TCR repertoire profiling reveals HLA-dependent and persistent T-cell expansions in synovial fluid of patients with spondyloarthritis
TCR repertoire profiling reveals HLA-dependent and persistent T-cell expansions in synovial fluid of patients with spondyloarthritis
Venous blood or synovial fluid was collected in EDTA Vacutainers (BD). For three patients samples were collected at two time points 9-23 months apart (two peaks of synovitis, with some remission in between). The rest of the samples were collected once during a synovitis episode. The samples were diluted three times in PBS and layered onto Ficoll (PanEco, Russia), then centrifuged at 300 g for 40 minutes at room temperature and washed 2 times with PBS. At least 3x10^6 of obtained MNCs were freshly lysed for total T-cell samples or used to isolate CD+ and CD8+ T-cell subsets. CD4+ T-cells were isolated from MNCs using anti-CD4 magnetic beads (Dynabeads, ThermoFisher), and then supernatant containing unbound cells was used to isolate CD8+ T-cells using anti-CD8 magnetic beads (Dynabeads, ThermoFisher). The obtained cell subsets were lysed with TRIzol reagent (Invitrogen). Total RNA was extracted with Trizol following manufacturer protocol Libraries of TCRbeta chains were prepared as previously described (Zvyagin et al., Leukemia, 2017). In brief, total RNA was used for cDNA synthesis with 5'RACE template switch technology to introduce universal primer binding site, first sample barcode and Unique Molecular Identifiers (UMI) at the 5' end of RNA molecules. Primers complementary to TCRbeta constant segments were used for cDNA synthesis initiation. cDNA was amplified in two subsequent PCR steps. During the second PCR step, second sample barcode and sequence adapters were introduced to the libraries.