1. Antibody-Mediated Rejection in Renal transplantation

2. Homo transplants differ not only in the strength of rejection, but also in the nature and location of the phenomena induced by rejection. This conclusion must not be regarded as a cause for discouragement. On the contrary, it should spur further research.
—Jean Hamburger

3. Antibody-mediated rejection
Alloantibodies are now appreciated as important mediators of acute and chronic rejection
Alloantibodies to HLA class I or II and other antigens expressed by endothelium cause a variety of effects on renal transplants, ranging from acute to chronic rejection, and even apparent graft acceptance (accommodation).
It differs in pathogenesis, or “nature,” from T cell–mediated rejection.

4. Antibody-mediated rejection
Alloantibodies preferentially attack a different “location,” namely
the peritubular and glomerular capillaries, in contrast to T cells, which characteristically infiltrate tubules and arterial endothelium.
Antibody-mediated rejection generally has a worse prognosis and requires a different form of therapy than the usual T cell–mediated acute rejection

5. Antibody-mediated rejection and C4d
C4d is a fragment of C4b, an activation product of the classic complement pathway
C4d remains covalently bound in the tissue for several days after complement activation
C4d deposition is strongly associated with circulating antibody to donor HLA class I or class II antigens
C4d is currently the best single marker of complement-fixing circulating antibodies to the endothelium.
Feucht et al Kidney Int 1993; 43:1333–1338. Mauiyyedi S et al. J Am Soc Nephrol 2002 ; 13: 779–787. Bohmig GA et al. J Am Soc Nephrol 2002; 13: 1091–1099

6. Complement activation pathways
Complement activation pathways. The classic pathway is relevant to antibody-mediated rejection

7. Antibody-mediated rejection
Four forms of antibody-mediated graft injury have been defined
Hyperacute rejection
Acute humoral rejection
Chronic humoral rejection
Accommodation

8. Stages of Antibody Mediated rejection
Stages I to II represent accommodation, stage III represents subclinical humoral rejection, and stage IV represents CHR.
The dashed lines for antibody and C4d deposition are meant to reflect the possibility of intermittent positivity over time.
Inevitability of progression is not meant to be implied by the term “stages.” The rate of progression is likely to be variable, and the early stages, I and II, are reversible. At any stage, the antibody/ C4d may become negative, in which case the process is inactive.
Colvin RB. J Am Soc Nephrol 18: 1046–1056, 2007

9. Hyperacute Rejection

10. Definition Hyperacute rejection
Hyperacute rejection arises within minutes or hours in presensitized patients who have circulating HLA, ABO, or other alloantibody-to-donor endothelial surface antigens

11. Hyperacute Rejection
The pathology of hyperacute rejection overlaps completely with AHR.
Deposition of C4d occurs in PTC and glomeruli, just as in AHR
Early biopsies may be negative for C4d, presumably because of lack of access of C4 to the site (vasoconstriction) or enough time for sufficient amount to be deposited.

12. Hyperacute rejection
Mediated by Rapid thrombotic occlusion of the vasculature of the transplanted allograft
Occurs within minutes to hours after host blood vessels are anastomosed to donor vessels
Mediated predominantly by IgG antibodies directed toward foreign protein molecules, such as MHC molecules
Result from prior exposure to alloantigens from blood transfusions, pregnancy, or previous transplantation

13. Acute Humoral Rejection

14. Acute humoral rejection
Definition
Rapid loss of graft function (days), any time after transplantation
An acute loss of graft function that often arises in the first few weeks after transplantation
Cannot be distinguished from cell-mediated rejection on clinical grounds

15. Acute humoral rejection
Antibody-mediated rejection frequently undiagnosed
Lack of typical morphologic characteristics
C4d immunohistochemistry
C4 is an element of the complement cascade
Its degradation product (C4d) adheres to endothelial cells
Can be detected by pathologist

16. Acute humoral rejection
Classification of rejection as either “cellular” or “humoral” is flawed
Significant overlap exists
1 Montgomery R, Zachary AA. Transplantation 2000;70:887–894, 2 Becker YT et al , Am J Transplant 2004;4:

17. Diagnostic criteria for acute antibody-mediated rejection (AHR)
Morphologic evidence of acute tissue injury acute tubular injury
neutrophils and/or mononuclear cells in PTC and/or glomeruli and/or capillary thrombosis fibrinoid necrosis/intramural or transmural inflammation in arteries
Immunopathologic evidence for antibody action C4d and/or (rarely) immunoglobulin in PTC Ig and complement in arterial fibrinoid necrosis
Serologic evidence of circulating antibodies to donor HLA or other anti-donor endothelial antigen
Cases that meet only two of the three numbered criteria are considered suspicious for AHR. Acute cellular rejection may also be present.
Colvin RB. J Am Soc Nephrol 18: 1046–1056, 2007

