Fig 2: Outcome of PD-1-PD-L1 Engagement at T Cell-APC Interface(A) Jurkat-PD-1OST 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-1OST 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-1OST 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-1OST 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.

Fig 3: SHP-1 Can Replace SHP-2 for PD-1 Coinhibition in Jurkat T Cells(A) Immunoblot analysis of equal amounts of proteins from total lysates of Raji (WT), Raji PD-L1, Raji SHP-2–, and Raji PD-L1 SHP-2– cells probed with antibodies to SHP-1 (right panel), SHP-2 (left panel), or VAV1 (loading control). Left margin, molecular size in kilodaltons. Data are representative of two independent experiments.(B) Raji, Raji PD-L1, Raji SHP-2–, and Raji PD-L1 SHP-2– cells were analyzed using flow cytometry for expression of HLA-DR, PD-L1, PD-L2, CD80, CD86, and HVEM. Gray shaded curves correspond to isotype-matched control antibody (negative control), and data are representative of two independent experiments.(C) Jurkat-PD-1OST and Jurkat-PD-1OST SHP-2– cells were stimulated with Raji SHP-2– cells or Raji PD-L1 SHP-2– cells that have been preincubated in the absence (–) or presence (+) of SEE and lysed for 2 min after the initial contact. Immunoblot analysis of equal amounts (90%) of lysates from the specified conditions subjected to affinity purification (AP) on Strep-Tactin-Sepharose beads, followed by elution of proteins with D-biotin, and probed with antibody to anti-SHP-1, anti-SHP-2, and phosphorylated proteins (Anti-p-Tyr). Also shown is immunoblot analysis of equal amounts (10%) of total lysates of the specified cells probed with anti-PD-1 antibody (loading control). Left margin, molecular size in kilodaltons (kDa). Data are representative of two independent experiments.