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andBTN2A1null2), with similar results also
from a distinct LM-MEL-75BTN2A1-mutant
cell line (Fig. 1C and fig. S3). This was in-
dependent of BTN3A1 expression, which was
essentially unchanged between parental LM-
MEL-62 andBTN2A1nulllines (Fig. 1D and
fig. S3A). Additionally, Vg9Vd 2 +TCR tetramer
reactivity ofBTN3A1nulllines was comparable
to that of parental lines (fig. S3B). Reintroduc-
tion of BTN2A1 into either LM-MEL-62
BTN2A1null1orBTN2A1null2cells restored
Vg9Vd 2 +TCR tetramer reactivity, whereas
transfection withBTN3A1had no effect (Fig.
1D). Thus, BTN2A1 expression is essential for
Vg9Vd 2 +TCR tetramer reactivity.
We next generated a panel of BTN2A1-
reactive mAbs, which exhibited varying degrees

of cross-reactivity to BTN2A2 (87% ectodo-

main homology) but not to BTN3A2 (45%

ectodomain homology) (fig. S4, A to C). These

mAbs stained parental LM-MEL-62, but most

failed to bind to LM-MEL-62BTN2A1nulllines,

confirming their reactivity to BTN2A1 (fig. S4, D

and E). Most of the anti-BTN2A1 mAbs fully or

partially blocked Vg9Vd 2 +TCR tetramer stain-

ing on LM-MEL-62, LM-MEL-75, and 293T cells

(Fig. 1E), suggesting that BTN2A1 is a ligand

for the Vg9Vd 2 +gdTCR.

To explore whether BTN2A1 selectively binds

to Vg9Vd 2 +gdT cells, we produced fluorescent

BTN2A1 ectodomain tetramers (fig. S5), which

stained a subset of CD3+TcellswithinPBMCs,

but no other cell type (Fig. 2A). The BTN2A1

tetramer+cells weregdTCR+,butnotabTCR+

(Fig. 2A). BTN2A1 tetramer labeled essentially

all Vg 9 +Vd 2 +and Vg 9 +Vd 1 +gdT cells, but no

Vg 9 −Vd 1 +gdT cells, suggesting that the Vg 9

domain of the TCRgchain is associated with

reactivity (Fig. 2B). Furthermore, Förster reso-

nance energy transfer (FRET) between fluo-

rescent BTN2A1 tetramer and anti-CD3emAb

( 22 ) indicated that BTN2A1 tetramer was bind-

ing within ~10 nm of thegdTCR (Fig. 2C). To

directly assess whether BTN2A1 binds Vg 9 +

gdTCR, we performed surface plasmon reso-

nance (SPR) to measure interactions between

soluble BTN2A1 andgdTCR ectodomains. Con-

sistent with the pattern of BTN2A1 tetramer

reactivity among primarygdT cells, soluble

BTN2A1 bound Vg9Vd 2 +TCR (TCR #6) with an

affinity ofKD=40mM, similar to what is observed

Rigauet al.,Science 367 , eaay5516 (2020) 7 February 2020 2of13

C1R MOLT-4 MEG-01 Jurkat THP-1 Ramos K562 293T LM-MEL-75LM-MEL-62 abTCR tet. #3

abTCR tet. #4

abTCR tet. #5

abTCR tet. #6

abTCR tet. #7

Control tet.

SAv. control

A

C WT

LM-MEL-75

LM-MEL-62

BTN2A1null

ab TCR tetramers

WT 2A1null1 2A1null2

BTN2A1

ab TCR tetramer (#6)

BTN3A

ab TCR tetramer (#6)

WT+2A1 2A1null1+2A1 2A1null2+2A1 WT WT+3A1 2A1null1 2A1null1+3A1 2A1null2 2A1null2+3A1

BTN2A1null2

D

E LM-MEL-62 LM-MEL-75 293T

abTCR tetramer (#6)

Isotype

Hu34C

268

267 227

231

228

259

243

Nothing (control tet. stain)

Pre-incubated with:

ab TCR tetramers

0

5

10

15

0

0

2

4

6

51015

BTN2A1

>0.05

FDR:

-log

10

(p

-value)

<0.05

SPPL3

log 2 (fold change)

CPM:

B

• 
  
  
  
  • 
    
  
  
  
  

WT BTN2A1null1

Fig. 1. Vg9Vd 2 +gdT cell receptor tetramer staining is dependent on
BTN2A1.(A)Vg9Vd 2 +TCR tetramer staining of various cell lines. Colored
histograms depictgdTCR tetramers #3 to #7; gray, irrelevant control (mouse
CD1d–a-GalCer) tetramer; unfilled, streptavidin (SAv)–PE control. (B)Volcano
plot depicting log 2 (fold-change) versus–log 10 (pvalue) for each gRNA, between
unsorted and Vg9Vd2 TCR tetramer #6loLM-MEL-62 cells, where magenta depicts
significant differences [false discovery rate (FDR) < 0.05]. CPM, counts per million.
(C)Vg9Vd 2 +TCR tetramer staining of LM-MEL-62BTN2A1nulland LM-MEL-75
BTN2A1nullcells compared to parental (wild type, WT) cells. Color scheme as in (A).

(D) Anti-BTN2A1 mAb (clone 231, yellow), anti-BTN3A1/3A2/3A3 mAb (clone 103.2,

blue), and Vg9Vd 2 +TCR tetramer (#6) staining (dark green) on parental and

BTN2A1null1 or null2LM-MEL-62 cells transfected with eitherBTN2A1orBTN3A1.

*gdTCR tetramer staining is depicted twice. (E)Vg9Vd 2 +TCR tetramer #6

staining of LM-MEL-62, LM-MEL-75, and HEK-293T cells, after preincubation of

cells with a panel of anti-BTN2A1 mAb (colored histograms), compared to isotype

control unfilled. Lower histograms (gray) depict control staining with irrelevant

mouse CD1d–a-GalCer tetramer. tet, tetramer. Data in (A), (C), (D), and (E) are

representative of two independent experiments 293T, HEK-293T.

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