The effect of mutations in the MHC class I peptide binding groove on the cytotoxic T lymphocyte recognition of the K^b‐restricted ovalbumin determinant

Abstract

The H-2Kb-restricted cytotoxic T lymphocyte (CTL) response directed against ovalbumin (OVA) is specific for a region contained within the sequence OVA253-276. In this study we have characterized this response by examining the class I-restricted presentation of OVA peptides by the naturally occurring Kb mutant (Kbm) glycoproteins Kbm1, Kbm3, Kbm5, Kbm8, Kbm10, Kbm11 and Kbm23. To facilitate this study we derived a series of somatic cell hybrid targets expressing the various Kbm class I molecules. Experiments using bulk OVA-specific CTL from C57BL/6 mice demonstrated that all the Kbm molecules except for Kbm1 and Kbm8 could present OVA peptides for effective T cell recognition. Clonal analysis revealed a more complex and relatively diverse pattern of CTL recognition of the Kbm/peptide combinations. This diversity is unlikely to result from the existence of multiple, independent Kb-restricted T cell determinants within OVA, since all CTL tested were specific for a single region between residues 259 and 273. Examination of the fine specificity of Kbm presentation identified individual changes at residues 77, 80 and 116 which affected T cell recognition. The results imply that these changes do not inhibit peptide binding since some clones could recognize peptide presented by a particular Kbm molecule, while other clones could not. All three residues reside within the peptide-binding cleft of the class I protein and are not expected to directly contact the T cell receptor. Although we did not formally demonstrate that OVA binding by Kbm vs. Kb is quantitatively identical, our results are best explained by postulating that the changes at residues 77, 80 and 116 indirectly affect T cell recognition by altering peptide conformation. Taken together our results suggest that changes within the class I binding site can profoundly modify peptide presentation without significantly inhibiting peptide-class I association.

Positive Selection Is Limited by Available Peptide-Dependent MHC Conformations

“…This was confirmed directly with the K b -specific Abs 5F1 and 100.30. These Abs recognizes both K b and K bm8 on spleen cells or tumor cells, where a variety of peptides are displayed (32). However, when the MHC molecules were stabilized at the cell surface with single peptides, we found that in some cases the epitope was completely destroyed on K b , but not on K bm8 , or vice versa.…”

“…We wanted to determine whether this defect was due to the absence of an appropriate selecting self peptide during thymic development or whether OVA-specific receptors are unable to interact with peptide/K bm8 due to structural differences imposed by the mutations. Although the dominant OVA epitope, OVAp, can bind K bm8 , it is not stimulatory to K b -restricted CTL when presented by K bm8 (27,32). This suggests that K bm8 may be structurally unrecognizable despite the high level of similarity between the K b and K bm8 alleles.…”

“…The amino acid differences between K b and K bm8 lie in the peptide binding groove, and indeed, it has been shown that the two molecules present distinct peptide repertoires (25). This has pointed to a role for the bound peptide in cases where K b and K bm8 function differently (27,32,39), but it is known that the MHC-bound peptide can influence the conformation of the ␣1 and ␣2 residues (40,42,43). Thus, we considered the possibility that OVAp-like peptides force the ␣1 and ␣2 helices to lie in different conformations in K bm8 compared with K b , thus altering the structure of the peptide- MHC complex.…”

“…Despite the fact that the endogenously processed OVA 257-264 SIINFEKL (OVAp) binds K bm8 , it is unable to stimulate OVAspecific CTL in the context of K bm8 (27,32). Before searching for peptides that would restore positive selection of OVA-specific receptors, we sought to determine whether peptides could be targets for OVA-specific CTL in the context of K bm8 , and how frequent they are.…”

Type I Interferons Mediate the Innate Cytokine Response to Recombinant Fowlpox Virus but Not the Induction of Plasmacytoid Dendritic Cell-Dependent Adaptive Immunity

“…Control mice were produced in-house as F1 progeny from successful matings of normal C57BL/6J (B6) and SV129 mice purchased from Laboratory Animal Services ([LAS] University of Adelaide, S. A., Australia). OT-I T-cell receptor (TCR) transgenic (Tg) mice (8), which contain Tg CD8 ϩ T cells that recognize the dominant chicken ovalbumin peptide consisting of residues 257 to 264 (OVA [257][258][259][260][261][262][263][264] ) in the context of H2-K b , and B6 bm1 mice, which harbor a single natural mutation in the binding groove of H-2K b and are therefore unable to present the H-2K b -restricted OVA 257-264 peptide to CD8 ϩ T cells (39), were both kindly provided by W. R. Heath (Walter and Eliza Hall Institute of Medical Research, Melbourne, Victoria, Australia). Congenic CD45.1 ϩ B6.SJL mice were purchased from the Animal Resource Centre (Canning Vale, W. A., Australia).…”