Supplementary Figure 1Histological staging criteria for acute GvHD

 Hepatic GVHD1. Minimal periductal lymphoid cell infiltrates (5-10 cells)2. Mild periductal lymphoid cell infiltrates (10-20 cells)3. Moderate periductal lymphoid cell infiltrates (20-40 cells), with intraepithelial lymphoid cells, single cell hepatocellular necrosis4. Moderate to severe periductal lymphoid cell infiltrates (>40 cells), with intraepithelial lymphoid cells; single cell hepatocellular necrosis; some bridging of portal zones with lymphoid infiltrates Gastric GVHD1. Squamous portion - small numbers of lymphocytes adjacent to basal epithelium2. Squamous portion - small number of lymphocytes adjacent to basal epithelium and small numbers of intraepithelial lymphocytes; glandular portion -intraepithelial lymphocytes with degeneration of Parietal cells3. Squamous portion - small to moderate number of lymphocytes adjacent to basal epithelium and small to moderate numbers of intraepithelial lymphocytes; glandular portion - intraepithelial lymphocytes with prominent degeneration of Parietal cells4. Squamous portion - small to moderate number of lymphocytes adjacent to basal epithelium and small to moderate numbers of intraepithelial lymphocytes; glandular portion - intraepithelial lymphocytes with prominent degeneration of Parietal cells; mucosal erosion/ulceration Intestinal GVHD1. Minimal to small numbers of infiltrating lymphocytes into crypt epithelium; rare degenerative epithelial cells2. Small numbers of infiltrating lymphocytes into crypt epithelium; increased numbers of mitoses; rare to small numbers of degenerative epithelial cells in some crypts3. Larger numbers of infiltrating lymphocytes into crypt epithelium; increased numbers of mitoses; small numbers of degenerative epithelial cells in most crypts; +/- mucosal ulceration4. Larger numbers of infiltrating lymphocytes into crypt epithelium; increased numbers of mitoses; most crypts have evidence of degeneration of crypt epithelial cells; +/- mucosal ulceration Cutaneous GVHD1. multifocal mild epidermal hyperplasia and hypekeratosis, minimal numbers of intraepithelial lymphocytes 2. multifocal mild epidermal hyperplasia and hypekeratosis, small numbers of intraepithelial lymphocytes, and rare degenerated epithelial cells 3. multifocal mild to moderate epidermal hyperplasia and hypekeratosis, small to moderate numbers of intraepithelial lymphocytes, and small to moderate numbers of degenerated epithelial cells 4. multifocal moderate epidermal hyperplasia and hypekeratosis, moderate numbers of intraepithelial lymphocytes, and moderate numbers of degenerated epithelial cells, microabscesses, skin erosion and/or ulceration

Supplementary Figure 2: Ruxolitinib does not impair post-transplant donor myeloid reconstitution and full donor chimerism.

BM Only

GVHD + vehicle

GVHD+Ruxolitinib 90 mg/Kg

GVHD+Ruxolitinib 45 mg/Kg

a b

0

0,5

1,0

1,5

2,0

Don

or C

D3+

cel

ls (

*10^

3)/u

l blo

od

p=0.0003

p=0.01

p=NS

p=NS

p=NS

Day +14 post- BMT Day +30 post- BMT

c d

0

1

2

3

4

Don

or G

r-1+

cel

ls (

*10^

3)/u

l blo

od

NS

p=0.02

p=0.09

Day +14 post- BMT Day +30 post- BMT

p=NS

0

1

2

3

4D

onor

B22

0+ c

ells

(*1

0^3)

/ul b

lood

p< 0.05

Day +14 post- BMT Day +30 post- BMT

p<0.01

0

0,5

1,0

1,5

2,0

Don

or T

er11

9 ce

lls (

*10^

3)/u

l blo

od

Day +14 post- BMT Day +30 post- BMT

p=NS

p=NS

p=NS

p=0.01

p=NS

Supplementary Figure 3: GVT effect against myeloid leukemia RMB-1 cell line is maintained in the presence of ruxolitinib

0

20

40

60

80

100

p=0.01 p=0.003

p=0.2

% B

one

Mar

row

RM

B-1

cel

ls

BM Only+RMB-1

GVHD+ vehicle+ RMB-1

GVHD+Ruxolitinib 45 mg/Kg+ RMB-1

0

20

40

60

80

100

p=0.0002 p=0.002

p=0.1

% S

plee

n R

MB

-1 c

ells

a b

Supplementary Figure 4: Ruxolitinib at the dose of 90 mg/Kg prevents acute GVHD without affecting T cell alloreactivity

a

c

d

b

*

% S

plee

n C

D8+

IFN

-γ+ c

ells** p=0.2

% S

plee

n C

D4+

IFN

-γ+ c

ells

p=0.3

% S

plee

n C

D4+

IL-1

7+ c

ells

e

0

25

50

75

100

***

***

Naive T (CD62L+CD44-) CM T (CD62L+CD44+) EM T (CD62L-CD44+)

% S

plee

n T

cel

ls

NS

NSNS

*

0

20

40

60

80

100

120

Total Spleen cells CD3+ cells CD4+ cells CD8+ cells

Cel

ls p

er S

plee

n (*

10^6

)

BM onlyGVHD+VehicleGVHD+Ruxolitinib 90 mg/Kg

% S

plee

n T

reg

cells

p=0.3

0

1

2

3

4

5

6 p=0.07

0

1

2

3

4

5

6** p=0.3

p=0.06

0

0,1

0,2

0,3

0,4

0,5

0

0.5

1.0

1.5

a b

0

1,0

2,0

3,0

BM only GVHD+ Vehicle

GVHD+Ruxolitinib 45 mg/Kg

# C

D4+

IFN

-γ+ s

plee

n ce

lls (

10^5

) ** p=0.8**

0

1,0

2,0

3,0

4,0

5,0

BM only GVHD+ Vehicle

GVHD+Ruxolitinib 45 mg/Kg

# C

D8+

IFN

-γ+ s

plee

n ce

lls (

10^5

) ** p=0.4**

0

0,1

0,2

0,3

0,4

0,5

GVHD+ Vehicle

GVHD+Ruxolitinib 45 mg/Kg

# T

h17

spl

een

cells

(10

^5)

p=0.4

Supplementary Figure 5: Ruxolitinib effect at 45 mg/Kg on absolute numbers of alloreactive T cells, Th17 and Treg cells.

a

b c

GVHD+ Vehicle

GVHD+Ruxolitinib 45 mg/Kg

# T

reg

spl

een

cells

(10

^5)

p=0.2

0

0,5

1,0

1,5

Supplementary Figure 6: Representative Images of Ruxolitinb effect on T cells and macrophage infiltration of GVHD organs

BM only GVHD + Vehicle GVHD + Ruxolitiniba b

c

BM only GVHD + Vehicle GVHD + Ruxolitinib

BM only GVHD + Vehicle GVHD + Ruxolitinib