BTLA inhibits Jurkat cells more potently than does PD-1. (A) Left: Cartoons depicting co-culture assays in which indicated types of Jurkat cells were stimulated by SEE-loaded Raji (PD-L1–mCherry⁺) cells. Right: Flow cytometry histograms showing PD-1 surface expressions in the indicated Jurkat T cells. (B) Scatter plots showing relative IL-2 levels in the medium of indicated Jurkat-Raji co-cultures. IL-2 levels were normalized to that of Jurkat (PD-1^FF–mGFP⁺) cells for each replicate (see Materials and methods). (C and D) Same as A and B, except PD-1^FF–mGFP, PD-1^WT–mGFP, and PD-L1–mCherry were replaced with BTLA^FFFF-mGFP, BTLA^WT-mGFP, and HVEM-mRuby, respectively. (E) Left: Representative IBs showing phospho-CD3ζ (anti-pY142) and phospho-CD28 (coimmunoprecipitated p85) in the lysates of the indicated co-cultures, with the duration of stimulation before lysis indicated (see Materials and methods). The asterisk indicates nonspecific bands. WCL, whole cell lysate. Right: Quantification graphs of phospho-CD3ζ and phospho-CD28, incorporating results from three independent experiments. In each replicate, data were normalized to the highest phosphorylation level under Jurkat (PD-1^FF–mGFP⁺) or Jurkat (BTLA^FFFF-mGFP⁺) conditions, respectively. (F) Same as E, except PD-1^FF–mGFP, PD-1^WT–mGFP, and PD-L1–mCherry were replaced with BTLA^FFFF-mGFP, BTLA^WT-mGFP, and HVEM-mRuby, respectively. Data in this figure are presented as means ± SD. *, P < 0.05; ****, P < 0.0001. Student’s t test for B and D (n = 6); two-way ANOVA for E and F (n = 3). ns, not significant.