18. Patterns of Rejection in ABO Incompatible Transplants
AMR
Cellular
Accommodation

19. Therapeutic Options For The Treatment Of AMR
Antibody Reduction
Immunomodulation
Plasmapheresis/IA
IVIg
IVIg
ATG
IL-2R blockers
Fk 506, Rapamycin
MMF/DSG
CAMPATH?
B-cell Modulation
Splenectomy
Anti-CD20
Cytoxan

20. Antibody Reduction Therapy
High dose IVIG (1-2 gms/kg)
Mechanism:
Anti-idiotypic networks probably important
Many putative immunomodulatory pathways identified
Advantages:
In vitro test for predicting efficacy
Ease of administration?
Disadvantages:
Non-responders
Different techniques required to follow DSA titers
Less rapid Ab removal, unproven for high-titer DSA
Toxicity & batch-to-batch variability
Unproven for ABOi Tx

21. Antibody Reduction Therapy
Plasmapheresis/Low Dose IVIg (100 mg/kg)
Mechanism:
Rapid reduction in anti-HLA or isoagglutinin Ab
Induces donor specific unresponsiveness (HLA) or accommodation (ABOI)
Advantages:
Predictable kinetics of plasmapheresis
No evidence of “nonresponders”
Able to easily follow DSA levels during/after therapy
Disadvantages:
DSA may rebound between treatments or if discontinued
Treatment may be prolonged and immunosuppressive
Expensive and resource intensive

22. Rituximab Monoclonal anti-CD20 antibody (found on B cells)
May improve outcomes in antibodymediated acute rejection episodes
1 Montgomery R, Zachary AA. Transplantation 2000;70:887–894, 2 Becker YT et al , Am J Transplant 2004;4:

23. B-Cell Modulation Anti-CD20 Mechanism:
Rapid ablation of the peripheral B-cell compartment
Advantages:
Probably reduces precursor cells responsible for clonal expansion during AMR
May produce more effective antibody reduction when combined with plasmapheresis or IVIG
Well-tolerated, little apparent toxicity
Effect on the immune system is temporary (6-months)
Disadvantages:
Plasma cells persist in the spleen
May not, on its own, reduce DSA titers during AMR
Immunosuppressive

24. Case Study: AMR in (+) Cytotoxic XM with High Titer Anti-HLA DSA
DSA titer
512
Cr 6.5
PP/ CMVIg
PRA
128
100
Cr 4.4
100
100
100
120 98 90
Cr 2.1
Cr 1.5
Cr 1.6 85 80
100 70
DSA titer 80
PRA 60 64 58 50 60
Anti-CD20
CD20=0
CD19=0
CD20=23.7 40 40 32 32 30 Tx 20 20 16 16 16 16 16 8 8 8 8 8 8 10 4 4 4 2 4 4 2 2 1 1 1
-19
-17
-15
-12 -8 -7 -6 -5 -4 -3 -2 -1 +2 +3 +4 +5 +6 +7 +8 +9
+11
+12
+13
+16
+18
+19
Days from Transplant

25. B-cell Modulation Splenectomy Mechanism:
Reduces plasma cells, precursor cells, B-cell immune surveillance capabilities
Advantages:
Can be performed using minimally invasive techniques
May produce more effective antibody reduction when combined with plasmapheresis or IVIG
Disadvantages:
Life-long risk of sepsis from encapsulated bacteria
Does not appear on its own to reduce DSA titers
Effect on immune system is permanent

26. The Effect of Splenectomy on Anti-Blood Group Ab
PP/IVIg
512
256
Splenectomy 4 3
Anti-A Titers (1:X)
Serum Creatinine
(mg/dL)
128 Tx 2 64 1 32 16
-28
-23
-21
-18
-16-15
-14
-12
-10 -6 -4 -3 -2 -1 3 5 7 8 10 12 14 17 21 25 27 31
32 34 40 42 45
Day With Respect to Transplant

27. Targets of Strategies for Antibody Removal
Plasmapheresis/IVIG
Plasma cells
Splenectomy
Clonal Expansion
B-cells &
Pre B-cells
Anti-CD20

