CTLA-4 inhibition of AICD in T-cell hybridoma transfected with human CTLA-4.

(A) Fluorescent activated cell sorter (FACS) profile of TCRζ/CD3, human CTLA-4, and murine CD28 (mCD28) surface expression on transfectants. DC27.10 cells were stably transfected with a vector (denoted Vector), human CTLA-4 (denoted CTLA-4 WT), and CTLA-4 mutant (denoted Y201-F). (B) CTLA-4 inhibits AICD in CTLA-4 transfectants. Cells were stimulated on a 96-well plate coated with anti-CD3 (145-2C11) at a concentration of 0.5 μg/mL and with anti–CTLA-4 (10 μg/mL). Anti-TNP control antibody was added to keep a final concentration of 10.5 μg/mL with anti-CD3 alone. AICD was determined by PI as described in “Study design.” ■ indicates media; ▪, αCD3; and ▧, αCD3 + αCTLA-4. (C) CTLA-4 inhibits AICD in a concentration-dependent manner. Anti-CD3 was used at a concentration of 0.5 μg/mL, while anti–CTLA-4 was used at concentrations of 2, 5, and 10 μg/mL. Anti-TNP control antibody was added to keep the final concentrations equivalent. ▪ indicates vector; ▧, CTLA-4 WT; and ■, Y201F. (D) CTLA-4 inhibits AICD induced by anti-CD3 in conjunction with anti-CD28. Anti-CD3 was used at 0.5 μg/mL; anti-CD28 at 5 μg/mL; and anti–CTLA-4 at 5 μg/mL. Anti-TNP control antibody was added to keep the final concentrations equivalent. ■ indicates media; ▪, αCD3; ▨, αCD3 + αCD28; ▧, αCD3 + αCD28 + αCTLA-4.