The Value of Online Algorithms to Predict T-Cell Ligands Created by Genetic Variants

<div><p>Allogeneic stem cell transplantation can be a curative treatment for hematological malignancies. After HLA-matched allogeneic stem cell transplantation, beneficial anti-tumor immunity as well as detrimental side-effects can develop due to donor-derived T-cells recognizing polymorphic peptides that are presented by HLA on patient cells. Polymorphic peptides on patient cells that are recognized by specific T-cells are called minor histocompatibility antigens (MiHA), while the respective peptides in donor cells are allelic variants. MiHA can be identified by reverse strategies in which large sets of peptides are screened for T-cell recognition. In these strategies, selection of peptides by prediction algorithms may be relevant to increase the efficiency of MiHA discovery. We investigated the value of online prediction algorithms for MiHA discovery and determined the <i>in silico</i> characteristics of 68 autosomal HLA class I-restricted MiHA that have been identified as natural ligands by forward strategies in which T-cells from <i>in vivo</i> immune responses after allogeneic stem cell transplantation are used to identify the antigen. Our analysis showed that HLA class I binding was accurately predicted for 87% of MiHA of which a relatively large proportion of peptides had strong binding affinity (56%). Weak binding affinity was also predicted for a considerable number of antigens (31%) and the remaining 13% of MiHA were not predicted as HLA class I binding peptides. Besides prediction for HLA class I binding, none of the other online algorithms significantly contributed to MiHA characterization. Furthermore, we demonstrated that the majority of MiHA do not differ from their allelic variants in <i>in silico</i> characteristics, suggesting that allelic variants can potentially be processed and presented on the cell surface. In conclusion, our analyses revealed the <i>in silico</i> characteristics of 68 HLA class I-restricted MiHA and explored the value of online algorithms to predict T-cell ligands that are created by genetic variants.</p></div

Binding motifs for HLA class I alleles.

<p>Binding motifs for HLA class I alleles.</p

Predicted HLA class I binding affinity for MiHA and allelic variants.

<p>HLA class I binding affinity as predicted for MiHA and their allelic variants by NetMHCpan 2.8. Predicted affinity (1/affinity (nM); upper) and %-Rank (1/%-Rank; lower) are shown for all MiHA with allelic variants (n = 57) divided into two groups based on whether the polymorphic amino acid is an anchor residue (n = 12; left) or TCR contact residue (n = 45; right). Default thresholds for SB and WB peptides are indicated by red lines. The data show that predicted HLA class I binding for the 12 MiHA with polymorphic amino acids at anchor positions was significantly higher than for their allelic variants (p = 0.0005 using Wilcoxon signed rank test). For the MiHA with polymorphic amino acids at TCR contact residues (n = 45), predicted HLA class I binding as compared to their allelic variants was higher for 9 MiHA with the variant residue immediately adjacent to the anchor at position 2 (p = 0.0039 using Wilcoxon signed rank test), but similar for the remaining 36 antigens (p = 0.1965 using Wilcoxon signed rank test).</p

<i>In silico</i> analysis of minor histocompatibility antigens.

<p><i>In silico</i> analysis of minor histocompatibility antigens.</p

HLA class I-restricted minor histocompatibility antigens.

<p>HLA class I-restricted minor histocompatibility antigens.</p

Predicted stability of the peptide-HLA class I complex, proteasomal cleavage, TAP transport and their integration.

<p>(A) Peptide-HLA class I complex stability as predicted by NetMHCstab 1.0 with default settings for all MiHA for which HLA class I restriction alleles are available in the algorithm (n = 46), HLA-A*02:01-restricted MiHA (n = 15) and HLA-B*07:02-restricted MiHA (n = 18). Indicated are absolute numbers of MiHA that are predicted as highly stable (HS; black bars), weakly stable (WS; light grey bars) or non-stable (NS; dark grey bars) complexes. The data show that NetMHCstab 1.0 accurately predicted 29 of the 46 MiHA, including 9 HLA-A*02:01-restricted MiHA and 14 HLA-B*07:02-restricted MiHA. (B) Proteasomal cleavage at the C-terminus as predicted by NetChop 3.1 for all different MiHA peptides (n = 60) and for MiHA that are predicted to bind to HLA-A*02:01 (n = 13) or HLA-B*07:02 (n = 17) by NetMHCpan 2.8. Whole protein sequences were fed into the algorithm and default settings were used to predict proteasomal cleavage. Indicated are absolute numbers of peptides with predicted cleavage at the C-terminus for MiHA (left) and the reference set of peptides (right). No significant difference was observed in proportion of peptides with predicted cleavage at the C-terminus between MiHA and reference peptides (80% for MiHA <i>versus</i> 70% for reference peptides, p = 0.3141 using Fisher’s exact test). (C) Affinity for the TAP transporter as predicted by TAPPred with default settings for all different MiHA peptides (n = 60) and for MiHA that are predicted to bind to HLA-A*02:01 (n = 13) or HLA-B*07:02 (n = 17) by NetMHCpan 2.8. Indicated are absolute numbers of peptides with high (black bars), intermediate (light grey bars) and low (dark grey bars) affinity for TAP for the MiHA (left) and the reference peptides (right). No significant difference was observed in proportion of peptides with high or weak affinity for TAP between MiHA (43% high, 43% intermediate and 13% low affinity) and the reference peptides (54% high, 39% intermediate and 7% low affinity). (D) Epitope prediction by NetCTLpan 1.1 with default settings for the total set of MiHA (n = 65) and for HLA-A*02:01-restricted MiHA (n = 15) and HLA-B*07:02-restricted MiHA (n = 18) (left) as compared to reference peptides (right). Indicated are absolute numbers of peptides that are predicted as epitopes (black bars) or non-epitopes (grey bars). For HLA-A*02:01, the proportion of peptides that are predicted as T-cell epitopes is similar between MiHA and reference peptides (33% <i>versus</i> 21%, p = 0.3338), whereas for HLA-B*07:02, the proportion of peptides that are predicted as T-cell epitopes is higher for MiHA than for reference peptides although it did not reach statistical significance (72% <i>versus</i> 46%, p = 0.0514).</p

Predicted HLA class I binding affinity.

