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 ComplementMediated

Cell Mediated (FcR)

Farkash and Colvin, Nat Rev Nephrol 8:255, 2012

Classical complement pathway activation:Antibody + Antigen C1

C4 C4a + C4b

C4d binds covalently to local

siteHelmut Feucht Clin Exp Immunol 86:464, 1991

Mannose binding lectin/MASP1

Role of C4d in antibody-mediated rejection

Detection of C4d is crucial for diagnosing antibody mediated rejection

Microcirculation inflammation in AMR

CD3 CD68

HeartKidney

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

Days post-transplant106 182 225 352 371

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

Gaston et al Transplant 90:68,2010

C4d-DSA-

C4d-DSA+

C4d+DSA-

C4d+DSA+

N=173

DeKAF StudyBiopsies for late graft dysfunction

Months post-bx

unsupervised Principal Component Analysis

Banff lesionsGraft Survival

Limited specificity of microcirculation inflammation

Fahim et al. Am J Transplant. 2007 Feb;7(2):385-93.

no rejection (n=453)

borderline (n=105)

TCMR (n=76)0

1020304050607080

14.1

45.7

68.4

% c

ases

with

cap

illar

itis

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” - + + + 1 (3)

“BD” - + - + 9 (24)

“CD” - - + + 1 (3)

“D” - - - + 1 (3)

Sis et al. AJT 2007; 7: 1743-1752

73% of Tg cases show some signs of humoral rejection

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

Potential causes for C4d negativity

C4d+ ABMR

C4d negative ABMR

Complement activationComplement dependent cell injuryEndothelial activationRecruitment and activation of leukocytes

Is C4d deposited in low amounts (Thus not detectable by current methods)Treatment effects?

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 ComplementMediated

Cell Mediated (FcR)

Farkash and Colvin, Nat Rev Nephrol 8:255, 2012

endothelial genes are increased in AMR

Red arrows indicate genes that are known to be involved in endothelial cell activation

N ABMR TCMRGene Symbol Normalized Signal Normalized Signal Normalized Signal

VWF 1.05 115.36 6.39 738.30 3.39 419.08CAV1 1.17 206.07 5.35 857.90 2.99 494.11RHOJ 0.99 27.60 2.94 85.27 1.65 53.58MCAM 1.12 192.50 2.93 476.93 2.02 326.45CDH5 1.00 75.34 2.58 201.54 1.61 123.20SELE 1.06 23.25 2.23 55.26 1.20 28.66

PALMD 0.93 78.15 2.12 182.35 1.36 122.61PECAM1 0.93 294.26 2.00 638.35 1.60 514.29

KLF4 1.11 236.31 1.52 318.70 1.00 216.59CYYR1 1.04 162.34 1.52 238.68 1.15 182.55CD34 1.05 138.33 1.50 198.87 1.12 148.89TEK 1.04 190.45 1.47 275.89 1.04 198.66

SOX18 1.02 13.48 1.46 19.92 1.08 14.41ZNF521 0.92 37.82 1.38 55.56 0.80 33.75RASIP1 0.98 80.80 1.37 114.53 0.97 80.99HOXD4 1.02 372.75 1.33 495.27 1.02 378.73RAI14 0.95 273.72 1.21 347.63 0.87 256.99

PODXL 1.11 1489.49 1.06 1404.16 0.74 1067.37DLC1 0.95 273.83 0.98 287.39 0.74 221.40FGD5 0.88 212.06 0.97 233.58 0.71 170.90FOXF2 1.02 12.59 0.86 10.61 0.99 12.25EMCN 1.01 700.89 0.85 600.32 0.64 459.15KDR 0.94 307.06 0.77 244.78 0.60 197.55

CETP 1.04 26.61 0.72 18.27 1.06 29.67MAOB 0.97 1450.68 0.69 1031.30 0.52 795.14

Welch t testFDR 0.05

Sis et al. AJT 2009;9:2312-23

Also not in our strictdefinition of ENDAT list, but increased in ABMR:

CDH13Duffy blood groupSOX7THBDMALL

Endothelial Cell-Associated Transcripts correlate with pathologic

features of AMR (in 173 biopsies)

Endothelial TranscriptsCorrelation coefficient p

C4d deposition .376 p<0.001Peritubular capillaritis .252 0.002

PTCBMML .266 0.004g .248 0.001

i .358 p<0.001t .135 NSv .092 NS

cg .261 0.001mm .173 0.02ci .330 p<0.001ct .286 p<0.001cv .014 NSah -.050 NS

Sis et al. AJT 2009;9:2312-23

n=81

Inci

denc

e of

tran

spla

nt g

lom

erul

opat

hy (*

%)

No Ab n=30

Ab with no E n=21

Ab with E n=30

C4d+ Transplant GlomerulopathyC4d Negative Transplant Glomerulopathy

0102030405060

no Ab Ab with noENDAT

Ab with ENDAT

6.7%19%

43%

C4d is negative in 60% of chronic active ABMR biopsies

Sis et al. Am J Transplant. 2009 Oct;9(10):2312-23.

No Abn=30

Ab with no En=21

Ab with En=30Tr

ansp

lant

Glo

mer

ulop

athy

scor

e ( c

g, m

ean

+ 95

% C

I)

n=81

p=0.01

p=0.53

Cum

ulat

ive

Surv

ival

No No AbAbAbAb with no Ewith no EAbAb with Ewith E

Post-biopsy time (months)

p=0.001p=0.001

Ab with E

No Ab or Ab with no E

60% C4d negative

Transcripts selectively associated with DSA:Endothelial and NK cell transcripts

Hidalgo et al. AJT 2010; 10: 1812–1822

NK

endothelial

CD56 ABMR

CD56 TCMR

CD68 ABMR

CD68 TCMR

CD3 ABMR

CD3 TCMR

0

1

2

3A. B.

C. D.

E.

C4d+ ABMR

C4d- ABMR

TCMR

CD56+ CD68+ CD3+

Mea

n nu

mbe

r of p

ositi

ve c

ells

in

five

per

itubu

lar c

apill

arie

s

p=0.006 p=0.03 p=0.09

CD3CD68CD56

NK cells and macrophages in antibody mediated peritubular capillaritis

Hidalgo et al. AJT 2010; 10: 1812–1822

Hirohashi and Colvin et al. Am J Transplant. 2012 Feb;12(2):313-21.  Akiyoshi and Colvin at al. Human Immunology Volume 73, Issue 12 2012 1226 - 1232

Role of complement and NK cells in antibody mediated rejection

NK cell

stain

AMR AMR + anti NK

A molecular classifier for diagnosing AMR

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

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.

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

*

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*

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 C4d30 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

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

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

Limited specificity of capillaritis

no rejection (n=453) borderline (n=105) TCMR (n=76)0

10

20

30

40

50

60

70

80

14.1

45.7

68.4

% c

ases

with

cap

illar

itis

Gibson et al. Am J Transplant. 2008 Apr;8(4):819-25.

AMR and vasculopathy