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Humoral rejection: What the pathologist needs to know

Humoral rejection: What the pathologist needs to know. Heinz Regele Clinical Institute of Pathology. Banff classification of renal allograft rejection. ATN. Capillaritis. Arterial necrosis. DSA. C4d. MHC I. +. +. or. or. MHC II. anti-C4d. C4. C2. C4a. C2b. C3a. C1qrs. C3.

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Humoral rejection: What the pathologist needs to know

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  1. Humoral rejection: What the pathologist needs to know Heinz Regele Clinical Institute of Pathology

  2. Banff classification of renal allograft rejection ATN Capillaritis Arterial necrosis DSA C4d MHC I + + or or MHC II anti-C4d

  3. C4 C2 C4a C2b C3a C1qrs C3 C3b C4b C2a C3 Convertase Allograft Endothelial cell Factor I C4b MCP (CD46) C4c C4d inactive C4d is a marker of antibody mediated rejection Active

  4. Sensitivity of C4d Staining for Alloantibodies Sensitivity of C4d deposits in PTCfor circulating anti-HLA-antibodies 31-95% No C4d detectable in up to 76% of recipients with circulating antibodies! Mauiyyedi, JASN 2002; Böhmig, JASN 2002; Koo Transplantation 2004

  5. Circulating DSA without C4d • Variable Sensitivity of serological assays • Non complement-activating alloantibodies • Insufficient sensitivity of C4d detection method?

  6. C4d detection by IF on frozen sections vs. IHC on paraffin sections 26 biopsies with diffuse staining by IF on frozen sections C.A. Seemayer et al, NDT 2007

  7. C4d scoring adjusted for staining method Suggestion by the Banff Conference 2007 K Solez et al, AJT 2008

  8. In 2 Patients severe rejection reversible by IA 4 allografts lost C4d neg/FCXM neg N = 20 C4d pos N = 16 C4d neg/FCXM pos N = 22 1 allograft lost C4d Staining and FCXM (Flow-Cytometry X-Match) of Corresponding Sera 113 Biopsies of 58 Renal Allograft Recipients G.A. Böhmig et al, JASN 2002

  9. Specificity of C4d Staining for Alloantibodies Specificity of C4d deposits in PTCfor circulating anti-HLA-antibodies 93-96% No alloantibodies detectable in 10-25% of recipients with C4d deposits! Lederer, KI 2001; Mauiyyedi, JASN 2002; Böhmig, JASN 2002; Koo, Transplantation 2004; Smith, JHLT 2005

  10. C4d deposits without circulating DSA • Adsorption of alloantibodies within the graft? • Non-HLA alloantibodies • Complement activation by ischemia/reperfusion?

  11. Complement Activation by Ischemia/Reperfusion C4d deposits in heart allografts are associated with morphologic signs of ischemic injury W.M. Baldwin et al, Transplantation 1999 • Activation of complement via the classical pathway occurs in experimental ischemia/reperfusion injury in • Heart • Skeletal muscle • Bowel

  12. Complement Activation by Ischemia/Reperfusion in the Kidney? Complement activation in (experimental) renal ischemia occurs via the alternative but not via the classical pathway No C4d deposits induced by ischemic injury in early post-transplant kidney allograft biopsy M. Haas et al, Transplantation 2002 C4d in cardiac allografts correlates with alloantibody: 21/24 BX from Pat with alloantibody are C4d positive and only 7/60 BX in alloantibody-negative recipients show C4d deposits R.N. Smith et al, JHLT 2005

  13. Banff classification of renal allograft rejection Glomerulopathy Capillaropathy Intimal Fibrosis DSA C4d MHC I + + or or MHC II anti-C4d

  14. Tissue injury Graft dysfunction I n t e n s i t y Antibody C4d Diagnostic threshold Serology?Protocol Biopsy? Biopsy T i m e Development of chronic antibody mediated rejection (months-years)

  15. C4d deposition in stably functioning grafts C4d deposition was observed in 4,4% of 551 renal allograft protocol biopsies but had no negative impact on outcome (median follow-up 3,5 years) M. Mengel et al., AJT 2005 80% of protocol Bx in ABO-incompatible grafts were C4d positive. C4d is not correlated with injury in most ABO-incompatible grafts. Haas et al., AJT 2006

