PD-1 recruits SHP2 but not SHP1, whereas BTLA recruits both in T cells. (A) One-sided volcano plot showing MS-identified proteins in GST–PD-1INT or GST-BTLAINT pull-down samples (see Materials and methods). SH2 domain-containing proteins are labeled. (B) Representative IBs showing the levels of phosphotyrosine (pY IB) and GFP (GFP IB) in PD-1–mGFP or BTLA-mGFP immunoprecipitate (GFP IP), as well as the coprecipitated SHP1, SHP2, p85, CSK, ZAP70, SAP, and GRB2 from the indicated co-culture lysates, with the duration of stimulation before the lysis indicated (see Materials and methods). GAPDH IB indicates the input of each sample. (C) Coprecipitated SHP1 and SHP2 in B were quantified using purified recombinant SHP1 or SHP2 standards and plotted as SHP1 versus SHP2 recruitments against the stimulation time. (D) OT-1–PLB assays showing the recruitment of SHP1 or SHP2 by PD-1 or BTLA in OT-1 cells. Leftmost are cartoons depicting indicated types of OT-1 cells interacting with PLB containing pMHC, CD86, and either PD-L1 or HVEM. Middle left are representative TIRF images of the indicated channels. Scale bars, 5 µm. Middle right are plots of fold intensities along the yellow diagonal line in the overlaid images calculated as described (Yokosuka et al., 2012). Rightmost are PCCs showing the colocalization scores of mGFP microclusters versus mCherry microclusters (see Materials and methods). **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. Student’s t test (n = 20). WCL, whole cell lysate.
