Figure 3.

Outcome of PD-1-PD-L1 Engagement at T Cell-APC Interface

(A) Jurkat-PD-1^OST cells were stimulated with Raji cells that have been preincubated in the absence (−) or presence (+) of 200 ng/mL SEE and lysed 2 min after the initiation of cell-cell contact. Immunoblot analysis of equal amounts of lysates from the specified conditions probed with antibody to phosphorylated proteins (Anti-p-Tyr) or with phospho-tyrosine-specific antibodies directed against SLP76 pY128, ZAP70 pY493, LAT pY171 or VAV1 (loading control). Left margin, molecular size in kilodaltons (kDa). Data are representative of three independent experiments.

(B) Expression of CD3, CD28, PD-1, PD-L1, PD-L2, BTLA, and HVEM at the surface of Jurkat cells and Jurkat-PD-1^OST cells, analyzed using flow cytometry.

(C) Expression of HLA-DR, PD-L1, PD-L2, CD80, CD86, and HVEM at the surface of Raji cells and Raji-PD-L1 cells, analyzed using flow cytometry. In (B) and (C), gray shaded curves correspond to isotype-matched control antibody (negative control), and data are representative of two independent experiments.

(D) IL-2 production by Jurkat and Jurkat-PD-1^OST cells stimulated for 24 h with either Raji or Raji-PD-L1 cells in the absence (0) or presence of the specified amounts of SEE. Data are representative of three independent experiments, and mean and SEM are shown.

(E) Jurkat-PD-1^OST cells stimulated at 37°C with Raji or Raji-PD-L1 cells preincubated in the absence (–) or presence (+) of 200 ng/mL SEE and lysed 2 and 5 min after the initiation of cell-cell contact. Immunoblot analysis of equal amounts of lysates from the specified conditions subjected to affinity purification on Strep-Tactin-Sepharose beads and probed with antibody to phosphorylated proteins (Anti-p-Tyr). Also shown are loading controls probed with anti-PD-1 antibody. Left margin, molecular size in kilodaltons (kDa). Data are representative of two independent experiments.