BTN2A1 or BTN3A1 B30.2 domains were buf-
fer exchanged into PBS and adjusted to a final
concentration of 100mM. HMBPP (Cayman
Chemical) and IPP were adjusted to final con-
centrations of 1.9 and 2 mM, respectively, and
serially injected into the cell in 2-mlincre-
ments, after an initial 0.4-ml injection that was
discarded from the analysis. Data were ana-
lyzed with Microcal Origin software.
Confocal microscopy
LM-MEL-75 WT,BTN2A1null,BTN3A1nullcells
were cultured overnight in RPMI-1640 (Thermo
Fisher) supplemented with 10% (v/v) FCS (JRH
Biosciences), penicillin (100 U/ml), streptomycin
(100mg/ml), Glutamax (2 mM), sodium pyruvate
(1 mM), nonessential amino acids (0.1 mM), and
HEPES buffer (15 mM), pH 7.2 to 7.5 (all from
Invitrogen Life Technologies), plus 50mM
2-mercaptoethanol (Sigma-Aldrich) and al-
lowed to adhere to chamber well slides (Lab-
Tek, Thermo Fisher). The next day, cells were
washed and incubated with human Fc recep-
tor block (Miltenyi Biotec) diluted with Opti-
MEM (Thermo Fisher) on ice for 20 min. Cells
were washed and stained with anti-BTN2A1-
AF647 (clone 259), anti-BTN3A-PE (clone 103.2),
and anti-pan-HLA class I-AF488 (clone W6/32,
BioLegend) diluted in Opti-MEM on ice for
20 min. Cells were fixed with 1% paraform-
aldehyde (Electron Microscopy Sciences) in
PBS for 20 min, then mounted with ProLong
Gold AntiFade (Thermo Fisher) and covered
with a #1 coverslip (Menzel-Gläser) overnight.
Each reagent was titrated to determine the
optimal dilution factor. Z-stack, single-tile im-
ages with 76.9 nm lateral and 400 nm axial
voxel size and 1024 × 1024 voxel density were
acquired on a LSM780 laser scanning confocal
microscope with an inverted 20× (0.8 numer-
ical aperture) objective, PMT detectors, and
Zen software (Zeiss). Fluorochromes were ex-
cited with 488-, 561-, and 633-nm laser lines.
Images were deconvoluted with Huygens Pro-
fessional (Scientific Volume Imaging) and
analyzed with Imaris (Oxford Instruments)
software. Regions of interest defining the
imaged cells were made on the basis of the
brightfieldchannel,andtheImarisColoc
module was used to calculate Pearson corre-
lation coefficients of voxels with intensity
thresholds set for each analyzed channel on
the basis of negative controls for each stain.
Immunoblotting
Cells were washed in PBS and lysed in Pierce
RIPA buffer (Thermo fisher) in the presence of
complete protease inhibitor cocktail (Roche).
Proteinquantificationincelllysateswasper-
formed with Pierce BCA protein assay kit
(Thermo fisher). Samples were run on NuPAGE 4
to 12% Bis-Tris protein gels (Invitrogen Life
Technologies), and proteins were resolved
by immunoblotting with the iBlot system
(Invitrogen Life Technologies). Primary anti-
bodies anti-BTN2A1 (0.2mg/ml, Sigma Prestige)
and GAPDH (0.04mg/ml, Cell Signaling Tech-
nology) were detected with IRDye 680RD goat
anti-rabbit IgG secondary antibody (0.1mg/ml,
Licor). Polyvinylidenedifluoride (PVDF) mem-
brane was scanned with an Odyssey scanner.Statistical analyses
For comparison of two independent groups, a
nonparametric Mann–WhitneyUtest was used.
For the comparison of more than two inde-
pendent groups, a Kruskal–Wallis test with a
Dunn’s post-test was used. For comparison
of two paired groups, a Wilcoxon test was used.
For comparison of more than two paired groups
that were normally distributed (determined
by a Kolmogorov–Smirnov test), a repeated-
measures ANOVA and Dunnett’smultiple
comparison test were performed with individ-
ual variances computedfor each comparison;
otherwise, a Friedman test with a Dunn’spost-
test was used. Allpvalues (or FDR values for
Fig. 1B) less than 0.05 were considered statis-
tically significant.REFERENCES AND NOTES- J. Rossjohnet al., T cell antigen receptor recognition of
antigen-presenting molecules.Annu. Rev. Immunol. 33 ,
169 – 200 (2015). doi:10.1146/annurev-immunol-032414-
112334 ; pmid: 25493333 - P. Constantet al., Stimulation of human gamma delta T cells
by nonpeptidic mycobacterial ligands.Science 264 , 267– 270
(1994). doi:10.1126/science.8146660; pmid: 8146660 - Y. Tanakaet al., Natural and synthetic non-peptide antigens
recognized by human gamma delta T cells.Nature 375 ,
155 – 158 (1995). doi:10.1038/375155a0; pmid: 7753173 - L. Zhao, W. C. Chang, Y. Xiao, H. W. Liu, P. Liu, Methylerythritol
phosphate pathway of isoprenoid biosynthesis.Annu. Rev.
