Fig. 2: Significant inter-allelic differences are observed in Tm distributions.
The length distributions of peptides bound to either HLA-A*02:01 or HLA-B*07:02 identified in the thermostability assay and the immunoprecipitation of pHLA complexes using established workflows9 were as expected from previous immunopeptidomics studies, with ligands being predominantly 9-mers. b Tm values for 1094 HLA-A*02:01-restricted peptides do not differ significantly based on their length (p = 0.016, Kruskal-Wallis test; individual length comparisons with Mann-Whitney U test with Bonferroni adjustment for multiple comparisons, p > 0.05). Tm values for 1354 HLA-B*07:02-restricted peptides do not differ significantly for all but one of the length comparisons, with a difference observed between the Tm distributions of 9mers and 10mers; p = 5.20*10-5, Kruskal-Wallis test; individual length comparisons with Mann-Whitney U test with Bonferroni adjustment for multiple comparisons, p > 0.05 for all but the 9mer and 10mer distributions with p = 1.20*10-6). Violin plot representation of data shows the median as center white dot and 25th percentile and 75th percentile as bounds of the center black box. c There is a good correlation between the Tm values for HLA-C*04:01-restricted peptides identified in either the C1R-A*02:01 or C1R-B*07:02 assay (PCC = 0.78) demonstrating high assay robustness. This correlation is further improved (PCC = 0.87) when removing outlier peptides (indicated in red) that have high binding affinity to other alleles expressed by the C1R cell line. d Tm distributions for HLA-A*02:01 (blue), HLA-B*07:02 (orange) and HLA-C*04:01 (green), expressed by the C1R cell lines studied show that there is a substantial difference in their variance (transparency is given to the bars to visualize their overlap and allele-specific distributions). e A significant difference in the Tm distribution for peptides restricted by HLA-A*02:01 (1094 peptides), HLA-B*07:02 (1354 peptides) and HLA-C*04:01 (359 peptides) is observed; p = 1.48*10-93, Kruskal-Wallis test; **p < 10-25, ***p < 10-50, Mann–Whitney U test with Bonferroni adjustment for multiple comparisons. Box plot representation of data shows the median as center, 25th percentile and 75th percentile as bounds of boxes, maximum as 75th percentile + 1.5 times the interquartile range and minimum as 25th percentile - 1.5 times the interquartile range. PCC: Pearson Correlation Coefficient.
