Figure 5.
Figure 5. PD-1 phosphorylation is predominantly regulated by the phosphatase activity of SHP2. (A) AND-TCR T cell hybridomas expressing PD-1–EGFP not transduced or transduced with phosphatase-dead SHP1 (SHP1CS) or SHP2 (SHP2CS) were stimulated with unpulsed or 5 µM MCC88-103 prepulsed DC-1 cells not expressing or expressing PD-L1 for 2 min. The cells were lysed, immunoprecipitated with anti-GFP, and blotted for phosphor-tyrosine (4G10) or PD-1. The bottom four rows show WCL blotted with 4G10, anti-SHP1, SHP2, or PD-1. A representative of three independent experiments is shown. (B) The cells in A were stimulated with DC-1 cells not expressing or expressing PD-L1 or PD-L2 with 0.3 µM MCC88-103 for 16 h. Concentration of IL-2 was measured by ELISA. A representative of two independent experiments is shown. Error bars represent SD. (C) AND-TCR T cell hybridomas expressing CTLA-4 were further transduced with SHP1CS or SHP2CS. The cells were stimulated by immobilized anti-CD3ε (2C11, 10 µg/ml) and control IgG or anti-CTLA-4 (UC10, 10 µg/ml) for 16 h. Concentration of IL-2 was measured by ELISA. The cell number initially prepared was measured by a Cell Counting kit. A representative of two independent experiments is shown. Error bars represent SD. (D) AND-TCR T cell hybridomas expressing both PD-1-EGFP and SHP2CS were stimulated with 5 µM MCC88-103 prepulsed DC-1 cells not expressing or expressing PD-L1 for the indicated times. The cells were lysed, immunoprecipitated, and blotted as in A. A representative of three independent experiments is shown. (E) AND-TCR T cell hybridomas expressing SHP2CS and WT or mutant PD-1-EGFP were stimulated with 5 µM MCC88-103 prepulsed DC-1 cells not expressing or expressing PD-L1 for 2 min. The cells were lysed and blotted as in A. A representative of three independent experiments is shown. (F) The cells in A were conjugated with 5 µM MCC88-103 prepulsed DC-1 cells not expressing or expressing PD-L1. At 2 min after contact, the cells were fixed, stained with 4G10, and imaged by confocal microscopy. Bars, 5 µm. The right graph shows the percent intensity of phosphotyrosine at the T cell–DC-1 interface against that of the entire T cell area. A representative of two independent experiments is shown. *, P < 0.001 with Student’s t test. Horizontal bars represent the mean of the percent intensity of phosphotyrosine at the interface.

PD-1 phosphorylation is predominantly regulated by the phosphatase activity of SHP2. (A) AND-TCR T cell hybridomas expressing PD-1–EGFP not transduced or transduced with phosphatase-dead SHP1 (SHP1CS) or SHP2 (SHP2CS) were stimulated with unpulsed or 5 µM MCC88-103 prepulsed DC-1 cells not expressing or expressing PD-L1 for 2 min. The cells were lysed, immunoprecipitated with anti-GFP, and blotted for phosphor-tyrosine (4G10) or PD-1. The bottom four rows show WCL blotted with 4G10, anti-SHP1, SHP2, or PD-1. A representative of three independent experiments is shown. (B) The cells in A were stimulated with DC-1 cells not expressing or expressing PD-L1 or PD-L2 with 0.3 µM MCC88-103 for 16 h. Concentration of IL-2 was measured by ELISA. A representative of two independent experiments is shown. Error bars represent SD. (C) AND-TCR T cell hybridomas expressing CTLA-4 were further transduced with SHP1CS or SHP2CS. The cells were stimulated by immobilized anti-CD3ε (2C11, 10 µg/ml) and control IgG or anti-CTLA-4 (UC10, 10 µg/ml) for 16 h. Concentration of IL-2 was measured by ELISA. The cell number initially prepared was measured by a Cell Counting kit. A representative of two independent experiments is shown. Error bars represent SD. (D) AND-TCR T cell hybridomas expressing both PD-1-EGFP and SHP2CS were stimulated with 5 µM MCC88-103 prepulsed DC-1 cells not expressing or expressing PD-L1 for the indicated times. The cells were lysed, immunoprecipitated, and blotted as in A. A representative of three independent experiments is shown. (E) AND-TCR T cell hybridomas expressing SHP2CS and WT or mutant PD-1-EGFP were stimulated with 5 µM MCC88-103 prepulsed DC-1 cells not expressing or expressing PD-L1 for 2 min. The cells were lysed and blotted as in A. A representative of three independent experiments is shown. (F) The cells in A were conjugated with 5 µM MCC88-103 prepulsed DC-1 cells not expressing or expressing PD-L1. At 2 min after contact, the cells were fixed, stained with 4G10, and imaged by confocal microscopy. Bars, 5 µm. The right graph shows the percent intensity of phosphotyrosine at the T cell–DC-1 interface against that of the entire T cell area. A representative of two independent experiments is shown. *, P < 0.001 with Student’s t test. Horizontal bars represent the mean of the percent intensity of phosphotyrosine at the interface.

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