Supplemental Figure legends following an allogeneic HSCT ... · PDF file Table 1.: Summay of outcomes comparing TrED recipients with PTCy recipients and GVHD controls following an

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

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

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

10

20

30

40

50

60

70

% T

re g

/ C D

4+

d d

0

10

20

30

40

50

60

70

% T

re g

/ C D

4+

No n-

ex pa

nd ed

Ex pa

nd ed No

n-

ex pa

nd ed

Ex pa

nd ed

******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

W ei

gh t i

n %

6 14 22 30 38 46 54 62 70 78 86 94

0

2

4

6

8

10

C lin

ic al

S co

re

0 20 40 60 80 10 0

12 0

0

20

40

60

80

100

Pe rc

en t s

ur vi

va l

GVHD PTCy TrED

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

GV HD

PT Cy

Tr ED

0

20

40

60

80

100

120

Tr eg

GV HD

PT Cy

Tr ED

0

20

40

60

80

100

120

Tr eg

BM donor T cell donor Recipient

*

GV HD

PT Cy

Tr ED

0

5

10

15

20

% T

re g

/ T ot

al C

D 4+

GV HD

PT Cy

Tr ED

0.0

0.2

0.4

0.6

0.8

1.0

Tr eg

/ To

ta l C

D 4+

ra tio

GV HD

PT Cy

Tr ED

0

5

10

15

20

% T

re g

/ T ot

al C

D 4+

GV HD

PT Cy

Tr ED

0.0

0.2

0.4

0.6

0.8

1.0

Tr eg

/ To

ta l C

D 4+

ra tio

ns

ns ***

*** ***

**

ns

ns

A

B

Supplemental Figure 4: Wolf et al.

GVHD PTCy TrED

Lu ng

GV HD

PT Cy

Tr ED

0

1

2

3

4

P at

ho lo

gi ca

l S co

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

D 8

/ L ym

ph oc

yt es

%

C D

4 / L

ym ph

oc yt

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

GV HD

PT Cy

TrE D

PT Cy

TrE D

PT Cy

TrE D

DN 1 (CD44+/CD25-) DN 2 (CD44+/CD25+) DN 3 (CD44-/CD25+) DN 4 (CD44-/CD25-)

0

20

40

60

80

100

120

D N

1 - D

N 4

*

** A B

Supplemental Figure 6: Wolf et al.

PT Cy

TrE D

PT Cy

TrE DD

0

20

40

60

80

100

120

D N

1 - D

N 4

ns

ns

BM

GV HD

PT Cy

Tr ED

0

10

20

30

40

50

% C

D 4

R TE

s

BM

GV HD

PT Cy

Tr ED

0

10

20

30

% C

D 8

R TE

s

BM

PT Cy

Tr ED

0

10

20

30

40

50

% C

D 8

R TE

s

CD4 CD8 CD19A

B

BM

PT Cy

Tr ED

0

20

40

60

80

% C

D 4

R TE

s

**

Supplemental Figure 7: Wolf et al.

BM

GV HD

PT Cy

Tr ED

0

20

40

60

80

% C

D 19

R M

E s

BM

PT Cy

Tr ED

0

20

40

60

80

% C

D 19

R M

Es