Effects of SHP1 and/or SHP2 deletions on PD-1 and BTLA signaling in Jurkat cells. (A) Representative IBs showing the levels of SHP1, SHP2, p85, and CD3ζ in WT Jurkat cells and BTLA^WT-mGFP–, BTLA^FFFF-mGFP–, PD-1^WT–mGFP–, or PD-1^FF–mGFP–transduced SHP1 KO, SHP2 KO, or SHP1/2 DKO Jurkat cells. GAPDH IB indicates the input of each sample. Asterisk labels nonspecific bands. (B) Flow cytometry histograms showing PD-1 (left) or BTLA (right) surface expression in parental Jurkat cells and PD-1^FF–mGFP–, PD-1^WT–mGFP–, BTLA^FFFF-mGFP–, or BTLA^WT-mGFP–transduced SHP1 KO, SHP2 KO, or SHP1/2 DKO Jurkat cells. (C) SHP1 KO Jurkat T cells transduced with PD-1^FF–mGFP, PD-1^WT–mGFP, BTLA^FFFF-mGFP, or BTLA^WT-mGFP were stimulated with SEE-loaded Raji (PD-L1–mCherry⁺) or Raji (HVEM-mRuby⁺) cells. Left: Representative IBs showing phospho-CD3ζ (anti-pY142) and phospho-CD28 (coimmunoprecipitated p85) in the lysates of the indicated Raji-Jurkat co-cultures, with the duration of the stimulation before lysis indicated (see Materials and methods). Right: Quantification plots of phospho-CD3ζ and phospho-CD28, incorporating results from three independent experiments. In each replicate, data were normalized to the highest phosphorylation level under SHP1 KO Jurkat (PD-1^FF–mGFP⁺) or SHP1 KO Jurkat (BTLA^FFFF-mGFP⁺) conditions, respectively. (D) Same as C, except using SHP2 KO Jurkat cells. (E) Same as C, except using SHP1/2 DKO Jurkat cells. (F–H) Scatter plots summarizing relative IL-2 levels in the medium of 6-h Jurkat-Raji co-cultures shown in C–E, respectively. For C–H, data are means ± SD from nine independent experiments. In each replicate, IL-2 levels of Jurkat (PD-1^WT–mGFP⁺) or Jurkat (BTLA^WT-mGFP⁺) were normalized to that of Jurkat (PD-1^FF–mGFP⁺) or to that of Jurkat (BTLA^FFFF-mGFP⁺) under the same SHP background, respectively (see Materials and methods). **P < 0.01, ****P < 0.0001. Two-way ANOVA for C–E (n = 3); Student’s t test for F–H (n = 9). ns, not significant; WCL, whole cell lysate.