B
D E
G
C
F
6.99
0
10,000
20,000
30,000
40,000 *
0
20
40
60
80 ***
0
2
4
6
8 *
IL-2
+ of
CD8
IL-10 T-cells (%)
+ of
CD8
T-cells (%)
Tim-3
+PD-1
+
of CD8
+TILs (%)
MFI of IFN
γ
Gata3–Gata3+ Gata3–Gata3+ Gata3–Gata3+
IFN
γ
Gata3
IFN
γ
IL-2
Tim-3
PD-1
Gata 3
Tim-3
IL-10
Gata3
IL-2
Gata3
0
0
103
102
103
104
104
105
0
103
104
105
105
0
0
103
102
103
104
104
105
105 0103104105
0
0
103
103
104
104
105
105
0103104105
0
103
104
105
0103104105
44.9 4.05
42.8 8.22
84.2 5.89
3.93 6.01
80.9 11.9
6.87 0.37
0.000
0.001
0.002
0.003
0.004
RE
Gata3
(^00510152025)
50
100
150
200
Days
Tumor size (mm
2 )
*
Control
LV
Gata3
LV
Gata3 del. PMEL → WT
Gata3
del.
Ctrl PMEL → WT
Ctrl
No cell transfer
82.9 6.11
1.75 9.28
78.4 9.15
2.16 10.3
Ctrl → WT Gata3 del. → WT
Control
→ WT
Gata3 del.
→ WT
0
20
40
60
80
0
20
40
60
80
(^100)
0
5
10
15
20
(^25) *
0
10
20
30
40
50
0
10
20
30
40
PMEL → WT
TNF
+α
of CD8
- TILs (%)
IFN
+γ
of
CD8
TILs (%)
IL-2
+of CD8
TILs (%)
IL-10
+of
CD8
TILs (%)
CtrlGata3 del.
PMEL → WT
CtrlGata3 del.
PMEL → WT
81.2 0.84
15.9 2.09
59.9 3.77
(^30) 6.35
0
103
104
105
0
103
104
105
0103104105
A
1.0
0.5
0.0
–0.5
–0.5 0.0 0.5 1.0
–1.0
–1.0
Dysfunction score
Activation score
SUDS3
Gata3
IKZF2
1
44444444 2
3333333333333333333333333
Figure 6. Gata3 Drives the Dysfunctional State in CD8+T Cells
(A) Gata3, a zinc-binding TF, ranks first in the dysfunction module of the TFs consistently differentially expressed across our datasets (black points).
(B and C) WT mice were implanted subcutaneously with B16F10 melanoma cells. TILs were isolated on day 15 and analyzed for Gata3 expression and T cell
function. (B) Representative flow cytometry data showing Gata3 expression gated on CD8+TILs. (C) Cytokine expression of Gata3+and Gata3CD8+TILs.
Statistical analysis was performed using paired Student’sttest. *p < 0.05, *p < 0.001.
(D) Targeted deletion of Gata3 using CRISPR-Cas9 genome editing. Naive CD8+T cells were sorted from pmel transgenic mice, infected with control or Gata3
lentivirus, and activated with plate-bound anti-CD3 and anti-CD28 antibodies in the presence of IL-2 (STAR Methods). Representative qPCR results showing
Gata3 mRNA level in control versus Gata3 lentivirus-targeted CD8+T cells.
(E) 1 3106 CRISPR-Cas9-targeted cells were transferred to WT mice (n = 5/group) bearing B16F10 melanoma tumors (day 5 post tumor grafting). Mean tumor
growth is shown. Data are representative of three independent experiments. Statistical analysis was performed using linear regression. p < 0.01.
(F and G) TILs were isolated on day 21 after tumor cell injection and analyzed for Tim-3 and PD-1 expression (F) and cytokine production (G) by flow cytometry.
Cell 166 , 1500–1511, September 8, 2016 1509