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

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Supplemental Figure 1: Wolf et al.

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

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Supplemental Figure 2: Wolf et al.

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Supplemental Figure 3: Wolf et al. Day 21 Day 30

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Supplemental Figure 4: Wolf et al.

GVHD PTCy TrED

Lung

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Naïve cells (CD62hiCD44lo)Memory cells (CD62loCD44hi)Remaining cells

Supplemental Figure 5: Wolf et al.

***

****

*

**

ns

**

Stats shown for naïve cells only

GVHDPTCy

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PTCyTrE

DPTCy

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

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Supplemental Figure 6: Wolf et al.

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Supplemental Figure 7: Wolf et al.

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