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. 2014:2014:246171.
doi: 10.1155/2014/246171. Epub 2014 Sep 1.

Soluble HLA technology as a strategy to evaluate the impact of HLA mismatches

Heike Kunze-Schumacher  1 , Rainer Blasczyk  1 , Christina Bade-Doeding  1
Affiliations

Soluble HLA technology as a strategy to evaluate the impact of HLA mismatches

Heike Kunze-Schumacher et al. J Immunol Res. 2014.

Abstract

HLA class I incompatibilities still remain one of the main barriers for unrelated bone marrow transplantation (BMT); hence the molecular understanding of how to mismatch patients and donors and still have successful clinical outcomes will guide towards the future of unrelated BMT. One way to estimate the magnitude of polymorphisms within the PBR is to determine which peptides can be selected by individual HLA alleles and subsequently presented for recognition by T cells. The features (structure, length, and sequence) of different peptides each confer an individual pHLA landscape and thus directly shape the individual immune response. The elution and sequencing of peptides by mass spectrometric analysis enable determining the bona fide repertoire of presented peptides for a given allele. This is an effective and simple way to compare the functions of allelic variants and make a first assessment of their degree of permissivity. We describe the methodology used for peptide sequencing and the limitations of peptide prediction tools compared to experimental methods. We highlight the altered peptide features that are observed between allelic variants and the need to discover the altered peptide repertoire in situations of "artificial" graft versus host disease (GvHD) that occur in HLA-specific hypersensitive immune responses to drugs.

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Figures

Figure 1
Figure 1
Assessment of _targetable allogeneic epitopes. Every ligand has to pass several filters, before being presented in an HLA molecule. The first is the individual expression profile. Proteins then undergo further proteasomal processing, where the individual cleavage profile determines the sequence of possible HLA ligands. The last filter is the individual HLA profile that differs between allelic variants.
Figure 2
Figure 2
Generation of peptide sequences from sHLA molecules. This figure gives a schematic overview of the steps towards the generation of peptide sequences obtained from sHLA molecules. The HLA heavy chain is given in blue; β2m is shown in yellow; and the peptide is shown in red. Cell culture supernatants containing sHLA molecules are passed over an N-hydroxysuccimide- (NHS-) activated HiTrap column coupled to mAb w6/32. Trimeric complexes (class I heavy chain, β2m and peptide) are eluted using a pH 2.7 elution buffer. Here, peptides from sHLA complexes can be differentiated into low and high binding peptides. The trimeric elution fractions are filtered through a 10 kDa cut-off membrane and the peptides detected in the flow through are considered to be of low affinity. The retentate containing dimeric (heavy chain and β2m) as well as trimeric complexes is then treated with 0.1% trifluoroacetic acid (TFA) to elute high binding peptides that can finally be separated by filtration through an additional 10 kDa cut-off membrane. Flow through fractions containing the low or high affinity peptides are subjected to mass spectrometric analysis using an Eksigent NanoLC Ultra 2D HPLC coupled to an orbitrap ion trap. Database queries can finally be carried out using Mascot software [25] incorporating the IPI human and the respective decoy databases.
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