PostTransplant Monitoring for the Development of AntiDonor HLA Antibodies

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 20043512678-2680
4
UNOS Update 2004 Nov-Dec pgs 20-23.
UNOS Update November-December 2004
5
American Journal of Transplantation 2002 2803
6
ALLOGRAFT SURVIVAL
Transplants performed between 1995-2004
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

• C4d is the stable (target bound) remnant of
  classical complement activation and can reveal
  humoral attacks against endothelial cells.

A J T 4 ( 3) cover March 2004
American Journal of Transplantation 2004
4331-318
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The Missing Link Complement C4d in graft
capillaries

• Prevalence of C4d
• Delayed renal function, pre-sensitized patients
  50 biopsies diffuse or focal staining of
  interstitial, peritubular capillaries
• Acute rejection 30 biopsies C4d
• Chronic rejection 60 biopsies C4d

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Capillary Staining for C4d
American Journal of Transplantation 3(6) front
cover, June 2003.
Racusen LC et al. Am J Transplant 2003 708-714.
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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

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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

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Post-Transplant Which way to go?

• Antibodies humoral arm of the immune system
• Cells cellular arm of the immune system

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Antibodies
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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)

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13th workshop
100
90
No HLA (806)
80
Percent Graft Survival
Overall (964)
70
HLA Antibody (158)
60
Plt0.0001
50
DECEASED DONOR
0
1
2
3
4
Years after Testing
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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.

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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

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Methods

• Study Samples
• 5,602 post-transplant sera collected from East
  Carolina University transplant patients.
• Specimens were processed, stored at -80oC until
  testing.
• An average of 12 samples per patient were tested
  for the presence of HLA antibodies.

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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 LabScreenTM 100
  Luminex.
• Increased fluorescence intensity reflects
  increased levels or strength of antibody.

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Demographics of ECU Study Patients
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Example of a Patient with a Functioning Graft
5/15/08
Cr

• Tested on 32 occasions for antibody
• Negative for Class I and Class II
  (green rectangles)

HLA Antibody
Strong HLA Antibody (High Levels)
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Other Functioning Patients
Years Posttransplant
5/14/08
4/11/08
7/22/08
5/27/08
1/07/08
7/1/08
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Other Functioning Patients
Years Posttransplant
2
1
3
4
5
5/19/08
4/1/08
3/18/08
NDSA
2/1/08
S6
NDSA
10/1/99
5/6/08
S8
NDSA
2/6/01
5/5/08
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Patients with Antibody but Functioning Graft
Years Posttransplant
1
2
3
4
5
TX date
DSA, NDSA
9/4/07
NDSA
DSA
5/1/08
NDSA
DSA
8/11/08
NDSA
DSA
6/3/08
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Patients with DSA and Graft Failure
Years Posttransplant
1
2
3
4
5
DSA
NDSA
DSA
NDSA
DSA
NDSA
DSA
DSA
R29
4/22/04
DSA
NDSA
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Months after transplant
Patient A30,-, B42,-, DR8,-, DQ7,- Donor
A3,30, B42,58, DR7,8, DQ2,7
10
20
30
40
50
DQ2
DQ2
SCr (mg/dL)
DSA B58, DQ2
Cyclosporine Level
B57
NDSA A23, A24, B53, B57, B63
SCr
B58
A23
A24
120
100
80
Cyclosporine
(ng/mL 10 )
40
40
20
0
30
20
(mg/bid)
Cellcept
10
0
16
Prednisone
(mg/bid 100)
12
8
50
(mg/qd )
30
10
0
10
20
30
40
50
Rebellato et al, Clinical Transplants 2006, p. 245
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Patient R24 A1,2B61,62 DR4,12DQ7,X
Donor A1,32B35,61DR12,7DQ2,7
Months post-transplantation
Cyclosporine (mg bid)
Rapamycin (mg qd)
Cellcept (mg bid)
Prednisone (mg qd) or Solumedrol
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Patient S209 A2,32B35,61DR7,8DQ2,4
Donor A2,3B27,44 DR1,4DQ5,7
Months post-transplantation
Cyclosporine (mg bid)
Cellcept (mg bid)
Prednisone (mg qd) or Solumedrol
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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.

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Immunosuppression Modulation to Reduce Anti-Donor
Antibody Levels after Kidney Transplantation

• Study Description
• Kidney transplant recipients enrolled in the
  post-txpt 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 wont exceed the manufacturers
  recommended.

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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).

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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.

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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.

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Immunosuppression Modulation to Reduce Anti-Donor
Antibody Levels after Transplantation

• Possible Study Candidates
• 265 stable recipients monitored
• 33 DSA (12)

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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
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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
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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).

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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.

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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