science.sciencemag.org/cgi/content/full/science.aay5516/DC1

Supplementary Materials for

Butyrophilin 2A1 is essential for phosphoantigen reactivity by γδ T cells

Marc Rigau, Simone Ostrouska, Thomas S. Fulford, Darryl N. Johnson, Katherine Woods, Zheng Ruan, Hamish E.G. McWilliam, Christopher Hudson, Candani Tutuka, Adam K. Wheatley, Stephen J. Kent, Jose A. Villadangos, Bhupinder Pal, Christian Kurts, Jason

Simmonds, Matthias Pelzing, Andrew D. Nash, Andrew Hammet, Anne M. Verhagen, Gino Vairo, Eugene Maraskovsky, Con Panousis, Nicholas A. Gherardin, Jonathan Cebon,

Dale I. Godfrey*†, Andreas Behren*†, Adam P. Uldrich*†

*These authors contributed equally to this work. †Corresponding author. E-mail: auldrich@unimelb.edu.au (A.P.U.); andreas.behren@onjcri.org.au (A.B.);

godfrey@unimelb.edu.au (D.I.G.)

Published 9 January 2020 on Science First Release DOI: 10.1126/science.aay5516

This PDF file includes:

Figs. S1 to S17 Tables S1 and S2 Caption for Database S1

Other Supplementary Materials for this manuscript include the following: (available at science.sciencemag.org/cgi/content/full/science.aay5516/DC1)

Database S1 as zipped archive

2

Fig. S1. Generation of soluble Vγ9Vδ2+ γδ TCR tetramers. (A) PCR for Vδ2 and Vγ9 on single-cell-sorted Vδ2+ γδ T cells from PBMCs. Negative controls depict PCR on empty wells from the same plate. (B) Paired γ-chain and δ-chain gene usage and CDR3 motifs from selected cells. Also shown for comparison is the prototypical clone G115. (C) Soluble γδ TCR construct design containing full-length ectodomains coupled to leucine zippers and an Avi-tag/His6-tag. (D) SDS–PAGE analysis of denatured soluble biotinylated and unbiotinylated Vγ9Vδ2+ γδ TCR, either alone or mixed with undenatured native streptavidin (SAv), showing incorporation of the biotinylated TCR δ-chain into a complex with native streptavidin. MW, molecular weight markers.

A

B

C

D

Negativecontrols

Vδ2

Vγ9

Clone

1

TRDV

TRDV2*03

TRDD

TRDD3

TRDJ

TRDJ1

CDR3δ

CACDPVQVTGGYKVDKLIF

TRGJ

TRGJP

CDR3γ

CALWEVHELGKKIKVF

TRGV

TRGV94 TRDV2*03 TRDD3 TRDJ1 CACDTVQRLGDRPTDKLIF TRGJP CALWEVQELGKKIKVFTRGV95 TRDV2*03 TRDD3 TRDJ1 CACDGILGDSHTDKLIF TRGJP CALWELAELGKKIKVFTRGV96 TRDV2*03 TRDD2+TRDD3 TRDJ1 CACDPLIGSERLGDTGIDKLIF TRGJP CALWESQELGKKIKVFTRGV97 TRDV2*02 TRDD3 TRDJ1 CACDRVLGDTRWTDKLIF TRGJP CALWEVHELGKKIKVFTRGV9

TRGC

Soluble γδ TCR

TRDV2 TRDC Fos Avi-tag His6-tag

TRGV9 Jun

δ chain-biotin+SAv

50 kDaδ chainγ chain

30 kDa

MW

ladd

er

γδTC

R #

3-bi

otin

γδTC

R #

3

γδTC

R #

3-bi

otin

+SAv

SAv

alon

eG115 TRDV2 TRDD3 TRDJ1 CACDTLGMGGEYTDKLIF TRGJP CALWEAQQELGKKIKVFTRGV9

3

Fig. S2. Identification of Vγ9Vδ2+ γδ TCR ligands using a whole genome CRISPR/ Cas9 knockout screen. (A) γδTCR tetramer #6lo LM-MEL-62 cells were sort-purified four consecutive times, from n=4 separate replicates. Histograms depict γδTCR tetramer #6 (blue) overlaid with control histograms (gray or red) after 1-2 weeks culture following each round of sorting. (B) Top 40 guide RNA gene targets within the γδTCR tetramer #6lo population, compared to control unsorted (“pre-sort”) LM-MEL-62 cells.

A

B

Pre-

sort

Firs

t sor

tSe

cond

sor

tTh

ird s

ort

Four

th s

ort

Replica 2Replica 1 Replica 3 Replica 4

6%4.1%3.5%1.3%

N.D.

