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. 2014 Oct 30;6(5):1290–1299. doi: 10.4161/mabs.29758

Figure 5.

Figure 5.

Absence of PD-1 mediated regulation of T cell function in CD28SA- stimulated T cells. (A) Schematic of the protocol used to investigate the functional significance of PD-1 pathway on NIB1412-activated CD4+ T cells. (B) Human PBMCs were stimulated for 48 h with plate-bound anti-CD3 mAb (CD3, 5 μg/ml); NIB1412 (NIB1412, 10 μg/ml); anti-CD3 mAb and NIB1412 (CD3 and NIB1412); control category included cells without any treatment (Control). Cells were then re-stimulated with anti-CD3 only (CD3,1 μg/ml) or with anti-CD3 and 10 μg/ml of rPD-L1 (CD3 and rPD-L1). Cells were harvested and stained with fluorochrome-conjugated anti-CD4 antibody, fixed, permeabilized, stained with fluorochrome-conjugated anti-IFNγ antibody and analyzed by flow cytometry. The CD4+ population is shown in light gray and the CD4− population in dark gray. The percentages of the CD4+ IFNγ+ cells are shown in the upper right quadrant. Results are representative of four independent experiments.