Inactivation of CALR does not intrinsically sensitize tumor cells to T cell–mediated killing but alters the MHC-I peptidome. (A) Coculture assay of OT-I T cells and control or Calr KO B16-OVA tumor. Tumor cells were either untreated or cocultured with preactivated OT-I T cells for 24 h at an effector-to-target ratio of 1:1. The number of tumor cells after coculture was measured by flow cytometry, and the percentage of remaining tumor cells relative to untreated control was shown. Data are presented as the mean ± SD and representative of two independent repeats. ns, not significant; *P < 0.05 by an unpaired t test. (B)In vitro competition assay based on tumor-CTL coculture. CTV-labeled LLC-OVA cells were mixed with either unlabeled cells transduced with control sgRNA (sgNTC) or sgRNA targeting Calr, then cocultured with preactivated OT-I T cells (at an effector-to-target ratio of 1:2), or left untreated. The proportion of unlabeled sgNTC or sgCalr tumor cell was determined by FACS, and then the proportion in the coculture group was compared with that in the untreated group, and shown as log2 fold change. Data are presented as the mean ± SD and representative of two independent repeats. **P < 0.01; ****P < 0.0001 by an unpaired t test. (C) Schematic diagram of in vivo competition assay using Calr KO and control LLC-OVA or B16F10 cells. Mixture of Calr KO and control cells (∼1:1 ratio) was inoculated in WT C57BL/6 mice, and Rag1 KO mice transferred with OT-I or Pmel-1 T cells, respectively. Rag1 KO mice without adoptive transfer were used as a control. Tumors were then collected, and the ratio of GFP+Calr KO to control cells was examined by FACS. (D) Summary of log2 fold change of percentages of LLC-OVA control or Calr KO cells in WT C57BL/6 mice (n = 8) and Rag1 KO mice transferred with OT-I T cells (n = 8–9). The proportion of GFP+ control or Calr KO cells in the mixed tumor was determined by FACS, then compared with the proportion obtained from Rag1 KO mice (without adoptive transfer) and shown as log2 fold change. Data are presented as the mean ± SD and analyzed by an unpaired t test. ***P < 0.001; ****P < 0.0001. (E) Summary of log2 fold change of percentages of Calr KO B16F10 cells in WT C57BL/6 mice (n = 9), and Rag1 KO mice transferred with Pmel-1 T cells (n = 5), as compared to the percentage in Rag1 KO mice (without adoptive transfer). Data are presented as the mean ± SEM and analyzed by an unpaired t test. ***P < 0.001. (F) Tumor volume of mixed LLC-OVA tumors at the day of tumor collection from Rag1 KO mice, with or without OT-I adoptive transfer, as in D. Data are presented as the mean ± SEM. *P < 0.05 by an unpaired t test. (G) Summary of log2 fold change of percentages of Calr KO B16F10 cells grown in WT C57BL/6 mice (n = 7–8), and Pmel-1 transgenic mice (n = 7), as compared to the percentage in Rag1 KO mice. Data are presented as the mean ± SEM and analyzed by an unpaired t test. ns, not significant; *P < 0.05. (H) Distribution of binding affinity of sgCalr-specific, common, and sgNTC-specific peptides identified by H2-Kb IP-MS, as in Fig. 3 F. The proportion of peptides of each group that fall into the indicated range of affinity was shown. Results are pooled from three biological replicates. (I) Level of H2-Kb bound to SIINFEKL measured by flow cytometry using the antibody 25-D1.16 in control and Calr KO B16F10 cells. Cells were pretreated with 20 ng/ml of IFN-γ for 18 h and then pulsed with SIINFEKL peptide (100 ng/ml) for 4 h prior to analysis. Data are presented as the mean ± SD and representative of two independent experiments. **P < 0.01 by an unpaired t test. (J) Level of H2-Kb bound to SIINFEKL measured by flow cytometry using the antibody 25-D1.16 in control and Calr KO MC38 cells. Cells were pulsed with SIINFEKL peptide (100 ng/ml) for 4 h prior to analysis. Data are presented as the mean ± SD and representative of two independent experiments. ***P < 0.001 by an unpaired t test. (K) Levels of H2Kb (using the antibody AF6-88.5) and SIINFEKL-bound H2Kb (using the antibody 25-D1.16) in LLC Calr KO or control cells after SIINFEKL pulsing. Cells were pretreated with 20 ng/ml IFN-γ for 16 h, then pulsed with 100 ng/ml SIINFEKL peptide for 4 h (or unpulsed), and analyzed by FACS. Relative exchange of H2Kb was calculated by normalizing the level of SIINFEKL-bound H2Kb to the level of total H2Kb, and shown as fold change relative to control cells. Data are presented as the mean ± SD and representative of two independent repeats. ****P < 0.0001 by an unpaired t test. (L)In vitro