Background: Pre-transplant regulation on the donor side(toward the recipient), as well as the recipient side(toward the donor) promotes long-term graft survival of a kidney allo-graft in a depletional protocol. In mice, donor-specific splenocytes transfusion (DST) and anti-CD40L monoclonal antibody (MR-1) induces tolerance. This treatment causes allo-specific regulation, which may include IL-10, TGF-b and IL-35 secreted by allo-specific regulatory T cells (T-regs). IL-35 has shown to play critical roles in the regulation of various immune responses. IL-35 is produced by FoxP3 positive T cells, and causes suppression of effector T cells, and expansion of non-Foxp3 T- and B-regs.

Hypothesis: IL-35 positive allo-specific T-regs formed in response to DST and MR-1 will migrate to tissues besides the spleen and lymph nodes, where they will be able to mediate allo-specific linked suppression, the pre-condition for bi-directional regulation after transplant of that organ.

Methods: CBA(H-2k) spleen cells were injected i.v. into C57/BL6[H-2b) on d.0. MR-1 was injected i.p. into the mice@ 125mg dose on d. 0,2, and 4. Mice were sacrificed d.14 and spleen, lymph nodes, and lungs were harvested; in the case of the lung leukocytes were obtained by collagenase digestion followed by Gentle-MACS dissociation. In order to investigate the tissue distribution of IL-35 positive allo-specific T-regs, we used a novel flow-cytometry assay for surface Ebi3 expression by Treg cells recently developed in Dario Vignali's lab. These results were correlated with trans vivo- delayed type hypersensitivity (tv- DTH) assays.

Results: The % of T-regs amongst total T-cells of the tolerized mice vs. control untreated B6 mice was 4.9 vs.2.7% in spleen, 6.0 vs. 3.0% in lymph nodes, 2.0 vs. 1.1% in Lung, respectively. The tolerized B6 mice had an abundant IL-35 positive T-regs in spleen, compared to comtrol B6 mice (1.2 vs. 0.19%, P=0.0089 by t test, n=5). In tv-DTH assays, self antigen was found to induce the highest level of linked suppression 14 d.after the treatment of DST and MR-1.

Conclusion: Surface IL-35 positive T-regs were detected in spleen and lymph nodes of tolerized mice. It is too early to tell if IL-35 positive T-regs migrate to the tissues and are associated with the induction of regulatory responses there. We'll present additional data on kinetics of appearance of IL-35 positive allo-specific T-regs in other organs, where they might contribute to bi-directional regulation in primarily vascularized orthotopic lung and heterothopic heart transplants.

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