SUPPLEMENTARY METHODS
Immunosuppressive regimens
All patients received induction therapy consisting of rabbit antithymocyte globulin
or
basiliximab. Target blood levels of maintenance immunosuppressive therapies were : i)
Ciclosporin: blood through level (T0) = 120 ng/mL, ii) Tacrolimus blood through level (T0)
between 7 and 8 ng/mL; iii) Mycophenolic acid: AUC0-12 between 30 and 60 µg/h/mL; iiii)
Everolimus: blood through level (T0) between 4 and 8 ng/mL iiiii) for patients maintained on
corticosteroids, a daily dose is of 0.1 to 0.2 mg/kg body weight.
Supplementary Table 1: Description of the time frame of index biopsy in the entire
cohort (test + validation cohorts)
Normal
allografts
before 1 year
Normal
allografts
after 1 year
Rejecting
allografts
before 1 year
Rejecting
allografts
after 1 year
Total
Test cohort
10
20
9
21
60
Validation cohort
10
12
16
15
53
Test + validation
20
32
25
36
113
Footnote: the cohort, is including rejection episodes occurring in the first year post transplant
(n=25/61, 41%) as well as rejection occurring after 1 year post transplant (n=36/61, 59%). In our
cohort, among the 36 late rejection episodes, 12 (33%) are T-cell mediated rejection and 24 (67%) are
antibody-mediated rejection.
Supplementary Table 2: Tissue distribution, known implications, regulations and
interactions of the fourteen miRNAs of interest (25, 27 , 28 , 29 , 30, 31, 32 , 33 , 34-
40).
Known implications in
heart/transplantation - related diseases
Known regulations/interactions
miRNAs mostly found in the endothelium
miR-92a
Apoptosis
eNOS, JUN/FOS pathway, integrin α5,
KLF2/4
miR-221
Hypertrophy, endothelial dysfunction
ICAM1, p27, c-kit, Ets-1
miR-296
Angiogenesis, hypertension, ischemia
VEGFR2
miR-126
Endothelial damage and injury, lipid
VCAM1, SPRED1, PIK3RS, CXCL12.
metabolism, angiogenesis
Reduced in coronary heart disease.
miRNAs involved in inflammatory response
miR-155
Cellular migration, cell survival (cytokines),
eNOS, RIP1, Ang2, Bcl6, Ets-1, HO-1.
necrosis, NK cells.
Increased in coronary artery disease
miR-142-3p
Inflammatory response
miR-451
Cell migration
Pi3K, ROS, Ago2, AMPK
miR-182
Present in PBMC
Increased in kidney transplanted patients
with AR
miR-10a
Atherosclerosis, shear stress
MAPK, NFκB Increased in kidney
transplanted patients with AR
miR-181b
Vascular inflammation, atherosclerosis
NFκB (importin α3), TGFβ
miR-181a
Acute cellular rejection, endothelial
Increased in CD4 cells
senescence
Cardiac-enriched miRNAs
miR-21
Ischemia, endothelial cell proliferation,
Spry1, ERK/MAPK, PI3K/AKT, eNOS,
pro-fibrosis, pro-apoptosis, hypertension,
BCL2/PTEN, Smad family, PPARα
shear stress
miR-31
Fibrosis
HIF-1, IL-2, e-selectin
miR-208a
Infarct, hypertrophy
α/β myosin, connexins
Supplementary Table 3: Grades of heart allograft rejection using ISHLT classifications
in the test and validation cohorts
NORMAL ALLOGRAFTS (n,%)
TEST
VALIDATION
COHORT
COHORT
(n=60)
(n=53)
p
30 (50%)
22 (41.5%)
0.4
REJECTING GRAFTS (n,%)
30 (50%)
31 (58.5%)
0.4
T-cell mediated rejection (n,%)
11 (18.3%)
14 (26.4%)
0.4
1R
3 (5%)
2 (3.8%)
2R
6 (10%)
9 (14%)
3R
2 (3.3%)
3 (5.7%)
19 (31.7%)
17 (32.1%)
pAMR1-I+
2 (3.3%)
2 (3.8%)
pAMR1-H+
7 (11.7%)
4 (7.6%)
pAMR2
10 (16.7%)
11 (20.7%)
pAMR3
0 (0%)
0 (0%)
0R and pAMR0
Antibody-mediated rejection (n,%)
1.0
Supplementary Table 4: Patient characteristics of the validation cohort
Recipient age, median (median, IQR [25 - 75])
NORMAL
REJECTING
ALLOGRAFTS
ALLOGRAFTS
(n=22)
(n=31)
P
value
27 [11 - 63]
39 [24 - 49]
0.9
17 (77%)
17 (55%)
0.1
1 (4%)
2 (6%)
1.0
Non-ischemic cardiopathy
15 (68%)
17 (55%)
0.4
Ischemic cardiopathy
3 (14%)
9 (29 %)
0.3
Valvular cardiomyopathy
0 (0%)
0 (0%)
1.0
Recipient gender, male, n (%)
Primary heart disease, n (%)
Congenital cardiopathy
Retransplant
0 (0%)
1 (3%)
1.0
3 (14 %)
2 (6%)
0.6
27 [18 - 43]
39 [29 - 48]
0.1
15 (68 %)
19 (61%)
0.8
208 [160 - 245]
218 [176 - 266]
0.7
Steroids
17 (77%)
26 (84%)
0.7
Calcineurin inhibitors
21 (95%)
30 (97%)
1.0
Mycofenolate acid
19 (86%)
28 (90%)
0.7
m-TOR inhibitors
4 (18%)
3 (10%)
0.4
Azathioprine
3 (14%)
0 (0%)
0.07
23 [3 - 44]
19 [11 - 33]
0.99
Miscellaneous
Donor age, median (median, IQR [25 - 75])
Donor gender, male, n (%)
Cold ischemic time, median (median, IQR [25 - 75])
Maintenance Immunosuppression (%)
Time between Tx and Index EMB,
Months IQR [25 - 75]
Supplementary Figure 1: Negative control of In situ hybridization using scrambled probe
control
Supplementary Figure 2: Dendrogram and unsupervised principal component
analysis.
A: Individual overview of the tissular expression of the 14 miRNAs of interest (Figure 1A). Each
variable(miRNA) in an individual patient is colored according to the threshold for each miRNA graded
from 0 to 3 according to quartiles.
B: Unsupervised principal component analysis (PCA). The PCA analysis examines the 60 heart
transplant patients using the 14 miRNAs of interest according to the heart allograft status: normal, Tcell mediated rejection and antibody-mediated rejection
Supplementary Figure 3: Sensitivity analysis: Diagnostic accuracy of miRNAs for
predicting T-cell mediated rejection (all cases from the test and the validation set)
Supplementary Figure 4: Sensitivity analysis: Diagnostic accuracy of miRNAs for
predicting antibody-mediated rejection (all cases from the test and the validation set)
Supplementary Figure 5: Sensitivity analysis: A: miRNA expression according to time
post transplantation. B: Diagnostic accuracy according to time post transplantation
A
footnote: ****: p<0.0001
B