*Purpose: Allogenic and autoimmune response against islets remain major obstacles to improve long term function of islet transplants. Inhibition of toll-like receptor 4 (TLR4) using small molecule inhibitor, resatorvid (TAK-242), improved outcomes in syngeneic islet transplants. Based on reports showing involvement of TLR4 signaling pathway in immunomodulation, we sought to investigate the effects of resatorvid on immune responses using mixed lymphocyte culture of peripheral blood mononuclear cells (PBMCs) and allogenic human islets.

*Methods: Human T cells were isolated from whole blood and stained with CellTrace Violet proliferation dye and stimulated with CD3/CD28 Dynabeads. Activation and proliferation of T cells was monitored 24 and 72 hours following exposure to beads. Human islets were isolated and cultured for at least 24 hours prior experiments and samples of islets were modified with an NHS cleavable linker incorporating cleavable TAK-242. 1 x 10⁵ human PBMCs were mixed with 15 IEQ of islets in the presence or absence of TAK-242 or its structural analog, MAP84. Following 24 and 48 hours of culture PBMCs were analyzed by flow cytometry and islets were removed and preserved in Histogel. Integrity of islets was observed by microscopy following H&E staining. Supernatant miRNA and inflammatory cytokine production was measured by RT-qPCR and Milliplex assay respectively.

*Results: T cells treated with TAK-242 showed decreased expression of CD69 and decreased proliferation when exposed to CD3/CD28 antibody. Decrease in expression of CD69 was observed both in CD4⁺ and CD8⁺ T cells. When islets were mixed with allogenic PBMCs, the presence of soluble TAK-242 administered freely or released from modified islet surfaces decreased the percentage of CD69⁺ T cells as well as the production of inflammatory cytokines IL-1β, IL-6, IP-10, and TNFα. More intact islets were observed with TAK-242, which released less damage associated miRNA-375 and miRNA-200c after 24 hours of culture.

*Conclusions: Inhibition of TLR4 with resatorvid is able to attenuate allogenic immune responses leading to less damage to allogenic islets in vitro. The results of this study further warrant the use of resatorvid in an in vivo model of allogenic islet transplantation. These results also further encourage the study of TLR4 and its role in signaling within immune cells as a possible target for immunomodulation in transplantation.

« Back to 2022 American Transplant Congress