Science - USA (2022-03-04)

Winkleret al.,Science 375 , eabi7377 (2022) 4 March 2022 7 of 12

CD79BIGLC2CD3DCCR7IKZF2IL2RACD8ACD8BGZMBGNLYMKI67TOP2AJCHAINCD1CEREGKYNU

LYZ

S100A8GPNMBCTSBLYVE1MRC1

SPP1TREM2CCL3IL1B

P2RY12CX3CR1

JUNB

HSPA1A

AIF1

pvM*

cDC

pvM 2

ExV

Mo1

MG

Mo2

Div

CD8 TC1

CD4 TC

pDC

Mo3

CD8 TC2

BC

pvM 1

NK

Treg

F

i.

PodocalyxinSMAIBA1P2RY12DAPI

IBA1+P2RY12 IBA1+P2RY12+

IBA1+P2RY12 IBA1+P2RY12+

256

32

4

(^050) Distance ( (^100) m) 150
log
(counts) 2
(^050) Distance ( (^100150) m) 200 250
2
8
32
128
log
(counts) 2
80
40
0
ns
Distance (
m)
0.4
0.2
0
-6 -3 036
IBA1+P2RY12 IBA1+P2RY12+
log 2 (cell ratio per image)
UMAP
UMAP
Immune
pvM
cDC
pvM 2
ExV
Mo1
MG
Mo2
Div
CD8 TC1
CD4 TC
pDC
Mo3
CD8 TC2
BC
pvM 1
NK
Treg
UMAP
UMAP
AVM
Condition
Control
A
% Exp.
250
50
75
Avg. Exp.
2.5
0
pvM
(11.4%)
cDC
(5.3%)
pvM 2
(4.3%)
ExV
(6.0%)
Mo1
(5.9%)(1.6%)MG
Mo2
(6.2%)
Div
(0.7%)
CD8 TC1
(14.4%)
CD4 TC
(19.2%)
pDC
(0.9%)
Mo3
(4.7%)
CD8 TC2
(2.3%)
BC
(1.3%)
pvM 1
(12.6%)
NK
(2.1%)
Treg
(1.0%)
AVM
(% Compositon)
Total cells = 41,207
pvM 2
(2.2%)
CD4 TC
(16.3%)
Treg
(0.7%) pvM
(15.9%)
BC
(5.4%)
cDC
(4.5%)
CD8 TC1
(12.8%)
CD8 TC2
(3.9%)
Div
(0.4%)
ExV
MG(7.0%)
(2.1%)
Mo1
(3.3%)Mo2 (4.3%)
Mo3
(2.6%)
NK
(5.0%)
pDC
(0.4%)
pvM 1
(13.1%)
Control
(% Compositon)
Total cells = 14,408
i. ii.
Cell number per mm
315000
10000
0
5000


Arteriovenous Malformation Nidus
pvM
cDC
pvM 2
ExV
Mo1
MG
Mo2
Div
CD8 TC1
CD4 TC
pDC
Mo3
CD8 TC2
BC
pvM 1
NK
Treg
AVM Control
10
0
5
Distance (
m) **
Immune cell proportion (%)
0 0.5 1.0
Density
B
C E
G
i.
ii.
Condition
AVM Control
Condition
D
0 1020304050
Myeloid
Lymphoid

• ns
  % of cells
  AVM Control
  Condition
  Fig. 4. Cerebrovascular inflammation with malformation.(A) UMAP visual-
  izations of coembedded immune cells states in control cerebrovasculature and brain
  AVMs (n= 5 donors per condition). (Top) Colored by condition. Control, gray;
  AVM, red. (Bottom) Colored by cell state. pvMfperivascular macrophage; pvMf,
  activated perivascular macrophage; Mo, monocyte; MG, microglia; ExV, ex vivo
  activated myeloid cells; cDC, conventional dendritic cells; pDC, plasmacytoid
  dendritic cells; CD8 TC, CD8+T cells; CD4 TC, CD4+T cells; Treg, regulatory T cells;
  NK, natural killer cells; BC, B cells; and Div, dividing immune cells. (B) Dot plot
  showing expression of immune cell markers. (C) Pie charts showing immune cell
  state proportions in (left) controls and (right) AVMs. (D) Bar graph with individual
  data points showing the proportion of myeloid and lymphoid immune cells captured
  in AVMs (red) and controls (gray).n= 5 donors per condition, mean ± SEM,
  two-tailedttest. P< 0.05; ns, not significant. (E) Bar graph of relative immune cell
  state proportions normalized to total cells sequenced in AVMs (red) and controls
  (gray). (F) Representative confocal microscopy analysis of endothelial cells
  [cyan, podocalyxin (PODOXL)], smooth muscle cells [blue,asmooth muscle actin
  (SMA)], macrophages [magenta, ionized calcium binding adaptor molecule 1
  (IBA1) encoded by the geneAIF1in (B)], and microglia [green, purinergic receptor
  P2Y12 (P2Y12)]. Scale bar, 1 mm. (i) Layered ameboid perivascular macrophages.
  (ii) Perivascular microglial response. Scale bars, insets, 50mm. (G) Quantification of
  (top left) abundance, (top right) cell ratio per image, and (bottom) perivascular
  distance of IBA1+P2R12−macrophages and IBA1+P2R12−microglia (n= 3 donors per
  condition; three nonadjacent sections per donor; 8 to 10 images per section).
  Mean ± SEM, two-tailedttest. **P< 0.01; ns, not significant.
  RESEARCH | RESEARCH ARTICLE