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

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

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

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

Improved HLA-class I restricted avidity of CD8αß expressing HA-2-TCR td CD4⁺ T-cells results in improved proliferation.

<p>(A) To study whether co-transfer of CD8 would also improve the peptide sensitivity of CD4⁺ T-cells transduced with a next generation HA-2-TCR, both mock and HA-2-TCR td CMV-specific CD4⁺ T-cells with or without co-transfer of different CD8 subunits as indicated in the figure were purified using flow cytometry based cell sorting and stimulated with unpulsed HLA-A2⁺ HA-2⁻ LCL IZA (white bars; LCL IZA), HLA-A2⁺ HA-2⁻ LCL-IZA pulsed with decreasing concentrations of HA-2 peptide (range 1 µM-10 pM) or HLA-A2⁺ HA-2⁺ LCL JYW (striped bars; LCL JYW). IFN-γ production was measured after 18 h of stimulation in duplicate, and a representative experiment out of 2 is depicted. The IFN-γ production of ΔCD8αß and wtCD8αß expressing HA-2-TCR_CC td CD4⁺ T-cells significantly higher (p-values <0.05) than CD8 negative or CD8αα expressing HA-2-TCR_CC td CD4⁺ T-cells is indicated with an asterisk. (B) To investigate their proliferative capacity, both mock and HA-2-TCR td CD4⁺ T-cells without CD8 or co-transferred with wtCD8α, wtCD8αß, or ΔCD8αß were purified based on markergene expression and CD8 cell surface expression and were either not stimulated (filled histograms) or stimulated with HLA-A2⁺ HA-2⁺ LCL-JYW (thick black line). Histograms depict PKH dilution measured 5 days after stimulation, and a representative example of 2 independent experiments is depicted.</p

In general, co-transfer of the extracellular domains of CD8α and ß is required and sufficient.

<p>To confirm the generality of the previous data, total CD4⁺ T-cells were transduced with codon optimized and cysteine modified HA-1-, HA-2- or PRAME-TCR (transduction efficiency 48%, 48% and 22%, respectively) either with or without co-transfer of different CD8 molecules, as indicated in the figure. One week after transduction, non-purified TCR td CD4⁺ T-cells were stimulated and tested for cytokine production using flow cytometry. HA-1- or HA-2-TCR td CD4⁺ T-cells were stimulated either with HA-1 or HA-2 peptide pulsed or unpulsed HLA-A2⁺ HA-1^- HA-2⁻ LCL-IZA, or HLA-A2⁺ HA-1⁺ HA-2⁺ LCL-MRJ, and PRAME-TCR td CD4⁺ T-cells were stimulated either with PRAME peptide pulsed or unpulsed HLA-A2⁺ PRAME⁻ melanoma cells, or HLA-A2⁺ PRAME⁺ melanoma cells. 5 h After stimulation, T-cells were permeabilized and stained with anti-NGF-R in combination with either anti-IFN-γ (upper panel), anti-IL-2 (middle panel) or anti-TNF-α (lower panel), and analyzed using flow cytometry. The percentage of markergene positive and CD8 positive T-cells producing cytokines after stimulation with antigen-negative cells (white bars; control), peptide pulsed cells (grey bars; pulsed peptide) or antigen-positive cells (black bars; endogenous peptide) is indicated.</p