Structural basis of K^bm8 alloreactivity: Amino acid substitutions on the β-pleated floor of the antigen recognition site

We have analyzed the functional significance of the four amino acid differences between the parental H-2K^b and mutant H-2K^bm8 glycoproteins. Six bm8 variants including single substitutions at residues 22, 23, 24, and 30 as well as paired substitutions at residues 23, 30 and 22, 24 were generated and transfected into L cells. Surface expression of these H-2K^b variants was analyzed using monoclonal antibodies which bind to well-defined H-2K^b epitopes. No alterations introduced into the conformational structure of H-2K^b by the amino acid substitutions were detected. The effect of these substitutions on CTL recognition was initially analyzed using the following bulk CTL: either H-2K^b anti-H-2K^bm8, H-2K^bm8 anti-H-2K^b, or third party anti-H-2K^b. The alloreactivity between H-2K^b and H-2K^bm8 is dominated by the amino acid substitution at residue 24 (Glu→Ser). The complete bm8 phenotype, however, also requires the additional substitution at residue 22 (Tyr→Phe). The H-2K^bm8 anti-K^b bulk CTL reacted with both variant H-2K^bm8 molecules containing single substitutions at amino acid positions 22 or 24 but not the variant molecule containing both substitutions. Further analysis using three individual H-2K^bm8 anti-K^b CTL clones indicated the complexity of the self K^bm8 phenotype. Clone 8B1.20 did not react to changes in residues 22 or 24. The 8B1.32 clone reacted with the change at residue 22 but not with the change at residue 24, although the 8B1.54 clone reacted with the change at residue 24 but not with the change at residue 22. The changes in residues 23 (Met→Ile) and/or 30 (Asp→Asn) did not impact significantly on the alloantigenic properties of K^bm8 as determined by both the bulk and cloned CTL populations. According to the three-dimensional class I structure the substitution at amino acid 24 is inaccessible to the TCR. The location of this substitution within the Ag recognition site implies that altered peptide binding, and not a disruption of MHC residues that interact with the TCR, is responsible for the alloreactivity between H-2K^b and H-2K^bm8.