mIHC specific analysis using phenoptrReports (R)

A. WT ROI #1-4
B. WT ROI #1
110
40 40 40 40
100
Mean Distance (Microns)

90

Distance (Microns)
30 30 30 30 80
70
20 20 20 20 60
50
10
40
10 10 10
30
20
0 0 0 0
10
C +
B +
F4 +
An 0+

C +
B +
F4 +
An 0+

C +
B +
F4 +
An 0+

C +
B +
F4 +
An 0+
ell

ell

ell

ell
0
4
8
0

4
8
0

4
8
0

4
8
0
yc

yc

yc

yc
D
D
22

/8

D
D
22

/8

D
D
22

/8

D
D
22

/8
C

ell
4+

8+

F4 +

An +
0

0
yc
D

D
22

/8
CD4 T cell distance CD8 T cell distance B cell distance Macrophage distance

C
B
to nearest: to nearest: to nearest: to nearest:
CD4 T cell distance
to nearest:
C. D. WT Infected Lung, ROI #1
CD4:Cell X interaction distance, WT ROI #1
Post spectral unmixing
10
(Sorted, based on distance
(Sorted, based on distance

20
Distance (microns)
to nearest CD4 T cell)

to nearest CD8 T cell)

30
Individual CD4 T cells (1 cell per row, n=299)

40
50
60
70
80
90
100
110
CD4+

CD8+

B220+

F4/80+
CD4+

CD8+

B220+

F4/80+

Phenotype map
Cell #674
Cell #1639
(Sorted, based on distance

CD4 T cell
(Sorted, based on distance
to nearest macrophage)

closest to a
to nearest B cell)

macrophage

Nearest
F4/80+ event
(105.9 μm)
Cell #1639
CD4 T cell
furthest from Nearest
a macrophage Cell #674 F4/80+ event
B220+
CD4+

CD8+

F4/80+

CD4+

CD8+

B220+

F4/80+

(4.7 μm)

E. CD4 T cell distance to nearest:

60
% of events

40

20

0
4+

8+

0+

0+

4+

8+

0+

0+
D

22

/8

22

/8
C

F4

F4
B

≤10 μm ≥30-110 μm