UnstainedγδTCR tetramer #6

Control mouse CD1d–α-GalCer tetramer

Rank Gene name Targeted DNA sequence log2FC log2CPM p-value FDR

1 BTN2A1 GGCCATCCTGCACCTCGTAG 13.485 14.819 3.024e-07 2.720e-03

2 SPPL3 CGAGTAGGTCTGCTCCGCCA 13.978 17.092 3.953e-07 2.720e-03

3 hsa-mir-507 GAAATATGTTTTGCACCTTT 14.973 17.599 8.096e-06 1.789e-02

4 ATE1 GGCACATTCCATGACAGTAC 15.100 18.108 8.111e-06 1.789e-02

5 IGF2R CGGACTGAAGCTGGTGCGCA 13.238 14.967 1.067e-05 1.789e-02

6 OR5M9 GAGTGATCATGTAAACCGCT 4.356 3.197 1.136e-05 1.789e-02

7 ACSS2 TCACATCAAAGTTGTACCGA 13.823 15.361 1.451e-05 1.789e-02

8 CEACAM1 CATGCCATTCAATGTTGCAG 15.004 18.496 1.478e-05 1.789e-02

9 RGAG4 TTCGGCGTGGTAACCACTTG 14.795 18.512 1.564e-05 1.789e-02

10 PET117 CTGCTGCTTCACATGTACGC 15.028 18.083 1.591e-05 1.789e-02

11 MANBAL GGAAGAGTCCGTACCGTAGC 13.837 13.453 2.039e-05 1.789e-02

12 ABL1 TGCACTCCCTCAGGTAGTCC 14.207 19.235 2.076e-05 1.789e-02

13 MAPK4 TGGAGACCATCCCTGTAATC 13.163 14.984 2.227e-05 1.789e-02

14 C1orf233 CACCGAGAACCTCATGCGCC 14.434 14.223 2.255e-05 1.789e-02

15 STRC TGCTCTTTAGCTATCCCTGG 13.545 14.522 2.424e-05 1.789e-02

16 EEFSEC CATCCCAACGAGAGATCCCT 14.660 15.404 2.580e-05 1.789e-02

17 TAS2R7 CACGCACCCCAGTAGAATCC 14.599 14.076 2.598e-05 1.789e-02

18 hsa-mir-7976 AATTTAGATCAAAAGCCTCA 14.694 12.738 3.018e-05 1.789e-02

19 MFSD10 ATCCCCTAGGACCCCCTCTA 14.158 12.043 3.114e-05 1.789e-02

20 MSMO1 GTGCAGTCATTGAAGATACT 13.909 14.818 3.431e-05 1.789e-02

21 ATP8A2 GGCGAGATGCTGAACGGCGC 4.845 2.943 3.602e-05 1.789e-02

22 C11orf82 CATGTCAGAAGTGCTTCTCT 13.249 13.988 3.763e-05 1.789e-02

23 hsa-mir-6772 TGGGCACAGAGTCAGGAGCA 13.862 14.656 3.880e-05 1.789e-02

24 SLC24A2 CTGAAGTTAATTCGAGTCTT 13.261 14.477 3.883e-05 1.789e-02

25 FNBP4 ACAAAACTAGTTTCAGCACC 14.981 17.307 3.911e-05 1.789e-02

26 FREM1 TGAATGAGCCAATAACCCAG 14.977 16.771 4.124e-05 1.789e-02

27 ACTRT3 ACTAACCGCTTGTCTAAACC 13.261 13.390 4.238e-05 1.789e-02

28 CCDC80 TAGGCCCAGTCTTCATCGTT 12.501 14.547 4.255e-05 1.789e-02

29 ASTN1 ATATCCCTTTGGTGCGCTGG 12.407 13.898 4.324e-05 1.789e-02

30 EIF4EBP2 CATGACTATTGCACCACGCC 12.130 14.537 4.409e-05 1.789e-02

31 APC GGATCTGTATCAAGCCGTTC 12.770 12.053 4.536e-05 1.789e-02

32 DNAJB12 TCTGGTTGAGGGACTCAATC 13.022 15.026 4.632e-05 1.789e-02

33 PROP1 CTGCCGACTCGCCCCTTCTT 14.399 14.744 4.697e-05 1.789e-02

34 TECPR1 CACCTCTGCCGTGTCCGCTC 13.112 14.926 4.729e-05 1.789e-02

35 KIAA2018 TACCTGCATTGTGTGTCTCC 13.818 17.786 4.776e-05 1.789e-02

36 RAB1B TGACGTTCTCGCTGGCATAG 14.324 17.617 4.870e-05 1.789e-02

37 NDUFAF1 ATTCTCCAGAGCTTAACCCA 11.723 11.584 4.960e-05 1.789e-02

38 BTN2A1 GCGACGCACCTGCCACTACG 11.319 11.662 5.121e-05 1.789e-02

39 IFNA1 + IFNA13 CCTGTGTGATGCAGGAGGAG 10.529 12.106 5.161e-05 1.789e-02

40 TMEM192 GCGTTGATGATACAGTCCTC 12.612 10.778 5.201e-05 1.789e-02

4

Fig. S3. Generation of BTN2A1 and BTN3A1 knockout cell lines. BTN2A1null and BTN3A1null LM-MEL-62 or LM-MEL-75 cells were generated via transient transfection of target cells with vectors encoding Cas9 and specific guide RNA, followed by bulk cell sorting. (A) Anti-BTN2A1 (clone 231, red) and anti-BTN3A1/3A2/3A3 (clone 103.2, blue) staining of each cell line overlaid with isotype controls. (B) Vγ9Vδ2+ γδ TCR tetramer #6 staining of each cell line (dark green) overlaid with irrelevant tetramer control (mouse CD1d–α-GalCer, gray). Data are representative of two similar experiments.

A

B

WT

LM-MEL-62

BTN2A1null2 BTN3A1null

BTN3A1null

BTN2A1null1

BTN3A

BTN2A1

Isotype (mouse IgG2a, κ)BTN2A1 (clone 231)

Isotype (mouse IgG1, κ)BTN3A (clone 103.2)

Control mouse CD1d–α-GalCer tetramerγδTCR tetramer #6

WT

LM-MEL-75

BTN3A1nullBTN2A1null

WT

LM-MEL-75

BTN3A1nullBTN2A1nullWT

LM-MEL-62

BTN2A1null2BTN2A1null1

BTN2A1null2BTN2A1null1WT

LM-MEL-62

BTN3Anull WT

LM-MEL-75

BTN3AnullBTN2A1null

γδTCR tetramer #6

5

Fig. S4. Generation of anti-BTN2A1 mAb. (A) Alignment of BTN2A1, BTN2A2, BTN3A1, and BTN3A2 ectodomains. Rasmol color schemeused. (B) Binding of anti-BTN2A1 mAb clones to plate-bound BTN2A1, BTN2A2, or BTN3A2ectodomains by ELISA, where heat maps depict absorbance. (C) Anti-BTN2A1 mAb reactivity tomouse NIH-3T3 cells transfected with full length human BTN2A1 (blue), BTN2A2 (green), or BTN3A1(pink), or untransfected cells (yellow). Data averaged from N=2 separate experiments. (D) Reactivityof selected anti-BTN2A1 clones (red) or isotype control (mouse IgG2a κ, clone BM4, gray) to LM-MEL-62 parental (“WT”), BTN2A1null1 and BTN2A1null2 cells, using a BV421-conjugated secondarypolyclonal Ab. The same isotype control is overlaid across each individual row. (E) Reactivity ofselected anti-BTN2A1 clones to LM-MEL-62 parental (“WT”), BTN2A1null and BTN3A1null cells usinga PE-conjugated secondary polyclonal Ab. A450, absorbance at 450 nm.

