Fig. 1. Systematic discovery of genes and
resistance of tumor cells
to Tcell–mediated killing.
(A) Screening strategy.
cells were transduced with
a genome-scale gRNA
library (four gRNAs/gene).
Edited B16F10 cells were
cocultured with activated
cytotoxic T cells followed
by Illumina sequencing of
Specific selection was
performed with Pmel-1
T cells (specific for gp100
melanoma antigen) or
OT-I T cells (specific for
Ova peptide). Control
selection was performed
with Tcells of irrelevant
specificity. (B) Top genes
for enriched gRNAs
from Pmel-1 screen.
Candidate genes were
plotted based on mean
log2 fold change of gRNA
counts compared to control selection and P values computed by MaGeCK (Model-based Analysis of Genome-wide CRISPR-Cas9 Knockout).
Dashed line indicates a FDR (false discovery rate) of 0.05. Annotated genes represent MHC class I (red), interferon (yellow), and Ras/MAPK
(blue) pathways. (C) Top genes for depleted gRNAs from Pmel-1 screen. Genes related to the PBAF form of SWI/SNF complex (red), NF-kB
pathway (blue), m TORC1 pathway (yellow), and known negative immune regulators (green) were annotated. (D) Selected pathways and
corresponding genes identified in the Pmel-1 screen. Color scale represents log2 fold change of average gRNA representation.
Pathways identified in the screen
Jak/Statpathway Antigenpresentation NegativeregulationofMAPK NF-κBpathway mTORC1pathway PBAFcomplex Glycolysis NADmetabolism
Genes with enriched gRNA
Genes with depleted gRNA
Log2 Fold Change
Log2 Fold Change
ARID2 expression level
0 500 1000 1500 2000
PBRM1 expression level
0 2000 4000 6000 8000
5 5 cor=-0.26
ARID2 vs. GZMB/CD8A PBRM1 vs. GZMB/CD8A
Survival (months) Survival (months)
0 0 100 100 200 200 300
CD8> 1 S.D. CD8< 1 S.D.
ARID2 vs. GZMB
-0.4 0.4 -0.20.20
PBRM1 vs. GZMB
-0.4 0.4 -0.2 0.2 0
Fig. 2. Expression of ARID2 and PBRM1 is negatively correlated with
Tcell cytotoxicity markers in TCGA data sets. (A) Correlation of ARID2
and PBRM1 mRNA levels with GZMB mRNA levels in indicated cancers.
Volcano plot showing the Spearman’s correlation and estimated significance
of ARID2 (left) or PBRM1 (right) with GZMB mRNA levels from RNA-seq
data across TCGA cancer types calculated by TIMER (Tumor Immune
Estimation Resource) and adjusted for tumor purity (32). Each dot represents
a cancer type in TCGA; red dots indicate significant correlations (P < 0.01).
(B) Analysis of ARID2 and PBRM1 mRNA levels in relation to GZMB and
CD8A as cytotoxicity and CD8 Tcell infiltration markers, respectively.
Spearman’s correlation of ARID2 (left) and PBRM1 (right) mRNA levels to
GZMB/CD8A mRNA ratio in the TCGA melanoma data set. (C) Correlation
of ARID2 expression level with survival of melanoma patients depending on
calculated level of CD8 T cell infiltration. All patients in the TCGA melanoma
study were divided according to the expression level of ARID2 (higher or lower
than mean expression value of all patients). The impact of ARID2 expression
level on survival is shown for patients whose tumors had higher (>1 SD) or
lower (<1 SD) expression of CD8 [(CD8A + CD8B)/2].