Inactivation of Calr induces an ER stress response and alters the peptidome presented on MHC-I. (A) Volcano plot of upregulated and downregulated genes in Calr KO versus control in LLC cells from RNA-seq data (n = 3 biological replicates). Genes related to ER stress response are highlighted. (B) Heatmap of upregulated and downregulated proteins in Calr KO versus control in LLC cells from proteomics data (n = 3 biological replicates). (C) Gene Ontology (GO) term analysis shows the top enriched pathway for upregulated genes (Calr KO versus control) from proteomics data as in B. (D) Venn diagram of MHC-I peptides in Calr KO and control in LLC cells identified by immunoprecipitation-mass spectrometry (IP-MS). Results are pooled from three biological replicates. (E) Volcano plot showing MHC-I peptides identified in both Calr KO and control LLC cells (n = 901). The x axis represents the log₂ fold change (Calr KO versus control) of each peptide, and the y axis represents the P value. (F) Top: Violin plot showing the log₂-transformed H2-Kb binding affinity (nM) of sgCalr-specific peptides (n = 56), sgNTC-specific peptides (n = 248), and peptides common to both groups (n = 860). The point represents the median log₂ affinity value of each group. Bottom: Table of the raw median affinity of sgCalr-specific, common, and sgNTC-specific peptides. sgCalr-specific peptides are defined as peptides unique to or over-presented in Calr KO cells, and sgNTC-specific peptides are defined as peptides unique to or over-presented in control cells. Affinity was predicted by netMHCpan 4.1. (G) Levels of SIINFEKL-bound H2Kb measured by flow cytometry using the 25-D1.16 antibody in Calr KO and control LLC cells upon pulsing or left unpulsed with SIINFEKL peptide (100 ng/ml, 4 h), with or without IFN-γ treatment (20 ng/ml) for 16 h. Data are presented as the mean ± SD and representative of two independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001 by an unpaired t test. (H) Levels of SIINFEKL-bound H2Kb measured by flow cytometry using the 25-D1.16 antibody in Calr KO and control LLC cells after pulsing with WT SIINFEKL (WT) and low-affinity variants SIINFEKV (V8) and SIINYEKL (Y5). Tumor cells were pretreated with 20 ng/ml IFN-γ for 16 h, then pulsed with peptides for 4 h at 200 ng/ml or 800 ng/ml as indicated. Data are presented as the mean ± SD and representative of two independent experiments. ns, not significant; **P < 0.01; ***P < 0.001; ****P < 0.0001 by an unpaired t test. (I) Boxplot demonstrating the binding affinity rank (BA rank) of HLA peptides identified from control or CALR KO A375 cells: sgCALR-specific peptides (n = 86), sgNTC-specific peptides (n = 166), and common peptides (n = 586). Affinity rank was predicted by netMHCpan 4.1. ns, not significant; ****P < 0.0001 by a Mann–Whitney U test. (J–L) HLA affinity analysis of peptides identified by HLA IP-MS by Shapiro et al. (2025) in CALR KO and WT cells. HLA-binding peptides were divided into three groups: those unique to or over-presented in CALR KO cells (sgCALR-specific, n = 1,403), unique to or over-presented in WT cells (WT-specific, n = 2,014), and common peptides (n = 13,648). The binding affinity (nM) of peptides presented on HLA-A*02:01 (J), HLA-B*40:01 (K), or HLA-C*03:04 (L) was predicted by netMHCpan 4.1 and shown as log₂-transformed values. The point represents the median of each group. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001 by a Mann–Whitney U test.