Post-Transplant Monitoring for the Development of Anti-Donor HLA Antibodies
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1 Post-Transplant Monitoring for the Development of Anti-Donor HLA Antibodies Lorita M Rebellato, Ph.D., D (ABHI) Associate Professor Department of Pathology The Brody School of Medicine at ECU Scientific Director of the Histocompatibility Laboratory Pitt County Memorial Hospital Greenville, North Carolina
2 Post-Transplant Monitoring for the Development of Anti-Donor HLA Antibodies Objectives: 1) Literature review of outcomes in renal transplantation 2) Describe the results of post-transplant follow up studies performed at the ECU 3) Describe our initial attempt to modulate antibody production in this transplant population
3 The First Successful Kidney Transplantation on December 23, 1954 Morris, P. J. N Engl J Med 2004;351:
4 UNOS Update 2004 Nov-Dec; pgs UNOS Update November-December 2004
5 American Journal of Transplantation 2002; 2:803
6 ALLOGRAFT SURVIVAL Organ 1 Year 5 Years 10 Years Deceased Donor Kidney 91% 68% 42% Living Donor Kidney 95% 79% 54% Liver 83% 67% 52% Heart 87% 72% 49% Lungs 84% 48% 19% Transplants performed between ; OPTN/SRTR Annual Report 2006
7 Factors Contributing to Chronic Failure of Renal Allografts
8 Humoral Contribution to Rejection/Failure of Grafts Immune system can use cellular and humoral mechanisms to respond to foreign antigens. Solid organ transplantation, histological studies during rejection showed lymphocytes infiltrating the graft, but no humoral effector molecules. Until recently, transplant rejection was believed to be cell mediated, and humoral rejection was believed to play no role.
9 The Missing Link: Complement C4d in graft capillaries Ten plus years after its first description by Feutch, capillary deposition of complement C4d in graft biopsies is recognized as a marker of antibody-mediated alloreactivity. Antibody Mediated Rejection: Graft dysfunction; presence of anti-donor HLA antibody in the circulation and C4d staining in the biopsy.
10 The Missing Link: Complement C4d in graft capillaries A J T 4 ( 3): cover ; March 2004 C4d is the stable (target bound) remnant of classical complement activation and can reveal humoral attacks against endothelial cells. American Journal of Transplantation 2004; 4:
11 The Missing Link: Complement C4d in graft capillaries Prevalence of C4d: 1) Delayed renal function, pre-sensitized patients: 50% biopsies diffuse or focal staining of interstitial, peritubular capillaries 2) Acute rejection: 30% biopsies C4d+ 3) Chronic rejection: 60% biopsies C4d+
12 Capillary Staining for C4d American Journal of Transplantation 3(6): front cover, June Racusen LC et al. Am J Transplant 2003;
13 Alloantibodies Associated With Poor Kidney Transplant Outcomes Donor-specific antibody (DSA) Anti-HLA Anti-endothelial cell antibodies Anti-human MHC Class I chain-related genes (MICA,B) Antibodies against tissue-specific antigens ABO isoagglutinins Non donor-specific antibodies Anti-HLA antibodies Anti-MICA antibodies
14 Alloantibodies: Mechanisms of Injury Alloantibody-induced Alloantibody-induced acute injury of the graft chronic injury Lytic activation of the C : Sub-lytic activation of C : Endothelial cell injury Activation of endothelium, Endothelial cell lysis platelets and MØ Activation of platelets Endothelial cell apoptosis and the clotting system Production of pro-fibrotic Enhancement of inflammation growth factors: by recruitment of neutrophils Basic fibroblast growth factor monocytes, T and B-cells Platelet-derived growth facto Thrombospodin-1
15 Post-Transplant: Which way to go? Antibodies: humoral arm of the immune system Cells: cellular arm of the immune system
16
17 Methods to Detect Anti-HLA Antibodies ELISA and Flow PRA Methods (solid Phase): Purified HLA antigens: 1) Antigens attached to an ELISA tray well 2) Antigens attached to beads and read in the flow cytometer (Flow PRA) or Luminex 3) Mixture of antigens or single antigens attached to wells or beads (for highly sensitized patients)
18 Percent Graft Survival P< th workshop No HLA (806) Overall (964) HLA Antibody (158) DECEASED DONOR Years after Testing
19 Post-Transplant Monitoring for the Development of HLA Antibody in Kidney Txpt Recipients: ECU Experience Study began: August 1999 Informed Consent 7 cc red top tube First 6 months: samples collected during clinic visits Letters sent to study patients During collection of routine labs; extra red top tube collected for the study.
