1

Antibody Mediated Rejection: Historical perspectives

and animal models

Robert B. Colvin, M.D.Massachusetts General Hospital

Harvard Medical School

Banff X

August 10, 2009

Banff, Alberta

Early Observations in Patients

1966 Hyperacute rejectionKissmeyer-Nielsen F, Olsen S, Petersen VP, and Fjeldborg O, Hyperacute rejection of kidney allografts, associated with pre-existing humoral antibodies against donor cells. Lancet, 2: 662-5,1966.

1967 Transplant glomerulopathyPorter KA, Dossetor JB, Marchioro TL, Peart WS, Rendall JST, and Terasaki PI, Human renal transplants. I. Glomerular changes. Lab Invest, 16: 153-81,1967.

1968 HLA antibodies post-transplantationMorris PJ, Williams GM, Hume DM, Mickey MR, and Terasaki PI, Serotyping for homotransplantation. XII. Occurrence of cytotoxic antibodies following kidney transplantation in man. Transplantation, 6: 392-9,1968.

Humoral Antibodies in Renal Allotransplantation in ManJeannet, Pinn, Flax, Winn, Russell, NEJM 282:111,1970

Chronic allograft arteriopathy

only in patients with de novo anti-donor antibodies

Dark Ages for Antibody1970-1990

No connection between antibodies and pathology

Antibodies regarded as epiphenomenon

T cells reigned

T Rex

Sunrise in Alberta 1990Antibodies affect outcome of acute rejection

and pathologic pattern

1990Halloran PF, Wadgymar A, Ritchie S, Falk J, Solez K, and Srinivasa NS, The significance of the anti-class I antibody response. I. Clinical and pathologic features of anti-class I-mediated rejection. Transplantation, 49: 85-91,1990.

1992Halloran PF, Schlaut J, Solez K, and Srinivasa NS, The significance of the anti-class I antibody response. II. Clinical and pathologic features of renal transplants with anti-class I-like antibody. Transplantation, 53: 550-5,1992.

1996Trpkov K, Campbell P, Pazderka F, Cockfield S, Solez K, and Halloran PF, Pathologic features of acute renal allograft rejection associated with donor-specific antibody. Analysis using the Banff grading schema. Transplantation, 61: 1586-92,1996.

2

Sunrises can be foggy in Canada

C4d Deposition detected in PTC in Rejection

Normal Acute Rejection

Cryostat sections, mAb to C4d, immunoperoxidase detectionFeucht et al 1991

“On the basis of covalent binding and amplification, it appears that staining of complement fragment C4d seems to be a practical tool for the demonstration of in situhumoral immune reactions that are not easily detectable otherwise.”

Helmut Feucht et al, 1991

Connect the dots

DSAC4dPTC

Graft Pathology

DSA = Donor Specific Alloantibody (HLA)

3

DSA correlated with C4d+ and polys in caps

C4d and Transplantation Publications by Year

0

10

20

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60

1990

1991

1992

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C4d, Clin --Feucht

Class I Ab, Clin, Path--Halloran

81

1990 1991 1992 1993 1994 1995 1996 1997 1998 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008

AHRCollins

CHR Mauiyyedi

86

“Chronic humoral rejection” = Transplant glomerulopathy or arteriopathy

+ C4d + DSA

38 pts 61% C4d+ 88% of C4d+ had DSA+

Documented:Transplant glomerulopathy, glomerulitisPTC capillaritis and BM multilamination

Introduced concept of evolution C4d+TG

C4d

Other key observations

C4d Deposition is transient (days)Indicates current antibody activityNickeleit V, Zeiler M, Gudat F, Thiel G, and Mihatsch MJ.

J Am Soc Nephrol, 13: 242-51,2002.

Minami K, Murata K, Lee CY, Fox-Talbot K, Wasowska BA, Pescovitz MD, and Baldwin WM, 3rd, Am J Transplant, 6: 923-32,2006 (rats).

ABOi grafts can have C4d without pathologyFidler ME, Gloor JM, Lager DJ, Larson TS, Griffin MD, Textor SC, Schwab TR, Prieto M, Nyberg SL, Ishitani MB, Grande JP, Kay PA, and Stegall MD, Am J Transplant, 4: 101-7,2004.

Haas M, Rahman MH, Racusen LC, Kraus ES, Bagnasco SM, Segev DL, Simpkins CE, Warren DS, King KE, Zachary AA, and Montgomery RA, Am J Transplant, 6: 1829-40,2006.

