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

Abstract

Co-stimulator blockade and trans inhibitory signaling, using agents such as CTLA-4-Ig and Fas ligand (FasL) respectively have been invoked as alternative strategies for suppressing pathogenic T cells. This study describes a novel hetero-bifunctional fusion protein, CTLA-4-FasL, designed to combine within a single protein both co-stimulator blocking and trans inhibitory signaling potentials. A chimeric expression cassette, in which the ectodomain coding sequences for CTLA-4 and FasL were linked in-frame, was used to produce a CTLA-4-FasL fusion protein. CTLA-4-FasL binding to both B7-1/B7-17-2-expressing Daudi B cells and Fas-expressing Jurkat T cells was documented by immunofluorescence 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 cells lines, which provided a membrane platform for the otherwise soluble CTLA-4-fusion protein. Moreover, in dual-chamber experiments, Daudi cells pre-coated with CTLA-4-FasL demonstrated Jurkat inhibitory activity that was cell-contact dependent. Significantly, when used to inhibit in vitro cellular proliferation of peripheral blood mononuclear cells, CTLA-4-FasL was ∼1000-fold more potent than the extensively characterized CTLA-4-Ig fusion protein. Furthermore, the degree of inhibition induced by CTLA-4-FasL substantially surpassed that observed for CTLA-4-Ig and a soluble FaL when used in combination. CTLA-4-FasL represents the first of a novel class of fusion proteins, designated here as 'trans signal converter proteins', that combine trans signal masking and direct trans signaling functions.