Ex vivo CRISPR/Cas9 screenings identify genes and pathways that regulate PD-1 expression of CD8+ T-cells.

a. Schematic view of ex vivo CRISPR/Cas9 screening in mouse primary CD8+ T-cells. b. Volcano plot showing results of ex vivo CRISPR/Cas9 genome-wide screenings. The p-values were calculated using the α-robust rank aggregation (α-RRA) algorithm in MAGeCK. c. Verification of candidate genes by individual gRNAs. The relative expression levels of surface PD-1 protein and PD-1 mRNA were measured by FACS as mean fluorescent intensity (MFI) and RT-qPCR, respectively. d. GSEA of significantly enriched KEGG pathways in genome-wide screening. The enrichment score (ES) and statistical significance were calculated using the clusterProfiler (version 3.12.0) R package.

In vivo CRISPR/Cas9 screenings with a custom gRNA library identify genes that regulate functions of CD8+ T-cells in tumor microenvironment.

a. Schematic view of in vivo CRISPR/Cas9 screening in mouse primary CD8+ T-cells. b. Volcano plot showing results of ex vivo CRISPR/Cas9 screening. The p-values were calculated using the α-RRA algorithm in MAGeCK. c. Volcano plot showing results of in vivo CRISPR/Cas9 screenings. The p-values were calculated using the α-RRA algorithm in MAGeCK.

B4GALT1 suppression in CD8+ T-cells activates TCR signaling and enhances T-cell functions.

a. CRISPR/Cas9 knockout of B4galt1 (sgB4galt1) in CD8+ T-cells increases expression of PD-1 before and after co-culture with B16F10-OVA cells. The MFIs of PD-1 were measured by FACS. The relative mRNA levels of PD-1 were measured by quantitative RT-qPCR. The p-values were calculated using a two-tailed Student’s t-test. b. The effect of B4galt1 knockout on PD-1 surface expression could be rescued by overexpression of either long- or short-isoform B4galt1. The p-values were calculated using a two-tailed Student’s t-test. c. CRISPR/Cas9 knockout of B4galt1 in CD8+ T-cells increases expression of TNFα and IFNγ after co-culture with B16F10-OVA cells. The relative mRNA levels were measured by quantitative RT-qPCR. The secreted TNFα and IFNγ in medium were measured by ELISA. The p-values were calculated using a two-tailed Student’s t-test. d. CRISPR/Cas9 knockout of B4galt1 in OT-I CD8+ T-cells increases in vitro specific killing activities on B16F10-OVA cells. The p-values were calculated using a two-tailed Student’s t-test. e. Schematic view of B4GALT1 knockdown in human NY-ESO-1 TCR-T-cells. f. Knockdown of B4GALT1 in human NY-ESO-1 TCR-T-cells by shRNA increases in vitro killing activities on A375 cells. The p-values were calculated using a two-tailed Student’s t-test. g. Knockdown of B4GALT1 in human NY-ESO-1 TCR-T-cells increases expression of TNFα and IFNγ after co-culture with A375 cells. The secreted TNFα and IFNγ in medium were measured by ELISA. The p-values were calculated using a two-tailed Student’s t-test. h. Heatmap demonstrating differentially expressed genes (DEGs) between B4galt1 knockout and control mouse OT-I CD8+ T-cells after co-culture. The genes in TCR signaling pathway are labeled on the left side. i. Volcano plot showing upregulated and downregulated genes (p-value<0.01) in B4galt1 knockout mouse OT-I CD8+ T-cells after co-culture. The genes in TCR signaling pathway are labeled with dark blue and dark red. Top genes and some genes in TCR signaling pathway are annotated. The p-value was calculated using the Wald test, and p.adjust was calculated using Benjamini-Hochberg with the R package DESeq2 (version 1.22.2). j. Bar graph showing KEGG pathways significantly changed in B4galt1 knockout mouse OT-I CD8+ T-cells after co-culture. The p-value was calculated using the clusterProfiler (version 3.12.0) R package. Data are shown as the mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001.

Knockout of B4galt1 in CD8+ T-cells enhances T-cell-mediated tumor immunotherapy.

a. Schematic view of B4galt1 functional test in tumor microenvironment. b. CRISPR/Cas9 knockout of B4galt1 in OT-I T-cells enhances growth control of B16F10-OVA tumors in vivo. The p-value was calculated using two-way ANOVA. c. Compared with control OT-I T-cells, the tumors were significantly smaller when B4galt1 knockout OT-I T-cells were transplanted. The p-value was calculated using a two-tailed Student’s t-test. d. CRISPR/Cas9 knockout of B4galt1 increases numbers of OT-I T-cells in B16F10-OVA tumors. The p-value was calculated using a two-tailed Student’s t-test. Data are shown as the mean ± SEM. *P < 0.05; **P < 0.01.

Systematic identification of direct substrates of B4GALT1 on T-cell surface.

a. Schematic view of recombinant Gal-1 pulldown and LC-MS experiments. b. Volcano plot showing identified Gal-1 binding proteins in control and B4galt1 knockout OT-I cells. Proteins among the top list were annotated and labeled with red (decreased in B4galt1 knockout) and green (increased in B4galt1 knockout). Proteins in TCR signaling pathway are underlined. The p-values were calculated using Limma in DEqMS (V1.8.0). c. Bar graph showing KEGG pathways significantly changed in B4galt1 knockout OT-I T-cells. The p-value was calculated using the clusterProfiler (version 3.12.0) R package. d. Western blot verification of pulldown hits in top list. e. N-glycome analysis with PNGase F suggests that CD8β is a direct substrate of B4GALT1. f. Compared with wild-type control, B4GALT1 knockout OT-1 T-cells showed stronger TCR-CD8 FRET signals. g. Schematic view of CD8β-CD3ε fusion construct. h. Overexpression of CD8β-CD3ε complex bypassed the effect of B4GALT1 on T-cell in vitro killing activities. All of the p-values were calculated by a two-tailed Student’s t test. Data are shown as the mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001; NS, not significant.

The expression levels of B4GALT1 and tumor infiltrated CD8+ T-cells in tumor microenvironment are associated with prognosis of human patients.

a. The association between B4GALT1 expression levels and overall survival for patients with different CD8A levels in TCGA-ACC, -LAML, -LUAD, and -READ cohorts. b. The association between CD8A expression levels and overall survival for patients with different B4GALT1 levels in TCGA-ACC, -LAML, -LUAD, and -READ cohorts. The p-values for all survival curves were calculated using two-sided Log-rank test.