Setoguchi K, Ishida H, Shimmura H, Shimizu T, Shirakawa H, Omoto K, Toki D, Iida S, Setoguchi S, Tokumoto T, Horita S, Nakayama H, Yamaguchi Y, and Tanabe K, Am J Transplant, 8: 86-94,2008.

4

Sunrise Banff 2001

2005 Chronic Ab Mediated Rejection

Solez K et al, Am J Transplant, 7: 518-26,2007.

2001 Acute Ab Mediated RejectionRacusen et al Am J Transplant, 3: 708-14,2003.

New Banff Categories

Solez K et al, Am J Transplant, 8: 753-60,2008.

2007 C4d deposition without active rejection

Technical Advances

Polyclonal anti-C4d for FFPE tissueUseful for evaluation of glomeruliRegele H, Exner M, Watschinger B, Wenter C, Wahrmann M, Osterreicher C, Saemann MD, Mersich N, Horl WH, Zlabinger GJ, and Bohmig GA. NDT, 16: 2058-66, 2001

Solid Phase Assays for HLA DSADonor cells not needed, sensitive, specificELISAKao KJ, Scornik JC, Small JC. Transplantation 1993; 55: 192–6.

LuminexFulton RJ, McDade RL, Smith PL, Kienker LJ, and Kettman JR.Clin Chem, 43: 1749-56,1997.

Terasaki et al AJT 7:408, 2007

Many studies by Terasaki, Worthington and others showed

de novo HLA antibodies a risk factor for graft loss

C4d has provided a missing link: antibody pathology

A t]

Incubate in fresh normal serumAlloAb

C4d

AntiC4d

C1q

AlloAb

HLA Class I HLA Class II

Complement fixing alloantibodies detected on Ag coated beads (FlowPRA, Luminex)

Wahrmann (Vienna) AJT 7:1033, 2007

FITC

More sensitive than cellular methods ~4x more presens detected

IgG-

IgG+C4d-

IgG+C4d+

C4d fixation by pretransplant sera on Class I beads correlates with outcome

N=338

Wahrmann (Vienna) AJT 7:1033, 2007

Strong correlation with C4d in bx

5

Intragraft events Immunoglobulin Gene Transcripts (IGT)vs. time post-transplant

IGT Score (log2)

Einecke (Edmonton) AJT 8:1434, 2008

intragraft synthesis of Ig is a feature of late graft dysfunction

Local Production of DSA by cells in renal allografts

Thaunat et al (Lyon), Transplant 85:1648, 2008

IgG DSALuminex

Perplexing Heterogeneity

Transplant Glomerulopathy (TG)

Antibody (DSA) 70% HLA Class II>I Ab(91% vs 61%)

C4d in PTC 32%PTC multilamination 91%

N=41 pts (3.1%), mean dx 5.5 yrs p-tx

Sis et al (Edmonton) AJT 7:1743, 2007

38% TG + DSA without C4dNon C fix Ab?

26% TG without C4d or DSANon Ab cause (T cell, TMA/CNIT)Residue of past Ab/C

Subsets of CHR revealed bycluster analysis

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TG, Class II DSA+

TG, Class II DSA-

Interstitial Inflammation

Interstitial Fibrosis

All Banff variables, DSA

Brad Farris et al (Boston) Banff 2009 Poster

6

Chronic disease evolves over time

Sequential events in CHR

C4d deposition predicts later transplant glomerulopathy

C4d TGBx <12 mo Bx > 12 mo

Neg 6%

Pos 46%

Regele et al (Vienna), JASN 13:2371,2002 (213 bx >12 m)

risk of TG after episode of AHR

Prevalence of TG in 1 yr protocol bxConventional 198 8%Presens HLAi 37 22% p<.015

ABOi 24 13%

• Prior AHR risk factor for TG (OR 17.5, multivariate)• Also for arteriopathy and interstitial fibrosis• Presensitized patients and possibly ABOi increased

risk

Gloor et al (Mayo) AJT 6:1841, 2006

Pathologic sequences observed in repeat biopsies

Collins et al, Mod Pathol, 2009

Postulated Stages of Humoral Rejection

TransplantTime (not to scale)

Blood: de novo antibodies

Graft: C4d

Graft: pathology

Clinical: graft dysfunction

I II III IV

Graft loss

Accommodation RejectionSubclinical ClinicalNo C4d With C4d

Colvin and Smith, Nature Rev Immunol 5:807, 2005

Other Organs

7

C4d Publications by Year

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10

20

30

40

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90

20082006

20042002

20001998

19961994

19921990

Year

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ub

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Kidney Heart Liver Lung Pancreas Bowel

281 52 29 21 5 2

Total 390

Kidney Heart Pancreas Lung Liver Bowel

Hyperacute rejection + + + + +

Acute humoral rejection + + +

Chronic humoral rejection +

Accommodation + +

Accepted Organ Specific Criteria for Antibody Mediated Graft Rejection

Lots of blanks!

