An HLA-E single chain trimer inhibits human NK cell reactivity towards porcine cells

HLA-E, when expressed by pig cells, could alleviate human natural killer (NK) cell-mediated rejection of porcine xenografts by providing a potent inhibitory ligand for human NK cells expressing CD94/NKG2A. Yet cell-surface expression of HLA-E on porcine epithelial (LLC-PK1) cells was not observed after transfection with an expression vector harboring HLA-E alone or in combination with an expression vector containing human β2m. A single chain trimer (SCT) of HLA-E consisting of, in the following order (from N- to C-terminus), the leader peptide of human β2m, VMAPRTLIL (an HLA-E-binding peptide), a 15 amino acid linker, mature human β2m, a 20 amino acid linker, and mature HLA-E heavy chain was engineered. Cell-surface expression and correct folding of HLA-E SCT was shown by FACS analyses of stably transfected LLC-PK1 cells. Untransfected LLC-PK1 cells were readily lysed by the NK cell lines NKL and NK-92, while LLC-PK1 cells expressing HLA-E SCT were almost completely protected. In addition, the HLA-E SCT recapitulates the peptide dependent properties of normal HLA-E trimeric complexes in that an HLA-E SCT with an hsp60 derived peptide, though expressed at the cell-surface, did not inhibit NK cell-mediated lysis. The HLA-E SCT, which conferred protection against NK cell-mediated killing, also inhibited NK cell IFN-γ secretion elicited by co-culture of NKL cells with LLC-PK1 cells. Thus, HLA-E SCT, in which all three components of a normal HLA-E protein complex are in one polypeptide chain, is immunologically functional as it is able to modulate NK cell cytotoxicity and cytokine secretion.