B

C

D E

Q F I V VGPT D P I L A T VGEN T T L RCH L SP EKN A EDMEV RWFR SQ F SPA V F V Y KGGR ERT E EQ ME E YRGRT T F V SKD I SRG SVQ F T V VGPAN P I L AMVGEN T T L RCH L SP EKN A EDMEV RWFR SQ F SPA V F V Y KGGR ERT E EQ ME E YRGR I T F V SKD I N RG SVQ F SV L GP SGP I L AMVGED AD L PCH L F PTM S A ET ME L KWV S S S L RQV VN V Y ADGK EV ED RQ SA P YRGRT S I L RDG I T AGK AQ F SV L GP SGP I L AMVGED AD L PCH L F PTM S A ET ME L KWV S S S L RQV VN V Y ADGK EV ED RQ SA P YRGRT S I L RDG I T AGK A

A L V I HN I T AQ ENGT YRC Y FQ EGR SYD EA I L H L V V AG L G SK P L I SMRGH ED GG I R L EC I SR GWY PK P L T VW RD P YGGV A PAA L V I HN V T AQ ENG I YRC Y FQ EGR SYD EA I L R L V V AG L G SK P L I E I K AQED G S I WL EC I SG GWY P E P L T VW RD P YGEV V PAA L R I HN V T A S D SGK Y L C Y FQ DGD F Y EK A L V E L K V A A L G SD L H VD V KGYKD GG I H L ECR ST GWY PQPQ I QW SNN KGEN I PTA L R I HN V T A S D SGK Y L C Y FQ DGD F Y EK A L V E L K V A A L G SN L H V EV KGY ED GG I H L ECR ST GWY PQPQ I QW SN A KGEN I PA

L K EV SMPD AD G L FMV T T A V I I RD K SV RNM S C S I NN T L L GQ K K E SV I F I P E S FMP SV SL K EV S I AD AD G L FMV T T A V I I RD K YV RN V S C SVNN T L L GQ EK ET V I F I P E S FMP SA SV EA PV V ADGV G L YA V A A SV I MRG S SGEGV S CT I R S S L L G L EK T A S I S I AD P F FR SAQV EA PV V ADGV G L Y EV A A SV I MRGG SGEGV S C I I RN S L L G L EK T A S I S I AD P F FR SAQ

BTN2A1BTN2A2BTN3A1BTN3A2

BTN2A1BTN2A2BTN3A1BTN3A2

BTN2A1BTN2A2BTN3A1BTN3A2

87%

96%

43% 45% homology

A

Hu3

4C22

622

722

822

923

023

123

223

323

423

523

623

723

823

924

024

124

224

324

424

524

624

724

824

925

025

125

225

325

425

525

625

725

825

926

026

126

226

326

426

526

626

726

826

927

0Ig

G2a

isot

ype

Blan

k

BTN2A1

BTN2A2

BTN3A2

Med

ian

fluor

esce

nce

inte

nsity

1

2

(A450 nm)

Mouse 3T3 fibroblasts transfected with: BTN2A1 BTN2A2 BTN3A1

0 103 104 105

2A1null1WT 3A1null

267266259236

227Hu34C1Isotype (mouse IgG2a, κ)No primary mAbBTN2A1 mAbIsotype (mouse IgG2a, κ)

228 231 233 243 259 267 268 Hu34C

WT

LM-M

EL-6

2BT

N2A

1null1

BTN

2A1nu

ll2

0 103 105

LM-MEL-62

Isot

ype

No

mAb

Hu3

4C 226

227

228

229

230

231

232

233

234

235

236

237

238

239

240

241

242

243

244

245

246

247

248

249

250

251

252

253

254

255

256

257

259

258

260

261

262

263

264

265

266

267

268

269

270

101

102

103

104

105 Untransfected

6

Fig. S5. Generation of BTN2A1 tetramers. (A) Construct design including BTN2A1 ectodomain (IgV and IgC domains; Gln29 to Ser245) fused to a C-terminal linker (amino acid sequence: GTGSGSGG), followed by Avi (biotin ligase)- and His6-tags (amino acid sequence: LNDIFEAQKIEWHEHHHHH). (B) SDS–PAGE of biotinylated BTN2A1, and control BTN3A1 ectodomains produced in HEK-293T cells. Right-hand lane depicts denatured BTN2A1–biotin complexed with undenatured native streptavidin (SAv.). (C) ELISA of plate-bound BTN2A1 ectodomain reactivity to anti-BTN2A1 clones Hu34C (green) and 231 (blue), compared to isotype control (clone BM4, red). Data in panel (C) representative of one experiment. MW, molecular weight markers.

A

CB

50 KDa40 KDa

30 KDa

MW

BTN

2A1

Biot

in

BTN

3A1

Biot

in

BTN

2A1

Biot

in +

SAv

00.

020.

040.

080.

16

0.31

0.62

1.25 2.5 5 10 20

BTN2A1 ectodomain (μg/ml)

Abso

rban

ce

0.6

0.4

0.2

Anti-BTN2A1 (clone Hu34C)Anti-BTN2A1 (clone 231)Isotype (mouse IgG2a, κ)

BTN2A1 Ectodomain Avi-Tag His6-Tag

7

Fig. S6. BTN2A1 is specifically recognized by Vγ9Vδ2+ γδ TCR tetramers. Vγ9Vδ2+ γδ TCR tetramer #6 (green), irrelevant control tetramer (mouse CD1d–α-GalCer, gray), or control streptavidin (SAv.) alone (yellow) staining on gated GFP+ mouse 3T3 cells following transfection with either human BTN2A1, BTN2A2, BTNL3 plus BTNL8, or BTN3A1 plus BTN3A2 (parent gating is depicted in the top row of density plots). Data are representative of two similar experiments.

