association (fig. S14). Thus, both the extra-
cellular and intracellular domains of BTN2A1
and BTN3A1 are closely associated.
Vg9Vd 2 +TCR recognizes at least two ligands
Given that BTN2A1 binds all Vg 9 +gdTCRs yet
only Vg9Vd 2 +gdT cells are pAg-reactive, we
hypothesized that Vd2isalsoinvolvedin
this interaction. A corollary of this hypothesis
could be that separate binding domains exist
on the Vg9Vd 2 +TCR: one responsible for bind-
ing BTN2A1, located within the germline-
encoded region of Vg9, and another that is
also responsible for pAg reactivity, incor-
porating Vd2 specificity. Mutations of Vg 9
residues Arg^20 ,Glu^70 ,andHis^85 (and to a
lesser extent Glu^22 )toAlaallresultedin
complete loss of BTN2A1 tetramer reactivity,
whereas none of the Vd2 mutations had this
effect (Fig. 6A). The side chains of these Vg 9
residues were in close proximity to one an-
other (Glu^70 to His^85 distance, 2.8 Å; His^85 to
Arg^20 distance, 5.1 Å) and located on the outer
faces of the B, D, and E strands, respectively, of
the ABED antiparallelbsheet of Vg9. Together
they formed a polar triad within the frame-
work region of Vg9 (Fig. 6B), consistent with
BTN2A1 binding to the vast majority of Vg 9 +
gdT cells (Fig. 2B). Thus, BTN2A1 appears to
bind to the side of Vg9, distal to thedchainand not in the vicinity of the complementarity-
determining region (CDR) loops that are
typically associatedwith Ag recognition.
We next examined which residues were im-
portant for mediating functional responses to
pAg. Jurkat cells transduced withgdTCR mu-
tants expressed similar amounts of CD3–gdTCR
complexontheirsurfaceandrespondedequiv-
alently to immobilized anti-CD3 mAb (fig. S15).
Mutations in each of the BTN2A1-binding triad
ofg-chain mutants abrogated pAg-mediated
Jurkat cell activation (Fig. 6B). However, mu-
tations in two additional residues, Arg^51 of
the Vd2-encoded CDR2 loop and Lys^108 of the
CDR3 loop of the TCRgchain, also abrogatedRigauet al.,Science 367 , eaay5516 (2020) 7 February 2020 6of13
Fig. 4. BTN2A1 and BTN3A1
are both necessary for pAg
presentation.(A) CD69 expres-
sion on G115 Vg9Vd 2 +TCR
(top row), control 9C2 Vg5Vd 1 +
TCR (middle), and parental
(TCR−) J.RT3-T3.5 (bottom row)
Jurkat cells after overnight
coculture with the indicated
APCs, in the presence (blue) or
absence (gray) of 40mM zole-
dronate. Numbers indicate the
median fluorescence intensity.
(B) Change in CD25 expression
(normalized to unstimulated
control for each sample) on
purified in vitro–expandedgd
T cells cocultured for 24 hours in
the presence (blue) or absence
(gray) of 4mM zoledronate with
CHO-K1 (hamster origin) or
NIH-3T3 (mouse origin) APCs
transfected with the indicated
combinations of (B)BTNL3,
BTNL8,BTN2A1,BTN3A1,and
BTN3A2or (C)BTN2A1DB30,
BTN3A1,andBTN3A2.(D)gd
T cells cocultured as in (B),
except in the presence of a 1:1
mixture of two populations of
APCs, each transfected sepa-
rately with combinations of
BTN2A1,BTN3A1, andBTN3A2.
Each symbol and connecting line
represents a different donor.
*p< 0.05, **p< 0.01 (Wilcoxon
paired test). Bar graphs depict
mean ± SEM. Data in (A) are
representative of one of three
similar experiments; data in (B)
to (D) representn=7to
9 donors per group pooled from
3 to 5 independent experiments.
BUnstimulatedZoledronateCD-10-6 -6 -6060
50
40
30
20
10Change in CD25, normalized to untransfected APC (%)L3 + L82A1 3A1 3A2
3A1 + 3A22A1 + 3A12A1 + 3A2
2A1 + 3A1 + 3A22A16 B302A16 B30 + 3A1
2A16 B30 + 3A2APC #1:
APC #2:2A1
3A12A1
3A22A1
3A1+3A22A16 B30 + 3A1 + 3A2* * * ***-10060
50
40
30
20
10-10060
50
40
30
20
10* ** * *
403020100403020100403020100Hamster (CHO-K1)Mouse (NIH-3T3)A29426724823413,0675428023525601853,570323313231129 161 137 137 153 138 153120 139 124 131 123 136131 161 138 142 160 140 147120 144 126 125 135 124 135122
CD69WTLM-MEL-75
BTN2A1null BTN3A1null WT BTN2A1null1 BTN3A1nullLM-MEL-62
No APCUnstimulated
Zoledronate0 103104105Jurkat G115+
(Vγ9Vδ 2 + TCR)Jurkat 9C2+
(Vγ5Vδ 1 + TCR)Jurkat parental
(TCR–)RESEARCH | RESEARCH ARTICLE