Hph-1-ctCTLA-4 suppresses T cell activation by inhibition of TcR-proximal signals. (A) Jurkat T cells or (B) mouse splenocytes were incubated with Hph-1-ctCTLA-4, and its inhibitory function on IL-2 secretion was stimulated by plate-bound anti-CD3 and soluble anti-CD28 antibodies or PMA/ionomycin was analyzed by ELISA. (C) After ctCTLA-4 or Hph-1-ctCTLA-4 was incubated with Jurkat T cells for 30 min, cells were activated with anti-CD3 and anti-CD28 antibodies for 5 min at 37 °C, cell lysates were analyzed by Western blot analysis with anti-Phospho-ERK, -JNK, -p38, -ZAP70, -ζ-chain antibodies, and anti-α-tubulin antibody was used as lysate control. (D and E) After each protein was incubated with (D) NFAT- or (E) AP-1-SEAP Jurkat T cells for 30 min, cells were stimulated with plate-bound anti-CD3 antibody and soluble anti-CD28 antibody for 24 h and chemiluminescent SEAP activity in the culture media was analyzed. Bars represent mean ± SD of three independent experiments. *, P < 0.05; **, P < 0.01 compared with the activated T cell group.