Biochem. 82 , 497–530 (2013). doi:10.1146/annurev-biochem-
052010-100934; pmid: 23746261 - A. Sandstromet al., The intracellular B30.2 domain of
butyrophilin 3A1 binds phosphoantigens to mediate activation
of human Vg9Vd2 T cells.Immunity 40 , 490–500 (2014).
doi:10.1016/j.immuni.2014.03.003; pmid: 24703779 - Y. L. Wuet al.,gdT cells and their potential for
immunotherapy.Int. J. Biol. Sci. 10 , 119–135 (2014).
doi:10.7150/ijbs.7823; pmid: 24520210 - J. Zheng, Y. Liu, Y. L. Lau, W. Tu,gd-T cells: An unpolished
sword in human anti-infection immunity.Cell. Mol. Immunol.
10 ,50–57 (2013). doi:10.1038/cmi.2012.43; pmid: 23064104 - L. Wang, A. Kamath, H. Das, L. Li, J. F. Bukowski, Antibacterial
effect of human V gamma 2V delta 2 T cells in vivo.J. Clin.
Invest. 108 , 1349– 1357 (2001). doi:10.1172/JCI200113584;
pmid: 11696580 - D. I. Godfrey, J. Le Nours, D. M. Andrews, A. P. Uldrich,
J. Rossjohn, Unconventional T Cell Targets for Cancer
Immunotherapy.Immunity 48 , 453–473 (2018). doi:10.1016/
j.immuni.2018.03.009; pmid: 29562195 - H. Wang, Z. Fang, C. T. Morita, Vgamma2Vdelta2 T Cell
Receptor recognition of prenyl pyrophosphates is dependent
on all CDRs.J. Immunol. 184 , 6209–6222 (2010).
doi:10.4049/jimmunol.1000231; pmid: 20483784 - C. T. Moritaet al., Direct presentation of nonpeptide prenyl
pyrophosphate antigens to human gamma delta T cells.
Immunity 3 , 495–507 (1995). doi:10.1016/1074-7613(95)
90178-7; pmid: 7584140 - C. Harlyet al., Key implication of CD277/butyrophilin-3
(BTN3A) in cellular stress sensing by a major humangdT-cell
subset.Blood 120 , 2269–2279 (2012). doi:10.1182/blood-
2012-05-430470; pmid: 22767497 - D. A. Rhodeset al., Activation of humangdT cells by cytosolic
interactions of BTN3A1 with soluble phosphoantigens and the
cytoskeletal adaptor periplakin.J. Immunol. 194 , 2390– 2398
(2015). doi:10.4049/jimmunol.1401064; pmid: 25637025
14. Z. Sebestyenet al., RhoB Mediates Phosphoantigen
Recognition by Vg9Vd2 T Cell Receptor.Cell Reports 15 ,
1973 – 1985 (2016). doi:10.1016/j.celrep.2016.04.081;
pmid: 27210746
15. S. Guet al., Phosphoantigen-induced conformational
change of butyrophilin 3A1 (BTN3A1) and its implication on
Vg9Vd2 T cell activation.Proc.Natl.Acad.Sci.U.S.A. 114 ,
E7311–E7320 (2017). doi:10.1073/pnas.1707547114;
pmid: 28807997
16. M. Salimet al., BTN3A1 DiscriminatesgdT Cell
Phosphoantigens from Nonantigenic Small Molecules via a
Conformational Sensor in Its B30.2 Domain.ACS Chem. Biol.
