Figure 4.

CD28 blockade induces checkpoint signaling. Expression of programmed death 1 (PD-1) and cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) on sorted naive T-cells 24 h after a secondary allogeneic mixed leukocyte reaction (MLR) without (−) or with (+) prior α-huCD28. Cells were gated according to: CD3⁺/live cells/CD4⁺/CTLA-4⁺⁺ or CD3⁺/live cells/CD4⁺/PD-1⁺CTLA-4⁺⁺. CTLA-4⁺⁺ and PD-1⁺CTLA-4⁺⁺ α-huCD28 pre-treated cells were analyzed for cellular proliferation by carboxyfluorescein succinimidyl ester (CFSE)/CPD dilution. (A) Representative dotplots of PD-1 and CTLA-4 expression on naive CD4⁺ T-cells in a secondary MLR (day 1). Numbers represent percent within the CD3⁺ cell population. Cumulative results of CTLA-4 and PD-1 expressions of naive T-cells with (▲) or without (○) prior α-huCD28 after a 1 day secondary allo-MLR. Points represent the mean of replicates from different recipient–donor pairs. (B) T-cells with prior CD28 blockade, highly expressing CTLA-4 or PD-1/CTLA-4, were analyzed for proliferation (CFSE^neg/CPD^neg, proliferated). Representative dotplots of CFSE/CPD staining are shown; bar graphs show the cumulative results of percent proliferated T-cells. (C) CD3⁺ T-cell lysates from a primary MLR with (+) or without (−) α-huCD28 for 0–96 h were examined by Western blot to detect phospho-protein phosphatase 2A (pPP2A), total protein phosphatase 2A (PP2A), Akt phosphorylated on threonine 308 (pAkt Thr) or serine 473 residues (pAkt Ser), total Akt, and vinculin as a control. For densitometric analysis (ratio of phosphorylated/total protein) the ImageJ program was used. *p < 0.05.