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% 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 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

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 E-CR1 – 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 K-alpha-1-tubulin Collagen V

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 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

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 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 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 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