CHARACTERIZATION OF CTLA-4 STRUCTURE AND EXPRESSION ON HUMAN T-CELLS

CTLA-4 is an adhesion receptor expressed on activated T cells. The ami no acid sequence of CTLA-4 is related to CD28, and although the functi on of CTLA-4 remains unknown, it shares several features with CD28, in cluding a common counter-receptor, B7, that is present on Ag-presentin g cells. In a recent study we found that CD28 and CTLA-4 were coexpres sed at the mRNA level on activated T cells but that only CD28 was expr essed on resting T cells. Here we show that within the T cell populati on, CTLA-4 expression is restricted to the subset of T cells that also express cell surface CD28. CTLA-4 mRNA expression can be induced on q uiescent T cells via phorbol ester-mediated activation of protein kina se C but not with calcium ionophore treatment alone. Phorbol ester-ind uced expression of CTLA-4 mRNA could be enhanced with calcium ionophor e treatment, and treatment of cells in this manner resulted in a recip rocal decrease in expression of CD28 mRNA. Ligation of CD28 with monoc lonal antibody also resulted in the specific and rapid induction of CT LA-4 mRNA. To study the expression of CTLA-4 at the protein level, a r abbit antiserum against a recombinant protein derived from CTLA-4 cDNA was generated. When activated T cells were labeled with [S-35]methion ine, the rabbit antiserum precipitated a 41- to 43-kDa protein from wh ole cell lysates. Similar results were found when detergent-soluble ly sates from I-125 surface-labeled resting and activated T cells were an alyzed by SDS-PAGE. Surprisingly, under the conditions tested, CTLA-4 migrated primarily as a monomer at the cell surface, and could not be shown to exist as a disulfide-bonded homodimer or as a heterodimer con sisting of CTLA-4 and CD28. These results suggest that B7 can bind to T cells via distinct receptor complexes consisting of either CD28 or C TLA-4, and that these complexes may potentially mediate distinct biolo gic functions. Further, the present results suggest that noncovalent i nteractions might mediate association of CTLA-4 and/or CD28 at the cel l surface.