Article Figures & Data
Figures
- Figure 1. An activation and inhibition assay amenable to single gRNA CRISPR-based genome editing.
(A) Schematics of the cellular assay to study activatory and inhibitory signaling pathways in Jurkat T cells (created with BioRender.com). (B) Representative flow plots of eGFP fluorescence levels measured by flow cytometry of Jurkat/control and Jurkat/PD-1 cells co-cultured with four different T-cell stimulator (TCS) conditions or cultured without stimulation (left panel). Mean eGFP fluorescence intensity from three replicate experiments in Jurkat/control and Jurkat/PD-1 cells co-cultured from four different TCS conditions or cultured without stimulation (right panel). Pairwise comparisons performed using independent t test. (C) Mean eGFP fluorescence intensity from three replicate experiments in Jurkat/control and Jurkat/PD-1 cells targeted by empty, RELA or NFKB1 sgRNAs after co-culture with TCS only. Pairwise comparisons performed using independent t test against empty-targeted samples within each Jurkat group. (D) Median eGFP fluorescence intensity from three replicate experiments in Jurkat/control and Jurkat/PD-1 cells targeted by empty, PTPN6 or PTPN11 sgRNAs after co-culture with TCS/PD-L1. Pairwise comparisons performed using one-way ANOVA, comparing against empty-targeted samples within each Jurkat group. (E) Median eGFP fluorescence intensity of Jurkat/PD-1 cells targeted by empty or PTPN11 sgRNAs co-cultured with TCS/CD86 or TCS/CD86/PD-L1, and eGFP fluorescence levels of Jurkat/PD-1 cells targeted with the same sgRNAs in the same conditions or without stimulation. Pairwise comparison performed using independent t test between TCS/CD86 and TCS/CD86/PD-L1 stimulated samples (left panel). Representative plots of eGFP fluorescence levels measured by flow cytometry for empty and PTPN11-targeted Jurkat/PD-1 cell line unstimulated or stimulated with TCS/CD86 and TCS/CD86/PD-L1 (right panel). In all cases, the number of replicates is three and P-value asterisks represent: ****P < 0.001, ***P < 0.001, **P < 0.01, *P < 0.05; ns, not significant.
- Figure S1. Assessment of Cas9-efficiency in Jurkat:control and Jurkat:PD1 cell lines.
Upper panel: schematic of sgRNA expression cassettes used to test Cas9 efficiency in Cas9-containing Jurkat cell lines. Polyclonal Cas9 lines were assessed for Cas9 efficiency using GFP-encoding lentivirus constructs containing either a gRNA targeting eGFP (gGFP) or not (“empty”). Two-dimensional flow cytometry plots of BFP and GFP fluorescence. BFP marks the presence of the lentiviral gRNA. Cells expressing Cas9 were efficient at targeting GFP as evidenced by the loss in GFP expression upon targeting with gRNA (GFP) construct.
- Figure 2. Arrayed CRISPR KO screen of SH2 signaling proteins in the two-cell signaling assay.
(A) Heatmap of the logarithm fold change of the eGFP median fluorescence intensity for each gRNA target and stimulation condition relative to the negative control (empty targeted). Data generated by calculating fold changes from mean MFIs from three replicates for each of the two separate targeting gRNAs. (B) eGFP fluorescence levels of Jurkat/PD-1 cells targeted with CSK, CBL, SLA or empty gRNA in five different T-cell stimulator (TCS) stimulation conditions (upper panel). eGFP fluorescence levels of Jurkat/PD-1 cells targeted with CSK, CBL, SLA or empty gRNA when unstimulated or stimulated in four different TCS stimulation conditions (upper panel). Pairwise comparisons between control and inhibition conditions performed using independent t test. (C) eGFP median fluorescence intensity of positive regulator (GRB2, LCK, LCP2, PLCG1, VAV1, ZAP70) gRNA-targeted Jurkat/PD-1 cells with two different gRNAs and under no stimulation or stimulation with TCS or TCS/CD86 using a low stimulation regimen. (A, D) Heatmap of logarithm fold change of the eGFP median fluorescence intensity for members of the SRC-family proteins (subset from (A)). (E) Fraction of cells expressing eGFP post stimulation with TCS/CD86 in Jurkat/PD-1 cells targeted with empty gRNA and wither LCK sgRNA or sgRNA targeting additional one other SRC family kinase sgRNA (BLK, FGR, FYN, HCK, SRC, YES1). Pairwise comparisons performed relative to LCK-only KO using one-way ANOVA. In all cases, the number of replicates is three and P-value asterisk represent: ***P < 0.001, **P < 0.01, *P < 0.05; ns, not significant.
