1. ERYTHROCYTE C4d: A MARKER OF ANTIBODY MEDIATED REJECTION
T. Mohanakumar, Ph.D.
Department of Surgery, Pathology & Immunology
Washington University School of Medicine

2. 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

3. ANTIBODIES IN ORGAN ALLOGRAFTS
Colvin RB et al, Antibody-mediated organ-allograft rejection. Nat Rev Immunol 2005;5(10):807-17

4. 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

5. INCIDENCE OF AMR Acute _ Heart-10-30% Lung: unknown Kidney: 20-30%
Chronic AMR
Heart: 20-40%
Kidney: 30-40%

6. 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

7. 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:

8. MONITORING FOR AMR Clinical Parameters Histology Serum Markers
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

9. 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.

10. 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:
E-C4d/E-CR1 ratio = (MFI E-C4d – MFI Isotype Control) / (MFI E-CR1 – MFI Isotype Control)
Cardiac transplant study, Lee KC et al. Transplant Proc 2008;40:
% E-C4d = % of total RBCs with > 2 S.D. positive mean fluorescence shift (MFIanti-C4d - MFIisotype control IgG ) compared to healthy controls.

11. E-C4d IN LUNG TRANSPLANTATION

12. CHRONIC LUNG ALLOGRAFT REJECTION : CORRELATION WITH DEVELOPMENT OF DSA
Sundaresan S et al. Transplantation 1998;65(5):648-53

13. CHRONIC LUNG ALLOGRAFT REJECTION; CORRELATION WITH DEVELOPMENT OF ANTIBODIES TO SELF-ANTIGENS
K-alpha-1-tubulin
Collagen V

14. 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

15. OBJECTIVE
Determine the E-C4d by flow cytometry ( a non-invasive, 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

16. 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

17. 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

18. INCREASE IN % E-C4d FOLLOWING LUNG TRANSPLANTATION
Control ( %) , LTx Recipients ( %)

19. 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 ( %), anti-HLA positive ( %)
and anti-HLA negative ( %)

20. 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+ ( %) Kα1T- ( %)
Col V+ ( %) Col V- ( %)

21. INCREASE IN % E-C4d IN LTx RECIPIENTS WITH C3d DEPOSITION IN BIOPSY
C3d positive ( %) C3d negative ( %)

22. 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.

23. LIMITATIONS
Small cohort of patients and therefore can only be viewed as encouraging preliminary results.

24. Acknowledgements Lung Transplant Physicians
Fellows
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