Unstransfected BTNL3+BTNL8 BTN2A1 BTN2A2 BTN3A1+BTN3A2

SAv. control

GFP

Forw

ard

scat

ter

γδTCR tetramer #6Control mouse CD1d–α-GalCer tetramer

0% 83% 57% 62% 76%

8

Fig. S7. Antagonist anti-BTN2A1 mAb specifically block pAg-mediated activation of Vδ2+ γδ T cells but not peptide-mediated activation of CD8+ αβ T cells. (A) Intracellular IFN-γ expression on gated Vδ2+CD3+ T cells (left) or CD8+CD3+ T cells (right) amongst PBMCs following in vitro challenge with either the pAg HMBPP (0.5 ng/ml) or zoledronate (4 μM) alone or in combination with CEF peptide mixture containing immunogenic peptides derived from cytomegalovirus, Epstein–Barr virus, and influenza (1 μg/ml) ± 10 μg/ml neutralizing anti-BTN2A1 mAbs (clones Hu34C, 236, 259, 267), anti-BTN3A molecules (clone 103.2) or isotype control (mouse IgG2a, k, clone BM4). (B) Representative gating (top row) and plots of IFN-γ staining on gated Vδ2+CD3+ T cells (middle row), or CD8+CD3+ T cells (bottom row). Data are representative of seven donors from two independent experiments.

B

A

Donor 1

Donor 2

Donor 3

Donor 4

Donor 5

Donor 6

Donor 7

103.

2

Hu3

4C 259

267

CEF

HM

BPP

Uns

timul

ated

Vδ2

Isot

ype

CD

8

236

IFNγ

HMBPP + CEF

0.16%

0.081%

82.8%

0.15% 3.17%

0.074% 81.8%

3.17% 2.60%

0.66% 33.9%

3.16% 3.12%

0.51%

3.18% 2.97%

54.4% 48.4%no

ant

ibod

y

81.6%

3.23%

Vδ2

CD8

Vδ2+

T cells

CD8+

T cells

T cells

Uns

timul

ated

HM

BPP CEF

Isot

ype

103.

2

236

259

267

noan

tibod

y0.000.050.100.150.200.25

1

2

3

4

Unst

imul

ated

ZOL

CEF

Isot

ype

103.

2

236

259

267

noan

tibod

y

0.000.050.100.150.200.25

1

2

3

4

HMBPP + CEF

Zoledronate + CEF

CD8+ T cells

IFN

-γ (%

)IF

N-γ

(%)

Hu3

4CH

u34C

U

nstim

ulat

ed

HM

BPP CEF

Isot

ype

103.

2

236

259

267

no

antib

ody0

20

40

60

80

100HMBPP + CEF

Zoledronate + CEF

Uns

timul

ated

ZOL

CEF

Isot

ype

103.

2

236

259

267

noan

tibod

y

0

20

40

60

80

Vδ2+ T cellsIF

N-γ

(%)

IFN

-γ (%

)

Hu3

4CH

u34C

9

Fig. S8. Jurkat G115 Vγ9Vδ2+ γδ T cell responses to zoledronate, HMBPP and IPP depend on BTN2A1. (A) CD69 induction on either Jurkat G115 Vγ9Vδ2 γδTCR+ or control Jurkat 9C2 Vγ5Vδ1 γδTCR+ Tcells following coculture with graded doses of the pAgs HMBPP, IPP, or zoledronate ± parental LM-MEL-75 APCs. (B) representative CD69 histograms and (C) expression levels following coculture ofJurkat G115 and Jurkat 9C2 T cell lines with either parental LM-MEL-75, BTN2A1null or BTN3A1null

APCs ± HMBPP (100 nM), IPP (100 μM) or zoledronate (40 μM). Data in (A) from one experiment;(B) and (C) pooled from N=4 independent experiments.

B

A

C

BTN2A1nullWT

CD69

CD69 (median fluorescence intensity)

BTN3A1null

LM-MEL-75

LM-MEL-75

Jurkat 9C2 control

Jurkat G115

Concentration (µM)

CD

69 (N

orm

alis

ed to

uns

timul

ated

con

trol)

(%)

No APC

40

20

0

10-6

10-5

10-4

10-3

10-2

10-1

100

102

101

10-6

10-5

10-4

10-3

10-2

10-1

100

102

101

40

20

0

Unstim.

Jurkat 9C2 control

Jurkat G115

IPP(100 µM)

Zoledronate(40 µM)

HMBPP(100 nM)

WTNo APC

050

0

1,00

0

1,50

0050

0

1,00

0

1,50

0 010

,000

20,0

0030

,0000

1,00

02,

000

3,00

04,

000

IPP (100 µM)Zoledronate (40 µM)HMBPP (100 nM)

IPPZoledronate

HMBPP

Unstimulated

BTN3A1null

BTN2A1null1

WTNo APC

BTN3A1null

BTN2A1null1

10

Fig. S9. BTN2A1 plus BTN3A1 engender mouse and hamster APCs with the capacity to present pAg to γδ T cells. (A) BTN2A1 (clone 231) versus BTN3A1/3A2/3A3 staining (clone 103.2), or isotype control staining(mouse IgG2a clone BM4), on NIH-3T3 cells transfected with the indicated combinations of BTNL3,BTNL8, BTN2A1, BTN3A1 and BTN3A2, or BTN2A1ΔB30. (B) CD25 expression on purified in vitro-expanded γδ T cells cocultured for 24 h in the presence (blue) or absence (gray) of 4 μM zoledronatewith CHO-K1 or NIH-3T3 APCs transfected with the indicated combinations of BTNL3, BTNL8,BTN2A1, BTN3A1 and BTN3A2, or BTN2A1ΔB30. Three groups on the right depict γδ T cells co-cultured in the presence of a 1:1 mixture of two populations of APCs, each transfected separately withthe indicated combinations of BTN2A1, BTN3A1, and BTN3A2. (C) Schematic of BTN2A1 andBTN2A1ΔB30 structures (left), and histograms depicting anti-BTN2A1 (clone 259, yellow), andγδTCR tetramer (#6, magenta) on NIH-3T3 cells transfected with BTN2A1 or BTN2A1ΔB30, overlaidwith relevant controls. Data represent n=7–9 donors per group pooled from 3–5 independentexperiments. TM, transmembrane domain;