- Figure S2. Volcano plots for z-scores derived for each stimulation context and for each set of gRNA libraries.
Targeted genes with absolute z-score more than 1.5 are labelled. Each gene was targeted with two different gRNAs and Set A and Set B refer to the two different sets of gRNA libraries.
- Figure S3. gRNAs from the arrayed screen, whose targeting led to decreased signaling under stimulation with signal 1 or 1 + 2.
(A) eGFP median fluorescence intensity of Jurkat/PD-1 cells targeted with empty gRNA or positive regulator-targeted gRNAs in three different T-cell stimulator stimulation conditions in both set A (upper panel) and set B (lower panel) of gRNAs. (B) eGFP fluorescence levels of Jurkat/PD-1 cells targeted with empty gRNA or PLCG1 or LCK gRNAs from both Set A and Set B. (C) eGFP fluorescence levels of Jurkat/PD-1 cells targeted by gRNAs as labelled under a low stimulation regimen. Worse-preforming gRNAs from high-stimulation regimen are depicted. (D) Expression of LCK on cells targeted either with sgRNA targeting LCK or empty control.
- Figure S4. Sorting strategy for genome-wide screen of Jurkat/PD-1 cells after co-culture with T-cell stimulator/PDL1 cells.
Control samples run on the same day show that PD1 mediated inhibition is in place as cells stimulated without the engagement of PDL1 show higher levels of eGFP expression (middle panel) compared with those with PDL1 engagement (upper panel). The gates depicted represent the cells that were sorted to identify positive regulators (lower eGFP) and negative regulators (higher eGFP).
- Figure 3. A genome-wide pooled CRISPR screen of T cell activation and inhibition using the two-cell signaling assay.
(A) The enrichment of gRNAs targeting each gene in cells with low eGFP (ordered alphabetically; left panel), and high eGFP (right panel) is quantified as the RRA-score. Each circle represents a specific gene. Only genes on the T-cell receptor signaling pathway and genes with false discovery rate < 10% are labelled and the green circles represent genes that are known to be related to the T cell function. Full data are available in Table S2. (B) Pathway enrichment of the hits from screen to identify positive regulators (low eGFP, upper panel) and negative regulators (high eGFP, lower panel). (C) Mean eGFP fluorescence intensity of parental Jurkat/control cells and Jurkat/PD1 cells targeted with ARPC4 sgRNA co-cultured with four different T-cell stimulator conditions or cultured without stimulation. (D) Representative flow plots showing CD25 expression levels in ARPC4, LCK and CSK-targeted CD8+ cells compared with non-targeted control CD8+ cells, in the course of killing A375 target cells. (E) Changes in integrated red area intensity of A375 cells co-cultured with ARPC4, LCK, CSK, and empty-targeted CD8+ cells or cultured alone. (F) Percentage of A375 cells remaining after co-culture with targeted CD8+ cells for 20 h. Pairwise comparisons performed using one-way ANOVA, comparing with empty-targeted conditions. In (C, D, E, F), the number of replicates is three and P-value asterisk represent the following: ***P < 0.001, **P < 0.01, *P < 0.05; ns, not significant.
- Figure S5. Investigation of the role of ARPC4 in T-cell activation.
(A) Western blot of ARPC4 and Cas9 in Jurkat/control and Jurkat/PD-1 cells targeted by empty and ARPC4 gRNAs. Both cells express FLAG-tagged Cas9, the expression of which was used as the loading control. (B) Expression of 41BB and CD69 on CD8+ parental cells or CD8+ ARPC4 targeted cells post activation with T-cell stimulator/CD86 for 24 h. Representative flow plots are depicted on the left panel and quantification using median fluorescence intensity on the right panel. (C) Schematic of the set-up of primary CD8+ cell-killing assay (created with BioRender.com). In the assay, NY-ESO-1 expressing A375 cells engineered to express mOrange was presented to kill by T-cells engineered to express 1G4. The engineered T-cells together with parental cells from which the 1G4 engineered cells were generated were used in the killing assay. Wells in which A375 cells were plated but no T-cells were added were used as a control. The cells were then left to perform killing for 20 h with images taken on the IncuCyte every 2 h. The integrated red intensity (the sum of all red fluorescence intensity values within the object multiplied by the pixel area) was calculated for each time point and each condition and plotted over the time course. (D) Western blot of ARPC4 in parental 1G4-transduced primary CD8+ cells and cells targeted with ARPC4 triple gRNA. GAPDH was used as the loading control. GAPDH was blotted on a separated blot but using the same lysate as that for ARPC4.
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