1. Prognostic significance of C4-positive vs. negative rejection Heinz Regele Department of Pathology Innsbruck Medical University

2. C4d-negative rejection Has all clinical and morphological features of antibody mediated rejection but lacks C4d in transplant biopsies Issues to discuss Clinical relevance (prognosis, diagnostic features) Biology

3. PMN NK-cell T-cell C1 C1 Mø Mø C4d C4d C4d C3b C3b MechanismsofHumoralAllograftRejection MAC MAC Allograft Endothelial cells Dual Role of Complement Biology C3, C5, C5b-9 Diagnostic marker C4d (C3?)

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

5. C4d- (N=42) 90% Total (N=93) 72% C4d (+) (N=8) 63% C4d+ (N=43) % Allograft suvival 57% 100 Months post TX 90 80 70 60 50 40 30 20 10 10 1 6 11 0 2 3 4 5 7 8 9 12 0 Capillary C4d deposition and allograft survival Renal C4d deposits in 93 patients with early allograft dysfunction Feucht et al, Kidney Int, 43:1333, 1993

6. 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 C4d neg/FCXM neg N = 20 C4d pos N = 16 C4d neg/FCXM pos N = 22 1 allograft lost G.A. Böhmig et al, JASN 2002

7. Tissue injury and outcome in DSA positive patients A. Loupy et al., AJT 2011

8. Microvascular injury and chronic ABMR ….C4d may not be a sufficiently sensitive indicator of activity, MI and DSA being more robust predictors of bad outcome..... A. Loupy et al., AJT 2011

9. C4d-negative DSA-associated microvascular injury • Antibody-mediated but complement-independent injury? • Sampling error? • Inadequate sensitivity of C4d detection? • Remnants of previously active ABMR?

10. Experimental evidence for C4d negative ABMR Recipients without adaptive immune system (RAG1 KO) MHC incompatible donor Anti-donor-MHC moAb Non complement fixing anti donor IgG cause chronic transplant arteriopathy (CTA). CTA even developed in RAG1-/-C3-/- double KO mice upon injection of DSA, strongly suggesting a complement independent mechanism of injury T. Hirohasi, AJT 2010 NK cells are essential for the development of DSA induced CTA in a FcgRIII dependent mechanism (in absence and presence of complement). DSA alone or in conjunction with macrophages only do not generate CTA. T. Hirohasi, AJT 2012 Jindra PT, Transplantation 2006

11. Current Opinion in Organ Transplantation 2010; 15: 42-48 Expression of endothelial cell associated transcripts (ENDATs) is present in all types of rejection but significantly higher in ABMR. Only 13/50 (26%) of kidneys with high ENDATs and DSA were C4d positive Only 38% of kidneys with high ENDATs and DSA that subsequently developed chronic ABMR were C4d positive

12. Reduced graft survival in C4d-negative ABMR A: DSA E: ENDAT C: C4d B. Sis et al., AJT 2009

13. C4d negative ABMR – the clinical approach What is the prevalence of DSA in C4d negative (micro)vascular injury in the general population (of TX-recipients)? What is the clinical course of C4d negative rejection without specific treatment? Which diagnostic features are associated with progression to chronic AMR and/or graft loss?

14. Prevalence of alloantibodiesin C4d-negative microvascular injury and in 42-100% of C4d negative glomerulopathy cases Alloantibodies are present in 38-70% of C4d negative glomerulitis cases Issa, Transplantation 2008; Sis, AJT 2007; Shimizu Clin Transpl 2009, Haas AJT 2011 Gaston, Transplantation 2010;Loupy AJT 2009

15. Renal TX12/00 – 2/05 (n=691) C4d negative ABMR – the clinical approach Biopsies for cause (n=481) C4d neg (n=378) C4d pos (n=75) C4d neg + mv lesions + serum (n=28) Regele et al, manuscript in preparation

16. DSA in C4d-negative vascular injury Anti-HLA antibodies Donor specific antibodies P=0.1 P=0.7 P=0.09 P=0.17 100 100 90 90 80 80 70 70 60 60 50 50 40 40 30 30 20 20 10 10 0 0 Neg Cont C4d-pos Cont C4d-neg mvi Neg Cont C4d-pos Cont C4d-neg mvi Regele et al, manuscript in preparation

17. Graft survival in C4d-negative vascular injury Death censored graft survival 1.0 C4d- (n=378) 0.8 C4d- mvi (n=28) 0.6 C4d+ (n=76) 0.4 0.2 0.0 0 1 2 3 4 5 6 7 8 P<0.0001 (C4d+ vs C4d-) Regele et al, manuscript in preparation

18. Summary C4d-negative ABMR Clinical observations and experimental evidence strongly support the concept of C4d-negative ABMR (Micro)vascular injury is a key diagnostic feature that should raise the suspicion and trigger the search for further evidence of ABMR Complement independent mechanisms seems to play a much more important role in chronic ABMR than in acute ABMR C4d-negative rejection tends to show a rather slow and indolent course Reliable diagnostic features of C4d-negative ABMR for therpeutic decisions in individual patients still need to be established