HLA-DM constrains epitope selection in the human CD4 T cell response to vaccinia virus by favoring the presentation of peptides with longer HLA-DM-mediated half-lives

Abstract

HLA-DM (DM) is a nonclassical MHC class II (MHC II) protein that acts as a peptide editor to mediate the exchange of peptides loaded onto MHC II during Ag presentation. Although the ability of DM to promote peptide exchange in vitro and in vivo is well established, the role of DM in epitope selection is still unclear, especially in human response to infectious disease. In this study, we addressed this question in the context of the human CD4 T cell response to vaccinia virus. We measured the IC(50), intrinsic dissociation t(1/2), and DM-mediated dissociation t(1/2) for a large set of peptides derived from the major core protein A10L and other known vaccinia epitopes bound to HLA-DR1 and compared these properties to the presence and magnitude of peptide-specific CD4(+) T cell responses. We found that MHC II-peptide complex kinetic stability in the presence of DM distinguishes T cell epitopes from nonrecognized peptides in A10L peptides and also in a set of predicted tight binders from the entire vaccinia genome. Taken together, these analyses demonstrate that DM-mediated dissociation t(1/2) is a strong and independent factor governing peptide immunogenicity by favoring the presentation of peptides with greater kinetic stability in the presence of DM.

FIGURE 1. Recognition of A10L peptides by a vaccinia virus-specific CD4 T cell line

(A) The specificity of the vaccinia-specific TCL was tested using IFNγ-ELISPOT. TCL was stimulated with vaccinia virus-infected CV-1 cell lysate (VV, red bar), non-infected CV-1 cell lysate (NI, green bar), and medium only (MED, blue bar), using irradiated autologous PBMC (irPBMC, left panel) or HLA-DR1 matched B-lymphoblastoid cells (LG2,right panel) as antigen presenting cells (APC). The p-value of responses between VV and NI is indicated. This data represent three independent experiments with 4 replicates each. (B, C) The VV-specific CD4 T cell line was stimulated with overlapping A10L peptides to test peptide immunogenicity, shown as spots per well (SPW) in IFNγ-ELISPOT assay using (B) autologous irPBMC (solid blue circle,) or (C) LG2 (solid green circle) as APCs. Medium (MED) was used as control (clear blue circle in (B) and clear green circle in (D) at the very right x-axis). Dashed line represented the threshold we set up for positive peptides (SPW is more than two fold of that of MED, and p value between that peptide and MED is less than 0.01). The positive peptides are labeled with p-value relative to medium control. (B) and (C) represent three independent experiments with at least two replicates each.

FIGURE 2. Different IC₅₀, dissociation kinetics, and DM-susceptibilities of A10L peptides bound to HLA-DR1

(A) Representative peptides with different IC₅₀ to HLA-DR1. IC₅₀ is reported as the 50% inhibition concentration calculated from the binding inhibition curve. (B–D) Representative peptides with different dissociation kinetics in the presence of various concentrations of DM. Dissociation half-lives in the absence of DM (intrinsic half-life) and in the presence of DM (DM-mediated half-life) were calculated from these dissociation curves. (E) k_off versus DM concentration for representative peptides. The DM susceptibility is calculated as the slope of the k_off versus DM concentration curve (using 0–1μM data only in cases where hyperbolic behavior was observed). The IC₅₀ and dissociation kinetics for all A10L peptides were listed in Table II, with fixed DM concentration at 1uM for dissociation kinetics. These data represent three independent experiments with two replicates each.

FIGURE 3. Analysis of IC₅₀, intrinsic half-life, DM-mediated half-life, and DM-susceptibility between epitopes and non-epitopes from A10L

The positive peptides selected in Fig. 1 were shown as epitopes (red squares), and the remaining negative peptides as non-epitopes (blue circles). Two-tailed unpaired t test between epitope and non-epitope was performed for (A) IC₅₀, (B) intrinsic half-life, (C) DM-mediated half-life, and (D) DM-susceptibility. p-value is indicated in each graph of (A–D). Correlation between DM-mediated half-life and IC₅₀ (E), and intrinsic half-life (F) were shown. The seven positive peptides were highlighted as red dots. Correlation coefficient (cc) is indicated in upper right of (E) and (F). DM-susceptibility =(k_off,DM-k_off,in)/[DM], where [DM]=1uM.

FIGURE 4. ROC analysis shows that DM-mediated half-life is a strong predictor of CD4 T cell epitopes from A10L

(A) Schematic contingency table showing calculation of true positive rate (sensitivity) and true negative rate (specificity). Sensitivity and specificity were calculated for (B) IC₅₀ (IC₅₀=800nM), (C) intrinsic half-life (t_1/2=45hr), (D) DM-mediated half-life (t_1/2DM=6hr), and (E) DM-susceptibility (DM-sus=0.1hr⁻¹uM⁻¹), with the selected threshold indicated in parenthesis. The ROC curves with all varying thresholds were plotted for (F) IC₅₀, (G) intrinsic half-life, (H) DM-mediated half-life, and (I) DM-susceptibility. The true positive peptides and true negative peptides set for this analysis were the same ones shown in Fig. 1. Area under curve (AUC) is indicated in upper right of each plot. The IC₅₀ and dissociation kinetics assays were repeated three independent times with two replicates each.

FIGURE 5. DM-mediated half-life distinguishes vaccinia epitopes from non-recognized peptides in a set of predicted HLA-DR1 binders

Binding properties of a selection of other vaccinia virus peptides predicted to bind to HLA-DR1 and shown to be recognized by a set of vaccinia-immune donors (epitopes, red) or not recognized (non-epitopes, blue). (A) IC₅₀, (B) intrinsic half-lives, (C) DM-mediated half-lives, and (D) DM-susceptibilities of epitopes and non-epitopes were shown with p-value indicated on the right panels. DM concentration was 1uM. The IC₅₀ and dissociation kinetics for each single peptide were listed in Table III. These data represent three independent experiments with two replicates each.

FIGURE 6. P9, IEDB and Syfpeithi prediction algorithms on peptide immunogenicity

Left panels: Two-tailed unpaired t test between epitopes (red squares) and non-epitopes (blue circles) were performed for (A) P9, (B) IEDB and (C) Syfpeithi predicted values. p-value is indicated on each graph. Right panels: ROC analysis for (A) P9, (B) IEDB and (C) Syfpeithi. Area under curve (AUC) is indicated in upper right of each plot. The positive and negative peptides selected for this analysis were the same as described above.