<p>(A) HLA class I binding affinity as predicted by NetMHCpan 2.8 for MiHA that have been identified as natural T-cell ligands by forward strategies. Results are shown for the total group of MiHA (n = 68) and for HLA-A*02:01-restricted MiHA (n = 15) and HLA-B*07:02-restricted MiHA (n = 18) (left) as compared to reference peptides with predicted binding affinity to HLA-A*02:01 (n = 906) or HLA-B*07:02 (n = 464) (right). Indicated are absolute numbers of peptides with strong predicted binding (SB; black bars), weak predicted binding (WB; light grey bars) and non-binding (NB; dark grey bars).The data show that the proportion of SB peptides in the group of MiHA is higher than in the reference set of peptides (54% <i>versus</i> 28% with p = 0.0581 for HLA-A*02:01; 65% <i>versus</i> 24% with p = 0.0005 for HLA-B*07:02 using Fisher’s exact test). (B) ROC curves for HLA class I binding affinity as predicted by NetMHCpan 2.8 for HLA-A*02:01 (left) and HLA-B*07:02 (right). Sensitivity and 1-specificity are shown on the Y- and X-axis, respectively. Curves for IC₅₀ (solid line) and %-Rank (dashed line) are plotted based on prediction data for MiHA and reference peptides. Sensitivity and specificity are indicated for default values for SB (≤0.5%-Rank or IC₅₀≤50 nM) and WB (≤2%-Rank or IC₅₀≤500 nM). For HLA-A*02:01, AUC values for %-Rank and IC₅₀ are 0.625 and 0.609, respectively (p = 0.0964 for %-Rank; p = 0.1486 for IC₅₀). For HLA-B*07:02, AUC values for %-Rank and IC₅₀ are 0.767 and 0.765, respectively (p = 0.0001 for %-Rank; p = 0.0001 for IC₅₀).</p

Predicted <i>in vivo</i> immunogenicity.

<p>(A) <i>In vivo</i> immunogenicity as predicted by the MHC I immunogenicity tool of the IEDB for the total group of MiHA (n = 65) and for MiHA with predicted binding to HLA-A*02:01 (n = 13) or HLA-B*07:02 (n = 17) by NetMHCpan 2.8. Indicated are immunogenicity scores for MiHA (left) and reference peptides (right). Designated are median immunogenicity scores (black horizontal lines) and thresholds of 0.27 and 0.22 to define immunogenic peptides for MiHA binding to HLA-A*02:01 or HLA-B*07:02, respectively (red lines). The data show a significant difference in proportion of immunogenic peptides between HLA-B*07:02-restricted MiHA and reference peptides (41% <i>versus</i> 10% with p = 0.0014 using Fisher’s exact test), but no significant difference between HLA-A*02:01-restricted MiHA and reference peptides (0% <i>versus</i> 10% with p = 0.3825 using Fisher’s exact test). (B) ROC curves for <i>in vivo</i> immunogenicity as predicted by the online tool of the IEDB for HLA-A*02:01 (solid line) and HLA-B*07:02 (dashed line) based on prediction data for MiHA and reference peptides. Thresholds with 90% specificity are indicated by the red vertical line.</p

Optimized Whole Genome Association Scanning for Discovery of HLA Class I-Restricted Minor Histocompatibility Antigens

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Optimized Whole Genome Association Scanning for Discovery of HLA Class I-Restricted Minor Histocompatibility Antigens

Patients undergoing allogeneic stem cell transplantation as treatment for hematological diseases face the risk of Graft-versus-Host Disease as well as relapse. Graft-versus-Host Disease and the favorable Graft-versus-Leukemia effect are mediated by donor T cells recognizing polymorphic peptides, which are presented on the cell surface by HLA molecules and result from single nucleotide polymorphism alleles that are disparate between patient and donor. Identification of polymorphic HLA-binding peptides, designated minor histocompatibility antigens, has been a laborious procedure, and the number and scope for broad clinical use of these antigens therefore remain limited. Here, we present an optimized whole genome association approach for discovery of HLA class I minor histocompatibility antigens. T cell clones isolated from patients who responded to donor lymphocyte infusions after HLA-matched allogeneic stem cell transplantation were tested against a panel of 191 EBV-transformed B cells, which have been sequenced by the 1000 Genomes Project and selected for expression of seven common HLA class I alleles (HLA-A∗01:01, A∗02:01, A∗03:01, B∗07:02, B∗08:01, C∗07:01, and C∗07:02). By including all polymorphisms with minor allele frequencies above 0.01, we demonstrated that the new approach allows direct discovery of minor histocompatibility antigens as exemplified by seven new antigens in eight different HLA class I alleles including one antigen in HLA-A∗24:02 and HLA-A∗23:01, for which the method has not been originally designed. Our new whole genome association strategy is expected to rapidly augment the repertoire of HLA class I-restricted minor histocompatibility antigens that will become available for donor selection and clinical use to predict, follow or manipulate Graft-versus-Leukemia effect and Graft-versus-Host Disease after allogeneic stem cell transplantation