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

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

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

4. No C4d detectable in up to 76% of recipients with circulating antibodies! Mauiyyedi, JASN 2002; Böhmig, JASN 2002; Koo Transplantation 2004 Sensitivity of C4d deposits in PTC for circulating anti-HLA-antibodies31-95% Sensitivity of C4d Staining for Alloantibodies

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

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

9. 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 Specificity of C4d deposits in PTC for circulating anti-HLA-antibodies93-96% Specificity of C4d Staining for Alloantibodies

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

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. No C4d deposits induced by ischemic injury in early post-transplant kidney allograft biopsy M. Haas et al, Transplantation 2002 Complement activation in (experimental) renal ischemia occurs via the alternative but not via the classical pathway Complement Activation by Ischemia/Reperfusion in the Kidney? 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. C4d Capillaropathy Intimal Fibrosis Glomerulopathy + or + DSA MHC I anti-C4d MHC II Banff classification of renal allograft rejection

14. T i m e T i m e I n t e n s i t y Tissue injury Development of chronic antibody mediated rejection Graft dysfunction Antibody C4d Diagnostic threshold Biopsy Serology? Protocol Biopsy? (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. Separate analysis of patients with excellent 1y graft function 1. GFR ≥60 ml/min (MC equation) 2. 24h protein excretion ≤0.5 g 3. no dysfunction/indication biopsy 4. no desensitization or rejection treatment 164 recipients with >1year graft function 1 year serial HLA Ab monitoring Follow-up: median 69 months 130 34 Humoral response in stably functioning grafts Bartel et al, Am J Transplant, in press

17. Excellent function during 1 st year (n= 34) Dysfunction during 1 st year (n=130) IgG HLA Ab in renal Tx recipients with excellent 1 year course No difference to non-stable patients ✔ Incidence ✔ Binding strength ✔ DSA frequency ✔ C4d-fixation in vitro Bartel et al, Am J Transplant, in press % Recipients Months 2612 Months 2612 0 10 20 30 40 50 0 10 20 30 40 50 P=0.4P=0.8 P=0.2 P=0.3P=0.7 P=0.5 % Recipients IgG alloreactivity Complement activating alloreactivity

18. Long-term outcome of 9 Ab+ recipients with excellent 1-year graft performance Bartel et al, Am J Transplant, in press Patient[IgG]FlowPRA[C4d]FlowPRA follow-up (months) GFR (ml/min) Proteinuria (g/24h) 31HLA I/II, DSAHLA I/II801180.2 59HLA I/II, DSAHLA I76660.52 76HLA I, DSAHLA I72700.15 82HLA I, DSA-7161 2.1** 105HLA I, DSA-65540.05 114HLA I, DSAHLA I 40*810.16 125HLA I, no DSA-62940.1 126HLA I, no DSA-62820.08 134HLA II, no DSAHLA II56980,05 *patient died with functioning graft due to pancreatic cancer **de-novo membranous glomerulonephritis

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

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

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. 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 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

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

23. Are capillary inflammatory lesions predictive of chronic rejection? 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 In protocol biopsies, PTCitis at 3 months was associated with chronic antibody mediated rejection at 12 months. E. Lerut et al., Transplantation 2007

24. Recipients without adaptive immune system (RAG1 KO, athymic) or MHC incompatible donor Ab induced C4d on EC 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…) T. Jindra, J immunol 2008; S. Uehara, AJT 2006; M. Koch Transpl Immunol, 2008, K. Solez, AJT 2008 Anti-donor-MHC moAb Molecular mechanisms of antibody/complement mediated EC injury

25. Summary C4d is a reliable marker of humoral rejection in dysfunctioning grafts. This might be due to accommodation or only transient and/or weak DSA reactivity Complement deposition and/or circulating DSA are far less predictive of bad outcome when observed in recipients with stable graft function. 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.