BTN2A1 (clone 259)Isotype (mouse IgG2a, κ)

γδTCR tetramer #6Control mouse CD1d–α-GalCer tetramer

Mou

se(N

IH-3

T3)

B UnstimulatedZoledronate

Ham

ster

(CH

O-K

1)

CD25

L3+L8 2A1 3A1 3A23A1+3A2

2A1+3A1

2A1+3A2

2A1+3A1+3A2

2A1ΔB30

2A1ΔB30+

3A1

2A1ΔB30+

3A2

2A1ΔB30+

3A1+3A2APC #1:APC #2:

2A13A1

2A13A2

2A13A1+3A2

C

L3+L8Untransfected 2A1 3A1 3A23A1+3A2

2A1+3A1

2A1+3A2

2A1+3A1+3A2

2A1ΔB30

2A1ΔB30+

3A1

2A1ΔB30+

3A2

2A1ΔB30+

3A1+3A2

BTN

2A1

BTN3A (103.2)

Isot

ype

Isotype (mouse IgG1)

A

IgV

IgCBTN2A1

B30.2

BTN2A1ΔB30.2

PILRβ-TM

IgV

IgC

4%4%4%7%5%8%3%36%4%23%4%4%4%7%3%

4% 7% 4% 4% 4% 14% 6% 34% 4% 10% 5% 5% 4% 4% 5%

11

Fig. S10. No detectable binding of HMBPP or IPP to intracellular B30.2 domain of BTN2A1. (A) Raw isothermal titration calorimetry traces and (B) binding isotherms of recombinant BTN2A1 (left column) or BTN3A1 (right column) B30.2 domains (100 μM), upon serial injections of the pAgs HMBPP (1.9 mM, magenta), IPP (2 mM, blue), or PBS buffer alone (green). Data shown from one of two independent experiments.

A

B

Time (min)

HMBPP

-4

-6

-4

-2

0

0 10 20 30 40

1 2 3 4 1 2 3 4

50 0 10 20 30 40 50

-3

-2

-1

0

Ener

gy (µ

cal/s

)BTN2A1 B30.2 domain

IPP PBS

HMBPP KD = 1.64 µMIPP KD = 813 µM

Inje

ctio

n he

at (K

cal/m

ol)

Molar ratio

BTN3A1 B30.2 domain

12

Fig. S11. Association between BTN2A1 and BTN3A1 on the cell surface is independent of intracellular B30.2 domains. Contour plots (top row) depict BTN2A1 (clone 259) versus BTN3A (clone 103.2) staining (magenta), overlaid with isotype control staining (mouse IgG1 clone MOPC-173 on the x-axis versus mouse IgG2a clone BM4 on the y-axis versus, gray) on mouse NIH-3T3 cells transfected with the indicated combinations of BTN2A1, BTN3A1, BTN3A1 and/or BTN2A1ΔB30. Histograms (second row) depict FRET signal in each staining condition. Data are representative of two independent experiments.

2A1Untransfected

control 2A1ΔB30 3A1 3A2 2A1+3A1 2A1+3A22A1ΔB30+

3A12A1ΔB30+

3A2

Isotype controls Anti-BTN2A1+anti-BTN3A

BTN3A (clone 103.2)

FRET

BTN

2A1

(clo

ne 2

59)

13

Fig. S12. Generation of CFP- and YFP-tagged butyrophilin constructs. (A) Design of full length BTN2A1, BTN3A1, BTNL3, and BTNL8 with either a “long” or “short” C-terminal flexible linker coupled to CFP or YFP, respectively. (B) Amino acid sequences of C-terminal linkers and CFP/YFP domains. (C) Representative plots depicting anti-BTN2A1 (clone 231) and anti-BTN3A molecules (clone 103.2) mAb staining (red) or isotype control staining (IgG1 versus IgG2a, gray) on mouse NIH-3T3 cells transiently transfected with each respective construct. (D) Representative plots depicting BTN2A1 (left) and BTN3A1 (right) surface expression on mouse NIH-3T3 cells transfected with WT BTN molecules (green), or CFP/YFP-tagged BTN molecules (blue).

A BTN2A1

EcoRIEcoRI Mfel Notl

Linker CFP or YFPBTN

BTN gene

IRESpMIG2

GFP or YFP

CFP

GFP

SalI

UntransfectedBTN2A1 WT

(GFP)BTN3A1 WT

(GFP) BTN2A1-YFP BTNL8-YFP BTN3A1-CFP BTNL3-CFP

SalI

YFP

Short/longlinker

YFPCFP CFP

BTN3A1 BTNL3 BTNL8

B

C

Short linker QLGSGGRGS

QLGSGGSGSGGSGSGGRGS

MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKLICTTGKLPVPWPTLVTTLGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYITADKQKNGIKANFKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLSYQSKLSKDPNEKRDHMVLLEFVTAAGITLGMDELYK

MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLSWGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYFSDNVYITADKQKNGIKANFKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLLEFVTAAGITLGMDELYK

Long linker

YFP(mVenus)

CFP(mTurquoise2)

UnstransfectedBTN2A1

BTN3A

BTN

2A1

BTN2A1 (clone 231) BTN3A (clone 103.2)

