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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