28. Acute De Novo AMR Occurs in 4-6% of transplants (80-100% fail)
By definition the current XM is negative
Risk factors include: + historic XM,
history of sensitizing event(s), high risk
donor/recipient combination
Historically suspected only after there is a poor
response to anti-lymphocytic agents
Diagnosis should be made by histology and
demonstration of the appearance of DSA

29. PP/CMVIg Treatment Protocol for Acute De Novo AMR
Plasmapheresis – single plasma volume exchange
Steroid bolus
-OR-
a-thymocyte globulin
IVIG – 100mg/kg following each PP treatment
(CMV hyperimmune globulin)
PP/Ig
PP/Ig
PP/Ig
PP/Ig
PP/Ig
Dx of
AMR 2 4 6 8
Time Relative to Initiation of Therapy
(Days)
Heparin
D/C FK 506
High Grade:
Johns Hopkins InKTP

30. De Novo AMR: Renal Allograft Function5 10 15
Serum Creatinine
(mg/dL)
Nadir
Rejection
1 Week
1 Month
Current *
* p<0.001
: 22 recipients
of deceased or live donor
kidney transplants
with AMR by Bx or DSA
treated with PP/IVIg
Mean f/u: 5 1/2 years
Median
Nadir Cr
(IQR)
Cr at AMR
1 week Cr
1 month Cr
Current Cr
3.3
(2.2 – 7.1)
6.4
(3.2 – 9.3) 4
(2.3 – 7.9)
1.9
(1.5 – 3.0)
1.5
(1.2 – 2.1)
Johns Hopkins InKTP

31. PP/CMVIg Treatment for De Novo AMR10 20 30 40 50 60 70 80 90
100
365
730
1095
Time (days)
Live donor
Deceased donor
100
100
Allograft Survival 90 90 80 80 70 70
p = NS
Live Deceased 60 60
p = NS 50 50
1-Year: % % 40 40 30 30 20 20
3-Year: % % 10 10
Add rate of failure with protocol necessitating Rituximab therapy (don’t include patients who received it for severe AMR)
365
365
730
730
1095
1095
Time (days)
Time (days)
5-Year: Overall 81.1%
Live donor
Deceased donor
Live donor
Deceased donor
Kaplan-Meier Estimate of Graft Survival for recipients who
developed de novo AMR and were treated with PP/CMVIg therapy
Johns Hopkins InKTP

32. Bx and DSA Proven De Novo AMRLD DD p n 5 13
Median Days to AMR
(Range) 11
(8-253) 9
(7-50)
0.25
Median Months F/U
13.6
(4-76)
11.2
(3-89)
0.77
Johns Hopkins InKTP

33. De Novo Renal Function Johns Hopkins InKTP15 12 9
Serum Creatinine (mg/dL) 6 3 LD DD
Creatinine at Biopsy
Creatinine 1 week
Creatinine 1 mo
Current Cr
P=NS for comparison between groups at each timepoint
Johns Hopkins InKTP

34. De Novo AMR Allograft Survival
100.00
90.00
80.00
70.00
60.00
Survival
(%)
50.00
40.00
30.00
20.00
10.00
0.00 6 12 18 24 30 36
Time (Months)
Live
Deceased
Johns Hopkins InKTP

35. Rejection and Clinical Outcomes Following (+) XM and ABOi
POSITIVE CROSSMATCH
ABO INCOMPATIBLE
# OF PATIENTS 86
# OF PATIENTS 28 1 2 3 31 12 5 1 2 3 4 1
Previous Txs
Previous Txs
AMR
CELLULAR REJECTION
27/86 (31%)
26/86 (30%)
AMR
CELLULAR REJECTION
3/28 (11%)
4/28 (14%)
SUBCLINICAL AMR
SUBCLINICAL CELLULAR
7/86 (8%)*
16/86 (19%)
SUBCLINICAL AMR
SUBCLINICAL CELLULAR
0/28 (0%)
7/28 (25%)
1-YEAR GRAFT SURVIVAL
3-YEAR GRAFT SURVIVAL
89.8%
80.9%
1-YEAR GRAFT SURVIVAL
3-YEAR GRAFT SURVIVAL
92.9%**
92.9%
1, 3, 6, 12 mos
**1 death WNE
1 Noncompliance
Johns Hopkins InKTP

36. (+) XM vs. De Novo AMR De Novo Desensitized p n 18 31
Median Days to AMR
(Range) 10
(7-253) 20
(2-634)
0.16
Median Months F/U
12.4
(3-89)
14.1
(0.6-65)
0.76
Johns Hopkins InKTP

