Figure 3.
Figure 3. SHP2, not SHP1, is transiently recruited to PD-1 microclusters. (A) AND-TCR T cell hybridomas expressing PD-1-Flag were stimulated by 5 µM MCC88-103 prepulsed DC-1 cells not expressing (−) or expressing PD-L1 (L1) or PD-L2 (L2) for 0 or 5 min. Cell lysate immunoprecipitated by anti-Flag or WCLs were blotted for SHP1, SHP2, or PD-1. A representative of five independent experiments is shown. (B) The cells in A were stimulated by nonexpressing or PD-L1–expressing DC-1 cells prepulsed with MCC88-103 at the indicated concentrations for 2 min. The cells were lysed, immunoprecipitated and blotted as in A. A representative of two independent experiments is shown. (C) The cells in A 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 four independent experiments is shown. (D) The PD-1–SHP2 association was analyzed as in C at much earlier time points indicated above. WCLs were blotted for SHP2, PD-1, phospho-PLCγ1, PLCγ1, phospho-Vav1, Vav1, phospho-Erk1/2, or Erk1/2. The graph shows fold intensities of SHP2 to PD-1 or phosphorylated PLCγ1, Vav1, or Erk to nonphosphorylated at each time point. A representative of two independent experiments is shown. (E) AND-TCR T cell hybridomas expressing PD-1–YPet (yellow) and ECFP-SHP1 (top) or -SHP2 (bottom, cyan) were plated onto an MCC88-103 prepulsed planar bilayer containing I-Ek–, ICAM-1–, CD80-, and PD-1–GPI and real-time imaged by confocal microscopy within 2 min after contact. FRET efficiency values presented as a pseudo-color scale. Histograms show fold fluorescent intensities of PD-1 (yellow) and SHP1 or SHP2 (cyan) on the two diagonal yellow lines in a DIC image. Bars, 5 µm. A representative of two independent experiments is shown. (F) AND-TCR T cell hybridomas expressing PD-1-YPet (yellow) and CFP for energy transfer CyPet-SHP1 (left, cyan) or -SHP2 (right, cyan) were conjugated with DC-1 cells not expressing (top) or expressing PD-L1 (middle) or PD-L2 (bottom) and real-time imaged by confocal microscopy at 2 min after contact. Histograms show fold fluorescent intensities of PD-1 (yellow) and SHP1 or SHP2 (cyan) on the diagonal yellow line in a DIC image. Bars, 5 µm. A representative of two independent experiments is shown.

SHP2, not SHP1, is transiently recruited to PD-1 microclusters. (A) AND-TCR T cell hybridomas expressing PD-1-Flag were stimulated by 5 µM MCC88-103 prepulsed DC-1 cells not expressing (−) or expressing PD-L1 (L1) or PD-L2 (L2) for 0 or 5 min. Cell lysate immunoprecipitated by anti-Flag or WCLs were blotted for SHP1, SHP2, or PD-1. A representative of five independent experiments is shown. (B) The cells in A were stimulated by nonexpressing or PD-L1–expressing DC-1 cells prepulsed with MCC88-103 at the indicated concentrations for 2 min. The cells were lysed, immunoprecipitated and blotted as in A. A representative of two independent experiments is shown. (C) The cells in A 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 four independent experiments is shown. (D) The PD-1–SHP2 association was analyzed as in C at much earlier time points indicated above. WCLs were blotted for SHP2, PD-1, phospho-PLCγ1, PLCγ1, phospho-Vav1, Vav1, phospho-Erk1/2, or Erk1/2. The graph shows fold intensities of SHP2 to PD-1 or phosphorylated PLCγ1, Vav1, or Erk to nonphosphorylated at each time point. A representative of two independent experiments is shown. (E) AND-TCR T cell hybridomas expressing PD-1–YPet (yellow) and ECFP-SHP1 (top) or -SHP2 (bottom, cyan) were plated onto an MCC88-103 prepulsed planar bilayer containing I-Ek–, ICAM-1–, CD80-, and PD-1–GPI and real-time imaged by confocal microscopy within 2 min after contact. FRET efficiency values presented as a pseudo-color scale. Histograms show fold fluorescent intensities of PD-1 (yellow) and SHP1 or SHP2 (cyan) on the two diagonal yellow lines in a DIC image. Bars, 5 µm. A representative of two independent experiments is shown. (F) AND-TCR T cell hybridomas expressing PD-1-YPet (yellow) and CFP for energy transfer CyPet-SHP1 (left, cyan) or -SHP2 (right, cyan) were conjugated with DC-1 cells not expressing (top) or expressing PD-L1 (middle) or PD-L2 (bottom) and real-time imaged by confocal microscopy at 2 min after contact. Histograms show fold fluorescent intensities of PD-1 (yellow) and SHP1 or SHP2 (cyan) on the diagonal yellow line in a DIC image. Bars, 5 µm. A representative of two independent experiments is shown.

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