SUPPLEMENTAL MATERIAL

(1)

1

SUPPLEMENTAL MATERIAL

Antibodies against ARHGDIB and ARHGDIB gene expression associate with kidney allograft outcome

Aleksandar Senev, MD; Henny G. Otten, PhD.; Elena G. Kamburova, PhD.; Jasper Callemeyn, MD;

Evelyne Lerut, MD, PhD; Vicky Van Sandt, PhD; Dirk Kuypers, MD, PhD.; Marie-Paule Emonds, MD, PhD; Maarten Naesens, MD, PhD

Table of contents:

Supplemental Table S1………..….………….2

Supplemental Table S2………..….………….4

Supplemental Table S3……….…..………..…..6

Supplemental Table S4.……….….……….………..….7

Supplemental Table S5.……….…….…..…..8

Supplemental Table S6.……….…….…..…..9

Supplemental Figure S1 ………10

Supplemental Figure S2 ………..….11

Supplemental Figure S3 ………..… 14

(2)

2

Supplemental Table S1. Univariate Cox proportional hazards analyses for death-censored graft failure according to the presence of non-HLA antibodies, with different thresholds for positivity using adjusted MFI values (N=422).

Directly coupled non-HLA proteins No. of patients

No. of

events HR 95% CI p-value Agrin

Adjusted MFI ≥ 500 7 1 0.78 0.11 - 5.65 0.78 APMAP

Adjusted MFI ≥ 500 29 6 1.31 0.57 – 3.04 0.54 Adjusted MFI ≥ 1000 11 2 1.06 0.26 – 4.34 0.93 ARHGDIB

Adjusted MFI ≥ 500 60 14 1.65 0.92 – 1.09 0.09 Adjusted MFI ≥ 1000 30 9 2.20 1.09 - 4.44 0.03 Adjusted MFI ≥ 1500 20 7 2.86 1.31 – 6.27 0.009 Adjusted MFI ≥ 2000 15 7 4.25 1.94 – 9.32 0.0003 AT1R

Adjusted MFI ≥ 500 378 57 0.74 0.37 – 1.50 0.41 Adjusted MFI ≥ 1000 238 29 0.56 0.35 – 0.92 0.02 Adjusted MFI ≥ 2000 92 12 0.76 0.40 – 1.41 0.38 Adjusted MFI ≥ 4000 40 4 0.55 0.20 – 1.50 0.24 ARHGEF6

Adjusted MFI ≥ 500 101 20 1.33 0.79 – 2.61 0.27 Adjusted MFI ≥ 1000 38 10 1.88 0.96 – 3.67 0.07 ETAR

Adjusted MFI ≥ 500 65 16 1.81 1.03 - 3.18 0.04 Adjusted MFI ≥ 1000 17 5 1.94 0.78 – 4.84 0.15 LMNB1

Adjusted MFI ≥ 500 27 6 1.47 0.64 – 3.41 0.37 Adjusted MFI ≥ 1000 4 1 1.28 0.18 – 9.19 0.81 LPLUNC1

Adjusted MFI ≥ 500 56 13 1.59 0.87 – 2.91 0.14 Adjusted MFI ≥ 1000 23 3 0.84 0.26 – 2.68 0.72 PECR

Adjusted MFI ≥ 500 134 18 0.75 0.44 – 1.29 0.30

Adjusted MFI ≥ 1000 76 8 0.59 0.28 – 1.23 0.16 PLA2R

Adjusted MFI ≥ 500 2 0 0.00 0.00 - . 0.99

PRKCZ

Adjusted MFI ≥ 500 116 18 1.01 0.59 – 1.73 0.97 Adjusted MFI ≥ 1000 77 9 0.75 0.37 – 1.51 0.42 TUBB4b

Adjusted MFI ≥ 500 102 21 1.54 0.92 – 2.59 0.10 Adjusted MFI ≥ 1000 56 14 1.91 1.06 – 3.44 0.03 Adjusted MFI ≥ 1500 35 11 2.69 1.41 – 5.15 0.002

(3)

3 Adjusted MFI ≥ 2000 22 6 2.20 0.95 – 5.08 0.07

Vimentin

Adjusted MFI ≥ 500 244 43 1.26 0.76 – 2.09 0.38 Adjusted MFI ≥ 1000 136 22 0.99 0.59 – 1.64 0.95

Statistically significant p-values after applying the Benjamini-Hochberg procedure for multiple testing with false discovery rate of 0.10 are indicated in bold font.

Adjusted MFI = Absolute MFI of the bead with the target non-HLA protein – absolute MFI of the empty bead

(4)

4

Supplemental Table S2. Univariate Cox proportional hazards analyses for death-censored graft failure according to non-HLA antibody with calculated ratios with the MFI value of the empty bead (N=422).

