Clustering of phosphatase outside TCRs microclusters is less effective for suppression of IL-2 production. (A) A diagram of the mPD-1–hCD22–mPD-1 YFYF–mSHP1 or –mSHP2 chimeras. The tyrosine substitution (Y225/148F) was inserted and EGFP was replaced by mSHP1 or mSHP2 in mPD-1–hCD22–mPD-1–EGFP chimeras as in Fig. 7 A. (B) AND-TCR T cell hybridomas were not transduced (−) or transduced with WT or YFYF mutant PD-1 or the YFYF mutant or the elongated PD-1 YFYF fused to SHP1 (YFYF, Igx5, Igx4, Igx2, or Igx1-SHP1) or SHP2 (YFYF, Igx5, Igx4, Igx2, or Igx1-SHP2) in A. The histograms show cell surface expression of the various PD-1 chimeras fused by SHP1 (left) or SHP2 (right). (C) The cells in B were stimulated with DC-1 cells expressing PD-L1 or PD-L2 with 0.3 µM MCC88-103 for 16 h. Immobilized 2C11 and PV-1 were used for control stimulation. Concentration of IL-2 was measured by ELISA. A representative of two independent experiments is shown. Error bars represent SD.