Post-Tx İmmün Monitorizasyon

Gerekli mi? Değil mi?

Caner SüsalCaner Süsal

Transplantasyon ImmünolojisiHeidelberg Üniversitesi, Almanya

Post-Tx Immune Monitoring

� T-cell reactivity

� Rejection-related proteins/peptides/genes

� Alloantibodies

Testing for T-Cell Reactivity

� Cylex ImmuKnow

� sCD30� sCD30

Cylex ImmuKnow

A Meta-Analysis Using the Cylex ImmuKnow Assay

Post-Tx: After PHA stim.,

Kowalski et al. Transplantation 2006

Post-Tx: After PHA stim., measurement of ATP by CD4+ T cells in a Luminometer

sCD30

CD30

CD40

NGFR

TNFR1

TNFR2

FAS

OX40

TNF/NGF Rezeptor Superfamilie

Cystein-reiche Domäne

4-1BB

Todesdomäne

Mechanism

Regulatory T cells suppress CD8+ memory T cell-mediated

allograft rejection via a CD30-dependent mechanism

(Dai 2004)

PostPost--Tx Changes in sCD30 Plasma LevelsTx Changes in sCD30 Plasma Levels

Sampling Time Points

Pre-Tx Day Post-Tx Days Patient Groups

0 3-5 7-9 12-14 17-19

UC 100%a 52±3%a,b 31±3%b,c 21±2%c,d 19±3%d

PP--values for pairwise comparisons:values for pairwise comparisons:aap<0.0001, p<0.0001, bbp<0.0001, p<0.0001, ccp<0.0001, p<0.0001, ddp=0.02, p=0.02, eep<0.0001, p<0.0001, ffp<0.0001, p<0.0001, ggp<0.0001, p<0.0001, hhp=0.002,p=0.002, iip=0.001,p=0.001, jjp<0.0001. All other comparisons: p=n.s. (Wilcoxon test)p<0.0001. All other comparisons: p=n.s. (Wilcoxon test)

ATN 100%e 67±2%e,f 44±3%f,g 29±2%g,h 25±4%h

R 100% 109±8%i 76±12%i,j 49±12%j 37±5%

Pelzl et al Transplantation 2003

Post-Tx sCD30 and Organ Transplantation

1. Slavcev Transpl Immunol 2005

2. Matinlauri Transplantation 2005

3. Yang Zhonghua Yi Xue Za Zhi 2005

4. Weimer Am J Transplant 2006

5. Kim Transplant Proc 2006

6. Dong Transpl Immunol 2006

7. Sengül Transplantation 2006

8. Fields Transplantation 2006

9. Langan Am J Transplant 2007

10. Wang Transplant Immunol 2007

11. Golocheikine Transpl Immunol 2008

12. Yang Transplant Proc 2008

13. Cervelli Transplant Proc 2009

14. Nafar Int Urol Nephrol 2009

15. Kamali Ex Clin Transplant 2009

80

100

120

ClCr [ml/min]

1-Year sCD30 and 2-Year Graft FunctionWeimer et al. AJT 2006

0

20

40

60

ClCr [ml/min]

1-Year sCD30 1-Year sCD30<60 U/ml ≥≥≥≥60 U/ml

P=0.02

HLA antibodies and soluble CD30 are associated with poor renal graft outcome

Langan et al. AJT 2007

Barakaldo Barcelona Berlin Budapest Cagliari Cape Town Cardiff Dallas Dresden Essen Freiburg Geneva Giessen Glasgow Goteborg Heidelberg Helsinki Innsbruck Izmir Karachi Lausanne Leicester Leuven Lexington

CTSProspective Serum Project

Leicester Leuven Lexington Liege Ljubljana Mannheim Marburg Medellin Mexico City Milan Munich New York Newcastle Phoenix Portland Porto Alegre Prague Quebec Reims Rijeka Rio de Janeiro Saint-Etienne Sao Paulo Seoul St. Gallen Strasbourg Stuttgart Sydney Tehran Torino Ulm Valencia Zagreb Zurich

mRNA Gene Expression

Aim: To identify the genes and gene clustersassociated with

� acute/chronic rejection,

� development of tolerance� development of tolerance

� Microarray or Taqman/Lightcycler RT-PCR

� Detect early response to the IS or transplant

� Biopsy, blood, urine samples

Proteomics

Aim: To identify the proteins/peptides associated with

� acute/chronic rejection,

� development of tolerance

� Mass spectrometry

� Blood and urine samples

Post-Tx AlloAb Monitoring

Author Type of Rejection C4d+No. of

Patients

Feucht 1993 Early Graft Dysfunction 54% 93

Association of C4d-Positivity in Biopsies With Acute/Chronic Rejection

Collins 1999 Acute humoral rejection 100% 10

Mauiyyedi 2001 Chronic rejection 61% 38

Regele 2002 Chronic allograft nephropathy 35% 189

Post-Tx Ab Monitoring

Aim:

