Journal: eLife

Article Title: Ex vivo and in vivo CRISPR/Cas9 screenings identify the roles of protein N-glycosylation in regulating T-cell activation and functions

doi: 10.7554/eLife.108724

Figure Lengend Snippet: ( a ) CRISPR/Cas9 knockout of B4galt1 (sgB4galt1) (sg2) 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 (n=6). The relative mRNA levels of PD-1 were measured by quantitative RT-qPCR (n=6). 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 (n=3). 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 (n=3). The secreted TNFα and IFNγ in medium were measured by ELISA (n=6). 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 (n=3). 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 (n=5). 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 (n=3). 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. All of these functional effects were biologically replicated at least twice. Data are shown as the mean ± SEM. *p<0.05; **p<0.01; ***p<0.001.

Article Snippet: B16F10 cell line (ATCC: CRL-6475) was provided by the laboratory of Dr. Ting Chen (National Institute of Biological Sciences, Beijing).

Techniques: CRISPR, Knock-Out, Expressing, Co-Culture Assay, Quantitative RT-PCR, Two Tailed Test, Over Expression, Enzyme-linked Immunosorbent Assay, In Vitro, Knockdown, shRNA, Control, Labeling, Functional Assay

Journal: eLife

Article Title: Ex vivo and in vivo CRISPR/Cas9 screenings identify the roles of protein N-glycosylation in regulating T-cell activation and functions

doi: 10.7554/eLife.108724

Figure Lengend Snippet: ( 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 (n=5 for control, n=7 for sgB4galt1). 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 (n=5 for control, n=7 for sgB4galt1). The p-value was calculated using a two-tailed Student’s t -test. The in vivo functional effects were biologically replicated at least twice. Data are shown as the mean ± SEM. *p<0.05; **p<0.01.

Article Snippet: B16F10 cell line (ATCC: CRL-6475) was provided by the laboratory of Dr. Ting Chen (National Institute of Biological Sciences, Beijing).

Techniques: Functional Assay, CRISPR, Knock-Out, Control, In Vivo, Two Tailed Test