Conversion of CD4+T cells to functioning and epigenetically stable induced regulatory T cells in patients with primary biliary cholangitis

Introduction

Primary biliary cholangitis (PBC), is a chronic, autoimmune liver disease. Regulatory T cells (Tregs) are a subset of CD4+ T lymphocytes whose activity is driven by the expression of a key transcription factor, Forkhead box P3 (FOXP3). A reduction in Treg frequency and functionality has been proposed as an underlying pathogenic mechanism of PBC. We aimed to characterise the natural Treg (nTreg) epigenetic profile in PBC patients. Additionally, we set out to induce functional and stable Tregs (SF-iTregs) from PBC-derived effector CD4 cells in vitro, via cyclin-dependent kinase (CDK8/19) inhibition.

Methods

CD4+ T cells were magnetically enriched from peripheral blood mononuclear cells (PBMCs) of PBC patients. CD4+T cells were activated by CD3+ activator beads only (without CD28 co-stimulation) and cultured in the presence of IL-2 and AS2863619, a CDK8/19 inhibitor. FoxP3, CTLA4 and Helios expression in SF-iTregs was assessed via flow cytometry and by bisulphite sequencing pre- and post-activation in the presence of Th1 polarising cytokines. nTreg and SF-iTreg suppressive function was investigated by measuring CellTrace Violet dye-labelled effector T-cell proliferation following co-culture with SF-iTregs in different dilutions. nTreg and SF-iTreg Foxp3 gene locus STAT5 binding, H3K27ac, and chromatin status was characterized by Chromatin immunoprecipitation followed by sequencing (ChIP-seq) and assay for transposase-accessible Chromatin sequencing (ATAC-seq).

Results

Deprivation of the CD28 signal with chemical inhibition of cyclin-dependent kinase 8/19 in CD4 T cells is instrumental to induce DNA hypomethylation in Treg signature genes. Our protocol allowed us to generate a >90% population of FOXP3 expressing cells with a 100-fold increase in the cell number over 2 weeks. ATAC-seq and ChIP-seq confirmed Treg specific epigenetic changes in the SF-iTregs, which were comparable to nTreg at the baseline. To resemble the liver inflammatory environment of PBC, we cultured nTreg and SF-iTregs in Th1-conditioned media containing IL-12 and IFN-γ for 6 days, demonstrating superior lineage stability in SF-iTregs compared with nTregs. In addition, SFiTreg maintain suppressive function in the inflamed environment.

Conclusions

We apply a novel technique to generate abundant, functional and stable induced regulatory T cells from peripheral blood conventional CD4+T cells in patients with primary biliary cholangitis. This approach would facilitate the production of phenotypically stable, functional induced Tregs from antigen-experienced disease-mediating T cells on a large scale, to apply as GMP cell-therapy in PBC.