37. (+) XM vs. De Novo AMR Outcomes3 6 9 12 15
Serum Creatinine (mg/dL)
PP/CMVIg Desensitized
De Novo Rejection
Creatinine at Biopsy
Creatinine 1 week
Creatinine 1 mo
Current Cr
P=0.04
P=0.01
P=0.002
P=NS between groups at current timepoint
Johns Hopkins InKTP

38. Allograft Survival After AMR (+) XM vs. De Novo
100.00
90.00
80.00
70.00
p=NS
60.00
Survival
(%)
50.00
40.00
30.00
Deaths: incompatible group = 7 (1-year = 81.5%, 3-year = 75.5%) , denovo group = 3 (1-year 94.4%, 3-year 81.9%)
20.00
10.00
0.00 6 12 18 24 30 36
Time (Months)
(+) XM
DeNovo
Johns Hopkins InKTP

39. Kaplan-Meier Estimate of Graft Survival (+) CDC XM @ Time of Tx vs (-) CDC XM10 20 30 40 50 60 70 80 90
100 %
Allograft
Survival
120
150
180
210
240
270
300
330
360
Time (Days)
+ Tx - Tx
1 Year
Graft Survival
p=NS
Tx - Tx
N= 14 N= 32
92.3% %
Johns Hopkins InKTP

40. Anti-CD20 Rescue Protocol
Inclusion Characteristics
Failure to Respond to Plasmapheresis/CMVIg Therapy
Poor or Incomplete Clinical Response
Persistence of High-Titer DSA
Persistence of Histologic Evidence of AMR
Initial Histologic Features That Portend Poor Outcome
and/or Graft Loss (Grade 2-3 AMR)
Study Group
Recipients of Deceased or Live Donor Kidneys
De novo AMR
AMR After Desensitization
Johns Hopkins InKTP

41. Renal Function Following Anti-CD20 Rescue
p=0.0003
p=0.01 10
p=0.07 9
p=0.25 8 7
17 recipients
undergoing a-CD20
rescue therapy
for AMR 6 5 4 3 2 1
Best
AMR
2 weeks
1 Month
Current
Best Cr
AMR Cr
2 week Cr
1 month Cr
Current Cr
Best
AMR
2 weeks
1 month
Current
1.8
(1.4 – 2.1)
4.3
(2.5 – 6.5)
3.4
(1.9 – 5.4)
2.1
(1.6 – 3.3)
1.7
(1.1 – 2.6)

42. Kaplan-Meier Estimate of Graft Survival for Anti-CD20 Rescue
100 75 %
Survival 50 25 1 2 3 4 5 6 7 8 9 10 11 12
Months Following Anti-CD20 Treatment
Johns Hopkins InKTP

43. Splenectomy Rescue N 4 Median Days to AMR (Range) (2-15)
Median Days to Splenectomy Following AMR Dx 1
(1-4)
Median SCr at Biopsy Dx
3.4
(1.5 – 6.0)
Median SCr 1 week
2.1
(0.8 – 5.8)
Median SCr 1 month
1.5
(0.7 – 2.3)
Median Current SCr
1.3
(1.2 – 2.6)
Allograft Survival
100%
Median Months Followup
6.9
(2.2 – 11.7)
Johns Hopkins InKTP

44. Paired Donation May Reduce the Incidence of AMR
Conventional KPD
Unconventional KPD A B
ABOi A
ABOi O B A
ABOi O
(+) XM A
# of KPD: 6 (12 patients)
# of KPD: 5 (13 patients)
Mean PRA: 14
Mean PRA: 58
6 mos Cr: 1.2 mg/dl
6 mos Cr: 1.1 mg/dl
AMR: 0%
Cellular 8%
AMR: 0%
Cellular 23%
Patient Survival: 100%
Patient Survival: 100%
Graft Survival: 91.7%
Graft Survival: 100%

45. Other Therapies Leflunomide FTY720 Efalizumab Alemtuzumab (Campath 1H)
An orally administered antimetabolite
Action against NF-B and JAK3, key mediators in cytokine generation
Inhibits CMV
FTY720
Reduces peripheral lymphocyte levels by causing sequestration in lymph nodes and Peyer patches
Efalizumab
Monoclonal anti-CD11a antibody
Blocks T cell adhesion
Alemtuzumab (Campath 1H)
Monoclonal antibody targeting CD52 (found on all lymphocytes)
Causes profound, prolonged lymphocyte depletion
Tolerance induction with full immunosuppression and bone marrow transplant

