- University of Helsinki
- Karolinska Institutet
- Cleveland Clinic
Background: Vigorous cell division increases the probability of acquiring mutations during DNA replication. Such may occur in T cells when they recognize their cognate antigen and proliferate. Conventionally, acquired somatic mutations have been associated with the development of malignancy. In T cells, somatic mutations may also lead to autoimmunity, as in large granular lymphocyte (LGL) leukemia with STAT3 mutations. Somatic STAT3 mutations have also been detected in patients with immune-mediated bone marrow failure (BMF) disorders without concomitant LGL lymphoproliferation. These BMF disorders, aplastic anemia (AA) and hypoplastic myelodysplastic syndromes (hMDS), are responsive to immunosuppressive therapy, indicating autoimmune nature of diseases. In AA and hMDS, cytotoxic T cell receptor (TCR) repertoire is skewed and clonal autoreactive cytotoxic T cells are thought to cause hematopoietic stem cell destruction. Because previous findings suggest that these immune-mediated BMF disorders and LGL leukemia share common features, we investigated whether also in AA and hMDS cytotoxic T cells accumulate acquired somatic mutations in genes that are important for cell survival and immune regulation.Methods: We sorted CD8+ and CD4+ T cell fractions from blood and BM samples of patients diagnosed with AA (n=24, median age 60) or hMDS (n=13, median age 64). TCR repertoire was analyzed with TCRB CDR3 deep sequencing or TCR Vb-antibody based flow cytometry, and all selected patients had at least one cytotoxic CD8+ T cell clone gt;7 CD4+ and CD8+ T cells were then sequenced with our custom gene panel covering 2000 essential genes of innate and adaptive immunity with mean sequencing depth of 270x. Results were compared to healthy controls (n=9, median age 48). For each subject, somatic mutations in CD8+ cells were called with VarScan2 using CD4+ cells as a "germline" control and vice versa. To reduce possible false positives, all detected mutations were evaluated visually in Integrative Genomics Viewers by two independent researchers in a blinded manner, with agreed and precise criteria to distinguish real and false mutations. Concordant results were validated with amplicon sequencing. The enrichment of cell specific somatic mutations in biological pathways among AA and MDS patients was compared to healthy controls.Results: Somatic mutations in CD4+ and CD8+ T cells were detected both in patients (29/35, 83 and controls (7/9, 78. The number of found somatic mutations was associated with the TCR clonality index in cytotoxic CD8+ T cells (r=0.76, plt;0.0001). Although single mutations were frequently found in healthy controls' T cells, the number of mutations per subject was significantly higher in patients' CD8+ T cells compared to controls (mean[range] of found somatic mutations per subject for each group: AA CD4 0.9[0-6] and CD8 3.5[0-10]; MDS CD4 0.7[0-2] and CD8 2.3[0-8]; controls CD4 1.1[0-3] and CD8 0.9[0-3]). The number of mutations did not correlate with age. In cases from which both blood and BM samples were available, mutations were detected in T cells derived from both compartments. Most of the genes that had somatic mutations in healthy controls had also mutations in AA and MDS patients (e.g. DNMT3A, TLR2). Although mutations were mostly distinct in individual patients, they accumulated in important immune regulatory pathways. When compared to healthy controls, AA and hMDS patients had more mutations in JAK-STAT signaling (SOCS7, STAT3, PTPN2), MAPK signaling (KRAS, PTPN5, TP53), other cancer-related pathways and in pathways related to viral carcinogenesis and HTLV-I infection. Notably, most of the BMF patients' T-cell mutations were in other genes than STAT3 or genes related to clonal hematopoiesis.Conclusions: Our findings confirm that a subset of immune-mediated BMF disorders may have similar pathogenesis to LGL leukemia highlighting the importance of JAK-STAT signaling pathway in immunoregulation and autoimmunity. Acquired somatic mutations in inflammatory cells leading to dysregulation of immune system may be the common denominator in the pathogenesis of autoimmune disorders and hematologic malignancies. Deep molecular depth analysis and functional studies on found somatic mutations can further illuminate the pathogenesis of immune-mediated BMF syndromes and other autoimmune disorders .Disclosures Maciejewski: Apellis Pharmaceuticals: Consultancy; Ra Pharma: Consultancy; Alexion Pharmaceuticals, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: Speaker Fees. Mustjoki: Novartis: Honoraria, Research Funding; BMS: Honoraria, Research Funding; Pfizer: Honoraria, Research Funding; Ariad: Research Funding; Celgene: Honoraria.↵* Asterisk with author names denotes non-ASH members.
|Tila||Julkaistu - 2017|
|OKM-julkaisutyyppi||A1 Julkaistu artikkeli, soviteltu|