competition coculture assay of Calr KO and control tumor cells with effector OT-I T cells. Calr KO or control tumor cells were first mixed with CTV-labeled control cells at 1:1 ratio, then pulsed with SIINFEKL peptide (100 ng/ml) for 4 h, and cocultured with OT-I T cells (or untreated) at an E:T of 1:2 (LLC and MC38) or 1:1 (B16F10) for 24 h. The percentage of unlabeled Calr KO or control cells was then determined by FACS. The fold change of the proportion of unlabeled Calr KO or control cell after coculture compared with that in the untreated group was calculated, and shown as the log2-transformed value. Data are presented as the mean ± SD and representative of two independent repeats. ***P < 0.001; ****P < 0.0001 by an unpaired t test. (M) Representative histograms of H2-Kb bound to SIINFEKL measured by flow cytometry in Calr KO and control LLC cells, as in Fig. 3 H. Cells were pretreated with 20 ng/ml IFN-γ for 16 h, then either unpulsed or pulsed with 800 ng/ml of WT (SIINFEKL), V8 (SIINFEKV), or Y5 (SIINYEKL) peptides for 4 h. (N) Western blot of control or CALR KO A375 and SW480 cells. GAPDH was used as a loading control. (O) Boxplot demonstrating the binding affinity rank (BA rank) of HLA peptides identified from control or CALR KO SW480 cells: sgCALR-specific peptides (n = 30), sgNTC-specific peptides (n = 64), and common peptides (n = 740). Affinity rank was predicted by netMHCpan 4.1. **P < 0.01; ****P < 0.0001 by a Mann–Whitney U test. (P) Table of the raw median affinity of sgCALR-specific, common, and WT-specific peptides identified by HLA IP-MS by Shapiro et al. (2025), as shown in Fig. 3, J–L. Affinity was predicted by netMHCpan 4.1. (Q) Coculture of control or CALR KO SW480 cells or A498 cells with Jurkat-expressing 1G4 TCR. Tumor cells were pulsed with 200 ng/ml SLLMWITQV (NY-ESO-1157-165 peptide variant) for 4 h, then cocultured with 1G4-Jurkat for 24 h at 1:1 ratio. The gMFI of CD69 on Jurkat cells was measured by flow cytometry. Data are presented as the mean ± SD. Significance was evaluated by an unpaired t test. *P < 0.05; ***P < 0.001. ACT, adoptive cell transfer. gMFI, geometric mean fluorescence intensity; E:T, effector-to-target ratio. Source data are available for this figure: SourceData FS2.
Figure S2.

Inactivation of CALR does not intrinsically sensitize tumor cells to T cell–mediated killing but alters the MHC-I peptidome. (A) Coculture assay of OT-I T cells and control or Calr KO B16-OVA tumor. Tumor cells were either untreated or cocultured with preactivated OT-I T cells for 24 h at an effector-to-target ratio of 1:1. The number of tumor cells after coculture was measured by flow cytometry, and the percentage of remaining tumor cells relative to untreated control was shown. Data are presented as the mean ± SD and representative of two independent repeats. ns, not significant; *P < 0.05 by an unpaired t test. (B)In vitro competition assay based on tumor-CTL coculture. CTV-labeled LLC-OVA cells were mixed with either unlabeled cells transduced with control sgRNA (sgNTC) or sgRNA targeting Calr, then cocultured with preactivated OT-I T cells (at an effector-to-target ratio of 1:2), or left untreated. The proportion of unlabeled sgNTC or sgCalr tumor cell was determined by FACS, and then the proportion in the coculture group was compared with that in the untreated group, and shown as log2 fold change. Data are presented as the mean ± SD and representative of two independent repeats. **P < 0.01; ****P < 0.0001 by an unpaired t test. (C) Schematic diagram of in vivo competition assay using Calr KO and control LLC-OVA or B16F10 cells. Mixture of Calr KO and control cells (∼1:1 ratio) was inoculated in WT C57BL/6 mice, and Rag1 KO mice transferred with OT-I or Pmel-1 T cells, respectively. Rag1 KO mice without adoptive transfer were used as a control. Tumors were then collected, and the ratio of GFP+Calr KO to control cells was examined by FACS. (D) Summary of log2 fold change of percentages of LLC-OVA control or Calr KO cells in WT C57BL/6 mice (n = 8) and Rag1 KO mice transferred with OT-I T cells (n = 8–9). The proportion of GFP+ control or Calr KO cells in the mixed tumor was determined by FACS, then compared with the proportion obtained from Rag1 KO mice (without adoptive transfer) and shown as log2 fold change. Data are presented as the mean ± SD and analyzed by an unpaired t test. ***P < 0.001; ****P < 0.0001. (E) Summary of log2 fold change of percentages of Calr KO B16F10 cells in WT C57BL/6 mice (n = 9), and Rag1 KO mice transferred with Pmel-1 T cells (n = 5), as