27
Figure 7.: Significantly higher frequencies of recent thymic/marrow emigrants (RTEs/RMEs) in TrED
recipients versus PTCy treated animals and GVHD controls early post-HSCT. A HSCT utilizing a B6
à BALB/c donor/recipient mouse model involving a complete MHC mismatch was performed on day 0.
Lethally irradiated (8.5 Gy) BALB/c mice received 5x106 TCD B6-RAG2p-GFP BM cells and spleen cells
from expanded (TL1A-Ig/IL-2; TrED group) or untreated B6-FoxP3rfp (GVHD and PTCy group) donor mice
adjusted to contain 1.1x106 total T cells. Cyclophosphamide was given on day 3 and 4 post-HSCT at 80
mg/kg ip. RTEs/RMEs were analyzed in PB by flow cytometry three weeks post HSCT (BM: n = 2; GVHD:
n = 5; PTCy and TrED n = 8). (A) Significantly higher frequencies of CD4+ and CD8+ RTEs as well as CD19+
B cells (RMEs) are detected in TrED recipients compared to PTCy treated animals and GVHD controls.
Data are expressed as means ± SEM and were analyzed by a two-tailed unpaired t test. *p<0.05; **p<0.01;
***p<0.001; ****p<0.0001 (B) A representative histogram from the TrED and the PTCy group is shown.
Table 1.: Summay of outcomes comparing TrED recipients with PTCy recipients and GVHD controls
following an allogeneic HSCT.
Supplemental Figure legends
Supplemental Figure 1.: Levels of donor Treg expansion. Donor mice were injected i.p. with TL1A-
Ig (50 µg) on days 1-4; rmIL-2 (1.5 µg) bound to the a-IL-2 mAb (JES6-5H4; 8ug) on days 4 and 6. Treg
expansion levels are shown for B6-FoxP3rfp mice (major model; A) (n = 6) and B10.D2 mice (minor
model; B) (n = 3). Data are expressed as means ± SEM and were analyzed by a two-tailed unpaired t
test. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001.
Supplemental Figure 2.: Comparable outcomes with lower amounts of PTCy. (A-C) A HSCT
utilizing a B6 à BALB/c donor/recipient mouse model involving a complete MHC mismatch was
performed on day 0. Lethally irradiated (8.5 Gy) BALB/c mice received 5x106 TCD B6-CD45.1 BM cells
and spleen cells from expanded (TL1A-Ig/IL-2; TrED group) or untreated B6-FoxP3rfp (GVHD and PTCy
group) donor mice adjusted to contain 1.1x106 total T cells. Cyclophosphamide was given on day 3 and
4 post-HSCT at 50 mg/kg ip. Weights (A), clinical scores (B) and survival (C) are shown (GVHD: n = 5;
TrED and PTCy: n = 10). Survival was analyzed by log-rank test (ns = not significant). TrED cells show
advantages over PTCy treatment early post-transplant even when using lower/clinically more relevant
amounts of Cy (50 mg/kg) and are comparable to 80 mg/kg in a major MHC mismatch model of pre-
clinical HSCT.
28
Supplemental Figure 3.: Treg assessment in blood three and four weeks post-HSCT. (A) Percent
Foxp3+ Tregs out of total CD4+ T cells (upper graphs) as well as Treg / CD4 ratios (lower graphs) are
shown for day 21 (left) and 30 (right). Data are shown as mean ± SEM; ANOVA with Bonferroni correction
was applied for multiple comparisons on day 21. Data are expressed as means ± SEM and were analyzed
by a two-tailed unpaired t test on day 30. *p<0.05; **p<0.01; ***p<0.001. Day 21: n = 6; day 30: GVHD: n
= 1; PTC: n = 3; TrED: n = 4. Data shown are from two experiments for day 21 and from one experiment for
day 30. (B) Treg engraftment over time shows a faster engraftment in TrED compared to PTCy
recipients on day 30. On day 21: GVHD: n = 2; PTCy and TrED n = 3. On day 30: GVHD: n = 1; PTCy:
n = 3: TrED: n = 4. Data are expressed as means ± SEM and were analyzed by a two-tailed unpaired
t test. *p<0.05.
Supplemental Figure 4.: Massons’s trichrome staining from recipient lungs in a Minor HSCT
mouse model on day 200 post-HSCT. Representative staining (chosen from 2 independent
experiments) exhibited multifocal areas of moderate chronic, active inflammation and fibrosis in the PTCy
compared to the TrED group which was within normal limits. Magnification = 200x. The pathology score is
shown on the right (n = 6-8). Data are shown as mean ± SEM; ANOVA with Bonferroni correction was
applied for multiple comparisons. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001. Data shown are from one
experiment. Scale bars: 100 µm.
Supplemental Figure 5.: Naïve/memory compartment over time (1-3 months post-HSCT). A HSCT
utilizing a B6 à BALB/c donor/recipient mouse model involving a complete MHC mismatch was
performed as described in Fig. 1. The naïve/memory compartment was analyzed by flow cytometry
(CD44/CD62L) in PB at 30, 60 and 90 days post-HSCT. The naïve compartments of CD4+ and CD8+
cells are significantly larger in TrED recipients compared to PTCy treated animals at 1 and 2 months
post HSCT. At 3 months post HSCT a significant difference is only seen in CD8+ cells. GVHD: n = 3;
PTCy and TrED: n = 8-10. Data are expressed as means ± SEM and were analyzed by a two-tailed
unpaired t test. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001.
