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A6.02 Somatic mutations in clonally expanded CD8+ T cells in patients with newly diagnosed rheumatoid arthritis
  1. T Kelkka1,*,
  2. P Savola1,6,*,
  3. H Rajala1,
  4. A Kuuliala2,
  5. K Kuuliala2,
  6. S Eldfors3,
  7. P Ellonen3,
  8. S Lagström3,
  9. RK Khajuria1,
  10. T Jaatinen4,
  11. R Koivuniemi5,
  12. H Repo2,
  13. J Saarela3,
  14. K Porkka1,*,
  15. M Leirisalo-Repo5,*,
  16. S Mustjoki6,*
  1. 1Hematology Research Unit Helsinki, University of Helsinki and Department of Hematology, Comprehensive Cancer Center, Helsinki University Hospital, Helsinki, Finland
  2. 2Department of Bacteriology and Immunology, Haartman Institute, University of Helsinki, Helsinki, Finland
  3. 3Institute for Molecular Medicine Finland (FIMM), University of Helsinki, Helsinki, Finland
  4. 4Clinical Laboratory, Finnish Red Cross Blood Service, Helsinki, Finland
  5. 5Department of Medicine, Division of Rheumatology, University of Helsinki and Helsinki University Hospital, Helsinki, Finland
  6. 6Department of Clinical Chemistry and Hematology, University of Helsinki, Helsinki, Finland
  7. *Equal contribution

Abstract

Background and objectives Clonally expanded CD8+ lymphocytes are known to exist in patients with rheumatoid arthritis (RA). Interestingly, in large granular lymphocyte (LGL) leukaemia (a haematological malignancy with indolent disease course), CD8+ lymphocytes expand and the expanded clones harbour somatic mutations. LGL leukaemia patients with STAT3 mutated CD8+ lymphocytes have significantly increased risk of developing autoimmune manifestations, RA being a common autoimmune co-morbidity.

As somatic mutations in CD8+ lymphocytes associate with autoimmunity in LGL leukaemia, we wanted to examine if similar phenomenon exists in newly diagnosed RA patients without known LGL lymphoproliferation.

Materials and methods Lymphocyte clonality was analysed by flow cytometry using vbβ -staining kit (Beckman Coulter) and the major clones were confirmed by TCR deep sequencing.

Deep sequencing of 986 selected immune-related genes was performed from CD8+ T cells using CD4+ lymphocytes as germline control. The coverage was 200–6000x enabling reliable calling of low frequency mutations. Candidate mutations were validated using targeted deep amplicon sequencing.

Results 43% (n = 31) of the newly diagnosed RA patients in our patient cohort (n = 68) had CD8+ lymphocyte expansions exceeding 10% of the total CD8+ T cells.

The largest clones were subjected to targeted deep sequencing. We identified somatic gene variants in five (n = 5) patients and in one healthy control. Most of these variants were non-synonymous coding single nucleotide changes in genes important for immune functions (C5, PADI4, IRF1, SLAMF6, BST1 and CLEC10A), or proliferation (PTPRO, PADI4, PROM1, CDK12 and CLIP2). Sequencing of FACS sorted CD8+ lymphocytes confirmed that the expanded clone harboured the identified mutations.

Conclusions In RA patients, somatic mutations accumulate in the CD8+ T cells during clonal expansion. Related findings have recently been reported in the myeloid lineage, where somatic variants are found to accumulate even in healthy subjects in a process called clonal haematopoiesis. Importantly, we focused on CD8+ lymphocytes that have undergone thymic education and thus we report acquired somatic variants in mature cytotoxic lymphocytes in a non-malignant set-up.

Although more research is needed to address the possible pathogenicity of the identified mutations, these findings provide an interesting link between autoimmunity and cancer.

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