Post-Tx İmmün Monitorizasyon Gerekli mi? Değil mi? Caner Süsal Caner Süsal Transplantasyon Immünolojisi Heidelberg Üniversitesi, Almanya
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
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
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
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
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?)
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%
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
5. Post-Tx Antikörper-Monitoring
(post-Tx Tage 0, 7, 30, 180, …)
CDC, ELISA, Luminex
Heidelberg Algorithm
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)
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)
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
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 -
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
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