A T cell immune suppression signature distinguishes European American auto-antibody positive (ANA+) healthy individuals

Anti-nuclear antibody (ANA) positivity is a principal feature of individuals with an autoimmune disease, yet up to one in five healthy individuals are ANA+ and most will never develop clinical autoimmunity. Here, we show that immune profiles in ANA+ healthy individuals are altered, and differ by race. A suppressive immune signature distinguished by reduced T cell numbers and altered gene expression of HLA class I, IFN-associated, and apoptosis pathways, characterized European American (EA) ANA+ healthy individuals. In contrast, African American (AA) ANA+ healthy individuals had more elements of activation with increased CD69 gene expression on T cells and heightened pro-inflammatory cytokines. Increases in STAT4, IFNGR2 and STAT1 T cell transcripts and decreases in TGFBR1 gene expression in monocytes correlated with T cell expansion and clinical SLE. Thus, a suppressive immune signature in EA ANA+ healthy individuals may avert clinical autoimmune disease, which is associated with activation of Type I and II IFN pathways and T cell expansion. PBMCs from 36 subjects (ANA-, ANA+ healthy or SLE patients) were stained with antibodies for CD3 (UCHT1), CD19 (SJ25-C1), HLA-DR (G46-6), CD14 (61D3), CD16 (3G8), CD56 (NCAM16.2), CD66b (G10F5) and CD66b-CD19-CD3+ T cells, CD66b-CD3-CD19+ B cells, CD66b-CD3-CD19-CD56-HLA-DR+CD14+CD16+/- monocytes were sorted using a FACS Aria III (BD Biosciences). RNA was isolated using TRIZOL reagent (Invitrogen/Thermo Fischer (Waltham, MA), purified with Direct-zol RNA microprep kit (Zymo Research, Irvine, CA), and quantitated using 2100 Bioanalyzer (Agilent, Santa Clara, CA). QuantSeq 3’ mRNA-sequencing library prep kit for Illumina (FWD) (Lexogen, Vienna, Austria) was used to create cDNA libraries, amplify and sequence 3 million reads/sample using NextSeq 550 (Illumina, San Diego, CA). Libraries were evaluated for poor sample quality using FASTQC (version 0.11.8), and adaptors and contaminating non-specific reads removed using BBTools (version38.56). Reads were aligned to the GENCODE release 28 transcriptome using STAR (version 2.5.3a). BAM files were then converted to a gene count matrix using StringTie (version 1.3.6). Examination of differentially expressed genes between African American and European American ANA-, ANA+ healthy, and SLE patients were completed between sorted T cells, B cells and monocytes using DESeq2 normalized files and visualizations created using DESeq2 vst transformed data.