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

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

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

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

5. Renal C4d deposits in 93 patients with early allograft dysfunction 0 10 20 30 40 50 60 70 80 90 100 04 1 3259 6 87 10 12 11 C4d- (N=42) Total (N=93) C4d (+) (N=8) C4d+ (N=43) 90% 72% 63% 57% Months post TX % Allograft suvival Capillary C4d deposition and allograft survival Feucht et al, Kidney Int, 43:1333, 1993

6. 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

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

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

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

10. Recipients without adaptive immune system (RAG1 KO) MHC incompatible donorAnti-donor-MHC moAb Experimental evidence for C4d negative ABMR Jindra PT, Transplantation 2006 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 Fc  RIII dependent mechanism (in absence and presence of complement). DSA alone or in conjunction with macrophages only do not generate CTA. T. Hirohasi, AJT 2012

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 B. Sis et al., AJT 2009 A: DSA E: ENDATC: C4d

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

15. Biopsies for cause (n=481) C4d neg + mv lesions + serum (n=28) C4d pos (n=75)C4d neg (n=378) Renal TX 12/00 – 2/05 (n=691) C4d negative ABMR – the clinical approach Regele et al, manuscript in preparation

16. DSA in C4d-negative vascular injury 0 10 20 30 40 50 60 70 80 90 100 0 10 20 30 40 50 60 70 80 90 100 Neg ContC4d-pos Cont C4d-neg mvi Neg ContC4d-pos Cont C4d-neg mvi P=0.1P=0.7 P=0.09P=0.17 Anti-HLA antibodiesDonor specific antibodies Regele et al, manuscript in preparation

17. Graft survival in C4d-negative vascular injury Death censored graft survival 876543210 1.0 0.8 0.6 0.4 0.2 0.0 P<0.0001 (C4d+ vs C4d-) C4d+ (n=76) C4d- (n=378) C4d- mvi (n=28) Regele et al, manuscript in preparation

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