Post on 14-Feb-2020
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.