HLA class I alleles are associated with peptide-binding repertoires of different size, affinity, and immunogenicity

Abstract

Prediction of HLA binding affinity is widely used to identify candidate T cell epitopes, and an affinity of 500 nM is routinely used as a threshold for peptide selection. However, the fraction (percentage) of peptides predicted to bind with affinities of 500 nM varies by allele. For example, of a large collection of ~30,000 dengue virus-derived peptides only 0.3% were predicted to bind HLA A*0101, whereas nearly 5% were predicted for A*0201. This striking difference could not be ascribed to variation in accuracy of the algorithms used, as predicted values closely correlated with affinity measured in vitro with purified HLA molecules. These data raised the question whether different alleles would also vary in terms of epitope repertoire size, defined as the number of associated epitopes or, alternatively, whether alleles vary drastically in terms of the affinity threshold associated with immunogenicity. To address this issue, strains of HLA transgenic mice with wide (A*0201), intermediate (B*0702), or narrow (A*0101) repertoires were immunized with peptides of varying binding affinity and relative percentile ranking. The results show that absolute binding capacity is a better predictor of immunogenicity, and analysis of epitopes from the Immune Epitope Database revealed that predictive efficacy is increased using allele-specific affinity thresholds. Finally, we investigated the genetic and structural basis of the phenomenon. Although no stringent correlate was defined, on average HLA B alleles are associated with significantly narrower repertoires than are HLA A alleles.

Figure 1. The predicted binding repertoire is highly variable among HLA alleles

(A) The repertoire size (cumulative percentage) of predicted binders among the 27 alleles considered in the study is shown. The peptides were considered to be binders if the binding affinity (IC₅₀) predicted by SMM algorithm was ≤ 500 nM. (B) The geometric mean of binding affinity (IC₅₀ predicted by SMM algorithm) of the top 1% peptides based on SMM IC₅₀ is shown.

Figure 2. Immunogenicity of predicted binders varies between HLA alleles

A) For each HLA allele 120 peptides were predicted to represent four percentile ranges: (i) 0 – 0.30, (ii) 0.30 – 1.25 (iii) 1.25 – 5.0 (iv) 5.0 – 15.0. For each percentile range 30 randomly selected peptides were pooled into 3 pools of 10 individual peptides [10μg/peptide]. Groups of 3 HLA transgenic mice between 8 and 12 weeks of age were immunized s.c with each pool diluted in 100 μl PBS emulsified in CFA. Two weeks post immunization the mice were sacrificed, and splenic CD8⁺ T cells were isolated and screened for IFNγ production. Data are expressed as mean number of SFC/10⁶ CD8⁺ T cells from three independent experiments. Error bars represent SEM. Responses against peptides were considered positive if the stimulation index (SI) exceeded double the mean negative control wells (CD8⁺ T cells plus APCs without peptide) and net spots were above the threshold of 20 SFCs/10⁶ CD8⁺ T cells experiments. Asterisks indicate peptides able to elicit a significant IFNγ response in two out of three individual experiments, according to the criteria described above. (B) Pools eliciting a significant IFNγ response were subsequently deconvoluted to identify individual epitopes. For each allele the number of identified epitopes per percentile is shown.

Figure 3. IC₅₀ threshold of epitopes derived from the IEDB

The cumulative epitope distribution is compared with the binding affinity (SMM IC₅₀ nM) for alleles for which at least 15 data points were available from the IEDB. While the affinity distribution for different alleles varied significantly, 500 nM was found to be a useful binding threshold. Overall, the median % fraction of epitopes identified by this threshold is 79%.

Figure 4

Correlation between cumulative % of predicted binders and binding affinity (IC₅₀) of the epitopes retrieved from IEDB at IC₅₀ 500nM among different alleles. Alleles with higher number of predicted binders had epitopes with stronger binding affinity.

Figure 5

Average number of peptides/allele required by different prediction strategies using a uniform threshold of IC₅₀ of 500 nM (black bar) or an allele specific IC50 threshold (white bar). The same set of alleles and DENV sequences from Figure 1 was utilized. Error bars represent SEM.

Figure 6

Average repertoire size of predicted binders restricted by HLA A and B alleles. HLA A alleles in general were found to be associated with broader predicted binder repertoires. Error bars represent SEM.