Figure 1.
Figure 1. PD-1 is translocated to TCR microclusters by PD-1–PD-L1 binding. (A) CD4+ T cells were purified from AND-Tg Pdcd1−/− Rag2−/− mice, stimulated with irradiated B10.BR whole splenocytes with 5 µM MCC88-103 peptide, and retrovirally transduced with PD-1-EGFP. The cells were plated onto an MCC88-103 (10 µM) prepulsed planar bilayer containing I-Ek–GPI (250/µm2), ICAM-1–GPI (100/µm2), and CD80-GPI (80/µm2) without (top) or with (bottom) PD-L1–GPI (150/µm2) and real-time imaged by TIRF microscopy (times are above images; Video 1). Bars, 5 µm. A representative of five independent experiments is shown. (B) Clustering and centripetal movement of PD-1 on the diagonal yellow line in A is presented as horizontal elements in kymographs. Bars, 5 µm. (C) The cells expressing PD-1–EGFP (green) in A were prestained with DyLight 649–labeled H57 Fab (red), plated onto a planar bilayer as in A, and real-time imaged by confocal microscopy at 2 (left) or 20 (right) 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 three independent experiments is shown. (D) The graph shows the percentage of TCR microclusters colocalized by PD-1 at 2 min after contact in C (n = 5). Error bars represent SD. (E) AND-Tg Rag2−/− CD4+ T cells were stimulated for 2 d, directly stained with DyLight 649–labeled H57 Fab (red) and DyLight 488–labeled anti–mPD-1 (RMA1-30; green), and imaged as in A. Bars, 5 µm. A representative of four independent experiments is shown. (F) The graph shows the percentage of TCR microclusters colocalized by PD-1 at 2 min after contact in E (n = 10). Error bars represent SD. (G) The cells expressing PD-1–EGFP (green) in A were stained with DyLight 549–labeled H57 Fab (red) and imaged on an MCC88-103 prepulsed planar bilayer containing I-Ek–GPI, ICAM-1–GPI, CD80-GPI, and Cy5-labeled PD-L1–GPI (150/µm2, cyan). Bars, 5 µm. A representative of two independent experiments is shown. (H) Images of cells expressing WT PD-1-EGFP in C at 20 s (left) or 20 min (right) after contact. The yellow squares (a and b) in the left panels are magnified in the right three panels. Yellow arrowheads, a TCR–PD-1 microcluster; red arrowheads, a TCR microcluster not colocalized by PD-1; green arrowheads, a PD-1 microcluster not colocalized by TCR. Bars, 5 µm. A representative of two independent experiments is shown.

PD-1 is translocated to TCR microclusters by PD-1–PD-L1 binding. (A) CD4+ T cells were purified from AND-Tg Pdcd1−/− Rag2−/− mice, stimulated with irradiated B10.BR whole splenocytes with 5 µM MCC88-103 peptide, and retrovirally transduced with PD-1-EGFP. The cells were plated onto an MCC88-103 (10 µM) prepulsed planar bilayer containing I-Ek–GPI (250/µm2), ICAM-1–GPI (100/µm2), and CD80-GPI (80/µm2) without (top) or with (bottom) PD-L1–GPI (150/µm2) and real-time imaged by TIRF microscopy (times are above images; Video 1). Bars, 5 µm. A representative of five independent experiments is shown. (B) Clustering and centripetal movement of PD-1 on the diagonal yellow line in A is presented as horizontal elements in kymographs. Bars, 5 µm. (C) The cells expressing PD-1–EGFP (green) in A were prestained with DyLight 649–labeled H57 Fab (red), plated onto a planar bilayer as in A, and real-time imaged by confocal microscopy at 2 (left) or 20 (right) 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 three independent experiments is shown. (D) The graph shows the percentage of TCR microclusters colocalized by PD-1 at 2 min after contact in C (n = 5). Error bars represent SD. (E) AND-Tg Rag2−/− CD4+ T cells were stimulated for 2 d, directly stained with DyLight 649–labeled H57 Fab (red) and DyLight 488–labeled anti–mPD-1 (RMA1-30; green), and imaged as in A. Bars, 5 µm. A representative of four independent experiments is shown. (F) The graph shows the percentage of TCR microclusters colocalized by PD-1 at 2 min after contact in E (n = 10). Error bars represent SD. (G) The cells expressing PD-1–EGFP (green) in A were stained with DyLight 549–labeled H57 Fab (red) and imaged on an MCC88-103 prepulsed planar bilayer containing I-Ek–GPI, ICAM-1–GPI, CD80-GPI, and Cy5-labeled PD-L1–GPI (150/µm2, cyan). Bars, 5 µm. A representative of two independent experiments is shown. (H) Images of cells expressing WT PD-1-EGFP in C at 20 s (left) or 20 min (right) after contact. The yellow squares (a and b) in the left panels are magnified in the right three panels. Yellow arrowheads, a TCR–PD-1 microcluster; red arrowheads, a TCR microcluster not colocalized by PD-1; green arrowheads, a PD-1 microcluster not colocalized by TCR. Bars, 5 µm. A representative of two independent experiments is shown.

This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal