Quantifying CD4+ T-cell Proliferation Suppression

Either ex vivo- or 7-day-cultured CD4⁺ T-cells were placed in flow cytometric suppression assays, as described previously [11 , 13 (link), 14 (link)]. Briefly, responder CD4⁺ T-cells were stained with CFSE, followed by culture 1 μg/ml of fixed anti-CD3 (eBiosciences, 16-0037-85) and 1μg/ml of fixed anti-CD28 (eBiosciences, 16-0289-85) in the presence or absence of ex vivo sorted autologous bulk CD8⁺ T-cells at 1:0, 1:1 and 1:0.5 CD4:CD8 cell concentrations. On day 7 of culture, cells were stained for anti-CD4 PE-Cy7 (BD, 557852), anti-CD3 AlexaFluor700 (BD, 557943), anti-CD8 Pacific Blue (Biolegend, 344718) and flow cytometrically assessed for CD4 proliferating fraction (CFSE dilution). Percent proliferation and percent suppression were calculated as described previously [13 (link)]. Other antibodies utilized include: CD45RO Pacific Blue, Biolegend 304216; CD45RO PE, Biolegend 304244; CD45RA Fitc, BD 555488; CD8 PE, BD 340046; CD8 BV786, BD 563823; CD4 BV786, BD 563877; CD4 APC, BD 561841; CD4 PE, BD 555347; CD3 APC, Biolegend 300412; CD25 PE, BD 555432; CD25 APC, BD 555434; CD25 PacBlue, Biolegend 356130. Cells were analyzed on a BD LSRII or Cytek Aurora. Data were analyzed with FlowJo software v10.