Cell - 8 September 2016

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 Gata3
  CD8+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