Directly coupled non-HLA proteins No. of patients

No. of

events HR 95% CI p-value Agrin

Absolute MFI/ MFI of the empty bead

Ratio ≥ 2 17 1 0.34 - 0.28

APMAP

Ratio ≥ 2 58 11 1.36 - 0.36

ARHGDIB

Ratio ≥ 2 92 22 1.93 1.16 - 3.22 0.01

Ratio ≥ 3 55 15 2.18 1.22 – 3.87 0.008

Ratio ≥ 4 33 10 2.56 1.30 – 5.02 0.006

AT1R

Ratio ≥ 5 236 31 0.72 - 0.19

Ratio ≥ 10 92 9 0.58 - 0.13

Ratio ≥ 15 38 3 0.50 - 0.24

ARHGEF6

Ratio ≥ 2 207 35 1.20 - 0.45

ETAR

Ratio ≥ 2 148 26 1.24 - 0.38

LMNB1

Ratio ≥ 2 69 7 0.58 - 0.18

LPLUNC1

Ratio ≥ 2 97 16 1.10 - 0.74

PECR

Ratio ≥ 2 292 45 0.99 - 0.81

Ratio ≥ 4 85 7 0.47 - 0.06

PLA2R

Ratio ≥ 2 6 0 0.00 - 0.98

PRKCZ

Ratio ≥ 2 188 67 1.06 - 0.81

TUBB4b

(5)

5

Ratio ≥ 2 156 29 1.50 - 0.10

Ratio ≥ 4 68 16 1.93 1.10 – 3.39 0.02

Vimentin

Ratio ≥ 2 403 64 1.63 - 0.50

Statistically significant p values after applying the Benjamini-Hochberg procedure for multiple testing with false discovery rate of 0.10 are indicated in bold font.

(6)

6

Supplemental Table S3. Adjusted multivariate Cox proportional hazards analyses for death- censored graft failure according to pretransplant non-HLA antibodies (N=422).

Antibodies No. of

patients

No. of

events HR 95% CI p-value

Anti-ARHGDIB antibody 422 66

Anti-ARHGDIB ≥ 500 aMFI (yes vs. no) 60 14 2.04 1.11 – 3.72 0.02 Anti- ARHGDIB ≥ 1000 aMFI (yes vs. no) 30 9 2.65 1.30 – 5.40 0.007 Anti- ARHGDIB ≥ 1500 aMFI (yes vs. no) 20 7 3.04 1.37 – 6.73 0.006 Anti- ARHGDIB ≥ 2000 aMFI (yes vs. no) 15 7 4.56 2.05 – 10.12 0.0002

Anti-TUBB4b antibody 422 66

Anti-TUBB4b ≥ 500 aMFI (yes vs. no) 102 21 1.53 0.89 – 2.61 0.12 Anti-TUBB4b ≥ 1000 aMFI (yes vs. no) 56 14 1.57 0.86 – 2.89 0.14 Anti-TUBB4b ≥ 1500 aMFI (yes vs. no) 35 11 1.88 0.96 – 3.70 0.07 Anti-TUBB4b ≥ 2000 aMFI (yes vs. no) 22 6 2.05 0.87 – 4.80 0.10

Statistically significant p-values are indicated in bold font.

All multivariate Cox models were adjusted for pretransplant DSA, repeat transplantation, delayed graft function, induction therapy, HLA-A/B/DR antigen mismatches and deceased donation.

Abbreviations: aMFI, adjusted MFI value.

(7)

7

Supplemental Table S4. Distribution of the patients with anti-PECR antibodies with a different thresholds for positivity between DSA

neg

AMR

h

and No AMR

h

groups (N=338).

Abbreviations: PECR, Peroxisomal trans-2-Enoyl-CoA reductase;

Anti-PECR antibodies DSAnegAMRh

(N=119)

NoAMRh

(N=219) p-value Test Different threshold for positivity

Adjusted MFI ≥ 500, n(%) 53 (44.5) 59 (26.9) 0.001 x²-test Adjusted MFI ≥ 1000, n(%) 34 (28.6) 32 (14.6) 0.002 x²-test Adjusted MFI ≥ 1500, n(%) 21 (17.6) 22 (10.0) 0.05 x²-test Adjusted MFI ≥ 2000, n(%) 17 (14.3) 12 (5.5) 0.005 x²-test

(8)

8

Supplemental Table S5. Adjusted multivariate Cox proportional hazards analyses for graft failure in patients with AMR

h

, according to pretransplant HLA antibodies, DSA and anti-ARHGDIB antibodies (N=203).

patients

No. of

Multivariate model-1

Anti-ARHGDIB abs (per 1000 MFI increment) 203 43 1.61 1.24 – 2.10 0.0004 Pretransplant HLA abs (yes vs. no) 109 33 4.12 1.87 – 9.08 0.0005

