PD-L1 inhibits the functions of mouse primary T cells deficient in both SHP1 and SHP2. (A) IBs validating the lack of SHP1 and SHP2 expression in dLck-Cre; Ptpn6^fl/fl; Ptpn11^fl/fl (SHP1/2 DKO) CD8⁺ T cells freshly isolated from dLck-Cre;Ptpn6^fl/fl;Ptpn11^fl/fl mice. β-actin and GAPDH IBs indicate the input of each sample. (B) Flow cytometry histograms showing PD-1 surface expression (black traces) in SHP1/2 DKO CD8⁺ T cells at 24, 48, and 72 h after stimulation. Gray traces correspond to isotype antibody–stained cells. (C) SHP1/2 DKO CD8⁺ T cells were stained with CTV dye and stimulated with protein G Dynabeads precoated with IgG alone, anti-CD3/anti-CD28/IgG, anti-CD3/anti-CD28/PD-L1–Fc^low, or anti-CD3/anti-CD28/PD-L1–Fc^high, respectively (see Materials and methods). Flow cytometry diagrams on the left show the degrees of CTV dilution as a consequence of cell proliferation at 24, 48, and 72 h under the indicated stimulation condition. The number denotes percentage of proliferating cells. Bar graphs on the right summarize the percentage of proliferating cells in three technical replicates. (D) Percentage of IL-2–positive cells 24 and 48 h after the indicated stimulation, measured by flow cytometry. (E) Percentage of IFN-γ⁺ cells 24 and 48 h after the indicated stimulation, measured by flow cytometry. (F–H) Same as C–E, except using WT CD8⁺ T cells freshly isolated from Ptpn6^fl/fl;Ptpn11^fl/fl mice. Data in this figure are presented as means ± SD. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. ns, not significant; Student’s t test (n = 3).