Fig. 5: PD-1 signaling activates SHP2 to induce dephosphorylation of γ_c^Y357.

a Association of γ_c with SHP2. Jurkat-PD-1 cells were stimulated with PHA (150 ng/mL) in the presence of plate-bound hPD-L1-Fc fusion protein or control hIgG1 (2 μg/mL) for the indicated times before co-immunoprecipitation and immunoblotting analysis with the indicated antibodies. b Effects of SHP2 on dephosphorylation of γ_c. HEK293 cells were transfected with the indicated plasmids for 24 h before co-immunoprecipitation and immunoblotting analysis with the indicated antibodies. c JAK3 mediates phosphorylation of γ_c^Y357. HEK293 cells were transfected with the indicated plasmids for 24 h before co-immunoprecipitation and immunoblotting analysis with the indicated antibodies. d IL-7 treatment induces γ_c^Y357 phosphorylation. Control or γ_c-deficient HPB-ALL cells were stimulated with IL-7 (100 ng/mL) for the indicated times before co-immunoprecipitation and immunoblotting analysis with the indicated antibodies. e SHP2-deficiency impairs PD-L1-induced γ_c^Y357 dephosphorylation. Control or SHP2-deficient Jurkat-PD-1 cells were stimulated with PHA (150 ng/mL) in the presence of plate-bound hPD-L1-Fc fusion protein for the indicated times before co-immunoprecipitation and immunoblotting analysis with the indicated antibodies. f SHP2-deficiency enhances IL-7-induced γ_c^Y357 phosphorylation. Control or SHP2-deficient HPB-ALL cells were stimulated with IL-7 (100 ng/mL) for the indicated times before co-immunoprecipitation and immunoblotting analysis with the indicated antibodies. g SHP2-deficiency promotes γ_c family cytokine-induced phosphorylation of STAT5^Y694/Y699. Control or SHP2-deficient HPB-ALL cells were stimulated with IL-9 (100 ng/mL) for the indicated times before immunoblotting analysis with the indicated antibodies (left panels). Control or SHP2-deficient CTLL2 cells were stimulated with IL-2 (400 IU/mL) for the indicated times before immunoblotting analysis with the indicated antibodies (right panels). h Effects of γ_c^Y357F mutant on the γ_c family cytokine-induced phosphorylation of STAT5 ^Y694/Y699. γ_c-deficient HPB-ALL cells were reconstituted with wild-type γ_c or γ_c^Y357F mutant and then stimulated with IL-7 (100 ng/mL) or IL-9 (100 ng/mL) for the indicated time lengths before immunoblotting analysis with the indicated antibodies. All the experiments were repeated for at least two times with similar results.