Correlation Between the CRM and Inflammatory Macrophage Scores Across 10 Renal Transplant Datasets (1,499 Graft Biopsies)

Supplementary figure and table legends

Supplementary Figure 1: Correlation between the CRM and inflammatory macrophage scores across 10 renal transplant datasets (1,499 graft biopsies). The CRM and inflammatory macrophage scores in each dataset are standardized (mean = 0, SD = 1). Gray bar: 95% confidence interval.

Supplementary Figure 2: (A). Correlation analysis of the 27-gene pro-inflammatory macrophage polarization signature across 104 diseases using Metasignature shows that many genes are significantly correlated with each other. (B) Enrichment of the 3-gene signature across 25 immune and epithelial cell types shows that the signature are enriched in inflammatory macrophages.

Supplementary Figure 3: M1 score in two independent validation datasets is higher in AR biopsies than in STA. STA, stable; AR, acute rejection.

Supplementary Figure 4: RT-qPCR of three genes resulting from LASSO analysis on discovery cohort. (A) Relative expression levels of each gene in AR (blue) samples vs. STA (orange) samples. All genes are significantly over-expressed in AR versus STA.

Supplementary Figure 5: AUCs of 3-gene signature in a lung dataset, consisting in 32 biopsies.

Supplementary Figure 6: Schematic overview of the longitudinal cohort (GSE25902).

Supplementary Figure 7: M1 score is higher in both ACR and AMR, compared to STA biopsies. ACR, acute cellular rejection; AMR, antibody-mediated rejection; STA, stable

Supplementary Table 1: Detailed description of datasets used in the discovery and direct validation cohorts. AR, acute rejection; STA, stable; HC, healthy controls; AMR, antibody mediated rejection; ACR, Acute cellular rejection; CAN, chronic allograft nephropathy; HEPC, hepatitis C; TOL, operationally tolerant; CNIT, calcineurin inhibitor toxicity; IF/TA, interstitial fibrosis/tubular atrophy; IF/TA+I, interstitial fibrosis/tubular atrophy plus inflammation; ADNR, acute dysfunction with no rejection. ISHLT, International Society for Heart & Lung Transplantation

Supplementary Table 2: Methodology utilized to identify subset genes. A generalized linear regression model with binary classification (AR vs STA) was employed to identify a subset of the inflammatory macrophage polarization genes most discriminative in > four out of eight discovery datasets

A B 4 C STA 3 STA AR AR STA 2 4 AR ed) ed)

2 ed) z z z 1 2 0 0

−1 0 CRM score (standardi CRM score (standardi CRM score (standardi Pearson correlation = 0.95 Pearson correlation = 0.77 P-value < 2.2E-16 −2 P-value < 2.2E-16 −2 Pearson correlation = 0.64 −2 P-value < 2.2E-16 −3 −2 0 2 −2.5 0.0 2.5 5.0 −2.5 0.0 2.5 5.0 M(IFN-gamma) score (standardized) M(IL4) score (standardized) M(IL10) score (standardized) Enrichment for 3−gene set A B −1 0 1 2 3 4 NK T−cell Band cell 11 HLAHLADRB1! DRB1 CD8+ T−cell−activated CCL4CCL4 0.80.8 CD4+ T−cell−activated CCL5 CCL5 T−cell FASFAS 0.60.6 NK cell IL15IL15 0.40.4 CCL8CCL8 T−reg CD86CD86 0.20.2 CD8+ T−cell BCL2A1BCL2A1 CD4+ T−cell CD40CD40 00 Neutrophil CCL20CCL20 ! 0.20.2 IL2RAIL2RA Eosinophils CCR7CCR7 Endothelial cell IL7RIL7R B−cell (plasma cell) CCL19 CCL19 MetaMyelocyte CXCL10CXCL10 B−cell CXCL11CXCL11 CXCL9CXCL9 Megakaryocyte IL15RAIL15RA Basophil TNF TNF Monocyte−CD16+ SPHK1SPHK1 B−cell (centrocyte) NOS2NOS2 SLC7A5SLC7A5 Dendritic cells (Plasmacytoid) CXCL16CXCL16 Monocyte IL6 IL6 Monocyte−CD16− CCL15 CCL15 ORM1 Dendritic cells (Myeloid) ORM1 PTX3 Dendritic cells

HLA CCL4 CCL5 F IL15 CCL8 CD86 BCL2A1 CD40 CCL20 IL2RA CCR7 IL7R CCL19 CXCL10 CXCL11 CXCL9 IL15RA TNF SPHK1 NOS2 SLC7A5 CXCL16 IL6 CCL15 ORM1 PTX3PTX3 AS F IL15 CCL8 CD86 BCL2A1 CD40 CCL20 IL2RA CCR7 IL7R CCL19 CXCL10 CXCL11 CXCL9 IL15RA TNF SPHK1 NOS2 SLC7A5 CXCL16 IL6 CCL15 ORM1 PTX3

