fig5

Figure 5. Flow cytometric characterization of ex vivo T cells: sex differences in the anti-inflammatory response to BIRT377. Naïve CD4 T cells were conditioned with (A-B) Th17 or (C-D) Treg inducing cytokines, with or without BIRT (500 ng/mL). After 4 days, all viable CD4 T cells were identified and analyzed for the expression of: (A) RORɣt (major transcription factor for Th17 cells) or (B) intracellular levels of pro-inflammatory cytokines IL-17A and TNF. BIRT377 treatment reduced RORɣt⁺ CD4 T cells (F_1,8 = 17.99, P = 0.002) and IL-17A protein production (F_1,8 = 24.3, P = 0.001) in females. (C-D) From Treg inducing culture, all viable CD4 T cells were analyzed for intracellular levels of (C) TGF-β1 and (D) IL-10. BIRT377 treatment increased intracellular TGF-β1 (F_1,5 = 12.85, P = 0.015) and IL-10 (F_1,6 = 10.57, P = 0.017) protein levels in females. (A-D) Representative flow cytometry plots are shown. Numbers represent the percentages of the (A) RORɣt or (B-D) cytokine positive CD4 T cells, where total CD4 T cells are taken as 100%. Corresponding isotype controls (stained with IgG, IgG2a or IgG2b fluorochrome conjugated antibody) for the intracellular staining are shown. Each experimental condition was run in 2-3-well replicates. Error bars represent variations in the well replicates. Data are representative of two independent experiments where T cells were pooled from n = 5 males or n = 5 females in each experiment. Viable cells were identified based on their light scatter properties (forward and side scatter plot) and viability dye staining. Viable cells were then gated for CD4 cell surface expression; only CD4⁺ T cells were included for further analysis. Positive staining for transcription factor and/or cytokines were determined based on staining with fluorochrome conjugated isotype controls (negative controls). *P values from post hoc comparisons ranges from P = 0.014 to P = 0.0005