A.

B6 #1 IL10KO #1
B.
40
15.70% 41.80% ***
Colored by:
30
viSNE

CD4+ expression
There are significantly

% CD4+
20 more CD4+ T cells in
IL10KO mice vs. B6 mice.

10

The CD4+ T cell island can be manually 0

gated and % of total events determined. B6 IL10

C. D.
30 B6 Non-significant clusters show
B6 (#1-5) IL10KO (#1-4) high inter-individual variation.
Colored by IL10KO
PhenoGraph

20 cluster ID

% of events
20
0 **
*
-20
tSNE2

-30 0 30-30 0 30
10
tSNE1 Only 2 of 29 PhenoGraph-
defined clusters are
By visual inspection, cluster #4, #5, #8,
identified as significantly
#17, and #25 appear to be different in
different between groups.
cellular abundance between B6 and IL10KO. 0

#4

#5

#8

5
#1

#2
E. B6 biased IL10KO biased
The proportion of events for each F. 100
#504
100
#524
100
#510
cluster from each experimental

% of events in cluster
% of events in cluster

% of events in cluster
100

B6 Biased
80 80 80
B6 condition can be determined.
60 60 60
IL10KO
40 40 40
The biasedness of some clusters
% of events in cluster

20 20 20
X-shift

seem to be driven by individuals. 0 0 0

B6 IL10 B6 IL10 B6 IL10

50
The % of events in each cluster for all #520 #495 #515

IL10KO Biased
individuals can be calculated and 100 100 100

% of events in cluster
% of events in cluster

% of events in cluster
statistical signficance determined.
80 80 80

60 60 60

40 40 40
Of the top 6 clusters most enriched in ** ***
20 20 20
0 either B6 or IL10KO mice, only 2
524
504
510
526
533
503
505
523
511
501
528
502
499
506
509
525
507
498
500
517
527
512
521
497
508
534
496
522
529
532
513
519
531
492
518
514
Input
530
490
493
516
491
494
515
495
520

0 0 0

Cluster ID were statistically significant. B6 IL10 B6 IL10 B6 IL10

G. B6 #1 IL10KO #1 H. * I. B6 #1 IL10KO #1
20 #18 #30 #18 #30
#37
#26 #26 B6 #37
#1 IL10KO #26 #26
#1
ns ns #2 #1 #2 #1
15
% of events

*
SPADE

**
10 *
#9 #12 #9 #12 ***
#31 #31
5
*
** Statistical analysis reveals more nodes
By visual inspection, nodes #1, #9, #12,
0 that are significantly different (#2, #18,
and #26 appear to be different in cellular
#30, #31, and #37) and some that do
1
2

7
8

0
6
#2
#1

#9

abundance between B6 and IL10KO.

#3
#1

#3
#3
#1

#2

Node ID not reach significance (#9, #12).

J. 82268 K. B6 Biased Clusters IL10KO Biased Clusters

#82263 #82265 #82267 #82243 #82252 #82256 #82266
IL10KO biased B6 Biased
82266 82267
1.0 1.0 *** 1.0 ** 1.0 1.0 1.0 1.0
**** **
Abundance
Abundance

Abundance

Abundance

Abundance

Abundance

Abundance

82260 82256 82264 82265 *** ****

0.5 0.5 0.5 0.5 0.5 0.5 0.5
****
82230 82257 82252 82250 82259 82263 82251
0.0 0.0 0.0 0.0 0.0 0.0 0.0
6

IL 6
O
6

O
B

B
B

B
K

K
K

82239 82254 82243 82236 82249 82255 82262 82261 82241

10

10
10

10

10

10

10
IL

IL

IL

IL

IL

IL
Citrus

82234 82232 82228 82247 82258

Citrus is the only algorithm which automatically identifies statistically significant differences.
82223 82229 82253 82238
Abundance values can be extracted from Citrus files to calculate an actual p value.
Cellular phenotype for each cluster can be defined by histogram overlays.
L. Tim3
CD4 CTLA4 GITR ICOS IRF4 Ki67 Lag3 PD1 Tbet
#82243

Background Expression Cluster Expression Figure 8

Kimball et al