The nature of peptides presented by an HLA class I low expression allele

Abstract

Background: The functional integrity of human leukocyte antigen low expression variants is a prerequisite for considering them as essential in the matching process of hematopoietic stem cell donors and recipients to diminish the risk of serious complications such as graft-versus-host disease or graft rejection. The HLA-A*3014L variant has a disulfide bridge missing in the alpha2 domain which could affect peptide binding and presentation to T cells.

Design and methods: HLA-A*3014L and HLA-A*3001 were expressed as truncated variants and peptides were eluted and subjected to pool sequencing by Edman degradation as well as to single-peptide sequencing by mass spectrometry. Quantitative analysis of binding peptides presented in vivo was performed by a flow cytometric peptide-binding assay using HLA-A*3001 and HLA-A*3014L-expressing B-LCLs.

Results: The truncated HLA-A*3014L protein was secreted in the supernatant and it was possible to elute and sequence peptides. Sequence analysis of these eluted peptides revealed no relevant differences to the peptide motif of HLA-A*3001, indicating that the Cys164Ser substitution does not substantially alter the spectrum of presented peptides. Strong binding of one of the shared in vivo identified HLA-A*3001/3014L ligands was confirmed in the peptide-binding assay.

Conclusions: This study is the first to demonstrate that HLA low expression variants are able to present peptides and, thus, can be considered as functionally active. When comparing peptide motifs, it is likely that HLA-A*3014L and HLA-A*3001 represent a permissive mismatch with low allogenicity in hematopoietic stem cell transplantation. These results indicate that surface expression, as well as peptide-binding data of HLA variants with similar disulfide bridge variations (e.g. HLA-A*3211Q) need to be considered as functionally active in an allogeneic hematopoietic stem cell transplantation setting as long as the opposite has not been shown. Otherwise a relevant but not considered HLA mismatch could result in a severe allogeneic T-cell response and graft-versus-host disease.

Figure 1.

Spectra of the undecameric peptide EITALAPSTMK eluted from HLA-A*3001 (A) and HLA-A*3014L (B). These spectra show the peptide summary for MS/MS fragmentation of the EITALAP-STMK peptide eluted from HLA-A*3001 (A) and HLA-A*3014L (B) produced using MASCOT software. Predicted masses for b and y fragment ions are listed, and observed ions are labeled in the respective spectra. N- and C-terminal peptide fragments present in all ESI-MS/MS spectra are labeled according to standard nomenclature.,

Figure 2.

Relative A*30/A*02 peptide-binding intensities for different HLA-expressing B-LCLs. The diagram shows the peptide-binding intensities of the investigated cell lines EBRCC-2296 (HLA-A*0201 homozygous), EBRCC-256 (HLA-A*0201/A*3001), Ulm-241539 (HLA-A*0201/A*3014L) achieved after acid-treatment and reconstitution using FITC-labeled A*3001/3014L-restricted (EITALAK(FITC)PSTMK) and A*0201-restricted (NLVPMK(FITC)VATV) peptides. Relative peptide-binding intensities of the HLA-A*30 lig-and compared to the HLA-A*0201 ligand were calculated for each of the three cell lines as followed: [double positive A*30 binding cells/double positive A*0201 binding cells]*100. Background expression of cells, which were cultured without the synthetic peptides, was used as the blank value.

Figure 3.

Homology-based model of HLA-A*3014L with the three shared peptide ligands. Modeling of the HLA-A*3001 and HLA-A*3014L structures was carried out using the SCWRL homology-based modeling server using the crystal structure of HLA-A1101 (1Q94) as a template. Peptide templates for 9-mer (1Q94), 11-mer (2BVO) and 12-mer (3BW9) were superposed and merged with the HLA-A*3014L model. Peptide mutagenesis was then performed using DeepView using the rotamer library to find the best side chain orientations with minimum steric clashes. Each model was then subjected to energy minimization as implemented in DeepView. The graphics program PyMOL (http://www.pymol.org) was used to generate the structure figures.