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

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

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

6. Detection of C4d is crucial for diagnosing antibody mediated rejection

7. Microcirculation inflammation in AMR
Kidney
Heart
CD68
CD3

8. Phenotype of glomerulits
CD15 – early AMR
CD68 – late AMR

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

10. 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.

11. Pathogenesis of Capillaropathy

12. Transplant-Capillaropathy

13. antibody-mediated injury and microcirculation inflammation
glomerulitis
glomerulopathy

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

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

16. 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

17. Graft Survival
Banff lesions
unsupervised Principal Component Analysis

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

19. 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:

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

21. 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?

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

23. 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:

24. 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:

25. 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 Oct;9(10):

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

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

28. 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
Hirohashi and Colvin et al. Am J Transplant Feb;12(2):  

29. 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 Apr;13(4):

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

31. * * * * * * * * * * * * * * * * * * * * * * * * * * *
Dean et al. Am J Transplant 2012; 12:

32. 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

33. C4d
C4d
30 minutes

34. 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. 

35. 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:

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

37. AMR and vasculopathy