BTN3A1

BTN3A1 (WT with no tag)BTN3A1-CFP

Isotype controls Anti-BTN2A1+anti-BTN3A

DUnstransfectedBTN2A1 (WT with no tag)BTN2A1-YFP

14

Fig. S13. Intracellular domains of BTN2A1 and BTN3A1 are associated in a manner unaffected by pAg. (A) Plots depict FRET versus donor fluorophore (CFP) on mouse 3T3 cells transfected with different combinations of butyrophilin molecules (top row) or single-transfected controls (second row). (B) FRET between the indicated combinations of CFP/YFP-tagged butyrophilin-transfected mouse 3T3 cells ± overnight challenge with HMBPP (100 ng/ml) or zoledronate (40 μM). (C) FRET between the BTN2A1 and BTN3A1 ectodomains, as measured by anti-BTN2A1 (clone 259) and anti-BTN3A1 (clone 103.2) co-staining, ± overnight challenge with HMBPP (100 ng/ml) or zoledronate (40 μM). All plots are pre-gated on transfected cells (CFP, or YFP, or both), except untransfected controls, as appropriate. Data in (A) representative of four independent experiments; (B) and (C) representative of two independent experiments.

A

B

C

Untransfected

CFP

CFP YFPBTNL3

BTNL3BTNL8

BTNL8

BTN3A1

BTN3A1

BTN2A1

BTN2A1

BTN2A1

BTN2A1

BTN3A1

BTN3A1

BTN3A1BTNL8

BTNL3BTN2A1

CFP

FRET

3A1-CFP 2A1-YFP 2A1-CFP 3A1-YFPL3-CFP +L8-YFP

3A1-CFP +2A1-YFP

2A1-CFP +3A1-YFP

L3-CFP +2A1-YFP

FRET

CFP

Unt

reat

edH

MBP

P(1

00 n

g/m

l)Zo

ledr

onat

e(4

0 µM

)

BTN2A1transfected

BTN3A1transfected

BTN2A1+BTN3A1transfected

FRET

BTN

3A

0 % 0 % 0 %

0% 0% 44%

0% 0% 46%

0% 0% 49%

0 % 0 % 0 %

97 %

0 %

82 % 81 % 13 % 20 %

0 % 0 % 0 % 0 % 87 % 44 % 42 % 4 %

0 % 0 % 0 % 0 % 81 % 65 % 39 % 2 %

Untreated

HMBPP(100 ng/ml)

Zoledronate(40 µM)

YFP

0 103 104 105

0 103 104 105

010

310

410

5

51 % 34 % 11 %0 % 0 % 0 % 0 % 90 %

010

310

410

5

15

Fig. S14. Intracellular domain association between BTN2A1 and BTN3A1 is disrupted by anti-BTN2A1 mAbs. Percentage of FRET+ cells between CFP or YFP-tagged BTN2A1 and BTN3A1 (blue) following incubation of transfected mouse NIH-3T3 cells with a panel of unconjugated anti-BTN2A1 mAb (10 μg/ml), or isotype control (mouse IgG2a, k, clone BM4). FRET levels of control BTN3A1+BTNL8 transfectants are also shown (red). Data for BTN2A1+BTN3A1 group are representative of two independent experiments, each performed with BTN2A1CFP+BTN3A1YFP and BTN3A1CFP+BTN2A1YFP transfectants (pooled together on graph); BTN3A1CFP+BTNL8YFP are from two independent experiments.

Isot

ype

226

227

228

229

230

231

232

233

234

235

236

237

238

239

240

241

242

243

244

245

246

247

248

249

250

251

252

253

254

255

256

257

258

259

260

261

262

263

264

265

266

267

268

269

270

Hu3

4C

0

20

40

60FR

ET (%

)BTN2A1+BTN3A1Control (BTN3A1+BTNL8)

16

Fig. S15. Normal γδTCR expression and responsiveness to anti-CD3 stimulation by Jurkat.G115 γδTCR mutants. (A) CD3ε/GFP co-expression on transfected HEK-293T cells with each of the Jurkat G115 γδTCRmutants. Gates depict cells used to determine BTN2A1 tetramer staining intensity. (B) RepresentativeBTN2A1 tetramer staining (red) and streptavidin alone control (gray) of each of the populations gatedin (A). (C) Representative CD69 induction on Jurkat G115 mutants in co-cultures containing LM-MEL-75 WT APCs with (blue) or without (gray) 40 μM zoledronate. (D) CD69 induction on JurkatG115 γδTCR mutants following overnight culture on platebound anti-CD3/anti-CD28 (10 μg/ml each,blue), or alone (gray). Data in (d) depict mean ± SEM of N=2 independent experiments.

A

B

C

D

CD

3

BTN2A1 tetramer

CD69

CD69

WTE5γ

R20γ

E22γ

T29γ

Y54γ

T57γ

K60γ

S62γ

S66γ

E70γ

E76γ

H85γ

N86γ

E88γ

Q90γ

K108γ

E28δ

R51δ

L97δ

9C2 T

CR

Parenta

l0

0 103 104 105

25

50

75

100

125

150

CD69

MFI

rela

tive

to W

T (%

)

