Quantifying Antigen-Specific CD4+ T Cells in Scleroderma

PBMCs from ATA-positive (n=11) and ATA-negative (n=11) patients with scleroderma were isolated by Ficoll density-gradient centrifugation (Ficoll-Paque Plus, GE Healthcare). The cells were resuspended to 1.5 × 10⁶ cells/well in RPMI medium supplemented with 5% autologous serum, as previously described (9 (link)). Briefly, PBMCs were pre-incubated with anti-human CD40 blocking antibody (1 ug/ml; G28.5, BioLegend) prior to stimulation for 18 hours with 2.5 uM of each candidate TOP1 peptide (Table 1; >95% purity, Genscript), or 1.45 ug/mL human recombinant TOP1 protein (Genscript). Following stimulation, cells were stained with BV510-conjugated anti-CD3 (OKT3, BioLegend), Pacific Blue-conjugated anti-CD4 (RPA-T4, BD Pharmingen), APC-H7-conjugated anti-CD8 (SK1, BD Biosciencies), PE-conjugated anti-CD154 (TRAP1, BD Pharmingen), and Live/Dead Fixable Stain (Molecular Probes). The percentage of live CD4+ T cells that upregulated CD154 was quantified by flow cytometry (FACSAria, BD Biosciences; FCS Express software, De Novo Software). A cutoff of greater than the 95^th percentile of the responses observed amongst the ATA-negative patients (%CD154+CD4+Tcells > 0.015%) was used to define a positive CD4+ T cell response to antigen stimulation.