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(dchain) and Lys^108 (gchain), all failed to
respond to parental APCs pulsed with agonist
anti-BTN3A1 mAb (Fig. 7). Thus, an interaction
between BTN2A1 and the Vg 9 +TCRgchain is
essential, but not sufficient, for BTN3A1-driven
gdT cell responses. This may explain why, in
earlier studies, the agonist anti-BTN3A1 mAb
failed to induce activation ofgdTcellsinco-
cultures with mouse-derived APCs transfected
with human BTN3A1 alone ( 5 ), because mice
do not express BTN2A1.
Accordingly, these mutant studies indicate
the existence of two separate interaction sites
on Vg9Vd 2 +TCRs necessary for pAg- and
BTN3A1-mediated activation. One site on the
side of the Vg9 domain is essential for both
BTN2A1 binding and for activation, whereas
the other site, incorporating both the Vd2-

encoded CDR2 andg-chain–encoded CDR3

loops, is required for pAg- and BTN3A1-mediated

activation. Thus, Vg9Vd 2 +gdT cells appear to be

selectively activated by pAg through a distinct,

dual-ligand interaction whereby BTN2A1 binds

to the Vg9 domain and another ligand, po-

tentially BTN3A1, binds to a separate interface

incorporating both the Vg9andVd2domains.

Concluding remarks

Our findings support a model whereby BTN2A1

and BTN3A1 associate on the cell surface and

are both required for pAg-mediatedgdTcell

activation. This model also suggests that after

pAg binds BTN3A1 through its intracellular

B30.2 domain, the BTN2A1–BTN3A1 complex

engages thegdTCR via two distinct binding

sites: BTN2A1 binds to Vg9 framework regions,

whereas another ligand—possibly BTN3A1—

binds to the Vd2-encoded CDR2 andg-chain–

encoded CDR3 loops on the opposite side of

the TCR. This represents a distinct model of

Ag sensing that is highly divergent from

canonical MHC-Ag complex recognition by

abT cells.

A previous study, using short hairpin RNA

knockdown, found no apparent role for BTN2A1

in pAg presentation ( 28 ). However, as the knock-

down efficiency was only 81% and BTN2A1

protein was not measured, residual BTN2A1

may have retained functionality. Until now,

BTN2A1 has been poorly characterized, with only

one earlier study identifying a glycosylation-

dependent receptor, CD209 ( 21 ). We found

that N-linked glycans were dispensable for

BTN2A1 binding to thegdTCR (fig. S16), making

Rigauet al.,Science 367 , eaay5516 (2020) 7 February 2020 8of13

R20a

H85a E70a

B

BTN2A1 tet. MFI

relative to WT (%)

BTN2A1 tetramer

WTE5AR20AE22AT29AY54AT57AK60AS62AS66AE70AE76AH85AN86AE88AQ90A

K108AE28AR51AL97A

9C2 control

0

25

50

75

100

125

150

SAv. alone

b-chain mutants

Unstimulated

Zoledronate

b-chain mutants

0

25

50

75

100

125

150

CD69 MFI relative to WT (%)

WTE5AR20AE22AT29AY54AT57AK60AS62AS66AE70AE76AH85AN86AE88AQ90A

K108AE28AR51AL97A

9C2 control

Jurkat parental

Top view Side view

Ca

Va 9 Va^9

Vb 2

R20a

H85a

E70a

K108a

E22a

BTN2A1 binding

Zoledronate reactivity

D

R51b

A C

a-chain mutants a-chain mutants

Fig. 6. Vg9Vd 2 +T cell receptors contain two distinct ligand-binding
domains.(A) BTN2A1 tetramer-PE (red) and control streptavidin-PE alone
(black) staining of gated GFP+CD3+HEK-293T cells transfected with single-
residue G115gdTCR alanine mutants (or control Jurkat 9C2gdTCR), normalized
to BTN2A1 tetramer staining of G115 WTgdTCR. (B) Cartoon view of the G115
gdTCR [Protein Data Bank (PDB) code 1HXM ( 25 )] Vg9 ABEDbsheet depicting
the side chains of R20, E70, and H85. (C) CD69 expression on Jurkat cells
expressing G115gdTCR alanine mutants (or 9C2gdTCR+or parentalgdTCR−
Jurkat cells), normalized to the activation levels of G115 WTgdTCR+Jurkat cells,
after overnight culture with LM-MEL-75 APCs in the presence (blue) or absence

(black) of 40mMzoledronate.(D) Surface of G115gdTCR [PDB code 1HXM ( 25 )]

depicting the residues important for BTN2A1 tetramer binding (top row) and

zoledronate reactivity (bottom row). Side chains of tested residues with >75% loss

of BTN2A1 binding or CD69 induction are shown in red; 50 to 75% reduction in

orange; <50% reduction in gray; Vd2, green; Vg9, blue; constant regions, white.

MFI, median fluorescence intensity; SAv, streptavidin alone control; Unstimulated,

unstimulated control. Data in (A) and (C) represent the mean ± SEM ofn=3

separate experiments. Single-letter abbreviations for the amino acid residues are as

follows: A, Ala; C, Cys; D, Asp; E, Glu; F, Phe; G, Gly; H, His; I, Ile; K, Lys; L, Leu;

M, Met; N, Asn; P, Pro; Q, Gln; R, Arg; S, Ser; T, Thr; V, Val; W, Trp; and Y, Tyr.

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