Cell - 8 September 2016

Metallothionein Deficiency Uncouples Co-inhibitory
Receptor Expression from a T Cell Dysfunction
Phenotype
We next analyzed the functional phenotype of CD8+TILs iso-
lated from WT andMT
/
tumor-bearing mice. Consistent with

retarded tumor growth, the effector function ofMT
/
CD8+

Tim-3+TILs was significantly improved, with higher production

of interleukin-2 (IL-2), tumor necrosis factora(TNF-a), and gran-

zyme B (Figures 2D andS2C) and a higher percentage of poly-

functional T cells (Figure 2E). However, despite the enhanced

0 5 10 15 20

0

100

200

300

400

Days

Tumor size (mm

2 )

Tim-3

+PD-1

+

of CD8

+TILs (%)

IFN

+γ

TNF

+α

IL-2

+

of CD8 (%)

Tu

mor size (mm

2 )

***

A

F

E

B

D

dLN C

10 5 2.5 1.25 0

0

5000

10,000

15,000

20,000

MT–/–

(^105) WT
104
103
102
101
100
cpm
TIL
10 2.5
cpm
0 5 10 15 20 25
0
50
100
150
200
Days
**
0
20
40
60
80
100 p= 0.078
0
20
40
60
80
TNFα+ of
Tim-3+CD8+
TILs (%)
IFNγ+ of
Tim-3+CD8+
TILs (%)
IL-2+ of
Tim-3+CD8+
TILs (%)

• 0
  5
  10
  15


• 20
  0
  20
  40
  60
  80
  0
  10
  20
  30
  (^40) * NS
  Polyfunctionality
  gp100 peptide (μM)
  MT–/–
  WT
  MT–/–
  Tim-3+ Tim-3–
  WT
  WT MT–/–
  MT–/–
  WT
  MT OT1
  Control OT1
  No cell
  Tim-3
  PD-1
  IFN
  γ
  Tim-3
  IL-2
  TNF
  α
  40.5 25.2
  3.62 30.8
  33.6 24
  3.78 38.6
  0
  0
  103
  103
  104
  104
  105
  105
  0
  103
  104
  105
  0103104105
  WT MT–/–
  WT MT–/–
  0
  103
  104
  105
  17.6 27
  37.7 17.7
  9.16 13.9
  43.1 33.8
  0
  0
  103
  103
  104
  104
  105
  105 0103104105
  22 21.8
  32.6 23.6
  18.9 13.4
  32.4 35.3
  44.3 45.1
  8.51 2.09
  38.2 45
  10.8 6.08
  MT–/–
  WT
  Figure 2. Metallothionein Deficiency Improves Anti-tumor Immunity and Reverses T Cell Dysfunction
  (A and B) Mice deficient in both MT1 and MT2 (MT
  /
  ) and WT littermate controls were implanted subcutaneously with B16F10 melanoma. (A) Mean tumor
  growth. Statistical analysis was performed using linear regression p < 0.001. (B) Tumor-draining lymph node (dLN, top) and tumor-infiltrating lymphocytes (TIL,
  bottom) were isolated from WT andMT
  /
  mice 15 days post-tumor inoculation and stimulated with tumor antigen gp100. On day 3, tumor-antigen-specific
  proliferation was measured by^3 H incorporation.
  (C) Naive OT-1 cells were sorted, activated, and infected with empty retrovirus (control OT1) or MT1 retrovirus (MT OT1) prior to transfer (1 3106 cells/mouse) into
  WT mice that were subsequently implanted with MC38-OVA tumor the next day. Mean tumor growth is shown. Statistical analysis was performed using linear
  regression. p < 0.01.
  (D and E)MT
  /
  CD8+TILs have increased functionality as compared to WT CD8+TILs. TILs were isolated and stimulated with PMA/ionomycin in the presence of
  brefeldin A for 4 hr prior to extracellular and intracellular staining and analysis by flow cytometry. p < 0.05.
  (F) Tim-3 and PD-1 expression in WT andMT
  /
  TILs. The DN, SP, and DP subpopulations are present in both the WT andMT
  /
  TILs.
  See alsoFigure S2.
  Cell 166 , 1500–1511, September 8, 2016 1503