Induced regulatory T cells via cyclin-dependent kinase inhibition from patients with primary biliary cholangitis are suppressive and stable in an IFN enriched environment and are epigenetically different from natural regulatory T cells

Background and Aims Dysfunction in regulatory T cells (Tregs) in primary biliary cholangitis (PBC) is well-documented. Studies using murine models have indicated the efficacy of Treg-based cell therapies in restoring liver tolerance in PBC. These therapies are effective when using Tregs from healthy animals, but not from PBC models, underscoring the impaired functionality of PBC-derived Tregs. Our research introduces a novel protocol aimed at generating stable and functional induced Tregs through the epigenetic modification of CD4+ T cells from the peripheral blood of PBC patients. The objective of our study is to assess the functionality and stability of our induced Tregs in comparison to natural Tregs from PBC patients, employing an ex-vivo wedge perfusion model. Method CD4+ T cells were magnetically enriched from peripheral blood mononuclear cells (PBMCs) of PBC patients. CD4+T cells were activated through TCR stimulation by CD3+ activator beads and cultured in presence of AS2863619 (4-[1-(2-methyl-1H-benzimidazol-5-yl)-1H-imidazo[4,5-c] pyridin-2-yl]-1,2,5-oxadiazol-3-amine dihydrochloride) and IL-2. FoxP3, CTLA4 and Helios expression was assessed in SF-iTregs via flow cytometry and by bisulphite sequencing pre- and post-activation in presence of Th1 polarising cytokines. The functionality of Tregs and SF-iTregs was investigated by in vitro suppression assays using CellTrace Violet dye-labelled effector T-cells, CD3 activator beads and IL-2. For the all three cell populations, nTreg, Tnaive, SF-iTregs, Foxp3 gene locus for STAT5 binding, H3K27ac, and chromatin status was characterized by Chromatin immunoprecipitation followed by sequencing (ChIP-seq) and assay for transposase-accessible Chromatin using sequencing (ATAC-seq). After labelling the cells with cell-tracker red, they were perfused under constant pressure in a liver wedge from a discarded donor liver for 12 hours. Results The chemical inhibition of cyclin-dependent kinase 8/19 and the deprivation of CD28 signal induced DNA hypomethylation in Treg signature genes. We obtained a 100-fold increase in the cell number and the product was suppressive and pure (>90% FOXP3 expressing cells). The SFiTregs were found to be more stable compared with nTreg when cultured for 6 days in Th1 polorising conditions. ATAC-seq and ChIP-seq confirmed that Treg specific epigenetic changes in the SF-iTregs were comparable to nTreg at the baseline. However, differentially accesible regions of SF-iTregs differ significantly when compared to nTreg. Furthemore, the cells were retained in the liver parenchyma significantly more than healthy and PBC controls when infused in a ex-vivo liver wedge. Conclusion We applied a novel technique to generate abundant, functional regulatory T cells from peripheral T CD4+ cells in patients with PBC. This approach would allow us to overcome the potential limit of using autologous ex-vivo expanded Tregs based therapy and generate a more stable product to infuse in the inflamed environment typical of the liver of PBC patients. Dysfunction in regulatory T cells (Tregs) in primary biliary cholangitis (PBC) is well-documented. Studies using murine models have indicated the efficacy of Treg-based cell therapies in restoring liver tolerance in PBC. These therapies are effective when using Tregs from healthy animals, but not from PBC models, underscoring the impaired functionality of PBC-derived Tregs. Our research introduces a novel protocol aimed at generating stable and functional induced Tregs through the epigenetic modification of CD4+ T cells from the peripheral blood of PBC patients. The objective of our study is to assess the functionality and stability of our induced Tregs in comparison to natural Tregs from PBC patients, employing an ex-vivo wedge perfusion model. CD4+ T cells were magnetically enriched from peripheral blood mononuclear cells (PBMCs) of PBC patients. CD4+T cells were activated through TCR stimulation by CD3+ activator beads and cultured in presence of AS2863619 (4-[1-(2-methyl-1H-benzimidazol-5-yl)-1H-imidazo[4,5-c] pyridin-2-yl]-1,2,5-oxadiazol-3-amine dihydrochloride) and IL-2. FoxP3, CTLA4 and Helios expression was assessed in SF-iTregs via flow cytometry and by bisulphite sequencing pre- and post-activation in presence of Th1 polarising cytokines. The functionality of Tregs and SF-iTregs was investigated by in vitro suppression assays using CellTrace Violet dye-labelled effector T-cells, CD3 activator beads and IL-2. For the all three cell populations, nTreg, Tnaive, SF-iTregs, Foxp3 gene locus for STAT5 binding, H3K27ac, and chromatin status was characterized by Chromatin immunoprecipitation followed by sequencing (ChIP-seq) and assay for transposase-accessible Chromatin using sequencing (ATAC-seq). After labelling the cells with cell-tracker red, they were perfused under constant pressure in a liver wedge from a discarded donor liver for 12 hours. The chemical inhibition of cyclin-dependent kinase 8/19 and the deprivation of CD28 signal induced DNA hypomethylation in Treg signature genes. We obtained a 100-fold increase in the cell number and the product was suppressive and pure (>90% FOXP3 expressing cells). The SFiTregs were found to be more stable compared with nTreg when cultured for 6 days in Th1 polorising conditions. ATAC-seq and ChIP-seq confirmed that Treg specific epigenetic changes in the SF-iTregs were comparable to nTreg at the baseline. However, differentially accesible regions of SF-iTregs differ significantly when compared to nTreg. Furthemore, the cells were retained in the liver parenchyma significantly more than healthy and PBC controls when infused in a ex-vivo liver wedge. We applied a novel technique to generate abundant, functional regulatory T cells from peripheral T CD4+ cells in patients with PBC. This approach would allow us to overcome the potential limit of using autologous ex-vivo expanded Tregs based therapy and generate a more stable product to infuse in the inflamed environment typical of the liver of PBC patients.