Science_-_7_February_2020_UserUpload.Net

Rigauet al.,Science 367 , eaay5516 (2020) 7 February 2020 3of13

A

C

BTN2A1 tetramer SAv. control

BTN2A1 tetramer

2

0.5

2

0.1

0.1

0.1

0 3 1

0 2 0

0 3 0

γδ T cells αβT cells B cells Monocytes Other

CD3ε BTN2A1 tetramer

SAv. control

PBMC

D

Donor 1

Donor 2

Donor 3

5.0

3.4 94 6

100

100

0.3 97

0.7 86 100

87 13

PBMC Vδ 1 + Vγ 9 – Vδ 1 + Vγ 9 + Vδ 2 + Vγ 9 +

γδ

TCR

CD3ε

V

δ1 TCR

Vδ2 TCR Vγ9 TCR BTN2A1 tetramer

(^761)
(^461)
69 1
B
Donor 4
Donor 5
Vδ 1 +Vγ 9 -
Vδ 2 +Vγ 9 +
Vδ 1 +Vγ 9 +
BTN2A1 tetramer

• (%)
  Vγ9Vδ 2 +(TCR #6) Vγ9Vδ 1 +(hybrid TCR) Vγ5Vδ 1 +(9C2 TCR)
  (^030609003060900306090)
  0
  50
  100
  Time (s)
  Response (RU)
  50
  100
  200
  3.1
  6.3
  12.5
  25
  Conc. (μM):
  98
  2%
  94% 99%
  25
  0
  50
  75
  100
  Co-stain
  CD3ε
  2A1 tet.
  SAv.
  FRET
  BTN2A1 tet.
  CD3ε
  1,520
  27
  13
  10
  γδ
  T cells B cells
  αβ
  T cells
  100
  75
  50
  25
  BTN2A1 tetramer 0


• 
  

  (%)
  Monocytes
  Other
  69%
  1% 2%
  6%
  1%
  FRET (MFI)
  1,500
  1,000
  500
  0
  2,000
  Vδ 1 + Vδ 2 +
  γδ T cells
  Concentration (μM)
  0 50 100 150 200 250
  0
  20
  40
  60
  80
  Response (RU)
  Bound
  (^0) 20 40 60 80
  1
  2
  Bound / Free
  KD = 40.2 ± 4 μM
  0 50 100 150 200 250
  Expt. 1
  Expt. 2
  0
  20
  40
  60
  80
  0
  1
  2
  20 40 60 80
  Bound
  Bound / Free
  KD = 50.4 ± 5 μM
  00
  Fig. 2. BTN2A1 binds Vg (^9) Vγ9Vδ 2 +(TCR #6) Vγ9Vδ 1 +(hybrid TCR)
  +gdT cell receptors.
  (A) BTN2A1 tetramer-PE (first column) or streptavidin-
  PE control (second column) versus CD3estaining on
  three representative human PBMC samples. Histograms
  depict BTN2A1 tetramer-PE staining (white) or
  streptavidin-PE control (gray) on gatedgdTcell
  (CD3+gdTCR+),abTcell(CD3+gdTCR−), B cell
  (CD3−CD19+), monocyte (CD3−CD19−CD14+)orother
  (CD3−CD19−CD14−) subsets. Box-and-whisker plots
  (right) depict the percentage of each cell lineage that
  binds to BTN2A1 tetramer in blood samples from
  different donors. (B) BTN2A1 tetramer (white histo-
  grams) overlaid with streptavidin-PE alone control (gray
  histograms) staining, on Vg 9 +Vd 2 +(orange), Vg 9 +Vd 1 +(pink), or Vg 9 −Vd 1 +(blue)
  T cells, with parent gating shown to the left. Box-and-whisker plots (right) depict
  the percentage of eachgdT cell subset that binds to BTN2A1 tetramer-PE in
  different donors. (C) FRET fluorescence (histogram overlays) between BTN2A1
  tetramer-PE and CD3e-APC on dual-stained (pink) or single-stained controls
  (orange and dark green, respectively), using purified in vitro–expanded Vd 2 +
  T cells. Box-and-whisker plots depict FRET median fluorescence intensity (MFI) in
  gdT cell subsets from different human donors. (D) Binding of soluble BTN2A1
  (200 to 3.1mM) to immobilized Vg 9 +Vd 2 +(“TCR #6,”left), Vg 9 +Vd 1 +(“hybrid,”
  middle), and Vg 5 +Vd 1 +(“9C2,”right)gdTCRs, as measured by surface plasmon
  resonance. Saturation plots (below) depict binding at equilibrium, and Scatchard
  plots.KD, dissociation constant at equilibrium ± SEM; SAv, streptavidin. Data
  represent (A)n= 8 donors pooled from two independent experiments; (B)n=8
  donors from two experiments; (C)n= 7 donors pooled from three independent
  experiments; (D)n= 2 separate experiments, one of which (Expt. 2) was
  performed in duplicate and averaged.
  RESEARCH | RESEARCH ARTICLE