Generation of anergic T cells after coculture with CTLA4-KDEL transfectants. The HC3 clone (10⁶) was cocultured with an equal number of HLA-DR1–expressing B cells, which were nontransfected, CTLA4-KDEL transfected, or mock-KDEL transfected. (A) After 3 days, the proliferation of the clones was measured by ³H-thymidine incorporation. □ indicates untransfected; ⋄, Mock-KDEL; ○, CTLA4-KDEL. (B) After 2 days the clone was isolated, and the expression of phosphorylated retinoblastoma protein, cyclin E, cyclin D2, Cdk4, and p27^kip1 proteins was analyzed by Western blotting. In addition, the coassociation of cdk2, Cyclin D2, cdk4 with other proteins was assessed by immunoprecipitation with appropriate Ab (IP Ab) followed by Western blotting and probing with anti–Cyclin E or Cyclin D2 Ab (blot Ab). The enzymatic activity of the proteins was determined using in vitro kinase reactions, with Histone H1 or a retinoblastoma–glutathione S transferase (Rb-GST) fusion protein. (C) From the primary coculture, the clones were then rested for 5 days before being stimulated with an equal number of unmodified HLA-DR1–expressing B cells prepulsed with HA peptide. The cultures were carried out in the absence (left) or presence (right) of 10 U/mL recombinant (r)IL-2. Proliferation of the T cells was determined by ³H-thymidine incorporation after 3 days. ▵ indicates no cells; □, untransfected; ⋄, Mock-KDEL; ○, CTLA-4 KDEL. The results are expressed as mean ± SD of triplicate wells of a single experiment. These data are representative of 3 independent experiments.