Supplementary Figure 8: Validating MARIA performance for predicting patient IgH HLA-DR presentation and immune response.

(a) NetMHCIIpan predicted HLA-DR presentation of lymphoma immunoglobulin correlated with experimentally identified HLA-DR immunoglobulin ligands. 18 MCL immunoglobulin sequences were analyzed by NetMHCIIpan(left, blue). The same 18 MCL samples were profiled with LC-MS/MS to determine the regions of immunoglobulin presented by HLA-DR. Predicted and observed presentation hot spots were significantly correlated on light chains (Spearman rho 0.48, p=3.8e-14, n=311). NetMHCIIpan prediction correlated with observed heavy chain presentation moderately (Spearman rho 0.10, p=0.02, n=1015). NetMHCIIpan predicted ligand numbers were normalized with the MS identified maximum ligand numbers for visualization purposes. (b) Precision-Recall curves of different models for identifying immunoglobulin (Ig) HLA-DR ligands. Curves depict the comparison of the precision/PPV (y-axis) for MARIA (blue curves) versus NetMHCIIpan (green curves) when considering a range of recall/sensitivity thresholds (x-axis). At 20% recall, MARIA achieved 56% and 31% precision for predicting Ig heavy chain (left panel) and light chain (right panel) presentation, respectively. In comparison, NetMHCIIpan achieved 13% and 16% at the same recall. Prevalence of 1% was used for all calculations. (c) Gating strategies of identifying alive CD4 T-cells after peptide stimulations. Analysis (d) was based on singlet lymphocyte populations with low 7-AAD and high CD4 levels. (d) Experimental validation of CD4 immunogenicity for candidate peptide neoantigens identified by MARIA. Peripheral blood mononuclear cells (PBMCs) were isolated from 3 MCL patients after immunization with autologous tumor vaccines. Patient PBMCs were re-stimulated with MARIA identified IgH neoantigens (>99.5th percentile) or control peptides for 30hrs. Antigen-specific T-cell activation was evaluated by cell surface CD137 induction. For 2 of 3 patients (MCL005 and MCL052), neoantigens induced specific CD4 T-cell activation with CD137+ levels comparable to positive controls (Pathogen Peptide Pool). These experiments were independently repeated in three patients with no technical replicates due to limited patient samples.