46. Summary
The diagnosis of AMR can now be made with a high level of certainty
There are therapeutic interventions for AMR with clinically proven efficacy
De novo AMR has a good long-term prognosis when treated with PP or IVIg
Results of PP or IVIg treatment for De novo AMR and AMR in the setting
of desensitization are comparable
A (+) cytotoxic XM at the time of Tx does not predict a worse outcome
AMR recalcitrant to PP/IVIg is associated with a lower graft survival rate
Results of emergent splenectomy at the time of severe AMR look promising
KPD may decrease AMR by lowering immunologic risk

47. Algorithm For Approach To AMR
De Novo AMR
AMR after (+) XM
AMR after ABOi
PP/IVIg
Severe AMR
Response
Incomplete Response
Anti-CD20
Splenectomy
Observe
Anti-CD20

48. Chronic antibody-mediated
rejection

49. Chronic antibody-mediated rejection (CHR)
Definition
Chronic antibody-mediated rejection (CHR)
Slow, progressive loss of graft function (months to years), often with proteinuria, hypertension
Colvin RB. J Am Soc Nephrol 18: 1046–1056, 2007

50. Chronic rejection Fibrosis and scarring in transplanted organ
Multifactorial etiology, not strictly immunologic
Inflammation, ischemia, and other processes play a role
Episodes of acute rejection are significant risk factors

51. Diagnostic criteria for CHRa
Histologic evidence of chronic injury (need 2 of 4)
arterial intimal fibrosis without elastosis
duplication of glomerular basement membrane
multilaminated PTC basement membrane
interstitial fibrosis with tubular atrophy
Evidence for antibody action/deposition in tissue
(e.g., C4d in PTC)b
Serologic evidence of anti-HLA or other anti-donor antibody
a-If only two of the numbered criteria are present, then the diagnosis is considered suspicious for CHR.
b-May be patchy in distribution.
Colvin RB. J Am Soc Nephrol 18: 1046–1056, 2007

52. Histology GBM duplication; mononuclear cells in glomeruli and PTC
intimal fibrosis
tubular atrophy and interstitial fibrosis
PTC basement membrane multilamination (by EM)

53. Chronic humoral rejection (CHR)
Transplant glomerulopathy
With duplication of the glomerular
basement membrane (GBM) and
accumulation of mononuclear cells
in glomeruli (periodic acid-Schiff
stain).
Immunofluorescence shows a
patch distribution of C4d
positivity in PTC.
Sis B, Mueller T et al ,. Am J Transplant [Suppl]: 469–470, 2006

54. Chronic humoral rejection (CHR)
The PTC have Prominent
multilamination of the GBM.
Electron microscopy shows
duplication of the GBM and
reactive endothelial cells.

55. C4d Positive PTC (often patchy) Glomeruli variably positive
Occasionally only glomeruli positive

56. Serology
Anti-donor HLA positive in majority, especially to MHC class II antigens.

57. CHR: Therapy
Outcome and optimal therapy not yet defined

58. Accommodation
Transplantation across an ABO barrier, which normally precipitates hyperacute rejection, has been done successfully using special protocols to deplete naturally occurring anti–blood group antibodies.
Remarkably, antibody (primarily IgM) returns in the circulation, sometimes in high titers, yet no obvious graft rejection occurs, a state termed “accommodation”
At a cellular level, accommodation may occur via multiple mechanisms, including internalization, downregulation, inactivation, and inhibition of the target antigen

59. Accommodation Histology Diagnosis Normal graft function
Normal, or minor changes

60. Accommodation C4d Serology Positive PTC; variable glomeruli
Common with ABO incompatibility;
occasionally with HLA antibodies
J Am Soc Nephrol 18: 1046–1056, 2007

61. Accommodation Light microscopy is normal
Electron microscopy shows no glomerular abnormality.
Prominent C4d deposition in PTC is present

62. Accommodation
Outcome and optimal therapy not yet defined

63. Antibody mediated graft rejection Summary
The detection of C4d deposition in capillaries of allografts has permitted the definition of two new forms of antibodymediated rejection, acute and chronic, as well as an apparent clinicopathologic state in which the endothelium undergoes no obvious pathologic change (accommodation).

64. Conclusion
Organ transplantation induces a fundamentally abnormal physiologic state
Steady improvement has been made in both prevention and treatment of organ rejection
The potential for further improvement – both incremental and revolutionary – is great