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CTLA-4 x Ig was originally designed as an immunosuppressive agent capable of interfering with the co-stimulation of T cells. In the present study, we demonstrate that CTLA-4 x Ig, in combination with TCR ligation, has the additional capacity to convert naive CD4+CD25- T cells into Foxp3+ regulatory T (T(reg)) cells, as well as to expand their numbers. The CD4+CD25+Foxp3+ T(reg) generated by CTLA-4 x Ig treatment in vitro potently suppress effector T cells. Extending this in vivo, we show that systemic administration of CTLA-4 x Ig increases the percentage of CD4+CD25(hi)Foxp3+ cells within mixed lymphocyte reaction-induced murine lymph nodes. Significantly, the in vitro conversion of naive CD4+CD25- T cells into T(reg) cells is antigen-presenting cell (APC) dependent. This finding, together with the further observation that this conversion can also be driven in vitro by an antibody that engages B7-2 ligand, suggests that CTLA-4 x Ig-driven T(reg) induction may be predicated upon active CTLA-4 x Ig to B7-2 signaling within APC, which elicits from them T(reg)-inducing potential. These findings extend CTLA-4 x Ig's functional repertoire, and at the same time, reinforce the concept that T cell anergy and active suppression are not entirely distinct processes and may be linked by some common molecular triggers.

A chimeric CD40.FasL (CD40-CD95L) protein was designed with the combined capacities to bind to two surface receptors on activated T cells, CD40 ligand (CD40L; CD154) and Fas receptor (CD95). CD40.FasL, once tethered to the cell surface via one of its ends, can transmit a signal via its other end. In principle, simultaneous triggering from both ends is possible, and thus there is the intriguing potential for 'auto-inhibition' if such dual triggering occurs on the same cell itself. Several lines of evidence support this mechanism: (i) CD40.FasL is cytotoxic to Fas receptor-positive cell lines of different cell lineages, (ii) CD40.FasL's function is potentiated when there is enforced expression of CD40L on target cells, (iii) CD40.FasL inhibition does not require intercellular contact, as demonstrated by soft agar clone formation and cell dilution analysis and (iv) introduction of exogenous CD40 into the system interferes with CD40.FasL inhibition. Taken together, these data are consistent with a 'loop-back' inhibitory mechanism within individual activated (CD40L and Fas receptor expressing) T cells causing suicide of these T cells. Significantly, this type of fusion protein provides a unique way to confine immunoinhibition to activated 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 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/B7-2-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 cell 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 approximately 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 FasL 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.

mAb with specificity for the alpha3 domain of HLA class I antigens, such as mAb TP25.99 and W6/32, are capable of inhibiting the proliferation of stimulated T cells in vitro by binding to their surface HLA class I antigens. The inhibitory potential of another HLA class I alpha3 domain-specific mAb, A1.4, was evaluated. In contrast to mAb TP25.99 and W6/32, which routinely inhibited superantigen (SEB) stimulation of T cells by >90%, mAb A1.4 at equivalent concentrations demonstrated only 20-50% inhibition. Univalent Fab fragments of all three mAb lacked inhibitory activity. Interestingly, however, by combining univalent W6/32 (or TP25.99) Fab fragments with intact, bivalent mAb A1.4 (at a non-inhibitory, sub-threshold concentration of 1 microg/ml), significant inhibition of SEB-driven T cell proliferation was obtained. Inhibition by the anti-HLA class I mAb W6/32 and TP25.99 was evident even when SEB was used in conjunction with paraformaldehyde-fixed HLA class I-, class II+ Daudi cells, suggesting that the inhibitory activity of these mAb results from direct HLA class I epitope engagement on the T cell. These findings suggest that effective antibody-mediated induction of the HLA class I inhibitory pathway within T cells is dependent upon two separable molecular triggers at the T cell surface. The first can be delivered by univalent mAb derivatives that engage one or more critical HLA class I epitope(s). The second requires intact mAb, though seems to be less selective as to the HLA class I specificity. This model may explain why some, but not all, anti-HLA class I mAb are inhibitory when used singly. Achieving synergies between a wider array of anti-HLA class I mAb and their derivatives may provide a path for more effectively tapping into the HLA class I inhibitory pathway in a therapeutic context.

By antisense-mediated inhibition of CD8 expression in T cell clones and expression of CD8 in non-T cell lines, we have produced several sets of CD8+/CD8- paired cell lines. These cellular reagents have allowed us to assess the effect of CD8 surface expression on the immunogenic potential of stimulator cells. We found that the presence of CD8 on stimulator cells markedly decreased their capacity to stimulate proliferative responses or to induce the generation of cytotoxic activity. The CD8 alpha chain, in the absence of the beta chain, was sufficient to mediate this inhibitory effect. Our findings support the notion that CD8 functions as an immunoregulatory ligand and that auxiliary cell surface molecules on stimulator cells can have profound effects on the immunogenic capabilities of these cells.