compared to the percentage in Rag1 KO mice (without adoptive transfer). Data are presented as the mean ± SEM and analyzed by an unpaired t test. ***P < 0.001. (F) Tumor volume of mixed LLC-OVA tumors at the day of tumor collection from Rag1 KO mice, with or without OT-I adoptive transfer, as in D. Data are presented as the mean ± SEM. *P < 0.05 by an unpaired t test. (G) Summary of log2 fold change of percentages of Calr KO B16F10 cells grown in WT C57BL/6 mice (n = 7–8), and Pmel-1 transgenic mice (n = 7), as compared to the percentage in Rag1 KO mice. Data are presented as the mean ± SEM and analyzed by an unpaired t test. ns, not significant; *P < 0.05. (H) Distribution of binding affinity of sgCalr-specific, common, and sgNTC-specific peptides identified by H2-Kb IP-MS, as in Fig. 3 F. The proportion of peptides of each group that fall into the indicated range of affinity was shown. Results are pooled from three biological replicates. (I) Level of H2-Kb bound to SIINFEKL measured by flow cytometry using the antibody 25-D1.16 in control and Calr KO B16F10 cells. Cells were pretreated with 20 ng/ml of IFN-γ for 18 h and then pulsed with SIINFEKL peptide (100 ng/ml) for 4 h prior to analysis. Data are presented as the mean ± SD and representative of two independent experiments. **P < 0.01 by an unpaired t test. (J) Level of H2-Kb bound to SIINFEKL measured by flow cytometry using the antibody 25-D1.16 in control and Calr KO MC38 cells. Cells were pulsed with SIINFEKL peptide (100 ng/ml) for 4 h prior to analysis. Data are presented as the mean ± SD and representative of two independent experiments. ***P < 0.001 by an unpaired t test. (K) Levels of H2Kb (using the antibody AF6-88.5) and SIINFEKL-bound H2Kb (using the antibody 25-D1.16) in LLC Calr KO or control cells after SIINFEKL pulsing. Cells were pretreated with 20 ng/ml IFN-γ for 16 h, then pulsed with 100 ng/ml SIINFEKL peptide for 4 h (or unpulsed), and analyzed by FACS. Relative exchange of H2Kb was calculated by normalizing the level of SIINFEKL-bound H2Kb to the level of total H2Kb, and shown as fold change relative to control cells. Data are presented as the mean ± SD and representative of two independent repeats. ****P < 0.0001 by an unpaired t test. (L)In vitro competition coculture assay of Calr KO and control tumor cells with effector OT-I T cells. Calr KO or control tumor cells were first mixed with CTV-labeled control cells at 1:1 ratio, then pulsed with SIINFEKL peptide (100 ng/ml) for 4 h, and cocultured with OT-I T cells (or untreated) at an E:T of 1:2 (LLC and MC38) or 1:1 (B16F10) for 24 h. The percentage of unlabeled Calr KO or control cells was then determined by FACS. The fold change of the proportion of unlabeled Calr KO or control cell after coculture compared with that in the untreated group was calculated, and shown as the log2-transformed value. Data are presented as the mean ± SD and representative of two independent repeats. ***P < 0.001; ****P < 0.0001 by an unpaired t test. (M) Representative histograms of H2-Kb bound to SIINFEKL measured by flow cytometry in Calr KO and control LLC cells, as in Fig. 3 H. Cells were pretreated with 20 ng/ml IFN-γ for 16 h, then either unpulsed or pulsed with 800 ng/ml of WT (SIINFEKL), V8 (SIINFEKV), or Y5 (SIINYEKL) peptides for 4 h. (N) Western blot of control or CALR KO A375 and SW480 cells. GAPDH was used as a loading control. (O) Boxplot demonstrating the binding affinity rank (BA rank) of HLA peptides identified from control or CALR KO SW480 cells: sgCALR-specific peptides (n = 30), sgNTC-specific peptides (n = 64), and common peptides (n = 740). Affinity rank was predicted by netMHCpan 4.1. **P < 0.01; ****P < 0.0001 by a Mann–Whitney U test. (P) Table of the raw median affinity of sgCALR-specific, common, and WT-specific peptides identified by HLA IP-MS by Shapiro et al. (2025), as shown in Fig. 3, J–L. Affinity was predicted by netMHCpan 4.1. (Q) Coculture of control or CALR KO SW480 cells or A498 cells with Jurkat-expressing 1G4 TCR. Tumor cells were pulsed with 200 ng/ml SLLMWITQV (NY-ESO-1157-165 peptide variant) for 4 h, then cocultured with 1G4-Jurkat for 24 h at 1:1 ratio. The gMFI of CD69 on Jurkat cells was measured by flow cytometry. Data are presented as the mean ± SD. Significance was evaluated by an unpaired t test. *P < 0.05; ***P < 0.001. ACT, adoptive cell transfer. gMFI, geometric mean fluorescence intensity; E:T, effector-to-target ratio. Source data are available for this figure: SourceData FS2.

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