Prevention/Diagnosis/Therapy of

Antibody-Mediated Rejection Antibody-Mediated Rejection

(ABMR)

Post-Tx Ab MonitoringCritical Issues

1. It is expensive; is it clinically relevant?

2. If yes, magnitude?How many of the graft losses are due to ABMR?due to ABMR?

3. Which test?

ABMR

� Early failures (rare)

� Highly sensitized patients

� Desensitized patients

� Patients with high alloreactivity and � Patients with high alloreactivity and

underimmunosuppression

� Late failures (frequent?)

Causes of Chronic Dysfunction/Failure

Nankivell, Chapman Transplantation 2006

Humoral Theory of TransplantationPaul I. Terasaki

“antibodies are responsible for all graft losses”

Terasaki AJT 2007Terasaki AJT 2007

Post-Tx DSA- Lachmann et al. 2009 -

DSA + NDSA = 30%

Weaknesses

1. Sera from different post-Tx time points

2. No consecutive series

3. Unclear if de novo or pre-existing Ab3. Unclear if de novo or pre-existing Ab

4. Elder Tx

HLA Class I or Class II Antibodies

(ELISA-AbScreen)

before…

1. Tx: 15%

2. Tx: 64%2. Tx: 64%

3. Tx: 84%

4. Tx: 92%

Were the antibodies induced

before or after rejection?

If before,

pre-existing or de novo antibody?

Impact of PostImpact of Post--Tx Alloantibodies on Tx Alloantibodies on Chronic Graft FailureChronic Graft Failure

Serial ten-year follow-up of HLA and MICA antibody production prior to kidney graft failure

The role of HLA antibodies in chronic allograft rejection was examined utilizing a unique

resource of sera collected annually and stored over a 12-year period from patients with

rejected or retained grafts. In patients selected for not having preformed HLA antibodies,

679 postoperative serial serum samples from 39 patients who rejected their grafts and 26

with functioning grafts were tested for HLA Class I and Class II antibodies by flow cytometry

and for MICA antibodies by cytotoxicity on recombinant cell lines. HLA antibodies were

Mizutani et al. AJT 2005

and for MICA antibodies by cytotoxicity on recombinant cell lines. HLA antibodies were

found in 72% of patients who rejected grafts, compared to 46% with functioning transplants

(p<0.05). In addition, the incidence of IgG HLA plus MICA antibodies was higher (77%)

among those with failed transplants than those with functioning transplants (42%) (p<0.01).

Finally, if patients with IgM anti-HLA antibodies were included, 95% of the 39 patients who rejected their grafts had HLA or MICA antibodies, compared to 58% with functioning grafts (p<0.01). Patients who rejected transplants had HLA and MICA antibodies more frequently than those with functioning grafts. These antibodies found in the

peripheral circulation, were not necessarily donor-specific, but their association with failure

is consistent with a causality hypothesis.

Pre-existing in X-Match overseen DSA

� Hyperacute rejection

� Accelerated/acute humoral rejection

� Delayed graft function

Pre-Tx HLA Antibodies and Early Adverse EventsCTS Prospective Serum Study / 2001-2007 / n=1098

ELISA

ARE DGF

OR p OR p

Class I 2.53 <0.001 1.78 0.023

Class II 1.12 0.69 1.31 0.27

ARE = acute rejection episode (first 3 mo post-Tx)DGF = delayed graft function OR = odds ratio

Süsal et al, Transplantation 2009

de novo DSA

5. Post-Tx Antikörper-Monitoring

(post-Tx Tage 0, 7, 30, 180, …)

CDC, ELISA, Luminex

Heidelberg Algorithm

Causes of Late Graft Failure- Halloran -

1. ABMR (63%)

2. Recurrent disease

3. P.3. P.

4. P.

5. P.

Underimmunosuppression asCause of Chronic Rejection

� Non-compliance (22%, Hansen 2007)

� Minimization of IS

⇒ T cell ↑ →→→→ DSA ↑ →→→→ Ab-mediated rejection⇒ T cell ↑ →→→→ DSA ↑ →→→→ Ab-mediated rejection

