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ERYTHROCYTE C4d: A MARKER OF ANTIBODY
MEDIATED REJECTION
T. Mohanakumar, Ph.D.
Department of Surgery, Pathology & Immunology
Washington University School of Medicine
ANTIBODY MEDIATED REJECTION
(AMR)


Allograft rejection caused by antibodies
Directed against
– Donor-specific HLA molecules
– Non-HLA tissue specific antigens



Myosin, Vimentin, Collagen V (Heart)
K-alpha-1-tublin, Collagen V (Lung)
Vimentin, Collagen IV (Kidney)
– Blood group ABO isoagglutinins
ANTIBODIES IN ORGAN ALLOGRAFTS
Colvin RB et al, Antibody-mediated organ-allograft rejection. Nat Rev Immunol 2005;5(10):807-17
ACTIVATION OF GRAFT ENDOTHELIUM BY
ANTIBODY AND COMPLEMENT
Colvin RB et al, Antibody-mediated organ-allograft rejection. Nat Rev Immunol 2005;5(10):807-17
INCIDENCE OF AMR

Acute
_ Heart-10-30%
– Lung: unknown
– Kidney: 20-30%

Chronic AMR
– Heart: 20-40%
– Lung: unknown
– Kidney: 30-40%
B CELLS IN REJECTING ALLOGRAFTS
Zarkhin V et al. Characterization of intra-graft B cells during renal allograft rejection.
Kidney Int 2008;74(5):664-73
DIAGNOSTIC CRITERIA FOR AMR


Clinical Evidence of Acute Graft Dysfunction
Histological Evidence of Acute Capillary Injury
– Capillary endothelial swelling or denudation with congestion
– Macrophages or neutrophils in capillaries
– Interstitial edema and/or hemorrhage

Immunopathologic Evidence for Antibody Mediated Injury
– Ig + C3d and/or C4d or C1q by immunofluorescence
– CD68 positivity for capillary macrophages/C4d capillary staining
– Fibrin in vessels

Serological Evidence for DSA or Anti-HLA antibodies
Takemoto SK et al. National conference to assess antibody mediated rejection in solid
organ transplantation. Am J Transplant 2004;4:1022-41
MONITORING FOR AMR

Clinical Parameters
– Heart: Diminished ventricular ejection fraction
– Lung: Diminished FEV1
– Kidney: Diminished GFR

Histology
– Ab and/or Complement deposition (C3d, C4d)

Serum Markers
– Donor specific antibody (DSA) to HLA
– Antibodies to self antigens
ERYTHROCYTE BOUND C4d (E-C4d)


Increased cell bound complement activation product,
C4d, detected on the surface of erythrocytes.
Have been shown to correlate with
– Disease activity in systemic lupus erythematous (SLE)
– Acute rejection after cardiac transplantation


E-C4d have increased half-life compared to serum C3
and C4d
More reliable tool compared to serum/biopsy C3d or
C4d.
METHODS OF E-C4d DETECTION


Erythrocytes from 5 cc fresh EDTA blood were analyzed by
indirect immunofluorescence using flow cytometry
Calculation of E-C4d :
– Surface expression E-C4d = MFI C4d/CR1 – MFI Isotype Control
SLE study: Manzi S et al, Arthritis Rheum 2004;50:3596-604
– E-C4d/E-CR1 ratio = (MFI E-C4d – MFI Isotype Control) / (MFI ECR1 – MFI Isotype Control)
Cardiac transplant study, Lee KC et al. Transplant Proc 2008;40:2638-2642
– % E-C4d = % of total RBCs with > 2 S.D. positive mean fluorescence
shift (MFIanti-C4d - MFIisotype control IgG ) compared to healthy controls.
E-C4d IN LUNG TRANSPLANTATION
CHRONIC LUNG ALLOGRAFT
REJECTION : CORRELATION WITH
DEVELOPMENT OF DSA
Sundaresan S et al. Transplantation 1998;65(5):648-53
CHRONIC LUNG ALLOGRAFT REJECTION;
CORRELATION WITH DEVELOPMENT OF
ANTIBODIES TO SELF-ANTIGENS
Collagen V
conc. of anti collagen
antibodies (microg/ml)
K-alpha-1-tubulin
800
600
400
200
0
normal
BOS -ve
BOS +ve
E-C4D IN ASSESSING AMR



