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pAg-mediated activation (Fig. 6C) ( 10 ). These
residues had no effect on BTN2A1 binding
(Fig. 6B) and were located on the opposite side
oftheTCRtotheputativeBTN2A1footprint
(~30 to 40 Å separation). However, they were in
close proximity to one another (~11 Å) (Fig. 6D),
thereby potentially representing a separate
binding interface necessary for pAg-mediated
activation via the Vg9Vd 2 +TCR, but not for
BTN2A1 binding. This second binding inter-

face explains the importance of (i) the Vd 2 +

TCRdchain through involvement of germline-

encoded residues and (ii) the invariant nature

of the CDR3gmotif among pAg-reactivegd

T cells, through engagement of specific resi-

dues within this loop.

Finally, we tested agonist BTN3A1 mAb

(clone 20.1)–mediated activation, which is

thought to mimic pAg-mediated signaling by

conformational modulation or cross-linking

of BTN3A1 ( 12 ). Agonist BTN3A1 mAb–pulsed

parental APCs induced Vg9Vd2 TCR+Jurkat

cell activation (Fig. 7), an effect that was not

observed with agonist BTN3A1 mAb–pulsed

BTN2A1nullAPCs, suggesting that BTN2A1 is

critical for BTN3A1-mediated activation ofgd

T cells. Furthermore, Jurkat cells expressing

TCRg-chain Ala mutants of the BTN2A1-

binding residues His^85 ,Arg^20 ,andGlu^70 ,as

well as BTN2A1-independent mutants of Arg^51

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

Fig. 5. BTN2A1 associates with
BTN3A1 on the cell surface.
(A) Z-stack confocal microscopy of
surface BTN2A1 (green, clone 259),
BTN3A (red, clone 103.2), and
pan-HLA (human leukocyte antigen)
class I (blue, clone W6/32) on
parental LM-MEL-75 (“WT”, top
row),BTN2A1null(middle row), and
BTN3A1null(bottom row) cells. Scale
bars, 10mm. (B) Graph depicts
Pearson correlation coefficients for
individual fields of view. Represent-
ative voxel density plots depicting
correlation between anti-BTN2A1
versus anti-BTN3A1/3A2/3A3
(“BTN3A”) (left); anti-BTN2A1 ver-
sus anti- HLA-A,B,C (middle); and
anti-BTN3A versus anti-HLA-A,B,C
(right). ***p< 0.001 using a
Kruskal–Wallis with Dunn’s post-
test. (C) Anti-BTN2A1 versus
BTN3A costaining (green), or single
staining (yellow and blue, respec-
tively), or mouse IgG1 versus mouse
IgG2a isotype control staining
(xandyaxis respectively, magenta)
on LM-MEL-75 cells using the
indicated mAb clones (top row).
Histograms (second row) depict
FRET fluorescence. (D) Percentage
of FRET+cells between
butyrophilinCFP/YFP-transfected
NIH-3T3 cells. Data are representa-
tive of [(A) and (B)] two pooled
independent experiments; (C) one
experiment; (D) three to four
independent experiments.

A

C

D

FRET

BTN3A (clone 20.1)

BTN2A1

(indicated clone)

BTN3A (clone 103.2)

BTN2A1

Voxel intensity

HLA-A,B,C

B

BTN2A1 BTN3A

BTN3A

HLA-A,B,C

WT

BTN2A1 BTN3A HLA-A,B,C Merged

BTN2A1

null

BTN3A1

null

Isotype BTN2A1 alone BTN3A alone BTN2A1+BTN3A

0

25

50

75

100

FRET (%)

YFP:

CFP:–

• 
  

L3

• 
  
  
  
  • L8
  
  
  
  

3A1

• 
  
  
  
  • 2A1
  
  
  
  

2A1

• 
  
  
  
  • 3A1
  
  
  
  

L3

L8

3A1

2A1

2A1

3A1

3A1

L8

L3

2A1

**

ns

BTN2A1and

BTN3A

BTN2A1 and

HLA-A,B,C

BTN3A and

HLA-A,B,C

-0.2

0.0

0.2

0.4

0.6

Pearson correlation

coefficient

***

***

BTN2A1

vs.BTN3A

BTN2A1

vs.HLA-A,B,C

BTN3A

vs.HLA-A,B,C

Voxel intensity

0103104105

Hu34C 231 259 267

0103104105

0

103

104

105

Hu34C 231 259 267

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