Fig. 2

The different order in which IFN-α and TCR activate CD4⁺T cells can lead to the activation of different STAT pathways. CD4⁺T cells purified from PBMCs of HDs were stimulated at different time points, then analyzed using phosflow technology. A Percentages of pSTAT1⁺ cells and pSTAT4⁺ cells after TCR stimulated for 20 min, 24 h, 48 h, or 120 h. B, C Percentages and MFI of pSTAT1⁺ cells, STAT3⁺ cells, STAT4⁺ cells, and pSTAT5⁺ cells under the 20-min stimulation of IFN-α with specified concentration without TCR. D After 48 h of TCR-stimulation, percentages of pSTAT1⁺cells, STAT3⁺cells, STAT4⁺cells, and pSTAT5⁺cells were measured after further stimulation with or without IFN-α (20 ng/ml) for 20 min. E Schematic diagram of the setting in-sequence or out-of-sequence for culture conditions. F Percentages of pSTAT1⁺ cells, STAT3⁺ cells, STAT4⁺ cells, and pSTAT5⁺ cells after stimulation under TCR, in-sequence or out-of-sequence condition for 5 days. G After 5 days of TCR-stimulation, percentages of pSTAT1⁺cells, STAT3⁺cells, STAT4⁺cells, and pSTAT5.⁺cells were measured after further stimulation with or without IFN-α for 20 min. Data are mean ± SEM of three independent experiments using different donors. un-stimul, un-stimulation; min, minutes; h, hours; d, days. *P < 0.05