Somatic Mutations in CD8+ T Cells in Patients with Chronic Immune Thrombocytopenia Are Associated with Increased Clonality and Cytotoxic Phenotype of CD8+ T Cells

Background The role of self-antigen-targeting T cells is well established in multiple autoimmune disorders. In immune thrombocytopenia (ITP), CD8+ cytotoxic T cells target both peripheral blood platelets and bone marrow megakaryocytes. In addition, CD4+ helper T cell imbalance and cytokine secretion leads to the release of autoantibodies by B cells favoring the destruction of platelets. In order to understand if somatic mutations play a role and drive the aberrant immune responses in ITP, we analyzed sorted CD4+ and CD8+ T cells with deep sequencing panel. Methods The study population consisted of 13 adult patients diagnosed with chronic ITP (median age 47 years) at least one year before the first sampling. The median number of lines of therapy was 4, (range 0-8). In addition, 11 healthy control samples were collected for T cell phenotyping and TCR analysis (median age 63 years). After separation of peripheral blood mononuclear cells, immunophenotyping by flow cytometry was performed for CD4+ and CD8+ T cells and both fractions were sorted using magnetic beads. The CD4+ and CD8+ clonality was analyzed by deep sequencing of TCRB CDR3 using Adaptive platform. Custom-made deep sequencing gene panel covering 2433 genes related to immunity and cancer was used to analyze somatic mutations from 11 ITP patients. Mean target coverage was 567X (range 405-757X). Somatic variants with over 2% variant allele frequency (VAF) were called using Varscan2-based bioinformatics tool and CD4+ and CD8+ fractions were used as each other's germline control. Candidate somatic variants were visually validated with Integrative Genomics Viewer. Results ITP patients harbored clonal TCR rearrangements both in CD8+ and CD4+ fractions. The median percentage of the largest T cell clone of all TCR rearrangements in an individual sample was 9.5% (range 1.8%-31.9%) in CD8+ cells and 7.1% (range 0.9-13.6%) in CD4+. The median clone size or clonality index did not differ between ITP patients and healthy controls. Age correlated with the largest rearrangement and clonality index both in CD4+ and CD8+ fractions in ITP patients (r=0.70, p=0.015 and r=0.60, p=0.043 for CD4+ and r=0.63, p=0.033 and r=0.77, p=0.0050 for CD8+), but no correlation was observed in healthy controls. The size of the maximum CD8+ TCR rearrangement and clonality index correlated positively with the percentage of highly cytotoxic CD8+CD57+ (r=0.80, p=0.0029 and r=0.90, p=0.0002) and terminally differentiated CD8+ effector m