Breakdown products of complement activation which are
bound to erythrocytes
Have been shown to correlate with disease status in SLE, used
as an adjunct to monitor SLE
Erythrocyte bound form is more stable and has an increased
half-life
OBJECTIVE


Determine the E-C4d by flow cytometry ( a noninvasive, simple and reliable method for diagnosing
AMR)
Correlate % E-C4d with
Development of DSA
 Development of Antibodies to self-antigens
 C3d staining in biopsy specimen

MATERIALS AND METHODS

22 post-lung transplant study patients
15 normal healthy adult volunteers

Antibodies to HLA (Luminex)

Antibodies to Self-Antigens (KA1T and Col V ,ELISA)
C3d deposition by Immunohistochemistry


MATERIALS AND METHODS

E-C4d: Immunofluorescence by flow cytometry
10ul fresh anticoagulated (EDTA) blood
– Murine monoclonal anti-human C4d or isotype control
– Fluorescein isothiocyanate conjugated goat anti-mouse IgG


% E-C4d was calculated by determining the percentage of
total RBCs with a positive mean fluorescence shift (MFIanti-C4d
- MFIisotype IgG1)
Two standard deviation from mean fluorescence shift obtained
in the control population was used as cutoff value to calculate
% E-C4d
INCREASE IN % E-C4d FOLLOWING LUNG
TRANSPLANTATION
p=0.02
Control (3.7+2.2%) , LTx Recipients (19.9+9.7%)
INCREASE IN % E-C4d IN LTx RECIPIENTS
WITH DSA
1 vs 2: p=0.02
1 vs 3: p=0.03
2 vs 3: p=0.1
1 vs 2+3: p 0.02
DSA positive (34.1+5.9%), anti-HLA positive (13.9+8.4%)
and anti-HLA negative (17.7+6.7%)
INCREASE IN % E-C4d IN LTx RECIPIENTS WITH
ANTIBODIES TO Kα1T AND/OR COL-V
Kα1T: p=0.02
Col V: p=0.03
Kα1T+ (23.0+10.5%) Kα1T- (3.4+1.4%)
Col V+ (22.9+9.7%) Col V- (3.4+1.4%)
INCREASE IN % E-C4d IN LTx RECIPIENTS WITH
C3d DEPOSITION IN BIOPSY
p=0.01
C3d positive (26.1+10.1%) C3d negative (15.5+6.8%)
SUMMARY AND CONCLUSION

Increase in % E-C4d are found in patients with
– DSA
– Antibodies to Kα1T and Col V
– C3d deposition in biopsy

% E-C4d may be useful as a simple method for
monitoring AMR following human lung transplantation.
LIMITATIONS

Small cohort of patients and therefore can only be
viewed as encouraging preliminary results.
Acknowledgements
Fellows
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Sudhir Sundaresan, MD
Scott I Reznik, MD
Michael A Smith, MD
Andres Jaramillo, PhD
Ryan Fields, MD
Trudie Goers, MD
Toru Higuchi, MD
Takahiro Maruyama, MD
Kishore Narayanan,PhD
Naohiko Fukami, MD, PhD
Deepti Saini, PhD
Ilias H. Basha,M.D
Sabarinathan Ramachandran, PhD
Ankit Bharat, MD
Anguswamy Nataraju,Ph.D
Swarup Trivedi,M.D,Ph.D
Masashi Takenaka,M.D
Lung Transplant Physicians
• G Alexander Patterson, MD
• Elbert P Trulock, MD
• Michael J Walter , MD
• Ramsey R Hachem, MD
Renal Transplant physicians
•Martin Jendrisak,M.D
•Surendra Shenoy,M.D
Lung Transplant co-ordinator
• Aviva Aloush
HLA
Laboratory