Novel Methods To Improve Islet Transplantation Engraftment And Treg Expansion

Objectives:Type 1 diabetes mellitus(T1DM)results from the autoimmune destruction of isletβcell and leads to a permanent reliance on exogenous insulin.Currently the only way to achieve insulin independence for T1DM patients isβcell replacement therapy such as islet transplantation.Barriers that impede the application of islet transplantation include a low engraftment rate and the life-long requirement for immune suppressants.Our preliminary data showed that islet co-transplantation with parathyroid(PTG)improved islet graft survival,but the actual function of PTG and its mechanism remains to be elucidated.The other barrier can potentially be overcome with the use of tolerance-inducing regulatory T cells(Treg)in place of systemic and long-term immune suppression.Polyclonal(PolyTreg)expanded byαCD3/CD28 beads and donor alloantigen-reactiveTregs(darTreg)expanded by donor derived antigen presenting cells called sBc,are two types ofTreg being tested in clinical trials of transplantation.However,all clinical manufacturing of PolyTreg or darTreg experience difficulties in low cell yield and are in need of improvement.This study aims to solve these 2 hurdles in islet transplantation by developing novel methods to improve islet engraftment,and ways to improve PolyTreg and darTreg expansion respectively.Approaches:1.First assess the ability of human PTG to protect islet survival in vitro under hypoxic conditions.Compare the difference in protein secretion panel between PTG and islet and validate the protective effect of a few selected factors.Then assess PTG’s ability to induce angiogenesis in vivo when co-transplanted with islets subcutaneously by macroscale vascular density and histological changes with immunofluorescence staining.2.Validate the ability of CD28SA in place ofαCD3/CD28 beads to stimulate PolyTreg expansion alone or with IL-6 and TNFα.Assess the lineage stability of beadless protocol(combination of CD28SA,IL-6 and TNFα)PolyTregs.Explore the metabolic features of beadless PolyTreg.Finally assess beadlessTreg’s in vitro suppressive function and in vivo ability to prevent or treat GVHD in NSG mice.3.Summarize clinical expansion data on darTreg expansion to identify potential factors influencing product yield variability.Analyze correlations between sBc characteristics and darTreg expansion.Enhance darTreg CD28 signal by addition of soluble or sBc cell surface-bound CD28 antibodies and compare differences in fold expansion.Explore cytokine secretion panel of sBc to identify other potential influence from sBc.Results:1.Human PTG co-culture with islets in vitro can reduce islet cell death in hypoxic environment(dead cell area percentage at 48hr control vs PTG:mean rank difference 274.6,p<0.0001).IL-6,OPN,PTHrP and PTH were identified from PTG culture supernatant,and through validation of adding individual factors into islet in vitro culture,IL-6 and PTH were found to improve islet cell survival(dead cell area percentage at 48hr control vs IL-6:mean rank difference 380.8,p<0.0001;control vs PTH mean rank difference 299.7,p=0.0002).PTG islet co-transplantation showed improved vessel density in NSG subcutaneous compared with islet alone(26.53%±3.439%vs 17.18%±2.675%,p=0.0101).Histological examination showed that PTG rapidly reconnected with host blood vessels and improved perfusion near co-transplanted islet graft.2.CD28SA single stimulation could expand PolyTreg,but with the addition of IL-6 and TNFα,fold expansion improved significantly compared with traditional bead stimulation(1081±409.6 vs 478.8±725.1,p=0.0302).This beadless protocol expanded PolyTregs showed similar lineage stability as the bead protocol,no evidence of effector T cell cytokine secretion,as well as more active energy metabolism and biosynthesis.Beadless PolyTreg also performed the same as beadTregs in the in vitro suppression assay and the ability to prevent or treat GVHD in vivo.3.Analysis of clinically manufactured darTreg showed significant difference in expansion potential within first 7 days upon sBc stimulation.Correlation analysis revealed a positive correlation between darTreg fold expansion and CD80,CD86 expression on sBc when using the sameTreg(CD80 R~2=0.8637,p=0.0073;CD86 R~2=0.9495,p=0.0010).Additional CD28 signal through soluble CD28 antibody negatively affected the allo-reactivity and stability of darTreg,while quantitatively planting CD28antibody on the surface of sBc lead to increased expansion during the first round of stimulation.sBc that were deemed good expanders showed an increase in chemokine secretion and decrease in IL-16.Conclusions:Human PTG co-transplantation with islets can improve islet engraftment by secreting survival factors,promoting angiogenesis and reconnecting blood vessels;CD28 in combination with IL-6 and TNFαas the new“Beadless”protocol can significantly improve PolyTreg expansion while maintaining its stability and suppressive function;darTreg expansion is impacted by sBc surface expression of CD80,CD86,and potentially chemokine and cytokine secretion.darTreg expansion can be improved by providing the appropriate anti-CD28 signal enhancement such as surface-bound CD28 antibody.Combing this novel method of islet transplantation with infusion of PolyTreg or darTreg expanded on a large scale,brings forth a promising treatment to cure T1DM with improved islet transplantation success rate and tolerance induction without immunosuppressants.