(ABMR)

Effect of Withdrawing or Reducing Maintenance IS After the Second Post-Tx Year

Opelz, Döhler 2008

Causes of Late Graft Failure- Halloran -

1. ABMR

2. Recurrent disease

3. CNI toxicity (<5%)

Antibody-Mediated RejectionABMR

� C4d-positive

� C4d-negative (suspicious for ABMR)(suspicious for ABMR)

Antibody-Mediated Microcirculation Injury is the Major Cause of Late Kidney Transplant Failure

Einecke et al. AJT 2009

Late Graft Failure

� C4d-positive ABMR 25%

� C4d-negative ABMR 37%(PRA + microcirculation injury)(PRA + microcirculation injury)

- Cosio et al 2010 -

C4d- AR

with capillaritis

without capillaritis

C4d+ ABMR

De novo DSA at the time of kidney transplant biopsy associates with

microvascular pathology and late graft failure

- Hidalgo et al. 2009 -

� Indication biopsy (post-Tx 7d to 31yr)

� 37% DSA

- 15% pre-ex DSA

- 22% de novo DSA (early: 4%; late: 34%)

� Mostly class II or class I and II PRA

� Rarely only class I PRA

- Hidalgo et al. 2009 -

Problem:

Indication biopsyIndication biopsy

Open Question

How often is DSA present in patients who never

develop ABMR?develop ABMR?

DSA by Luminex

� Non-DSA

� Denaturated antigen

� Non-HLA

� High resolution full HLA typing of � High resolution full HLA typing of donor ± recipient is required(HLA-A, -B, -C, -DR, -DQ, -DP)

Post-Tx İmmün Monitorizasyon

Gerekli mi? Değil mi?

Gerekli gibi gözüküyor,Gerekli gibi gözüküyor,

en azından klinik bulgusu olan hastalardaP

Ab-Monitoring in all Patients

Benefit/Risk Ratio: ?

nakil öncesi ve 3 ayda biryüksek risk grubunda daha sık

Antibody-Mediated Rejection- ABMR-

� Monitoring

- DSA

- Protocol biopsy

� Treatment

- Rituximab+IvIg

- Rituximab+IA/PPH

- Bortezomib

Therapy of ABMR

1. Depletion of plasma cells (bortezomib)

2. Inhibition of naive and memory B cells progressing to plasma cells (rituximab)

3. Suppression of the cell cycle and proliferation of both B and T cells (this can be done with current baseline T cells (this can be done with current baseline immunosuppression such as tacrolimus and MMF)

4. Elimination of circulating antibodies with IA/plasmapheresis

5. B-cell/C-inhibition by IvIg

6. Serial monitoring of HLA antibodies via solid phase assay

De novo DSA at the time of kidney transplant biopsy - Hidalgo et al. 2009 -

� de novo DSA correlated with- Microcirculation inflammation (glomerulitis, capillaritis)

- Damage (glomerulopathy, capillary basement membrane multilayering) multilayering)

- C4d staining

� de novo DSA did not correlate with

- Scarring

- Arterial fibrosis

- Tubulo-interstitial inflammation

Screening for De Novo Anti-Human Leukocyte Antigen Antibodies in Nonsensitized Kidney Transplant Recipients Does Not Predict Acute

Rejection- John et al. 2010 -

Strength of Antibody- Mizutani 2007 -

Smith Smith … Colvin et al, AJT 2008 … Colvin et al, AJT 2008 417 specimens from 143 renal417 specimens from 143 renal--transplanted monkeys with tolerance transplanted monkeys with tolerance induction protocols. 18 animals had chronic rejectioninduction protocols. 18 animals had chronic rejection

DSA, day 182DSA, day 182 C4d+ and TG, days 225, 352, 371C4d+ and TG, days 225, 352, 371

Problems

� DSA’s are absorbed onto the organ

� Good function despite DSA

3 Amino Acid DR-Epitopein der Peptidgrube

Cai et al. AMJT 2006

Healthy Male Blood Donors With HLA Antibodies

- Luminex SA Testing -

63%

12%

Morales-Buenrosto, Terasaki et al. 2008

Transplantation 2009

� [beta]2m-free, denatured antigen-specific antibodies negatively interfere with the

predictive value of intact antigen-specific antibodies

� To get a clinically meaningful result, we have to use, in addition to regular beads, “elution buffer”-treated beads

Half-Life of Deceased Donor TransplantsUNOS

1966-1975 7.5 years1987-1995 7.5 years1996-2006 8.1 years1996-2006 8.1 years