20 Methods Study Patients: IRB-approved protocol; 350 kidney transplant recipients studied Immunosuppression: 1) Thymoglobulin or anti-il2r monoclonal antibody (Zenapax or Simulect). 2) Calcineurin inhibitor (Neoral or Gengraf or Prograf) 3) Mycophenolate Mofetil 4) Steroids
21 Methods Study Samples: 5,602 post-transplant sera collected from East Carolina University transplant patients. Specimens were processed, stored at -80 o C until testing. An average of 12 samples per patient were tested for the presence of HLA antibodies.
22 Methods Antibody Detection Assay: Sera first screened for HLA Class I and II antibodies using LABScreen mixed beads. Samples that tested positive by screening were further tested by single antigen beads (One Lambda) and analyzed on the LabScreen TM 100 Luminex. Increased fluorescence intensity reflects increased levels or strength of antibody.
23 Demographics of ECU Study Patients
24 Example of a Patient with a Functioning Graft S7 Class I Class II 9/11/00 S9 3/13/01 S4 2/8/00 Years Posttransplant Tested on 32 occasions for antibody Negative for Class I and Class II (green rectangles) 1.0 5/15/ Cr 1.7 S2 5/11/99 S10 6/7/01 HLA Antibody Strong HLA Antibody (High Levels)
25 S7 Other Functioning Patients Years Posttransplant Class I Class II 9/11/ /14/08 S5 8/30/99 S9 3/13/01 S4 2/8/00 S2 5/11/99 S10 6/7/ /11/ /22/ /27/ /07/ /1/08
26 R12 6/3/00 R2 9/7/99 S1 5/27/99 S3 8/30/00 S6 10/1/99 S8 2/6/01 Other Functioning Patients Years Posttransplant NDSA NDSA NDSA 1.5 5/19/ /1/ /18/ /1/ /6/ /5/08
27 Patients with Antibody but Functioning Graft TX date R43 7/17/01 Years Posttransplant DSA, NDSA 5.5 9/4/07 R33 NDSA DSA 11/29/ /1/08 R15 11/23/00 NDSA DSA 1.8 8/11/08 R24 5/25/04 DSA NDSA 2.4 6/3/08
28 GF13 6/6/00 Patients with DSA and Graft Failure GF11 8/11/00 R19 10/24/02 Years Posttransplant HD HD DSA NDSA HD DSA NDSA DSA NDSA GF6 11/10/03 R29 4/22/04 GF16 5/15/00 HD DSA HD DSA HD DSA NDSA
29 Patient: A30,-, B42,-, DR8,-, DQ7,- Donor: A3,30, B42,58, DR7,8, DQ2,7 DSA: B58, DQ2 NDSA: A23, A24, B53, B57, B63 SCr Months after transplant DQ2 B57 B58 A23 A24 50 SCr (mg/dl) Cyclosporine Level Cyclosporine Cellcept Prednisone (ng/ml * 10 ) (mg/bid) (mg/bid * 100) (mg/qd ) Rebellato et al, Clinical Transplants 2006, p
30 Patient R24 A1,2;B61,62; DR4,12;DQ7,X Donor: A1,32;B35,61;D R12,7;DQ2,7 Cyclosporine (mg bid) SCr Class I Class II (DQ2) Months post-transplantation SCr (mg/dl) Cyclosp level (mg/ml*10) Rapamycin (mg qd) Cellcept (mg bid) Rapa Level (mg/ml) Prednisone (mg qd) or Solumedrol Solumedrol
31 Patient S209 A2,32;B35,61; DR7,8;DQ2,4 Donor: A2,3;B27,44; DR1,4;DQ5,7 Cyclosporine (mg bid) Class II (DQ7,8,9) Class I Months post-transplantation SCr SCr (mg/dl) Cyclosp Level (mg/ml*10) Cellcept (mg bid) Prednisone (mg qd) or Solumedrol Solumedrol
32 Immunosuppression Modulation to Reduce Anti-Donor Antibody Levels after Kidney Transplantation Study Rationale: Patients that develop DSA are at risk for rejection and graft failure. It takes time after antibodies are detected until graft loss. Our hypothesis is that antibody responses can be manipulated with an appropriate dose and type of immunosuppression.