Supplemental Figure 6.: Assessment of the DN CD44/CD25 subsets at 3 weeks and 1 month
post allogeneic HSCT by flow cytometry. A HSCT utilizing a B6 à BALB/c donor/recipient mouse
model involving a complete MHC mismatch was performed as described in Fig. 1. (A) At 3 weeks post-
HSCT no significant difference could be detected in DN3 (CD44-CD25+) and DN4 (CD44-CD25-)
subtypes between the TrED and the PTCy group (n = 3-4). (B) However, at 1 month post HSCT there
29
are significant differences detectable in DN3 (CD44-CD25+) and DN4 (CD44-CD25-) subtypes between
the TrED and the PTCy group (n = 6). Data are expressed as means ± SEM and were analyzed by a
two-tailed unpaired t test. *p<0.05; **p<0.01.
Supplemental Figure 7.: Assessment of recent thymic/marrow emigrants (RTEs/RMEs) at one
and two months post-HSCT. A HSCT utilizing a B6 à BALB/c donor/recipient mouse model involving
a complete MHC mismatch was performed on day 0. Lethally irradiated (8.5 Gy) BALB/c mice received
5x106 TCD B6-RAG2p-GFP BM cells and spleen cells from expanded (TL1A-Ig/IL-2; TrED group) or
untreated B6-FoxP3rfp (GVHD and PTCy group) donor mice adjusted to contain 1.1x106 total T cells.
Cyclophosphamide was given on day 3 and 4 post-HSCT at 80 mg/kg ip. RTEs/RMEs were analyzed
in PB by flow cytometry one (A) and two (B) months post-HSCT (n = 2-3). (A) One month post-HSCT
no significant differences were detectable between the TrED and the PTCy group in CD4+, CD8+ and
CD19+ cells. (B) Two months post-HSCT the only significant difference was evident in the CD8+
compartment between the 2 groups. Data are expressed as means ± SEM and were analyzed by a
two-tailed unpaired t test. *p<0.05; **p<0.01.
0
10
20
30
40
50
60
70
% T
reg
/ CD
4+
d d
0
10
20
30
40
50
60
70
% T
reg
/ CD
4+ Non-
expan
ded
Expan
ded Non-
expan
ded
Expan
ded
******A B
Supplemental Figure 1: Wolf et al.
6 14 22 30 38 46 54 62 70 78 86 94
60
70
80
90
100
110
120
Wei
ght i
n %
6 14 22 30 38 46 54 62 70 78 86 94
0
2
4
6
8
10
Clin
ical
Sco
re
0 20 40 60 80 100
120
0
20
40
60
80
100
Perc
ent s
urvi
val
GVHDPTCyTrED
ns
Supplemental Figure 2: Wolf et al.
A B C
Days post-HSCT Days post-HSCT Days post-HSCT
Supplemental Figure 3: Wolf et al. Day 21 Day 30
GVHD
PTCy
TrED
0
20
40
60
80
100
120
Treg
GVHD
PTCy
TrED
0
20
40
60
80
100
120
Treg
BM donor T cell donorRecipient
*
GVHD
PTCy
TrED
0
5
10
15
20
% T
reg
/ Tot
al C
D4+
GVHD
PTCy
TrED
0.0
0.2
0.4
0.6
0.8
1.0
Treg
/ To
tal C
D4+
ratio
GVHD
PTCy
TrED
0
5
10
15
20
% T
reg
/ Tot
al C
D4+
GVHD
PTCy
TrED
0.0
0.2
0.4
0.6
0.8
1.0
Treg
/ To
tal C
D4+
ratio
ns
ns***
******
**
ns
ns
A
B
Supplemental Figure 4: Wolf et al.
GVHD PTCy TrED
Lung
GVHD
PTCy
TrED
0
1
2
3
4
Pat
holo
gica
l Sco
re **
****
C
0
10
20
30
40
50
0
10
20
30
40
50
0
10
20
30
40
50
0
10
20
30
40
50
0
5
10
15
0
5
10
15
% C
D8
/ Lym
phoc
ytes
%
CD
4 / L
ymph
ocyt
es
Day 30 Day 60 Day 90
Naïve cells (CD62hiCD44lo)Memory cells (CD62loCD44hi)Remaining cells
Supplemental Figure 5: Wolf et al.
***
****
*
**
ns
**
Stats shown for naïve cells only
GVHDPTCy
TrED
PTCyTrE
DPTCy
TrED
DN 1 (CD44+/CD25-)DN 2 (CD44+/CD25+)DN 3 (CD44-/CD25+)DN 4 (CD44-/CD25-)
0
20
40
60
80
100
120
DN
1 - D
N4
*
**A B
Supplemental Figure 6: Wolf et al.
PTCyTrE
DPTCy
TrEDD
0
20
40
60
80
100
120
DN
1 - D
N4
ns
ns
BM
GVHD
PTCy
TrED
0
10
20
30
40
50
% C
D4
RTE
s
BM
GVHD
PTCy
TrED
0
10
20
30
% C
D8
RTE
s
BM
PTCy
TrED
0
10
20
30
40
50
% C
D8
RTE
s
CD4 CD8 CD19A
B
BM
PTCy
TrED
0
20
40
60
80
% C
D4
RTE
s
**
Supplemental Figure 7: Wolf et al.
BM
GVHD
PTCy
TrED
0
20
40
60
80
% C
D19
RM
Es
BM
PTCy
TrED
0
20
40
60
80
% C
D19
RM
Es