GG-01 Hypomethylation of STAT1 and HLA-DRB1 in CD8+ T cells is associated with type-I interferon-dependent HLA-DRB1 overexpression and activation of autologous CD4+ T cells in systemic lupus erythematosus

Background Systemic lupus erythematosus is a chronic autoimmune disease characterized by epigenetic dysregulation, and increased autoantibody and type-I interferon (IFN) production. The goal of this study was to explore possible pathogenic roles of CD8 +T cells in lupus through characterizing DNA methylation changes. Methods Genome-wide DNA methylation of lupus and age, sex and ethnicity-matched control CD8+ T cells was measured using the Infinium MethylationEPIC arrays. Data preprocessing and statistical analysis of differentially methylated CpG sites was performed using GenomeStudio (Illumina). Gene ontology analysis of differentially methylated sites was performed with DAVID. Measurement of HLA-DRB1 expression on the surface of lupus and control CD8+ T cells with and without interferon alpha (IFNα) was performed by flow cytometry. Co-incubation of IFNα-treated CD8+ T cells from lupus patients and controls with autologous naïve CD4+ T cells to assess effects on CD4+ T cell stimulation were performed. CD8+ T cell mRNA levels normalized to β-actin were quantified by qPCR. Results Lupus CD8+ T cells had 188 hypomethylated CpG sites compared to healthy matched controls. Among the most demethylated were sites associated with HLA-DRB1 (Δβ=−0.33) and STAT1 (Δβ=−0.15). The proportion of CD8+ T cells expressing HLA-DRB1 was significantly higher in lupus compared to controls. IFNα treatment upregulated cell surface expression of HLA-DRB1 on CD8+ T cells of lupus patients but not healthy controls. Co-incubation of naïve CD4+ T cells with IFNα-treated autologous CD8+ T cells led to increased expression of the stimulation marker CD69 on CD4+ T cells in lupus patients, but not in healthy controls. This effect can be abrogated using HLA-DR blocking antibodies. Lupus and control CD8+ T cells significantly increased STAT1 mRNA levels after treatment with IFNα. The expression of CIITA, a key interferon/STAT1 dependent MHC-class II regulator, is only induced by IFNα in lupus CD8+ T cells, but not healthy controls. Conclusions HLA-DRB1 and STAT1 loci are hypomethylated and epigenetically poised for overexpression in lupus CD8+ T cells in the presence of type-I interferon. IFNα-treated lupus CD8+ T cells stimulate autologous CD4+ T cells in vitro and blocking of HLA-DR on CD8+ T cells can reduce this effect. These data suggest a possible pathogenic role for CD8+ T cells in lupus that is dependent upon a high type-I interferon environment and epigenetic priming. Acknowledgements This work was supported by the National Institute of Allergy and Infectious Diseases of the National Institutes of Health grant number R01AI097134.