CTLA-4-Fas ligand functions as a trans signal converter protein in bridging antigen-presenting cells and T cells

Co-stimulator blockade and trans inhibitory signaling, using agents such as CTLA-4-Ig and Fas ligand (FasL) respectively have been invoked as alternat ive strategies for suppressing pathogenic T cells. This study describes a n ovel hetero-bifunctional fusion protein, CTLA-4-FasL, designed to combine w ithin a single protein both co-stimulator blocking and trans inhibitory sig naling potentials. A chimeric expression cassette, in which the ectodomain coding sequences for CTLA-4 and Fast were linked in-frame, was used to prod uce a CTLA-4-FasL fusion protein. CTLA-4-FasL binding to both B7-1/B7-2-exp ressing Daudi B cells and Fas-expressing Jurkat T cells was documented by i mmunofluorescence and flow cytometry, The capacity of CTLA-4-FasL to induce apoptosis in Jurkat targets was markedly enhanced by the addition of Daudi and other B7-1/B7-2(+) B cell lines, which provided a membrane platform fo r the otherwise soluble CTLA-4-fusion protein. Moreover, in dual-chamber ex periments, Daudi cells pre-coated with CTLA-4-FasL demonstrated Jurkat inhi bitory activity that was cell-contact dependent. Significantly, when used t o inhibit In vitro cellular proliferation of peripheral blood mononuclear c ells, CTLA-4-FasL was similar to 1000-fold more potent than the extensively characterized CTLA-4-Ig fusion protein. Furthermore, the degree of inhibit ion induced by CTLA-4-FasL substantially surpassed that observed for CTLA-4 -Ig and a soluble Fast when used in combination. CTLA-4-FasL represents the first of a novel class of fusion proteins, designated here as 'trans signa l converter proteins', that combine trans signal masking and direct trans s ignaling functions.