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
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
CD3
Heart
CD68
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
106
Stage I
Stage II
182
225
Stage III
352
Stage IV
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
DeKAF Study
Biopsies for late graft dysfunction
C4d-DSA-
C4d-DSA+
C4d+DSAC4d+DSA+
Months post-bx
N=173
Gaston et al Transplant 90:68,2010
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.
80
70
60
50
40
30
20
10
0
68.4
45.7
14.1
no rejection
(n=453)
borderline
(n=105)
TCMR (n=76)
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”
-
+
+
+
1 (3)
“BD”
-
+
-
+
9 (24)
“CD”
-
-
+
+
1 (3)
“D”
-
-
-
+
1 (3)
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
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 complementindependent 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
Gene Symbol
VWF
CAV1
RHOJ
MCAM
CDH5
SELE
PALMD
PECAM1
KLF4
CYYR1
CD34
TEK
SOX18
ZNF521
RASIP1
HOXD4
RAI14
PODXL
DLC1
Also not in our strict
FGD5
definition of ENDAT list,
FOXF2
but increased in ABMR:
EMCN
CDH13
KDR
Duffy blood group
CETP
SOX7
MAOB
THBD
MALL
N
Normalized
1.05
1.17
0.99
1.12
1.00
1.06
0.93
0.93
1.11
1.04
1.05
1.04
1.02
0.92
0.98
1.02
0.95
1.11
0.95
0.88
1.02
1.01
0.94
1.04
0.97
Signal
115.36
206.07
27.60
192.50
75.34
23.25
78.15
294.26
236.31
162.34
138.33
190.45
13.48
37.82
80.80
372.75
273.72
1489.49
273.83
212.06
12.59
700.89
307.06
26.61
1450.68
ABMR
Normalized
6.39
5.35
2.94
2.93
2.58
2.23
2.12
2.00
1.52
1.52
1.50
1.47
1.46
1.38
1.37
1.33
1.21
1.06
0.98
0.97
0.86
0.85
0.77
0.72
0.69
Signal
738.30
857.90
85.27
476.93
201.54
55.26
182.35
638.35
318.70
238.68
198.87
275.89
19.92
55.56
114.53
495.27
347.63
1404.16
287.39
233.58
10.61
600.32
244.78
18.27
1031.30
Red arrows indicate genes that are known to be
involved in endothelial cell activation
TCMR
Normalized
3.39
2.99
1.65
2.02
1.61
1.20
1.36
1.60
1.00
1.15
1.12
1.04
1.08
0.80
0.97
1.02
0.87
0.74
0.74
0.71
0.99
0.64
0.60
1.06
0.52
Signal
419.08
494.11
53.58
326.45
123.20
28.66
122.61
514.29
216.59
182.55
148.89
198.66
14.41
33.75
80.99
378.73
256.99
1067.37
221.40
170.90
12.25
459.15
197.55
29.67
795.14
Welch t test
FDR 0.05
Sis et al. AJT 2009;9:2312-23
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
p<0.001
t
.135
NS
v
.092
NS
cg
.261
0.001
mm
.173
0.02
ci
.330
p<0.001
ct
.286
p<0.001
cv
.014
NS
ah
-.050
NS
Sis et al. AJT 2009;9:2312-23
Transplant Glomerulopathy score ( cg, mean + 95% CI)
n=81
60
50
40
30
20
10
0
43%
19%
Ab
Abwith
with no
no E
n=21
ENDAT
p=0.53
No Ab
n=30
Ab with no E
n=21
Ab with E
n=30
No Ab or
Ab with no E
C4d positive
6.7%
No
Ab
no Ab
n=30
p=0.01
C4d negative
with
E
AbAb
with
ENDAT
n=30
C4d+ Transplant Glomerulopathy
C4d Negative Transplant Glomerulopathy
Sis et al. Am J Transplant. 2009 Oct;9(10):2312-23.
Cumulative Survival
Incidence of transplant glomerulopathy (*%)
C4d is negative in 60% of
chronic active ABMR biopsies
n=81
Ab with E
60%
C4d negative
No Ab
Ab with no E
Ab with E
p=0.001
Post-biopsy time (months)
Transcripts selectively associated with DSA:
Endothelial and NK cell transcripts
endothelial
NK
Hidalgo et al. AJT 2010; 10: 1812–1822
NK cells and macrophages in antibody mediated
peritubular capillaritis
A.
B.
3
p=0.03
p=0.006
p=0.09
C4d+ ABMR
Mean number of positive cells
in five peritubular capillaries
C4d- ABMR
TCMR
2
1
CD3+
TC
M
B
C
D
3
A
D
3
C
D
68
M
R
R
R
B
M
CD68 E.
C
A
CD68+
TC
M
R
R
C
D
68
TC
M
CD56+
D
56
C
D
56
CD56 D
.
C
C.
A
B
M
R
0
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.
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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, ginterferon), endothelial, and NK cell associated transcripts
as the molecular correlate
C4d
30 minutes
C4d
Objective
• To review the current knowledge of
mechanisms, diagnostics and clinical
management of patients with antibodymediated 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, ginterferon) in XM+ biopsies
Dean et al. Am J Transplant 2012; 12:1551-1563
Limited specificity of capillaritis
Gibson et al. Am J Transplant. 2008 Apr;8(4):819-25.
% cases with capillaritis
80
68.4
70
60
45.7
50
40
30
20
14.1
10
0
no rejection (n=453)
borderline (n=105)
TCMR (n=76)
AMR and vasculopathy