Introduction: We previously reported that donor specific splenocytes transfusion (DST) plus anti-CD40L costimulatory blockade treatment causes allo-specific regulation approximately 5 weeks after the tolerization. The regulation may include IL10, TGFb and IL35 (both Ebi3 and p35 subunit) secreted by allo-specific regulatory T (Treg) cells. IL35 is one of the key molecules of immune regulation and infectious tolerance, which suppress the proliferation of effector T cells, while expandingnon-Foxp3 regulatory T and B cells.

Hypothesis: 1) Foxp3⁺ Treg cells are the main source of surface Ebi3 (sEbi3) acquired by bystander CD4 T cells after DST plus costimulatory blockade. 2) IL35 will be secreted as components of exosomes by antigen-specific Treg cells.

Methods: CBA (H2^k) spleen cells were injected i.v. on day 0 into double reporter transgenic mice (C57BL/6; H2^b background), which expressed YFP under the Foxp3 and TdTomRed under the Ebi3 promoter [Ebi3⁺ mice]. Anti-CD40L blockade (MR-1) was injected i.p. into the mice of 125ug dose on day 0, 2 and 4. Mice were sacrificed on day 35, spleen and lymph nodes were harvested and purified exosomes. In order to investigate functions of IL35 containing exosome purified from tolerized mice, we used a novel flow-cytometry assay for sEbi3 expression by Treg cells, ELISA and tv-DTH linked-suppression assay.

Results: The tolerized Ebi3⁺ mice had much greater population of sEbi3⁺CD4 T cells in 24-hour culture in the presence of allo-specific CBA-antigen, predominantly among CD4 non-Treg (Foxp3^neg) cells (P<0.01). By ImageStream population microscopy analysis, the sEbi3 appeared to be secreted as exosomes by the Treg cells and captured by bystander CD4 non-Treg cells. ELISA was able to provide exosome detection, and CD81 was enriched in the exosomes isolated by ultracentrifugation. Furthermore, CD81⁺ exosomes could be captured in ELISA by CD39- or Ebi3-specific, but not by IL12/p35-specific coating antibodies. However, both p35- and Ebi3-specific antibodies could significantly (p<0.05) reverse suppression caused by adding the exosomes to a TT-specific third party recall response, indicating that both chains of IL35 were present and active in suppression.

Conclusions: IL35 contributes to donor specific tolerance via exosomes produced by regulatory cells and acquired by conventional CD4 T cells.

CITATION INFORMATION: Tomita Y, Bracamonte-Baran W, Jankowska-Gan E, Zhang Q, Vignali D, Burlingham W. Exosomes Containing IL35 Are Secreted by Donor-Specific Regulatory T Cells and Cause Linked-Suppression. Am J Transplant. 2017;17 (suppl 3).

« Back to 2017 American Transplant Congress