Cardiac Allograft Vasculopathy (CAV)

C4d+ bx intimaat one year IVUS

Poelzl et al (Innsbruck) Transplant Int 18:2303, 2005

Arteriopathy + C4d + DSA= CHR of heart?

CAV correlates with prior C4d+

80% with C4d CAV27% without C4d CAV

p<0.001

Rodriguez et al (Cleveland) AJT 5:2778, 2005

Even the liver can have AMR

Watson Kozlowski Nickeleit… (Chapel Hill NC) AJT 6:3022, 2006

HLA presensitized patient (HLA-B7): day zero biopsyDeveloped chronic damage (bridging fibrosis, ductular proliferation)Ischemia reperfusion control = neg

C4d

Bellamy (Edinburgh) Histopathol 50:739, 2007

C4d Rosetta Stone

Liver Liver Kidney

Combined kidney and liver transplant from same donor into presensitized recipientBoth rejected on day 18 and both C4d+

Bellamy (Edinburgh) Histopathol 50:739, 2007

Liver is less susceptible to CHR than KidneySimultaneous liver-kidney tx– DSA+

C4d - LiverC4d - Kidney

8

Endothelium: Target and Defender Anti-HLA antibodies trigger endothelial responses

Endothelial cell

Colvin, Smith, Nat Immunol Rev 2005

Apoptosis

C4d

Antibody mediated effects on endothelium

MacNK FcR

Phosphorylated signaling proteins In vivo markers of endothelial activation

Normal Rejection

What role do cells play?

Lee CY, Lotfi-Emran S, Erdinc M, Murata K, Velidedeoglu E, Fox-Talbot K, Liu J, Garyu J, Baldwin WM, 3rd, and Wasowska BA, Transplantation, 84: 1324-34,2007.

Endothelial cells in vitro

DSA alone DSA + Macs

MCP-1, KC, IL-6, Rantes, TIMP-1MCP-1, KC

FcRIII KO macrophagesless active, as were F(ab)2 DSA

Macrophages enhance cytokine productionby endothelial cells triggered by anti-MHC I Ab

9

Endothelial cells resist effects of antibody/complement

• bcl-xL in renal allografts from patients with donor specific HLA antibody

Salama (Hammersmith) Am J Transplant 1:260, 2001

• muc-1 gene expression/glom ABOiPark et al (Mayo) AJT 3:952, 2003

• CD55 (DAF) in stable vs unstable cardiac graftsGonzalez-Stawinski et al (Cleveland) JHLT 27:357, 2008

C4d+DAF+No dysfunction

C4d+DAF-Acute dysfunction

Gonzalez-Stawinski et al (Cleveland) JHLT 27:357, 2008

Capillary DAF+

identifies C4d+ biopsies with no graft dysfunction

DAF = decay accelerating factor, CD55

Endothelial Gene Expressionin kidneys with rejection and DSA

Transcripts include vWF, caveolin-1,

E-selectin, CD31, CD34…

• Distinct pattern vs DSA- rejection

• Highest levels assoc with C4d+

• Significantly in C4d- DSA+ cases

Sis et al (Edmonton) AJT in press

• Low sensitivity of C4d stain vsnon-C’ fixing effects of DSA

Experimental Studies

Animal Models of AHR/CHR

Xenograft rejectionPig to monkey/baboonRat heart to gal KO mouse

AHRRat (C6) (Baldwin)Mouse heart (Wasowska)

CHRMouse heart allograftMouse with human arteryMonkey renal allograft

AHR depends on complement fixation

Rahimi S, Qian Z, Layton J, Fox-Talbot K, Baldwin WM, 3rd, and Wasowska BA, Non-complement- and complement-activating antibodies synergize to cause rejection of cardiac allografts. Am J Transplant, 4: 326-34,2004.

