Chimeric  2 microglobulin/CD3  polypeptides expressed in T cells convert MHC class I peptide ligands into T cell activation receptors: a potential tool for specific targeting of pathogenic CD8+ T cells

Abstract

CD8(+) T cells are key mediators of transplant rejection and graft-versus-host disease and contribute to the pathogenesis of autoimmune diseases. We tested whether TCR ligands can be converted into T cell activation receptors, redirecting genetically modified T cells at pathogenic CD8(+) T cells. For this purpose we exploited the ability of the non-polymorphic beta(2) microglobulin light chain to pair with all MHC class I heavy chains. In this report we describe the design and expression in a T cell hybridoma of two modalities of beta(2) microglobulin polypeptides, fused with the transmembrane and intracellular portion of CD3zeta chain. In the absence of a particular antigenic peptide, the chimeric product associates with different endogenous MHC class I heavy chains and triggers T cell activation upon heavy chain cross-linking. When an antigenic peptide is covalently attached to the N-terminus of the chimeric polypeptide, transfectants express high level of surface peptide-class I complexes and respond to antibodies and target T cells in a peptide-specific manner. Our results provide the basis for a universal genetic approach aimed at antigen-specific immunotargeting of pathogenic CD8(+) T cells.

Adoptive Transfer of mRNA-Transfected T Cells Redirected against Diabetogenic CD8 T Cells Can Prevent Diabetes

“…We previously showed that expression of the peptide/b 2 m/anchor configuration resulted in exceptionally high occupancy of the restricting MHC-I product by the linked peptide. 9,40 To supplement the endogenous H-2K d in the polyclonal NOD CD8 T cells, we tested the addition of mRNA encoding H-2K d heavy chains. Figure 3A illustrates that the transfected polyclonal CD8 T cells have acquired the ability to kill the target CHIB2 cells and overexpression of H-2K d had no discernible effect on the killing activity.…”

“…For cloning of the four constructs encoding peptide/human b 2 m/mouse CD3-z, we used essentially the same cloning strategy we described previously. 9 The gene segment encoding the full leader sequence of human The resulting XbaI/BamHI fragment and a BamHI/EcoRI fragment encoding the hb 2 m/CD3-z portion of the chimeric polypeptide 9 were inserted into pGEM4Z/A64.…”

“…We have shown that b 2 m can serve as a versatile molecular scaffold for chimeric MHC-I/CD3-z T cell activation receptors. 9 A single b 2 m/CD3-z-based expression cassette enables covalent linking of any pre-selected peptide to the N terminus of b 2 m, so as to redirect T cells at autoreactive CD8 T cells of a given specificity.…”

Membrane-Anchored β2-Microglobulin Stabilizes a Highly Receptive State of MHC Class I Molecules

“…The use of the pBJ1-Neo expression vector and the construction of plasmid 21-2, encoding chimeric human ␤ 2 m (h␤ 2 m)/CD3, have been described elsewhere (29).…”

“…The original design of membrane-anchored h␤ 2 m has already been delineated (29). Briefly, the carboxyl-terminal methionine residue of h␤ 2 m was genetically linked to a peptide bridge, comprising the 13 membrane-proximal amino acids of the extracellular portion of HLA-A2, LRWEPSSNPTIPI (single-letter code), which encompasses the proline-rich connecting peptide.…”