Humoral rejection: What the pathologist needs to knowHumoral rejection: What the pathologist needs to know

Heinz RegeleHeinz Regele

Clinical Institute of PathologyClinical Institute of Pathology

Banff Banff classification of renal allograftclassification of renal allograft rejection rejection

C4d Capillaritis Arterial necrosisATN

+ or or+

DSA

MHC I

anti-C4d

MHC II

Allograft Endothelial cell

C3

C3a

C3b

C4 C2

C2a

C3 Convertase

C2bC4a

C4b

C4d is a marker of antibody mediated rejection

C1qrs

C4b

Active

C4d

C4c

inactive

Factor I

MCP(CD46)

No C4d detectable in up to

76%76% of recipients with circulating antibodies!

Mauiyyedi, JASN 2002; Böhmig, JASN 2002; Koo Transplantation 2004Mauiyyedi, JASN 2002; Böhmig, JASN 2002; Koo Transplantation 2004

Sensitivity of C4d deposits in PTC for circulating anti-HLA-antibodies

31-95%31-95%

Sensitivity of C4d Staining for AlloantibodiesSensitivity of C4d Staining for Alloantibodies

Circulating DSA without C4dCirculating DSA without C4d

•Insufficient sensitivity of C4d detection method?

•Non complement-activating alloantibodies

•Variable Sensitivity of serological assays

C4d detection by IF on frozen sections vs. IC4d detection by IF on frozen sections vs. IHCHC on paraffin sections on paraffin sections26 biopsies with diffuse staining by IF on frozen sections26 biopsies with diffuse staining by IF on frozen sections

C.A. Seemayer et al, NDT 2007C.A. Seemayer et al, NDT 2007

C4d scoring adjusted for staining methodC4d scoring adjusted for staining methodSuggestion by the Banff Conference 2007Suggestion by the Banff Conference 2007

K Solez et al, AJT 2008K Solez et al, AJT 2008

C4d posN = 16

C4d neg/FCXM posN = 22

C4d neg/FCXM negN = 20

C4d Staining and FCXM (Flow-Cytometry X-Match) of Corresponding SeraC4d Staining and FCXM (Flow-Cytometry X-Match) of Corresponding Sera 113 Biopsies of 58 Renal Allograft Recipients113 Biopsies of 58 Renal Allograft Recipients

In 2 Patients severe rejection reversible by IA 4 allografts lost

1 allograft lost

G.A. Böhmig et al, JASN 2002G.A. Böhmig et al, JASN 2002

No alloantibodies detectable in

10-25%10-25% of recipients with C4d deposits!

Lederer, KI 2001; Mauiyyedi, JASN 2002; Böhmig, JASN 2002; Koo, Transplantation 2004; Lederer, KI 2001; Mauiyyedi, JASN 2002; Böhmig, JASN 2002; Koo, Transplantation 2004; Smith, JHLT 2005Smith, JHLT 2005

Specificity of C4d deposits in PTC for circulating anti-HLA-antibodies

93-96%93-96%

Specificity of C4d Staining for AlloantibodiesSpecificity of C4d Staining for Alloantibodies

C4d deposits without circulating DSAC4d deposits without circulating DSA

•Complement activation by ischemia/reperfusion?

•Adsorption of alloantibodies within the graft?

•Non-HLA alloantibodies

Complement Activation by Ischemia/ReperfusionComplement Activation by Ischemia/Reperfusion

C4d deposits in heart allografts are associated with morphologic signs of ischemic injury W.M. Baldwin et al, Transplantation 1999

Activation of complement via the classical pathway occurs in experimental ischemia/reperfusion injury in

• Heart• Skeletal muscle• Bowel

No C4d deposits induced by ischemic injury in early post-transplant kidney allograft biopsy

M. Haas et al, Transplantation 2002

Complement activation in (experimental) renal ischemia occurs via the alternative but not via the classical pathway

Complement Activation by Ischemia/Reperfusion in Complement Activation by Ischemia/Reperfusion in the Kidney?the Kidney?

C4d in cardiac allografts correlates with alloantibody: 21/24 BX from Pat with alloantibody are C4d positive and only 7/60 BX in alloantibody-negative recipients show C4d deposits

R.N. Smith et al, JHLT 2005

C4d Capillaropathy Intimal FibrosisGlomerulopathy

+ or or+

DSA

MHC I

anti-C4d

MHC II

Banff Banff classification of renal allograftclassification of renal allograft rejection rejection

T i m eT i m e

I n

t

e

n

s i

t

yI

n

t e

n

s

i

t y

Tissue injuryTissue injury

Development of chronic antibody mediated rejectionDevelopment of chronic antibody mediated rejection

Graft dysfunction Graft dysfunction

AntibodyAntibody

C4dC4d

Diagnostic thresholdDiagnostic threshold

BiopsySerology?

Protocol Biopsy?

