Microvascular inflammation and endothelial cell activation in kidney antibody mediated rejection  Michael Mengel Department of Laboratory Medicine and Pathology University of Alberta, Edmonton, Canada

Three Pathways to Antibody-Mediated Injury Antibody Alone Complement Mediated Cell Mediated (FcR) Farkash and Colvin, Nat Rev Nephrol 8:255, 2012

Role of C4d in antibody-mediated rejection Classical complement pathway activation: Antibody + Antigen C1 C4 C4a + C4b Mannose binding lectin/MASP1 Tx 1377  C4d binds covalently to local site Helmut Feucht Clin Exp Immunol 86:464, 1991

Detection of C4d is crucial for diagnosing antibody mediated rejection

Microcirculation inflammation in AMR Kidney Heart CD68 CD3

Phenotype of glomerulits CD15 – early AMR CD68 – late AMR

Diagnosis of AMR Mengel M et al. Transpl Int. 2012 Jun;25(6):611-22

Follow up of C4d positive biopsies and the development of TX-Glomerulopathy Significant more often associated with Transplant Glomerulopathy (53% vs. 14%) Significant more often associated with Transplant Capillaropathy (71% vs. 13%) Significantly associated with progression of Transplant Glomerulopathy in follow-up biopsy (82% vs. 27%, median after 23 months) Regele et al.

Pathogenesis of Capillaropathy

Transplant-Capillaropathy

antibody-mediated injury and microcirculation inflammation glomerulitis glomerulopathy

Sequential development of CHR in non-human primates No CHR Stage I Stage II Stage III Stage IV 106 182 225 352 371 Days post-transplant Smith et al (Boston) AJT 8:1662, 2008

C4d versus microcirculation inflammation in biopsies prior to AMR treatment (1996-2001) Verghese et al. Clin. Transplant 2013 in press

Biopsies for late graft dysfunction DeKAF Study Biopsies for late graft dysfunction C4d-DSA- C4d-DSA+ C4d+DSA- C4d+DSA+ Months post-bx N=173 Gaston et al Transplant 90:68,2010 16 16

Graft Survival Banff lesions unsupervised Principal Component Analysis

Limited specificity of microcirculation inflammation Fahim et al. Am J Transplant. 2007 Feb;7(2):385-93. % cases with capillaritis Gibson et al. Am J Transplant. 2008 Apr;8(4):819-25.

The association of TG (D) (n=44) with antibody (A), PTCBMML (B), and C4d (C). TG phenotype* A B C D n (%) “ABCD” + 10 (27) “ABD” - 12 (32) “ACD” 2 (5) “AD” 1 (3) “BCD” “BD” 9 (24) “CD” “D” 73% of Tg cases show some signs of humoral rejection Sis et al. AJT 2007; 7: 1743-1752

Loupy et al: Subclinical progressive microcirculation injury in presensitized patients, despite C4d negativity 20

Potential causes for C4d negativity Complement activation Complement dependent cell injury Endothelial activation Recruitment and activation of leukocytes C4d+ ABMR Is C4d deposited in low amounts (Thus not detectable by current methods) Treatment effects? ? C4d negative ABMR ? Do HLA antibodies in a complement-independent way cause EC activation and subsequent inflammation and Fc receptor mediated graft injury?

Three Pathways to Antibody-Mediated Injury Antibody Alone Complement Mediated Cell Mediated (FcR) Farkash and Colvin, Nat Rev Nephrol 8:255, 2012

endothelial genes are increased in AMR Also not in our strict definition of ENDAT list, but increased in ABMR: CDH13 Duffy blood group SOX7 THBD MALL Welch t test FDR 0.05 Red arrows indicate genes that are known to be involved in endothelial cell activation Sis et al. AJT 2009;9:2312-23

Endothelial Transcripts Peritubular capillaritis Endothelial Cell-Associated Transcripts correlate with pathologic features of AMR (in 173 biopsies) Endothelial Transcripts Correlation coefficient p C4d deposition .376 p<0.001 Peritubular capillaritis .252 0.002 PTCBMML .266 0.004 g .248 0.001 i .358 t .135 NS v .092 cg .261 mm .173 0.02 ci .330 ct .286 cv .014 ah -.050 Sis et al. AJT 2009;9:2312-23

C4d is negative in 60% of chronic active ABMR biopsies with no E n=21 with E Transplant Glomerulopathy score ( cg, mean + 95% CI) n=81 p=0.01 p=0.53 Cumulative Survival Post - biopsy time (months) p=0.001 or 60% C4d negative n=81 Incidence of transplant glomerulopathy (*%) No Ab n=30 Ab with no E n=21 Ab with E C4d+ Transplant Glomerulopathy C4d Negative Transplant Glomerulopathy 6.7% 19% 43% Sis et al. Am J Transplant. 2009 Oct;9(10):2312-23.

Transcripts selectively associated with DSA: Endothelial and NK cell transcripts Hidalgo et al. AJT 2010; 10: 1812–1822

NK cells and macrophages in antibody mediated peritubular capillaritis C4d+ ABMR C4d- ABMR TCMR Mean number of positive cells in five peritubular capillaries CD56+ CD68+ CD3+ C. CD56 D. CD68 E. CD3 Hidalgo et al. AJT 2010; 10: 1812–1822

Role of complement and NK cells in antibody mediated rejection AMR AMR + anti NK NK cell stain Akiyoshi and Colvin at al. Human Immunology Volume 73, Issue 12 2012 1226 - 1232 Hirohashi and Colvin et al. Am J Transplant. 2012 Feb;12(2):313-21. 

A molecular classifier for diagnosing AMR Classifier score correlates with: Pathology (ptc, g, cg, I, cv, ah, ct, ci) Consensus amongst pathologists Presence of DSA outcome Sellares et al. Am J Transplant. 2013 Apr;13(4):971-83.

Sellares et al. Am J Transplant. 2013 Apr;13(4):971-83.

* * * * * * * * * * * * * * * * * * * * * * * * * * * Dean et al. Am J Transplant 2012; 12:1551-1563

Summary Donor-specific antibody acting on the allograft is associated with endothelial cell and local complement activation [ in most cases] DSA acting on the allograft is associated with microcirculation inflammation as the morphological correlate [notion: the antigen is expressed in the microcirculation] DSA acting on the allograft is associated with increased expression of inflammation (T cells, macrophages, g-interferon), endothelial, and NK cell associated transcripts as the molecular correlate

C4d C4d 30 minutes

Objective To review the current knowledge of mechanisms, diagnostics and clinical management of patients with antibody-mediated rejection. Since the vast majority of experience in this area has been accumulated in renal transplant patients, this group of patients will be the main focus of the presentation, but relevant lessons applicable to other types of organ transplants will be discussed as well. 

Intragraft gene expression in positive crossmatch kidney allografts Class – Comparisons: A and B: no significant differences in gene expression C and D: over-expression of inflammatory transcripts (T cells, macrophages, g-interferon) in XM+ biopsies Black error indicate class comparisons of gene expression. All cases are living donors. Dean et al. Am J Transplant 2012; 12:1551-1563

Limited specificity of capillaritis % cases with capillaritis Gibson et al. Am J Transplant. 2008 Apr;8(4):819-25.

AMR and vasculopathy