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: [email protected] (A.P.U.); [email protected](A.B.); [email protected] (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
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Supplementary Materials for...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
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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*†
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.
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
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
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.
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.
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;
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).
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.
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).
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
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.