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Rigauet al.,Science 367 , eaay5516 (2020) 7 February 2020 5of13

C

F

D

E

V

δ^2

+γδ

T cells (x10

-4

)

WT 2A1null1

0

1

2

3

Unstimulated 4

Zoledronate

No APC MEL-62

2A1null1

0

10

20

30

40

50

MEL-62

2A1null2

MEL-62

WT

No APC MEL-62

2A1null1

MEL-62

2A1null2

MEL-62

WT

CD25 (%)

CD3 (MFI x 10

-3

)

0

10

20

30

40

Relative number of

viable cells (%)

Time (h)

24 48 72

0

50

100

MEL-62 WT

MEL-62 WT + Zol

MEL-62 BTN2A1null1

MEL-62 BTN2A1null1 + Zol

*

100

50

25

0

75

IFN-

γ (

pg/ml)

101

100

102

103

104

Unstim. Unstim.

CD25 (%)

CD3+

CD28

HMBPP CD3+

CD28

HMBPP

No mAb

Isotype

Hu34C

236

266

227

267

*

Vδ 2 + Vδ 1 +

CD25

0%

0%

10%

13%

1%

4%

4%

2%

2%

3%

7.7K

4.4K

4.2K

7.7K

7.3K

7.2K

2.6K

2.6K

2.5K

2.6K

2.5K

2.5K

CD3ε

A Vδ 2 +γδ T cells B

0

10

20

30

40

50

CD25 (%)

Zol:–+++++

Isotype

mAb: – –

2A1 (Hu34C)

2A1 (259)2A1 (267)

–+++++

Isotype

––

2A1 (Hu34C)

2A1 (259)2A1 (267)

Vδ 1 +γδ T cells

0

2.5

5.0

7.5

10

CD3 (MFI x 10

-3

)

0

200

1,000

2,000

3,000

IFN-

γ (pg/ml)

–+++++

Isotype

––

2A1 (Hu34C)

2A1 (259)2A1 (267)

Zol:

mAb:

0

100

2,000

3,000

4,000

TNF (pg/ml)

5,000

4,000

1,000

38%

36%

***

***

**

***

***

***

***

***

**

ns

ns

ns

Fig. 3.gdT cell functional responses to pAg depend on BTN2A1.(A)CD25
expression and CD3emean fluorescence intensity (MFI) on Vd 2 +and control
Vd 1 +T cells gated among PBMCs cultured for 24 hours ± 4mMzoledronate
and ± 10mg/ml neutralizing anti-BTN2A1 mAb as indicated. p<0.01,
p< 0.001, by ANOVA. (B)IFN-gand TNF concentration in the culture super-
natants from (A). p< 0.01, p< 0.001, by Friedman test. (C)CD3MFIand
CD25 expression on purified in vitro–expanded Vd 2 +T cells cocultured with parental
orBTN2A1nullLM-MEL-62 APCs without (gray) or with (blue) 4mM zoledronate. Each
symbol represents a different donor. Bar graphs depict mean ± SEM of the donors,
each averaged from the technical replicates. (D)NumberofVd 2 +gdT cells in cocultures
of PBMCs with parental orBTN2A1null1LM-MEL-62 APC after a 2-day challenge with
1 mM zoledronate followed by maintenanceof nonadherent PBMCs for an additional
7 days in media containing IL-2. *p<0.05usingaMann–Whitney test. (E) Cell viability

(mean ± SEM) as determined using the metabolic dye MTS, normalized against input

cell number, of cocultures of parental (WT) or BTN2A1nullLM-MEL-62 targets with in

vitro–expanded Vd 2 +T cells, at the indicated time points ± 1mMzoledronate.*p<0.05

using a Mann–Whitney test between zoledronate-treated groups. (F) CD25 expression

(left) and IFN-gconcentration (right) after culture of purified in vitro–expanded

Vd 2 +T cells with HMBPP (0.5 ng/ml) or plate-bound anti-CD3 plus anti-CD28

(10mg/ml each) ± 10mg/ml neutralizing anti-BTN2A1 mAb. Data represent [(A) and

(B)]n= 8 donors pooled from two independent experiments; (C)n=2to3donors

pooled from three independent experiments, each performed withn= 1 to 4 technical

replicates indicated by different symbols; (D)n= 4 donors, each averaged from one

to five technical replicates acrossfive independent experiments; (E)n=4donors,

each averaged from two to six technical replicates across six independent experiments;

(F)n= 8 donors pooled from two independent experiments. Zol, zoledronate.

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