Unstimulated Zoledronate

Unstimulated Anti-CD3 + anti-CD28

SAv.-PE BTN2A1 tetramer

WT E5γ R20γ E22γ T29γ Y54γ T57γ K60γ S62γ S66γ E70γ

9C2 TCRE76γ H85γ N86γ E88γ Q90γ K108γ E28δ R51δ L97δ Parental

9C2 TCR

WT E5γ R20γ E22γ T29γ Y54γ T57γ K60γ S62γ S66γ E70γ

E76γ H85γ N86γ E88γ Q90γ K108γ E28δ R51δ L97δ

9C2 TCR

WT E5γ R20γ E22γ T29γ Y54γ T57γ K60γ S62γ S66γ E70γ

E76γ H85γ N86γ E88γ Q90γ K108γ E28δ R51δ L97δ Parental

0 103 104 105

0 103 104 105

WT E5γ R20γ E22γ T29γ Y54γ T57γ K60γ S62γ S66γ

GFP

E76γ H85γ N86γ E88γ Q90γ K108γ E28δ R51δ L97δ

E70γ

γ-chainaloneCD3 alone

δ-chainalone

9C2 TCR

21% 24% 24% 17% 24% 27% 25% 15% 19% 27%

4% 0.3% 0.2%

23%

18% 26% 25% 12% 24% 26% 24% 25% 25% 21%No DNA

0%

17

Fig. S16. N-linked glycans are not required for BTN2A1 binding to Vγ9Vδ2+ γδ TCR. BTN2A1 ectodomain with complex glycans was produced in mammalian Expi293F, and BTN2A1 ectodomain with simple glycans was produced in GNTI-defective HEK-293S cells. The latter was treated with endoglycosidase H in GlycoBuffer 3 overnight at room temperature according to manufacturer instructions (NEB) to yield deglycosylated BTN2A1. (A) SDS–PAGE of the different biotinylated BTN2A1 ectodomains. (B) Phycoerythrin-conjugated tetramers produced from each batch of biotinylated BTN2A1 ectodomain, or control streptavidin (SAv.) alone were used to co-stain parental (TCR−) J.RT3-T3.5 (top row), J.RT3-T3.5.9C2 Vγ5Vδ1+ γδ TCR (middle row), and Jurkat J.RT3-T3.5.G115 Vγ9Vδ2+ γδ TCR (bottom row) cell lines along with anti-CD3ε-allophycocyanin.FRET between BTN2A1 tetramer and anti-CD3ε (lower histograms) was also measured in eachsample. (C) Staining of glycosylated (complex or simple) BTN2A1 tetramer on a PBMC donor (left) orn=3 samples of purified and in vitro-expanded Vδ2+ γδ T cells (right hand plots).

B

C

A

Jurkat Parental

MW

.

Gly

cosy

late

d BT

N2A

1 (c

ompl

ex g

lyca

ns)

Deg

lyco

syla

ted

BTN

2A1

FRET

CD

3-AP

C

Jurkat 9C2 Vγ5Vδ1+ γδTCR

DeglycosylatedBTN2A1 tet.

GlycosylatedBTN2A1 tet.

(simple glycans)

GlycosylatedBTN2A1 tet.

(complex glycans)Streptavidin

alone

Jurkat G115 Vγ9Vδ2+ γδTCR

Purified in vitro-expanded Vδ2+ γδ T cellsPBMC

CD3ε

Vδ2

BTN2A1 tetramer BTN2A1 tetramer

CD3ε

Donor TL68Gatted Vδ2+ γδ T cells

0.31% 99.8% 99.9%99.9%

Donor RS7Donor RS6 Donor U1

SAv.-PE control

BTN2A1 tetramer SAv.-PE control

Gly

cosy

late

d BT

N2A

1 (s

impl

e gl

ycan

s)

Jurkat G115Vγ9Vδ2+ γδTCR

Jurkat 9C2Vγ5Vδ1+ γδTCR

Parental Jurkats75%

67%

100%

81% 75%83% 19%

63%

41%

100%

0%

0%

98%

0%

0%

0%

Glycosylated BTN2A1 (complex glycans)Glycosylated BTN2A1 (simple glycans)

30 kDa40 kDa

18

Fig. S17. BTN2A1 is expressed on circulating leukocytes. (A) Anti-BTN2A1 clone 259 (green) and clone 229 (blue), which are not cross-reactive to BTN2A2, staining of gated leukocyte subsets from two healthy PBMC donors, compared to isotype control (gray, IgG2a, k) or secondary alone (white). Histograms depict staining on: B cells (CD19+CD3−), CD4+ T cells (CD3+CD4+CD8−), CD8+ T cells (CD3+CD4−CD8+), γδ T cells (CD3+γδTCR+), MAIT cells (CD3+MR1-5-OP-RU tetramer+), NK cells (CD3−CD56+), and monocytes (CD14+). Parental LM-MEL-62 and BTN2A1null were included within the same experiment (lower histograms). (B) As per (A), except graphs depict mean fluorescence intensity (MFI) staining of n=4-5 donors. (C) Immunoblot analysis of BTN2A1 and control GAPDH on in vitro-expanded Vδ2+ γδ T cells from five independent donors, compared to parental LM-MEL-62 and BTN2A1null1 cells.

Don

or M

S33

Don

or M

S34

B cells CD4+ T cells

LM-MEL-62

62 kDa

49kDa

38kDa

28kDa

LM-MEL-62BTN2A1null1

CD8+ T cells MAIT cells MonocytesNK cellsγδ T cells

BTN2A1

BTN2A1

ASecondary alone control Isotype (mouse IgG2a, κ) BTN2A1 (clone 229) BTN2A1 (clone 259)

B C

BTN

2A1

(MFI

x10

-3)

Primary Vδ2+ γδ T cells ex vivoIn vitro-expanded Vδ2+ γδ T cells

20

15

10

5

0

LM-M

EL-6

2

LM-M

EL-6

2 BT

N2A

1null1

Mar

ker

Don

or 1

Don

or 2

Don

or 3

Don

or 4

Don

or 5

GAPDH

BTN2A1

In vitro-expanded Vδ2+ γδ T cells

Isotyp

e

BTN2A1 (

229)

BTN2A1 (

259)

19

Table S1. Antibodies used for flow cytometry. Target Target species Source species Clone name Fluorochrome Manufacturer Concentration

