Inactivation of CALR potentiates antitumor immunity and synergizes with anti-PD-1 therapy. (A) Schematic representation of the in vivo CRISPR screen design. Figure created using https://BioRender.com. (B) Scatterplot depicting the top enriched and depleted sgRNAs based on the mean log₂ fold change of sgRNA counts in the anti-PD-1 treatment group (200 μg/mice on days 6, 9, and 12; n = 15 mice) compared with the CD8⁺ T cell depletion group (n = 14 mice) in LLC tumors. Annotated genes represent MHC-I antigen presentation components (green), enriched genes (red), and depleted genes (blue). (C) GSEA of top 40 depleted hits identified from the in vivo screen. (D) Diagram illustrating pathways regulating the expression of MHC-I and the PLC. (E) Surface level of H2-Kb measured by flow cytometry in LLC and B16F10 cells upon IFN-γ stimulation (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; ****P < 0.001 by ordinary one-way ANOVA (compared with control cells). (F) Tumor volume measurements of control (transduced with control sgRNA), Erap1 KO, Pdia3 KO, and Calr KO LLC tumors in WT C57BL/6 mice (n = 8–9 per group). *P < 0.05; **P < 0.01; ****P < 0.001 by two-way ANOVA. (G) Tumor volume measurements of control or Calr KO B16F10 tumors in WT C57BL/6 mice (n = 4 per group). Data are presented as the mean ± SEM and representative of two independent experiments. **P < 0.01 by two-way ANOVA. (H) Tumor volume measurements of control or Calr KO B16F10 tumors in WT C57BL/6 mice (n = 6 per group) with anti-PD-1 treatment (200 μg/mice on days 4, 7, and 10). Data are presented as the mean ± SEM. **P < 0.01 by two-way ANOVA. GSEA, gene set enrichment analysis.