  16. Humoral response in stably functioning grafts 164 recipients with >1year graft function 1 year serial HLA Ab monitoring Follow-up: median 69 months Separate analysis of patientswith excellent 1y graft function1. GFR ≥60 ml/min (MC equation)2. 24h protein excretion ≤0.5 g3. no dysfunction/indication biopsy4. no desensitization or rejection treatment 34 130 Bartel et al, Am J Transplant, in press

  17. IgG HLA Ab in renal Tx recipients with excellent 1 year course Complement activating alloreactivity 50 50 IgG alloreactivity 40 40 P=0.2 P=0.4 P=0.8 30 30 % Recipients % Recipients P=0.5 P=0.3 P=0.7 20 20 10 10 0 0 2 6 12 2 6 12 Months Months No difference to non-stable patients ✔Incidence ✔Binding strength ✔DSA frequency ✔C4d-fixation in vitro Excellent function during 1st year (n= 34) Dysfunction during 1st year (n=130) Bartel et al, Am J Transplant, in press

  18. Long-term outcome of 9 Ab+ recipients with excellent 1-year graft performance *patient died with functioning graft due to pancreatic cancer **de-novo membranous glomerulonephritis Bartel et al, Am J Transplant, in press

  19. Banff classification of renal allograft rejection DSA C4d MHC I + but no morphological lesions MHC II anti-C4d

  20. Accommodation, acquired resistance of the graft against persisting alloimmune reactions ? ? Alloantibody and/or complement in stable grafts Subclinical rejection, with a high risk of chronic allograft damage and accelerated graft loss Transient/weak immune response very low risk of graft loss

  21. Duration and intensity of humoral injury Most serologic and biopsy studies on the impact of antibodies and complement on chronic allograft damage are based on single measurements and rarely provide data on antibody binding strength/titer. Persistence of circulating antibody (in serial samples) significantly increased the risk of subsequent graft loss, was however in a few recipients also compatible with continuous stable function. van den Berg-Loonen et al., Clin Transplants 2006 In 65 patients with DSA (26 with stable function and 39 failed) strength of DSA binding was strongly associated with late graft failure. K. Mizutani et al., AJT 2007

  22. Accommodation, acquired resistance of the graft against persisting alloimmune reactions Alloantibody and/or complement in stable grafts Subclinical rejection, with a high risk of chronic allograft damage and accelerated graft loss Transient/weak immune response very low risk of graft loss

  23. Are capillary inflammatory lesions predictive of chronic rejection? In protocol biopsies, PTCitis at 3 months was associated with chronic antibody mediated rejection at 12 months. E. Lerut et al., Transplantation 2007 10/10 recipients with subclincal AMR showed accumulation of immune cells in peritubular capillaries (PTCitis) and 8/10 had glomerulitis. Subclinical AMR is associated with increase of cg, ci and ct in follow up Bx. M. Haas et al., AJT 2006

  24. Molecular mechanisms of antibody/complement mediated EC injury Recipients without adaptive immune system (RAG1 KO, athymic) MHC incompatible donor Anti-donor-MHC moAb or No early graft loss due to acute ABMR Chronic vascular injury after 2-16 weeks in hearts and kidneys (CTA and chronic Glomerulopathy) EC response partially independent of C-activation; activates distinctive signalling pathways (akt, mTor…) Ab induced C4d on EC T. Jindra, J immunol 2008; S. Uehara, AJT 2006; M. Koch Transpl Immunol, 2008, K. Solez, AJT 2008

  25. Summary C4d is a reliable marker of humoral rejection in dysfunctioning grafts. Complement deposition and/or circulating DSA are far less predictive of bad outcome when observed in recipients with stable graft function. This might be due to accommodation or only transient and/or weak DSA reactivity Sequential testing for detection of persisting DSA reactivity and assessment of concomitant histological lesions may help identifying patients with subclinical rejection and subsequent risk of accelerated graft loss. New, additional markers for prospective discrimination between accommodation and rejection are required to further refine risk assessment.

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