dendritic cells (CD11c+) were detected in LN or spleen of KO mice, but innate lymphoid cells 2 (ILC2) were increased, while ILC1 and ILC3 were decreased in LN. Importantly, Treg were increased in the LN of KO mice, particularly in the T cell zones, and were highly localized around PNAd+ cells of the high endothelial venules, which were also significantly increased in numbers. In tacrolimus treated recipients, KO mice had significantly prolonged allograft survival compared to WT mice (mean survival time (MST) 89 vs 27.5 days, p<.002). KO mice treated with a single dose of anti-CD40L had a trend for increased allograft survival compared to WT controls (MST 155 vs 91 days, p=0.07).

*Conclusions: Laminin α5 depletion changed LN to a more tolerogenic structure, including increased laminin α4:α5 ratio, increased Treg with a cortical distribution, and altered innate lymphatic subsets.These differences modulated immune responses toward the allograft, so that in calcineurin inhibitor or costimulatory blockade based immunosuppressive regimens laminin α5 deficiency resulted in marked prolongation of cardiac graft survival.

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