AS M2 macrophage ! DRB1 M1 macrophage

Std. Deviations (Z−score) GSE53605 GSE76882 4 2 ● ● STA ● ● STA ● ● AR AR ●● ● ● ● ●● 1 ●●● ●● ●● ● ●● 2 ●● ● ●● ● ●● ● ● ● ●●●● ●● ●● 0 ● ● ● ● ● ● ● ●● ● ● ● ● ●● ●●● ● ● ●● ●●● ●●● ●● ●● ● ● ●● ●● ● ● ● ●●● ●●● ● ● ●● −1 ●● 0 ●●●● ●● ● ●●● ●● ●● ●●●●● ● ●●●● ● ●●●● ● ●●● ●●● ●●● ●● ●●● ●●●●●● ● ●● ● ●●●● ●●●● ●● −2 ●●● ●●●

−2 −3 STA AR STA AR p = 6.60353791088933e−06 p = 6.25529816225218e−16 CXCL11 CCL19 CD86

• • • ct ct ct Δ Δ Δ - - - 2 2 2 • • • p < 2.2E-16 p = 0.016 p = 0.009 M1 R 1.00

0.75

0.50 v ositi P 0.25 ue r T

A 0.00 0.00 0.25 0.50 0.75 1.00 False Positiv Longitudinal cohort (GSE25902)

120 renal allograft biopsies

24 biopsies from 96 biopsies from AR patients 48 patients

72 longitudinal 24 biopsies from stable biopsies patients from 24 patients

24 biopsies at 6 24 biopsies 24 24 biopsies at time months after months after of implantation implantation implantation GSE36059 GSE48581 1.1e−08 1.9e−11 7.1e−07 2e−05 1.7e-03 1.1e−11 4.9e-03 9.1e-04

2 M1 Score

−2 STA ABMR Mixed TCMR STA ABMR Mixed TCMR Enrichment for 3−gene set A B C −1 0 1 2 3 4 CD86 CXCL11 CCL19 NK T−cell Megakaryocyte 4 Band cell 11 Plasma cell HLAHLADRB1! DRB1 CD8+ T−cell−activated CCL4 Basophil CCL4 0.80.8 2 CD4+ T−cell−activated CCL5CCL5 Neutrophil T−cell FASFAS 0.60.6 CD4+ T cell NK cell IL15IL15 0 0.40.4 Eosinophils CCL8CCL8 T−reg NKT cell CD86CD86 0.20.2 CD8+ T−cell BCL2A1BCL2A1 −2 CD4+ T cell (activated) CD4+ T−cell CD40CD40 00 Band cell Neutrophil CCL20CCL20 ! 0.20.2 NK cell −4 IL2RAIL2RA Eosinophils CCR7CCR7 CD8+ T cell Endothelial cell IL7R IL7R CD8+ T cell (activated) B−cell (plasma cell) CCL19CCL19 T cell MetaMyelocyte CXCL10CXCL10 Treg CXCL11CXCL11 B−cell CXCL9CXCL9 Endothelial cell Megakaryocyte IL15RA IL15RA MetaMyelocyte Basophil TNFTNF B cell Monocyte−CD16+ SPHK1SPHK1 Plasmacytoid Dendritic cells B−cell (centrocyte) NOS2NOS2 SLC7A5SLC7A5 B cell (centrocyte) Dendritic cells (Plasmacytoid) CXCL16 CXCL16 CD16- Monocyte Monocyte IL6 IL6 Monocyte−CD16− CCL15 Monocyte CCL15 ORM1 Dendritic cells Dendritic cells (Myeloid) ORM1 PTX3 Dendritic cells HLA ! CCL4 CCL5 F IL15 CCL8 CD86 BCL2A1 CD40 CCL20 IL2RA CCR7 IL7R CCL19 CXCL10 CXCL11 CXCL9 IL15RA TNF SPHK1 NOS2 SLC7A5 CXCL16 IL6 CCL15 ORM1 PTX3PTX3 CD16+ Monocyte AS CCL5 F IL15 CCL8 CD86 BCL2A1 CD40 CCL20 IL2RA CCR7 IL7R CCL19 CXCL10 CXCL11 CXCL9 IL15RA TNF SPHK1 NOS2 SLC7A5 CXCL16 IL6 CCL15 ORM1 PTX3 AS Myeloid Dendritic cells M2 macrophage DRB1 M2 macrophage M1 macrophage M1 macrophage Std. Deviation (Z−score) GSE53605 GSE76882

STA STA 2 AR AR

M1 Score 0

−2 −2

STA AR STA AR p = 2.905e−05 p = 1.134e−08 CXCL11 CCL19 CD86

• • • ct Δ - - Δ ct - Δ ct 2 2 2 • • • p < 2.2E-16 p = 0.016 p = 0.009 M(IFNg) R 1.00

0.75

0.50 ositi v

ue P 0.25 r T

A 0.00 0.00 0.25 0.50 0.75 1.00 False Positiv Longitudinal cohort (GSE25902)

120 renal allograft biopsies

24 biopsies from 96 biopsies from AR patients 48 patients

72 longitudinal 24 biopsies from stable biopsies patients from 24 patients

2 M(IFNg) Score

−2 STA AMR Mixed ACR STA AMR Mixed ACR