it unlikely that CD209 plays a role in this in-
teraction. Although little is known about the
expression pattern of BTN2A1, RNA analysis
predicts broad expression on immune cells.
We confirmed that BTN2A1 is expressed on
circulating B, T, and NK cells, as well as
monocytes and Vg9Vd 2 +gdT cells (fig. S17),
potentially explaining howgdT cells can pres-
ent pAg to themselves ( 11 ).
Recent studies revealed that human BTNL3
and BTNL8 coassociate and are stimulatory to
human Vg 4 +gdT cells, with BTNL3 interact-
ing with a germline-encoded region of the
g-chain variable domain termed the HV4 loop
( 29 – 31 ). Likewise, mouse BTNL1 and BTNL6
are linked and important for intestinal Vg 7 +gd
T cell function and appear to bind to a similar
region of thegdTCR ( 29 – 31 ), and a similar
ligand-binding domain was also recently iden-
tified within Vg9( 31 ). The BTN2A1–Vg9bind-
ing interface encompasses a similar docking
site, although it exhibits greater dependency
on the outer face of the ABEDbsheet of the
Vg9 TCR that extends beyond the HV4 loop.
This suggests that the BTN2A1-binding foot-
print on Vg9 may be located farther away from
the CDR loops and closer to the Cgdomain.
Given the tendency of butyrophilin molecules
to dimerize [e.g., BTN3A1 can form stable
V-shaped homodimers, and also heterodimers
with BTN3A2 ( 15 ), and BTNL3–BTNL8 heter-
odimers ( 30 )], it is possible that the associa-
tion between BTN2A1 and BTN3A1 represents
a direct interaction, although the molecular
basis for this remains to be determined.
Compared to other Ag-presenting molecules
(MHC and MHC-like molecules), the recog-
nition of heteromeric butyrophilin complexes
represents a fundamentally distinct class of
immune recognition. It is not yet known how
pAgaltersthiscomplextoinduceantigenicity,
but it may involve butyrophilin dimer or multi-
merremodelingand/orconformationalchanges
to BTN2A1 and BTN3A1. Other associated mole-
cules such as ABCA1 ( 32 )maybedirectlyre-
quired, although on the basis of our data, these
would need to be conserved across humans,
mice, and hamsters.
In conclusion, this study substantially ad-
vances our understanding of how pAgs are
sensed bygdT cells. In light of the mixed
outcomes in numerous clinical trials that have
utilizedgdTcellsforanticancertherapy
through pAg stimulation ( 9 ), it will be impor-
tant to reexamine those data to determine
whether BTN2A1 expression holds prognostic
and/or therapeutic value. Our findings also
suggest that BTN2A1 may represent a direct
target for agonistic and/or antagonistic in-
tervention ingdTcell–mediated immuno-
therapy for infectious disease, cancer, and
autoimmunity.Methods
Human samples
Healthy donor-derived human PBMCs were ob-
tained from the Australian Red Cross Lifeblood
under ethics approval 17-08VIC-16 or 16-12VIC-
03, with ethics approval from University of
Melbourne Human Ethics Sub-Committee
(1035100) or Olivia Newton John Cancer Re-
search Institute (ONJCRI) Austin Health Hu-
man Research Ethics Committee (H2012-04446)
and isolated by density gradient centrifuga-
tion (Ficoll-Paque PLUS GE Health care) and
red blood cell lysis (ACK buffer, produced in-
house). Established cell lines were routinely
verified asMycoplasma-negative using the
MycoAlert test (Lonza).Flow cytometry
Human cells were pelleted (400g), washed,
and incubated at 4°C with phosphate-buffered
saline (PBS) supplemented with 2% fetal calf
serum (FCS) containing human Fc receptor
block (Miltenyi Biotec). Mouse NIH-3T3 cells
were incubated with anti-CD16/CD32 (clone
2.4G2, produced in-house). Cells were then
incubated with 7-aminoactinomycin D (7-AAD,
Sigma) or LIVE/DEAD viability markers (ThermoFisher) plus antibodies (table S1). BTN2A1 and
BTN3A were detected using monoclonal anti-
bodies generated in-house (see below). Anti-
BTN2A1 mAb or matched isotype control (clone
BM4, produced in house) were conjugated to
Alexa Fluor-647 by amine coupling (Thermo
Fisher), and anti-BTN3A (clone 103.2) was
conjugated to R-phycoerythrin (PE) (Prozyme)
using sulfo-SMCC heterobifunctional cross-
linker. In some experiments, unconjugated
anti-BTN2A1 mAbs were detected with goat
anti-mouse polyclonal secondary antibody BV421
or PE (BD-Pharmingen), with a subsequent
blocking step (5% normal mouse serum). Cells
were also stained with tetrameric Vg9Vd 2 +TCR,
BTN2A1, MR1–5-OP-RU, or mouse CD1d–a-
GalCer ectodomains (produced in house, see
below), or equivalent amounts of streptavidin
conjugate alone (BD). Each reagent was titrated
to determine the optimal dilution factor. All data
were acquired on an LSRFortessa II, FACSCanto
(BD),orCytoFLEXLX(BeckmanCoulter)and
analyzed with FACSDiva and FlowJo (BD)
software. All samples were gated to exclude
unstable events, doublets, and dead cells using
time, forward-scatter area versus height, and
viability dye parameters, respectively.gdT cell isolation and expansion
In some experiments,gdT cells were enriched
by magnetic-activated cell sorting (MACS)
using PE-Cy7-conjugated anti-gdTCR followed
by anti-phycoerythrin–mediated magnetic bead
purification (Miltenyi Biotec). After enrichment,
CD3+Vd 2 +gdT cells were further purified by
sorting with an Aria III (BD). EnrichedgdTcells
were stimulated in vitro for 48 hours with plate-
bound anti-CD3e(OKT3, 10mg/ml, Bio-X-
Cell), soluble anti-CD28 (CD28.2, 1mg/ml, BD
Pharmingen), phytohemagglutinin (0.5mg/ml,
Sigma), IL-15 (50 ng/ml, PeproTech), and recom-
binant human IL-2 (100 U/ml, PeproTech),
followed by maintenance with IL-2 and IL-15
for 14 to 21 days. Cells were cultured in complete
medium consisting of a 50:50 (v/v) mixture ofRigauet al.,Science 367 , eaay5516 (2020) 7 February 2020 9of13
Fig. 7. Agonistic activity of
anti-BTN3A1 mAb clone
20.1 depends on BTN2A1.
CD69 expression on Jurkat cells
expressing Vg9Vd 2 +TCR
(clone G115), or the indicated
G115gdTCR mutants, or control
Vg5Vd 1 +TCR (clone 9C2)
after coculture with either
parental LM-MEL-75 (“WT”)or
BTN2A1nullAPCs preincubated
with anti-BTN3A (clone 20.1,
10 mg/ml, blue histograms) or
isotype control (mouse IgG1,
10 mg/ml, gray). Data are
representative of one of two
separate experiments.
CD69LM-MEL-75 LM-MEL-75 BTN2A1nullIsotypeBTN3A1 (20.1)436444710457Glu70a
367405353382His85a
423507
439526Arg20a
589450
4,454679WT 9C2 control
476552
447452G115 abTCR0103 104105481400
851450Lys108a
404344
354345Arg51bRESEARCH | RESEARCH ARTICLE