Cell phenotyping using flow cytometry software (FlowJo®)

A. WT #1
B. 4
Parent: All events WT #1 CD4KO #1 CD8KO #1

Entire Cell CD4 (Opal 520) Total

10
CD4+ CD8+ CD4+ CD8+ CD4+ CD8+
10
4 CD4 T cells 1.77% CD4 T cells 0.05% CD4 T cells 0.0%
3 22.6% 0.05% 18.0%
10

3
Nuclear Area (pixels)

10

2
10

2
10 Non T cells Non T cells Non T cells
1
49.2% 69.0% 70.2%
10

1
10

0 CD8 T cells CD8 T cells CD8 T cells

10
13.4% 28.3% 0.0%
0
10
0 1 2 3 4 0 1 2 3 4 0 1 2 3 4
10 10 10 10 10 10 10 10 10 10 10 10 10 10 10

4 Entire Cell CD8 (Opal 690) Total

10

Parent: CD4 T cells Parent: Non T cells Parent: Non T cells, B220-
Nucleus DAPI Total

4
4
WT #1 WT #1 WT #1
Entire Cell CD4 (Opal 520) Total

10 10
3
10
20

Entire Cell Area (pixels)

3
10 3
10
2
10 15

Count
10
2
10
1
10 10

1 T regs B cells
10
3.76% 12.1% Macrophages
0 1
10 10
5.0 8.42%
0 1 2 3 4
10 10 10 10 10
0
10
Entire Cell Area (pixels) 10
0
0

0 1 2 3 4 0 1 2 3 4 0 1 2 3 4
10 10 10 10 10 10 10 10 10 10 10 10 10 10 10

Entire FoxP3 (Opal 650) Total Entire Cell B220 (Opal 570) Total Entire Cell F480 (Opal 540) Total

C. 60 D. 10
4
Input: FlowJo-defined CD4 T cells

CD4 T cells solely

% of DAPI+ Events

identified by FlowJo
Entire Cell CD4 (Opal 520) Total

CD4 T cells 54.7%

3 15
40 Tregs
10

Count
10
CD8 T cells
2
10
B cells 5.0

20 Macrophages 0

1 0 1 2 3 4
10 CD4 T cells identified 10 10 10 10 10
by inForm & FlowJo Entire Cell CD4 (Opal 520) Total
45.3%
CD4 T cells identified by inForm & FlowJo
0 10
0 inForm CD4 Confidence %
CD4 T cells solely identified by FlowJo
0.0 100.0
C O ( 4)
IF KO 4)

O 4)

)
=4
4K =

8 n=
γK (n=

0 20 40 60 80 100
D T (n

(n

inForm CD4 Confidence %

W

D
N
C

CD4+/CD8- events identified

E. Input: All events

by biaxial gating were
classified as multiple
phenotypes by inForm®
F. This cell (gray circle) was
given a high confidence value
as a CD8 T cell. It is in close
4
contact with a CD4 T cell.
Entire Cell CD4 (Opal 520) Total

10 Input: inForm defined CD8 T cells

CD4 T cells 4
10
3
22.6% CD4+ CD8+ CD4
Entire Cell CD4 (Opal 520) Total

10 14.9% 1.2K
CD8
3
10
2
CD4 T cells
10
10.7% 900

2
10
1
10 600

Colored by inForm
Cell Y Coordinate

638 DAPI+ events

0 CD4 Confidence % 10
1
10
CD4 T cells 300
1 99.78 Non T cells
0 1 2 3 4
T regs 4.76% CD8 T cells
10 10 10 10 10 0 52.1%
CD8 T cells 10 0
Entire Cell CD8 (Opal 690) Total 0 1 2 3 4 0 300 600 900 1.2K
10 10 10 10 10
B cells
Entire Cell CD8 (Opal 690) Total Cell X Coordinate
Macrophages
Other
Colored by inForm CD8 Confidence %

34.71 97.11