Anti-ARHGDIB abs (per 1000 MFI increment) 203 43 1.55 1.17 – 2.05 0.002

Pretransplant DSA (yes vs. no) 74 24 3.07 1.45 – 6.47 0.003

Multivariate model - 3

Anti-ARHGDIB ≥ 500 aMFI and DSA 203 43

None 113 14 1 – –

Anti-ARHGDIB abs 16 5 2.54 0.89 – 7.26 0.08

DSA 65 19 2.94 1.34 – 6.47 0.007

DSA and anti-ARHGDIB abs 9 5 9.96 3.00 – 33.05 0.0002

None 120 16 1 – –

Anti-ARHGDIB abs 9 3 2.85 0.80 – 10.21 0.11

DSA 70 21 2.88 1.36 – 6.12 0.006

None 122 16 1 – –

Anti-ARHGDIB abs 7 3 4.33 1.22 – 15.43 0.02

DSA 71 22 2.95 1.39 – 6.26 0.005

None 124 16 1 – –

Anti-ARHGDIB abs 5 3 6.63 1.86 – 23.65 0.004

DSA 72 22 2.94 1.38 – 6.26 0.005

Statistically significant p values are indicated in bold font.

All multivariate Cox models were adjusted for repeat transplantation, delayed graft function, induction therapy, HLA-A/B/DR antigen mismatches and deceased donation.

Abbreviations: abs, antibodies; aMFI, adjusted MFI; HLA, human leukocyte antigens; DSA, donor-specific HLA antibodies.

(9)

9

Supplemental Table S6. Adjusted multivariate Cox proportional hazards analyses for death- censored graft failure in the patients without AMR

h

, according to presence of pretransplant HLA antibodies, DSA and anti-ARHGDIB antibodies (N=219).

patients

No. of

Anti-ARHGDIB abs (per 1000 MFI increment) 219 23 1.15 0.86 – 1.55 0.35

Pretransplant HLA abs (yes vs. no) 50 10 3.18 1.24 – 8.14 0.02

Anti-ARHGDIB abs (per 1000 MFI increment) 219 23 1.01 0.73 – 1.41 0.94

Pretransplant DSA (yes vs. no) 9 5 12.52 3.44 – 45.59 0.0001

Multivariate model - 3

Anti-ARHGDIB abs ≥500 aMFI and DSA 219 23

None 177 15 1 – –

Anti-ARHGDIB abs 33 3 1.07 0.31 – 3.74 0.91

DSA 7 4 12.99 3.31 – 51.06 0.0002

Anti-ARHGDIB abs ≥1000 aMFI and DSA 219 23

None 195 16 1 – –

Anti-ARHGDIB abs 15 2 1.70 0.39 – 7.45 0.48

DSA 7 4 13.40 3.44 – 52.27 0.0002

All multivariate Cox models were adjusted for repeat transplantation, delayed graft function, induction therapy, HLA-A/B/DR antigen mismatches and deceased donation.

Abbreviations: abs, antibodies; aMFI, adjusted MFI.

(10)

10

Supplemental Figure S1. Distribution of measured MFI values per bead in all pretransplant sera

(N=422). All individual A) absolute and B) adjusted MFI values together with the medians and

interquartile ranges are shown, using a scatter dot plot for IgG beads (red dots), empty beads

(green dots), transferrin beads (green dots), and 13 beads with non-HLA proteins (blue dots).

(11)

11

Supplemental Figure S2. Comparison of adjusted MFI values per non-HLA target microsphere

between different groups. The black lines illustrate the median values, with whiskers indicating

variability outside the upper and lower quartiles of the median. The groups were compared using

Mann-Whitney U test. The significant p-values that remain significant after applying Benjamini-

Hochberg correction for multiple testing with a false discovery rate of 0.10 are displayed on the

graph in the figures.

(12)

12

A) Comparison of adjusted MFI values between AMR

h

and NoAMR

h

groups.

(13)

13

B) Comparison of adjusted MFI values between DSA

neg

AMR

h

and NoAMR

h

groups.

(14)

14

Supplemental Figure S3. Occurrence of rejection episode within the first 5 years after transplantation in the study population grouped according to different thresholds for anti- ARHGDIB status (N=422). The p-value is calculated with Gray’s test. The cumulative incidence should be interpreted cautiously as this is the representation of a selected cohort, with enrichment of the dataset with cases with AMR

h

, which is thus importantly overrepresented in comparison to it natural incidence.

A) Occurrence of AMR

h

episodes within the first 5 years after transplantation.

(15)

15

B) Occurrence of TCMR episodes within the first 5 years after transplantation.

(16)

16

C) Occurrence of any rejection (TCMR and/or AMR

h

) episodes within the first 5 years after

transplantation.

(17)

17

D) Occurrence of cAMR

h

episodes within the first 5 years after transplantation.

(18)

18

SUPPLEMENTAL MATERIAL