(months-years)

C4d deposition in stably functioning graftsC4d deposition in stably functioning grafts

C4d deposition was observed in 4,4% of 551 renal allograft protocol biopsies but had no negative impact on outcome (median follow-up 3,5 years)

M. Mengel et al., AJT 2005

80% of protocol Bx in ABO-incompatible grafts were C4d positive. C4d is not correlated with injury in most ABO-incompatible grafts.

Haas et al., AJT 2006

Separate analysis of patientswith excellent 1y graft function

1. GFR ≥60 ml/min (MC equation) 2. 24h protein excretion ≤0.5 g

3. no dysfunction/indication biopsy

4. no desensitization or rejection treatment

164 recipients with >1year graft function1 year serial HLA Ab monitoring

Follow-up: median 69 months

130

34

Humoral response in stably functioning grafts

Bartel et al, Am J Transplant, in press

Excellent function during 1st year (n= 34)

Dysfunction during 1st year (n=130)

IgG HLA Ab in renal Tx recipients with excellent 1 year course

No difference to non-stable patients✔ Incidence✔ Binding strength✔ DSA frequency✔ C4d-fixation in vitro

Bartel et al, Am J Transplant, in press

% R

ecip

ien

tsMonths

2 6 12Months

2 6 120

10

20

30

40

50

0

10

20

30

40

50

P=0.4 P=0.8 P=0.2

P=0.3 P=0.7 P=0.5

% R

ecip

ien

ts

IgG alloreactivityComplement

activatingalloreactivity

Long-term outcome of 9 Ab+ recipientswith excellent 1-year graft performance

Bartel et al, Am J Transplant, in press

*patient died with functioning graft due to pancreatic cancer **de-novo membranous glomerulonephritis

Banff Banff classification of renal allograftclassification of renal allograft rejection rejection

C4d

+

DSA

MHC I

anti-C4d

MHC II

bbut no morphological lesions ut no morphological lesions

Alloantibody and/or Alloantibody and/or complement in stable complement in stable

grafts grafts

Transient/weak immune response very low risk of graft loss

Subclinical rejection, with a high risk of chronic allograft damage

and accelerated graft loss

Accommodation, acquired resistance of the graft against

persisting alloimmune reactions

?

?

Duration and intensity of humoral injuryDuration and intensity of humoral injury

Most serologic and biopsy studies on the impact of antibodies and complement on chronic allograft damage are based on single measurements

and rarely provide data on antibody binding strength/titer.

In 65 patients with DSA (26 with stable function and 39 failed) strength of DSA binding was strongly associated with late graft failure.

K. Mizutani et al., AJT 2007

Persistence of circulating antibody (in serial samples) significantly increased the risk of subsequent graft loss, was however in a few recipients also compatible with continuous stable function.

van den Berg-Loonen et al., Clin Transplants 2006

Alloantibody and/or Alloantibody and/or complement in stable complement in stable

grafts grafts

Transient/weak immune response very low risk of graft loss

Subclinical rejection, with a high risk of chronic allograft damage

and accelerated graft loss

Accommodation, acquired resistance of the graft against

persisting alloimmune reactions

Are capillary inflammatory lesions predictive of chronic rejection? Are capillary inflammatory lesions predictive of chronic rejection?

10/10 recipients with subclincal AMR showed accumulation of immune cells in peritubular capillaries (PTCitis) and 8/10 had glomerulitis. Subclinical AMR is associated with increase of cg, ci and ct in follow up Bx.

M. Haas et al., AJT 2006

In protocol biopsies, PTCitis at 3 months was associated with chronic antibody mediated rejection at 12 months.

E. Lerut et al., Transplantation 2007

Recipients without adaptive immune system

(RAG1 KO, athymic) or

MHC incompatible donor

Ab induced C4d on EC No early graft loss due to

acute ABMR

Chronic vascular injury

after 2-16 weeksin hearts and kidneys

(CTA and chronic Glomerulopathy)

EC response partially independent of

C-activation; activates distinctive signalling

pathways (akt, mTor…)

T. Jindra, J immunol 2008; S. Uehara, AJT 2006; M. Koch Transpl Immunol, 2008, K. Solez, AJT 2008

Anti-donor-MHC moAb

Molecular mechanisms of antibody/complement mediated EC injury Molecular mechanisms of antibody/complement mediated EC injury

SummarySummary

C4d is a reliable marker of humoral rejection in dysfunctioning grafts.

This might be due to accommodation or only transient and/or weak DSA reactivity

Complement deposition and/or circulating DSA are far less predictive of bad outcome when observed in recipients with stable graft function.

Sequential testing for detection of persisting DSA reactivity and assessment of concomitant histological lesions may help identifying patients with subclinical rejection and subsequent risk of accelerated graft loss.

New, additional markers for prospective discrimination between accommodation and rejection are required to further refine risk assessment.