12 , 2631–2643 (2017). doi:10.1021/acschembio.7b00694;
pmid: 28862425
17. Y. Yanget al., A Structural Change in Butyrophilin upon
Phosphoantigen Binding Underlies Phosphoantigen-
Mediated Vg9Vd2 T Cell Activation.Immunity 50 ,
1043 – 1053.e5 (2019). doi:10.1016/j.immuni.2019.02.016;
pmid: 30902636
18. L. Staricket al., Butyrophilin 3A (BTN3A, CD277)-specific
antibody 20.1 differentially activates Vg9Vd2 TCR clonotypes and
interferes with phosphoantigen activation.Eur. J. Immunol. 47 ,
982 – 992 (2017). doi:10.1002/eji.201646818;pmid: 28386905
19. F. Riañoet al., Vg9Vd2 TCR-activation by phosphorylated
antigens requires butyrophilin 3 A1 (BTN3A1) and additional
genes on human chromosome 6.Eur. J. Immunol. 44 ,
2571 – 2576 (2014). doi:10.1002/eji.201444712;
pmid: 24890657
20. A. Behrenet al., The Ludwig institute for cancer research
Melbourne melanoma cell line panel.Pigment Cell Melanoma Res.
26 ,597–600 (2013). doi:10.1111/pcmr.12097;pmid: 23527996
21. G. Malchereket al., The B7 homolog butyrophilin BTN2A1 is a
novel ligand for DC-SIGN.J. Immunol. 179 , 3804–3811 (2007).
doi: 10 .4049/jimmunol.179.6.3804; pmid: 17785817
22. P. Batardet al., Use of phycoerythrin and allophycocyanin for
fluorescence resonance energy transfer analyzed by flow
cytometry: Advantages and limitations.Cytometry 48 ,97– 105
(2002). doi:10.1002/cyto.10106; pmid: 12116371
23. M. E. Birnbaumet al., Deconstructing the peptide-MHC
specificity of T cell recognition.Cell 157 , 1073–1087 (2014).
doi:10.1016/j.cell.2014.03.047; pmid: 24855945
24. A. J. Roelofset al., Peripheral blood monocytes are responsible
for gammadelta T cell activation induced by zoledronic acid
through accumulation of IPP/DMAPP.Br. J. Haematol. 144 ,
245 – 250 (2009). doi:10.1111/j.1365-2141.2008.07435.x;
pmid: 19016713
25. T. J. Allison, C. C. Winter, J. J. Fournié, M. Bonneville,
D. N. Garboczi, Structure of a human gammadelta T-cell
antigen receptor.Nature 411 , 820–824 (2001). doi:10.1038/
35081115 ; pmid: 11459064
26. A. P. Uldrichet al., CD1d-lipid antigen recognition by thegd
TCR.Nat. Immunol. 14 , 1137–1145 (2013). doi:10.1038/ni.2713;
pmid: 24076636
27. P. Vantouroutet al., Heteromeric interactions regulate
butyrophilin (BTN) and BTN-like molecules governinggdT cell
biology.Proc. Natl. Acad. Sci. U.S.A. 115 , 1039–1044 (2018).
doi:10.1073/pnas.1701237115; pmid: 29339503
28. S. Vavassoriet al., Butyrophilin 3A1 binds phosphorylated
antigens and stimulates humangdT cells.Nat. Immunol. 14 ,
908 – 916 (2013). doi: 10 .1038/ni.2665; pmid: 23872678
29. R. Di Marco Barroset al., Epithelia Use Butyrophilin-like
Molecules to Shape Organ-SpecificgdT Cell Compartments.
Cell 167 , 203–218.e17 (2016). doi:10.1016/j.cell.2016.08.030;
pmid: 27641500
30. D. Melandriet al., ThegdTCR combines innate immunity with
adaptive immunity by utilizing spatially distinct regions for
agonist selection and antigen responsiveness.Nat. Immunol.
19 , 1352–1365 (2018). doi:10.1038/s41590-018-0253-5;
pmid: 30420626
31. C. R. Willcoxet al., Butyrophilin-like 3 directly binds a human
Vg 4 +T cell receptor using a modality distinct from clonally-
restricted antigen.Immunity 51 , 813–825.e4 (2019).
doi:10.1016/j.immuni.2019.09.006; pmid: 31628053
32. B. Castellaet al., The ATP-binding cassette transporter A1
regulates phosphoantigen release and Vg9Vd2 T cell activation
by dendritic cells.Nat. Commun. 8 , 15663 (2017).
doi:10.1038/ncomms15663; pmid: 28580927
33. J. Holstet al., Generation of T-cell receptor retrogenic mice.
Nat. Protoc. 1 , 406–417 (2006). doi:10.1038/nprot.2006.61;
pmid: 17406263
34. J. Jounget al., Genome-scale CRISPR-Cas9 knockout and
transcriptional activation screening.Nat. Protoc. 12 , 828– 863
(2017). doi:10.1038/nprot.2017.016; pmid: 28333914
Rigauet al.,Science 367 , eaay5516 (2020) 7 February 2020 12 of 13
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