Engineering A Novel Treg Population to Control Autoimmune Diabetes

Purpose Type 1 diabetes (T1D) in an autoimmune disorder where T cells attack  islet cells, which are the pancreatic cells responsible for insulin production. T1D affects about 1 in 500 children in the United States and is one of the most common chronic diseases in pediatrics. Currently, insulin is the most effective treatment for T1D However, it does not change the underlying disease, and patients still experience many complications throughout their lifetimes. With a significant increase in the prevalence of T1D in children and adolescence in the past two decades, it’s important to explore therapies that alter the immune response as a possible way to reverse or even prevent the progression and development of this disease. One approach being investigated is to use regulatory T cells (Tregs), which can suppress the function of autoreactive T cells, as treatment for autoimmune disease. However, using a patient’s own naturally occurring Tregs (nTregs) as therapy poses several challenges. Therefore, we developed a novel way to generate human engineered Tregs (eTregs) from conventional T cells (Tconvs) by expressing both FOXP3 and HELIOS in both CD4+ and CD8+ Tconvs. We hypothesize that these novel eTregs will be as, or more, effective in their suppressive abilities against islet-specific effector T cells and be more stable under inflammatory conditions compared to naturally occurring Tregs. Methods We isolated CD8+ T cells from healthy human donors through negative selection using magnetic beads. From these cells we created cytotoxic T cells (Tconvs) through lentiviral transduction of a vector that expressed a TCR specific for an islet-specific antigen (IGRP) presented in HLA-A2:01 and mCherry as a fluorescent marker of transduction. The presence of IGRP-specific TCR was confirmed using dextramer staining. These Tconvs were co-cultured with BetaLox5 cells, an immortalized human cell line derived from islet cells. The response of the CD8+ Tconvs was determined by measuring effector cytokine release. We also created autologous natural Tregs (nTregs) and tested the ability of these cells to reduce the CD8+ Tconv response against BetaLox5Unpaired T test analysis was used to measure the statistical difference in cytokine production between co-culture conditions. Summary of Results IGRP-specific T cells responded to the human islet cell line BetaLox5 in vitro, demonstrated by elevated IFN-γ and TNF- production after co-culture. This response was decreased when nTregs were present in co-culture, as the amount of IFN-γ and TNF- in co-culture supernatant was significantly lower. Conclusions We were able to create a model to test the immunosuppressive capacity of Helios+FOXP3+ eTregs compared to nTregs against islet-specific autoreactive T cells. We also showed that nTregs decrease the response of these cytotoxic T cells when co-cultured with BetaLox5 cells. We are now comparing the immunosuppressive ability of our eTregs to nTregs in this assay, as well as comparing their stability under inflammation.