Supplementary Figure 3: Relationship between gene expression levels and HLA ligand presentation.

(a) Comparison of gene expression levels of HLA-DR ligands and non-ligands in JeKo-1 cell line. Distributions of RNA-Seq estimated gene expression level (TPM) from JeKo-1 cell line HLA-DR ligands (red, n=5720), whole transcriptome (green, n=23165), and non-ligand genes (blue, n=22000) were plotted in a logarithmic space. HLA-DR ligands have significantly higher gene expression levels than the whole transcriptome (Mann-Whitney U test, **=p<1e-5), non-ligands have significantly lower gene expression levels than the whole transcriptome (Mann-Whitney U test, **=P<1e-5). In (a)-(c), Violin curves represent probability distribution function of gene expression, black boxes represent middle two quartiles, and white dot represents median. (b) Effects of correcting blood and ECM genes in estimating ligand gene expression levels in MCL patients. We reassigned gene expression values of blood particle or extracellular (ECM) associated genes to 50 TPM. Statistical significantly lower numbers of MCL patient HLA-DR ligand genes have low expression levels (0.1 TPM) after correction of blood and ECM genes (green, Mann-Whitney U test, **=P<1e-5, n=34049). (c) Comparison of gene expression levels of HLA-I ligands and non-ligands in JeKo-1 cell line and MCL patients. Distributions of RNA-Seq estimated gene expression level (TPM) from HLA-I ligands (red, n=60169 and 5555) and random protein-coding genes (green, n=23165 and 23165) were plotted in a logarithmic space. HLA-I ligands have significantly higher gene expression levels than the random protein-coding genes in both JeKo-1 cell lines (Mann-Whitney U test, **=P<1e-5) and MCL patients (Mann-Whitney U test, **=P<1e-5). Lowly expressed HLA-I ligands (<0.1TPM) are weakly enriched for blood micro-particles (FDR-corrected hypergeometrical test, q-value < 0.05). (d) Influence of filtering decoy peptide gene expression values on predictive power of gene expression. Gene expressions alone can differentiate between presented peptides and random decoys with 0.81 AUC. However, the predictive decreases as we removes lowly expressed genes. Gene expression does not differentiate MCL HLA-DR ligands and decoy peptides with >25 TPM gene expression values (0.51 AUC, n=3300 for ligands and n=10000 for decoy peptides). (e) Performance of MARIA on MCL validation set using various gene expression profiles. For 6 MCL patients, MARIA was run with three different gene expression profile dictionaries: patient-matched RNASeq, external MCL RNASeq, and shuffled external MCL RNASeq. MARIA predictive powers are not different between using patient-matched and external MCL RNASeq dictionaries (P=0.3, n=6). MARIA predictive power decreases when using a shuffled RNASeq dictionary (P=0.0002, n=6). P-values were determined with two-tailed paired t-test. (f) Performance of MARIA using tissue-matched or tissue-mismatched gene expression profile. For predicting ligands presented by melanoma HLA-II (n=10513), using tissue-mismatched RNASeq dictionaries decreases MARIA by less than 1% AUC. However, using a shuffled RNASeq dictionary profile data decreased MARIA performance by 7% AUC. SKCM: Skin cutaneous melanoma, GBM: Glioblastoma multiforme, BRCA: Breast invasive carcinoma, LUSC: Lung squamous cell carcinoma. (g) Influence of expression level thresholds for correction of extracellular matrix (ECM) genes on model performance. Depicted is the relationship between cross-validation AUC at various correction levels (b, TPM thresholds) for genes associated with extracellular matrix (Gene Ontology Cellular Compartment accession GO:0031012). Asterisks capture significant differences in cross-validation AUCs when comparing lower TPM thresholds than the mean level (TPM~50) that we had originally selected (** indicates Mann-Whitney U test p=0.0002 for TPM0 vs. TPM50, n=10; * indicates Mann-Whitney U test P=0.001 for TPM10 vs. TPM50, n=10). Models using thresholds between 20-100 TPM yielded highly similar validation AUC scores.