33 Immunosuppression Modulation to Reduce Anti-Donor Antibody Levels after Kidney Transplantation Study Description: Kidney transplant recipients enrolled in the posttxpt monitoring protocol. Recipients actively making donor-specific HLA antibody post-transplantation will be asked to participate in this study. Additional dose of a antiproliferative drug will be given. Dose won t exceed the manufacturers recommended.
34 Immunosuppression Modulation to Reduce Anti-Donor Antibody Levels after Kidney Transplantation Maintenance Immunosuppression: 1) Steroids 2) Calcineurin Inhibitors: (Cyclosporine and Tacrolimus) 3) Antiproliferative agents: Azathioprine; MPA (Mycophenolate mofetil (MMF) and Enteric-coated Mycophenolate sodium (EC- MPS).
35 Immunosuppression Modulation to Reduce Anti- Donor Antibody Levels after Transplantation Maintenance Immunosuppression: Needed to prevent rejections Typically the doses are slowly decreased over time to help lower the overall risk of infection and malignancy. Lowering immunosuppression dose is needed to diminish the side effects that are often associated with a particular drug.
36 Immunosuppression Modulation to Reduce Anti- Donor Antibody Levels after Transplantation Study Goal: By increasing the dose of the antiproliferative drug MPA, we will reduce the DSA strength to ZERO.
37 Immunosuppression Modulation to Reduce Anti- Donor Antibody Levels after Transplantation Possible Study Candidates: 265 stable recipients monitored 33 DSA (12%)
38 Example of a patient treated with higher dose MPA to reduce DSA Recipient HLA: HLA-A2,32; B35,61: DR7,8; DQ2,4 Donor HLA: HLA-A2,3; B27,44; DR1,4; DQ5,7 Status post-txpt LSM Class I LSM Class II SA Class II Five months Neg Neg Neg 629 Highest Raw Seven months Eighteen months Neg Pos DQ7, 8, Neg Pos DQ7, 8, Baseline Study Pos NDSA Pos Weak DQ7, Weeks Weak pos Pos Neg 402
39 Example of a patient treated with higher dose MPA to reduce DSA Recipient HLA: HLA-A34,66; B44,63; DR1,15; DQ5,6 Donor HLA: HLA-A29,68; B51,58; DR8,X; DQ4,X Status post-txpt LSM Class I LSM Class II SA Class II Four months Pos (NDSA) Neg Neg 877 Highest Raw One year Pos (NDSA) Pos DQ2, 4, Three years (Baseline) Eight weeks Twenty-one Weeks Pos (NDSA) Pos DQ2, 4, 7, DR12,16 Pos (NDSA) Pos (NDSA) Pos DQ2, 4, 7, DR12, 16, DP Pos DQ2, 4,
40 Summary 93% of patients with graft failure had HLA antibodies at the time of failure. 33% had pre-existing Ab 75% developed donor specific Ab 14% of patients with a functioning graft developed de novo antibodies and 12% developed donor specific Ab. HLA-DQ antibodies were prevalent (mostly DSA).
41 Conclusions HLA antibodies are produced before renal failure and before the elevation of serum creatinine. Our data support further studies to evaluate prospective monitoring for HLA antibody. Prospective monitoring may improve our ability to prevent antibody mediated acute rejection episodes and antibody mediated chronic rejection. Understanding and modifying the antibody response is critical to extending the longevity of transplanted organs.
42 Acknowledgments Department of Pathology Kim Briley, Dr. Paul Catrou, PCMH HLA Laboratory Staff Specimen Acquisition Staff Department of Surgery Drs. Carl Haisch and Kathryn Verbanac ECU Division of Nephrology Eastern Nephrology Associates Terasaki Foundation Miyuki Ozawa, Dr. Paul Terasaki
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