A.

CD11b CD11c Gr-1 PD-L1 Tbet

Gated on: CD4+
Some markers identified visually by

B6 #1
T cells in FlowJo
viSNE are not significantly different.
viSNE

**
IL10KO #1
B.
** 150

Median Intensity
***
ns

*
100

viSNE plots Altered location of CD4+ T cells, according 50

display only to tSNE1 and tSNE2, suggest B6
CD4+ T cell islands IL10KO 0
changes in phenotype of CD4+ T cells.
CD11b CD11c Gr-1 PD-L1 Tbet

C. B6
D. Focused analysis on CD4+ T cell clusters
CD11c CTLA4
Lag3
PhenoGraph defined six
subtypes of CD4+ T cells. PhenoGraph-defined
PhenoGraph

clusters differ in expression

Naive CD4+ T cells, (cluster #16) across multiple parameters.
% CD4+ T cells =16.3% Regulatory CD4+ T cells, (cluster #7)
CD11b ICOS IRF4
Effector CD4+ T cells A, (cluster #12)
IL10KO Effector CD4+ T cells B, (cluster #8)
Effector CD4+ T cells C, (cluster #28)
Effector CD4+ T cells D, (cluster #18) Certain cellular markers
are exclusively positive in
Effector CD4+ T cells A, (cluster #12)
Average of all events
IL10KO mice show IL10KO dominant clusters
Effector CD4+ T cells B, (cluster #8)
increased CD4+ T cells Effector CD4+ T cells C, (cluster #28) (cluster #8 and #28).
and altered distribution
Cluster #524

Individual Events
B6 biased

of CD4+ T cell subtypes. CD4+ T cell clusters that differ in

% CD4+ T cells =30.2%
abundance between conditions.

E. F. Line graphs of a cluster’s median

expression can be used to identify
cell type and phenotype subset.
Cluster #520

Individual Events

8 Cluster #515 Ki6

7 CD1
1b
MH
-1
Median Expression

Sca CI
S

PD1
6
The inner ring indicates core
ICO
IL10KO biased
X-shift

CD3
IRF4

4 CD4+ T cells
markers expressed on all
Tim3

Lag3
#515
1 L

2
44
PD

CD4+ T cells.
CD

Tb CD4 TR
Average of all events e

H.
t GI
Tbet
Tim3
Lag3
IkBa
CD19
NKp46
Ki67
PDL2
CD64
PDL1
IRF4
Sig F
FoxP3
KLRG1
Bcatenin
CD127
CD11c
GITR
MHCI
CD69
CD8
CD11b
CD25
Gr-1
p4eBP1
CD3
CTLA4
PD1
Sca1
CD45
CD44
CD4
CD117
MHCII
ICOS

CTLA4

100 Parameters

8 Cluster #520 IRF

4
-1 MH
Sca
CD

CI
Cellular expression
Median Expression

6
The outer ring defines
MHCII
Gr-1

across all parameters 4

Dendritic cells

X-shift can define the average #520

accessory phenotypes,
showing data from
CD

2
44
11

cellular expression across all

CD

with characteristics
Ki

CD11b
a
c

IkB
67

ten individual cells

parameters (here the most unique to this cell cluster,
Tbet
Tim3
Lag3
IkBa
CD19
NKp46
Ki67
PDL2
CD64
PDL1
IRF4
Sig F
FoxP3
KLRG1
Bcatenin
CD127
CD11c
GITR
MHCI
CD69
CD8
CD11b
CD25
Gr-1
p4eBP1
CD3
CTLA4
PD1
Sca1
CD45
CD44
CD4
CD117
MHCII
ICOS

(in cluster #520) Parameters

IL10KO biased cluster). not consistently present
highlights conserved,
among all cell subsets.
variable phenotypes.

G. CD11b CD11c Gr-1 PD-L1 Tbet H. Node #1, CD4+ T cells

#26
5 ** ns
B6 #1

#1
ns
**
Median Intensity

Changed color on a node 4 ns

indicates altered expression
SPADE

#26 3
IL10KO #1

#1 between conditions.
2
B6
1
1.0 6.5 0.5 6.8 0.3 4.5 0.8 5.8 0.0 4.3
Median expression values IL10KO
for a single node can be 0
Selected CD4+ T cell nodes assessed for significance. CD11b CD11c Gr-1 PDL1 Tbet

K.
Number of model features #82228 IRF4 #82254 IRF4 #82228, PDL1 #82267, CD11b

I. J.
82233 82253

0 2 2 4 4 5 14 26 45 93 206 382 634

**** **** **** ****
Median Expression
Median Expression

Median Expression
Median Expression

82256

3 3 4 6
100

Cross Validation Error Rate Citrus Settings: 82248

3
82239

Feature False Discovery Rate Events sampled per file: 9,141

82261
2 2 4
cv.min
cv.1se
Minimum cluster size: 5%
Group assignment: Correct IRF4 2
PDL1
82265
cv.fdr.constrained
Cluster characterization: Medians 1 1 2
1
82264
Model Cross Validation Error Rate

82228 82249

cv.min model has 10% CD11b

75

82254 82250

0
82258
0
Citrus

0
82267
0

cross validation error rate

6

O
IL 6
O

IL 6
O

IL 6
O

82268 82222
B
B

B
K

82263
K

82260
10
10

10

10

82259
IL

82266
50

82237

82251
82262
82257
#82228

#82254

#82228

#82267

82252

82247

82255
25

82245
82241
IRF4 IRF4 PDL1 CD11b

82217
82220
Population: Background Cluster

Citrus identified 4 features that predict

0

Figure 9
7.15 6.36 5.56 4.77 3.97 3.18 2.38 1.59 0.79 0.00
Regularization Threshold
differences between experimental groups. Kimball et al