Fc receptor block Human Unknown N.A. None Miltenyi Biotec 1:40

Fc receptor block Mouse Rat 2.4G2 None In-house 1:50

7-AAD Mouse/human N.A. N.A. Not applicable Sigma 3 μg/ml

LIVE/DEAD marker Mouse/human N.A. N.A. Near-IR, Violet ThermoFisher 1:1,000

CD3ε Human Mouse UCHT1 APC BD-Pharmingen 1:50

CD3ε Human Mouse UCHT1 BUV395 BD-Pharmingen 1:100

CD3 Human Mouse SK7 APC-Cy7 BioLegend 1:100

γδTCR Human Mouse 11F2 PE-Cy7 BD-Pharmingen 1:50

CD19 Human Mouse SJ25C1 APC-Cy7 BD-Pharmingen 1:100

CD4 Human Mouse RPA-T4 FITC BD-Pharmingen 1:20

CD8α Human Mouse SK1 APC, PE BD-Pharmingen 1:100-200

CD56 Human Mouse HCD56 BV605 BioLegend 1:100

TCR Vδ1 Human Mouse TS8.2 FITC Invitrogen 1:200

TCR Vδ2 Human Mouse B6 BV711 BioLegend 1:150-400

TCR Vγ9 Human Mouse B3 APC BioLegend 1:400

CD14 Human Mouse M5E2 BUV805 BD-Pharmingen 1:400

CD45 Human Mouse HI30 AF700 BioLegend 1:150

CD25 Human Mouse M-A251 PE BD-Pharmingen 1:50

CD69 Human Mouse FN50 PE-Cy7 BD-Pharmingen 1:100

CD69 Human Mouse FN50 PE BD-Pharmingen 1:50

IFN-γ Human Mouse 4S.B3 PerCP-Cy5.5 Biolegend 1:100

Isotype control IgG1, κ N.A. Mouse MOPC-21 Unconjugated, PE BioLegend 10 µg/ml

Isotype control IgG2a, κ N.A. Human–m. IgG2a BM4 Unconjugated, AF647 In house 2 µg/ml

BTN2A1 Human Human–m. IgG2a See Supp. Fig. 4B Unconjugated, AF647 In house 2 µg/ml

BTN3A1/3A2/3A3 Human Mouse 20.1 Unconjugated, PE In house 2 µg/ml

BTN3A1/3A2/3A3 Human Mouse 103.2 Unconjugated, PE In house 0.3 µg/ml

pan-Immunoglobulin Mouse Goat polyclonal BV421, PE BioLegend 1:40

MR1–5-OP-RU tetramer Human BV421 In-house 2 µg/ml

TCR tetramers Human PE In-house 5 µg/ml

BTN2A1 tetramer Human PE In-house 5 µg/ml

mouse CD1d tetramer Mouse PE In-house 5 µg/ml

20

Table S2. Primers used for PCR and site-directed mutagenesis. Purpose Name Sequence (5′ to 3′)

Single-cell PCR round 1 TRDV2_External TGGGCAGGAGTCATGTCAG TRDC_Rev1 GCAGGATCAAACTCTGTTATCTTC TRGV9_External GGCTCTGTGTGTATATGGTGC TRGC_Rev1 CTGACGATACATCTGTGTTCTTTG Single-cell PCR round 2 TRDV2_Fwd_soluble ATACCGGTGCCATTGAGTTGGTGCCT TRDC_Rev_soluble TGTTCCGGATATCCTTGGGGTAGAATTCCTTCA TRDV9_Fwd_soluble ATACCGGTGCAGGTCACCTAGAGCAAC TRDC_Rev_soluble CAGCAATTGAAGGAAGAAAAATAGTGGGCTTG Site-directed mutagenesis E28Aδ_Fwd ATGAAGGGCGCAGCCATCGGC E28Aδ_Rev GCTACACCGCAGTGTGGC R51Aδ_Fwd CTTCATCTACGCAGAGAAGGACATCTACGG R51Aδ_Rev GTCATGGTGTTGCCCTGG L97Aδ_Fwd CTGTGACACAGCTGGAATGGGCGGCGAG L97Aδ_Rev GCGCAGTAGTAGCTGCCC E5Aγ_Fwd GGACATCTGGCACAGCCCCAG E5Aγ_Rev AGCGCCATACACACACAG R20Aγ_Fwd CAAGACCGCCGCACTGGAATGC R20Aγ_Rev CTCAGTGTCTTGGTGCTG E22Aγ_Fwd GCCAGACTGGCATGCGTGGTG E22Aγ_Rev GGTCTTGCTCAGTGTCTTGG T29Aγ_Fwd GTCCGGCATCGCAATCAGCGC T29Aγ_Rev ACCACGCATTCCAGTCTGG Y54Aγ_Fwd GTCCATCAGCGCCGATGGCACC Y54Aγ_Rev ACCAGGAACTGGATCACTTC T57Aγ_Fwd CTACGATGGCGCCGTGCGGAA T57Aγ_Rev CTGATGGACACCAGGAACTGG K60Aγ_Fwd CACCGTGCGGGCAGAGAGCGGC K60Aγ_Rev CCATCGTAGCTGATGGACAC S62Aγ_Fwd GCGGAAAGAGGCCGGCATCCCTTC S62Aγ_Rev ACGGTGCCATCGTAGCTG S66Aγ_Fwd CGGCATCCCTGCTGGCAAGTT S66Aγ_Rev CTCTCTTTCCGCACGGTG E70Aγ_Fwd GGCAAGTTCGCGGTGGACAGAATC E70Aγ_Rev AGAAGGGATGCCGCTCTC E76Aγ_Fwd AGAATCCCCGCGACAAGCACC E76Aγ_Rev GTCCACCTCGAACTTGCC H85Aγ_Fwd ACTGACCATCGCCAACGTGGAAAAGCAG H85Aγ_Rev GTGCTGGTGCTTGTCTCG N86Aγ_Fwd GACCATCCACGCCGTGGAAAAGCAG N86Aγ_Rev AGTGTGCTGGTGCTTGTC E88Aγ_Fwd CACAACGTGGCAAAGCAGGATATC E88Aγ_Rev GATGGTCAGTGTGCTGGT Q90Aγ_Fwd CGTGGAAAAGGCGGATATCGCC Q90Aγ_Rev TTGTGGATGGTCAGTGTG K108Aγ_Fwd AGAGCTGGGCGCGAAAATCAAGGTGTTCG K108Aγ_Rev TGTTGGGCTTCCCACAGG

21

Dataset S1: Raw count files in csv file format, and analysis script in R script format, for the whole genome knockout screen depicted in Fig. 1B Provided as separate zip file.