IMMUNE REGULATION AND TOLERANCE II

T.W. He¹, P. Zhou², B.C. Xue¹, Y. Liu³, C. Li⁴, Z.K. Chen^1
1Institute Of Organ Transplantation, Tongji Hospital, Tongji Medical College, HUST, Wuhan/CHINA, ²Institute Of Organ Transplantation, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan/CHINA, ³Institute Of Organ Transplantation, Tongji Hospital, Wuhan/CHINA, ⁴Institute Of Organ Transplantation, Tongji Hospital, Tongji Medical College, Wuhan/CHINA

Body: Background. We have demonstrated that blockade of TLR signaling can promote tolerance to allografts in MyD88 knockout mice. However, it is unknown whether MyD88 may act as a therapeutic target of small molecular compound inhibitor to induce tolerance to allografts. Previously, we demonstrated that STTJ, a small peptidomimetic molecule which inhibits the dimerization of MyD88, alleviates cardiac allograft rejection in mice. In this study, we investigate if short term administration of STTJ can induce tolerance in the more strengint skin transplant model in mice, and the mechanism. Methods. BALB/c (H-2^d) to C57BL/6 (H-2^b) skin transplants were grouped by treatments, as shown in Fig 1. STTJ was administered post-transplant (250 mg/kg/d, i.p.) on day 0-3, 5, 7, 9, 11, 13, 15, and MR1 (200ug/d, i.p.) on day 0, 1, 3, 7, 14. Splenocytes were harvested on day 170 for Elispot and flow cytometry analysis. For Elispot, recipient cells were stimulated with MMC-treated donor (Balb/c) or third-party (C3H) splenic or self (C57BL/6) cells. Naive C57BL/6 splenocytes stimulated with MMC-treated Balb/c cells were used as control. BALB/c BMDCs were stimulated by TLR agonists (LPS, CpG, Poly I:C) and cardiac homogenate, in the presence and absence of STTJ for 12 hours. CD80/CD86 on DCs were analyzed by flow cytomety. C57BL/6 T cells were co-cultured with DCs for 5 days in the medium containing CpG with or without STTJ at different doses. CD3⁺/CD44⁺/Ki-67⁺ cells were analyzed by flow cytometry. Results. STTJ inhibited the upregulation of CD80/CD86 on DCs stimulated by TLR agonists. STTJ dose-dependently reduced the CD3⁺/CD44⁺/Ki-67⁺ cells in vitro. Short term STTJ with MR1 promotes permanent acceptance of fully allogeneic skin grafts for >150 days. Elispot showed donor specific tolerance in the long-term recipients, with normal IL-4 response, but with significantly reduced IFN-g secretion. STTJ administration was also associated with impaired Th1/Th17 immune responses and upregulated proportion of CD4⁺CD25⁺Foxp3⁺ Treg in the splenocytes in vivo. Conclusion. The MyD88 inhibitor STTJ leads to effective blocking to pan-TLR signaling, indicating that MyD88 is a critical therapeutic target for tolerance induction. Short-term STTJ administration promotes robust tolerance to skin allograft in mice. The inhibited activation of DCs and T cells, the increased CD4⁺CD25⁺Foxp3⁺ Treg and impaired Th1/Th17 responses may relate to the acceptance of the grafts. This is the first discovery of a novel therapeutic MyD88 inhibitor, and the first therapeutic small molecular compound targeting on innate immune system. We also first demonstrate that MyD88 can be a critical therapeutic target with vast clinical potentials. (This research is funded by National Natural Sciences Foundation of China no. 30772041. Corresponding Author: Ping Zhou).

Disclosure: All authors have declared no conflicts of interest.