Figure 7.
Figure 7. Colocalization of PD-1 and TCRs at microclusters is required for PD-1–mediated T cell suppression. (A) A diagram of the EGFP-tagged mPD-1–hCD22–mPD-1 or mPD-1–hCD4–mPD-1 chimeras. The murine PD-1 IgV domain was attached to the second to sixth (Igx5), third to sixth (Igx4), fifth and sixth (Igx2), or sixth (Igx1) IgC domains of hCD22 or the stalk region of hCD4 (Igx0). The cytoplasmic tail of hCD22 or hCD4 is exchanged by that of murine PD-1 containing two tyrosine motifs, ITIM and ITSM, and further tagged with EGFP at the C terminus of mPD-1. (B) AND-TCR T cell hybridomas were not transduced (−) or transduced with EGFP-tagged WT PD-1 (WT) or elongated mPD-1 chimeras shown in A. The histograms show expression of cell surface PD-1 (top) or EGFP (bottom). (C) AND-Tg Pdcd1−/− Rag2−/− CD4+ T cells were reconstituted with the EGFP-tagged elongated PD-1, mPD-1–hCD22/CD4–mPD-1-EGFP constructs (Igx0–5; green) in A, stained with DyLight 649–labeled H57 Fab (red), plated onto an MCC88-103 prepulsed planar bilayer containing I-Ek–, ICAM-1–, and CD80- (top) plus PD–L1-GPI (bottom six rows), and real-time imaged by confocal microscopy at 2 min after contact. Histograms show fold fluorescent intensities of TCR-β (red) and PD-1 (green) on the two diagonal yellow lines in the merged images. Bars, 5 µm. A representative of two independent experiments is shown. (D) The left graph shows the percentage of TCR microclusters colocalized by WT or elongated PD-1 in C (n = 10). The right graph shows the percentage of cells forming >50% of TCR microclusters colocalized by PD-1 in C (n = 50). Error bars represent SD. (E) AND-TCR T cell hybridomas expressing the EGFP-tagged elongated PD-1 chimeras in B were stimulated with DC-1 cells expressing PD-L1 or PD-L2 with 0.3 µM OVA88-103 for 16 h. Immobilized anti-CD3ε (2C11) and anti-CD28 (PV-1) were used for control stimulation. Concentration of IL-2 was measured by ELISA. A representative of three independent experiments is shown. Error bars represent SD. (F) The cells in B were stimulated with 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 SHP2 or PD-1. WCLs (bottom six rows) were blotted for PD-1, phospho-Vav1, Vav1, phospho-PLCγ1, PLCγ1, phospho-Erk1/2, or Erk1/2. A representative of three independent experiments is shown. (G) The cells in B were further transduced with SHP2CS and stimulated as in F. The cells were lysed, immunoprecipitated with anti-GFP (top two rows), and blotted with 4G10 (top) or anti-GFP (middle). WCLs (bottom) were blotted for SHP2. A representative of two independent experiments is shown.

Colocalization of PD-1 and TCRs at microclusters is required for PD-1–mediated T cell suppression. (A) A diagram of the EGFP-tagged mPD-1–hCD22–mPD-1 or mPD-1–hCD4–mPD-1 chimeras. The murine PD-1 IgV domain was attached to the second to sixth (Igx5), third to sixth (Igx4), fifth and sixth (Igx2), or sixth (Igx1) IgC domains of hCD22 or the stalk region of hCD4 (Igx0). The cytoplasmic tail of hCD22 or hCD4 is exchanged by that of murine PD-1 containing two tyrosine motifs, ITIM and ITSM, and further tagged with EGFP at the C terminus of mPD-1. (B) AND-TCR T cell hybridomas were not transduced (−) or transduced with EGFP-tagged WT PD-1 (WT) or elongated mPD-1 chimeras shown in A. The histograms show expression of cell surface PD-1 (top) or EGFP (bottom). (C) AND-Tg Pdcd1−/− Rag2−/− CD4+ T cells were reconstituted with the EGFP-tagged elongated PD-1, mPD-1–hCD22/CD4–mPD-1-EGFP constructs (Igx0–5; green) in A, stained with DyLight 649–labeled H57 Fab (red), plated onto an MCC88-103 prepulsed planar bilayer containing I-Ek–, ICAM-1–, and CD80- (top) plus PD–L1-GPI (bottom six rows), and real-time imaged by confocal microscopy at 2 min after contact. Histograms show fold fluorescent intensities of TCR-β (red) and PD-1 (green) on the two diagonal yellow lines in the merged images. Bars, 5 µm. A representative of two independent experiments is shown. (D) The left graph shows the percentage of TCR microclusters colocalized by WT or elongated PD-1 in C (n = 10). The right graph shows the percentage of cells forming >50% of TCR microclusters colocalized by PD-1 in C (n = 50). Error bars represent SD. (E) AND-TCR T cell hybridomas expressing the EGFP-tagged elongated PD-1 chimeras in B were stimulated with DC-1 cells expressing PD-L1 or PD-L2 with 0.3 µM OVA88-103 for 16 h. Immobilized anti-CD3ε (2C11) and anti-CD28 (PV-1) were used for control stimulation. Concentration of IL-2 was measured by ELISA. A representative of three independent experiments is shown. Error bars represent SD. (F) The cells in B were stimulated with 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 SHP2 or PD-1. WCLs (bottom six rows) were blotted for PD-1, phospho-Vav1, Vav1, phospho-PLCγ1, PLCγ1, phospho-Erk1/2, or Erk1/2. A representative of three independent experiments is shown. (G) The cells in B were further transduced with SHP2CS and stimulated as in F. The cells were lysed, immunoprecipitated with anti-GFP (top two rows), and blotted with 4G10 (top) or anti-GFP (middle). WCLs (bottom) were blotted for SHP2. A representative of two independent experiments is shown.

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