Background: CD40-CD40L costimulatory blockade plus donor-specific splenocyte transfusion (DST) induces tolerance concurrent with regulatory T cell (Treg) induction in the lymph node (LN). Treg are associated with the cortical ridge (CR) within the tolerant LN, a region of lymphocyte entry via high endothelial venules (HEV) and antigen presentation. HEV basement membranes (BM) contain laminins, stromal fibers that affect lymphocyte vascular egress and are remodeled by matrix metalloproteinases (MMP). The sphingosine 1-phosphate receptor-1 (S1P1) inhibitor FTY720 supports tolerance by sequestering lymphocytes in the LN, whereas blocking HEV entry with anti-CD62L inhibits tolerance. We hypothesized that tolerance induction requires a unique form of lymphocyte trafficking through and localization around HEV.

Methods: Tolerance was induced in C57BL/6 mice with BALB/c DST + anti-CD40L mAb. Tolerant mice received FTY720, anti-CD62L mAb, or MMP inhibitors (MMI). Adoptively transferred alloantigen specific T cells were tracked with vital dyes. ER-TR7, PNAd, CD3, and Foxp3 mAbs were used for immunohistochemistry of LN and HEV structures.

Results: Tolerance induction resulted in increased complexity of ER-TR7+ fibers around the abluminal HEV surface. The numbers of total and antigen specific T cells entering HEV, and Treg associated with HEV, were also increased as early as 4 hours following tolerization. In contrast, immune and naÏve HEV were morphologically similar, with similar numbers of total T cells, antigen specific T cells and Treg entering and associated with HEV. FTY720 enhanced the tolerogenic phenotype and the association of Treg with the HEV. Anti-CD62L prevented the tolerogenic phenotype, resulting in a decreased complexity of the ER-TR7+ fibers, and impeded lymphocyte entry and Treg association with HEV. MMI decreased the number of antigen specific T cells associated with HEV, while increasing the number of T cells located in the lumen of HEV, suggesting control of lymphocyte LN entry by BM remodeling.

Conclusions: Tolerance induction results in the unique migration of antigen specific T cells and Treg in the CR of the LN, which encompasses HEV. These changes begin within 4 hours of tolerization and affect both the morphology of HEV and how lymphocytes enter the LN. These findings suggest tolerance induction commences acutely following antigen exposure by establishing a permissive LN microenvironment. Further, HEV are not only entry points to the LN, but also early mediators of alloantigen specific lymphocyte fate decisions.

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