Characterization of CTLA-4 Antigen Expression Identification of Molecules Composing Intracellular CTLA-4 Multiprotein Complex

Rhim, Dae Cheol; Chung, Yong Hoon

Abstract

BACKGROUND: CTLA-4 (Cytotoxic T Lymphocyte associated Antigen 4, CD152) has been known as a homologue of CD28, an accessory molecule providing a key costimulatory signal for successful antigen-driven activations of T lymphocyte. Most of biochemical and cell biological characteristics of the CD152 protein remain unknown while those of CD28 have been characterized in detail.
METHODS
In this study CD152 expression in both CD4+ and CD8+ PBLs was studied by using flow cytometry. And intracellular CD152 multiprotein complex was purified and used for generating antibodies recognizing proteins composing of intracellular CTLA-4 multi protein complex.
RESULTS
Level of surface expression of this molecule was peaked at 2 days of PHA stimulation in flow cytometric analysis. 40~45% of PHA blast cells were CD152+ in both of two subsets at this stage and the level of expression were equivalent in both two subsets. Contrary to this surface expression, intracellular expression was peaked at day 3 and it was preferentially induced in CD8+ cells and about 60% of CD8+ cells were CD152+ at this stage. High molecular weight (> 350 kD) intacellular CD152 protein complex purified by using preparative electrophoresis were immunized into rabbits and then 3 different anti-P34PC4, anti-P34PC7 and anti-P34PC8 antibodies were obtained. Using these 3 antibodies two unknown antigens associated with intracellular CD152 multiprotein complex were found and their molecular weights were 54 kD and 75 kD, respectively. Among these, the former was present as 110 kD homodimer in non- reducing condition.
CONCLUSION
It seemed that 34 kD intracellular CD152 molecule forms high molecular weight multiprotein complex at least with 2 proteins of 75 kD monomer and 110 kD homodimer.