B10.A hearts B6 Ig KO

Complement fixing IgG2b DSA triggered immediate rejection at 10 days, but non-C’ fix IgG1 did not.

Mouse heart allografts

10

AJT 7:57, 2007Control

B10.BR heart B6/RAG1-/-anti-H-2Kk

Chronic Arteriopathy can be caused by DSA Passive Mab to donor class I B10.BR heart B6.RAG1-/- given anti-H-2Kk x28 d

C4d

CD3F4/80(Mac)

Uehara et al AJT 27:5,2007

Non-complement fixing antibody (IgG1) can initiatechronic allograft arteriopathy

Anti-H-2Kk

IgG1(n=8)

No Rx(n=5)

Anti-H-2Kk

IgG2a (Allograft)

(n=10)

Anti-H-2Kk

IgG2a (Isograft)

(n=4)

80

60

40

20

0

100

24.8

5.7

52.3

5.5

Neo

intim

alIn

dex

p<0.01

p<0.005

p<0.005 p<0.05

Hirohashi et al, submittedMab RAG1-/-B6 recip with H-2Kk heart grafts

Anti-H-2Kk mAb B10BR hearts in B6IgG1 B6.RAG1-/- IgG2a B6.C3-/-RAG1-/-

Control

Hirohashi, ATC 2008

Chronic Allograft Arteriopathyin C3 deficient RAG1-/- recipients

Mab B6.C3-/-RAG1-/- recip with H-2Kk heart grafts Hirohashi et al submitted

DSA-IgG2a No Ab

Alloantibody can cause one feature of chronic humoral rejection without complement (NK/FcR?)

May be relevant to cases with DSA and chronic rejection without C4d

11

Human arterial segment transplanted into immunodeficient mouse (SCID/Beige)

Lorber, Tellides, Pober (Yale)Transplant 67:897,1999

Thomsen (Toulouse)-mesenteric

JHLT 25:675, 2006

Galvini (Toulouse)

in press AJT

Anti-HLA class I arteriopathy in 6 wk

Non-human Primate Renal Allografts

Knechtle

Larsen, Kirk

Thomas

Jonker

Wieczorek, Mihatsch, Nickeleit

Cosimi, Kawai

Variants of mixed chimerism protocolAll off immunosuppression after day 30

48% developed anti-donor alloantibodies29% C4d+ 22% TG+

143 Recipients269 biopsies (protocol/indication)

5 nephrectomies143 autopsies

CHR in Cynomolgus Renal AllograftC4d+, DSA+

4498 d371

Smith et al, AJT 6:1790, 2006

Similarity to human CHR

PTC BM multilamination

GBM duplication

Smith et al, (Boston) AJT 8:1662, 2008

Renal allograft loss due to rejection- N=143Non-human primates off immunosuppression

12

Sequential Development of CHR-Monkeys

No CHR Stage I Stage II Stage III Stage IV

Days post-transplant

106 182 225 352 371

Smith et al (Boston) AJT 8:1662, 2008M4498

High Noon for AMR

Future Questions

Basic

Regulation of B cell function

Endothelial mechanisms

Mechanism of accommodation

Role of complement and FcR/cells

Clinical

Natural history

Monitoring Ab, intervention

New therapies (Plasma cell, B cell, C’)

Late Graft Failure

AbominableAntibodyT Rex

(this guy is bigger)

Transplant Research MGH

Terry Strom

Mo Sayegh

Harvard Collaborators

CTOT-C

Genomics of Tx

ITN

CTOT

Multicenter Trials

Catherine Adams

Bernard Collins

Nicole Brousiades

Tricia Delle Pella

Diana Taheri

Brad Farris

Sandro Alessandrini

R. Neal Smith

Colvin Lab/Pathology

Donna Fitzpatrick

Susan Saidman

Histocompatibility

Fred Preffer

Megan Sykes

Jay Fishman

Frank Delmonico

Tatsuo Kawai

Win WIlliams

Elliot Heher

Nelson Goes

Nina Tolkoff-Rubin

Joren Madsen

Jim Markman

Ben Cosimi

Clinical

Masa Miyayama

Tommy Hirohashi

Joren Madsen

Cuffy Chase

Paul Russell

Mouse

Kazu Yamada

Akira Shimizu

David Sachs

Pig

Joren Madsen

Gilles Benichou

Svetlan Boskovic

David Sachs

Tatsuo Kawai

Ben Cosimi

Monkey