Emanuele Cozzi Transplantation Immunology Unit Padua University Hospital

1. De novo appearance of anti-HLA antibodies in a population of first renal allograft recipients Emanuele Cozzi Transplantation Immunology Unit Padua University Hospital

2. Objective of the study • Evaluate the kinetics of appearance of DS and NDS anti-HLA antibodies and study their possible correlation with an early deterioration of graft function in a group of patients transplanted in 9 Italian Renal Transplant Centres • This cohort of patients should form the basis for future interventional studies

3. ItalianCentersactivelyinvolved in the study • Padua • Bologna • Brescia • Florence • Novara • Rome • Turin • Treviso • Bari • Paul Terasaki (ONE LAMBDA - Terasaki Foundation) • Robert Montgomery (Johns Hopkins, Baltimore)

4. Study design: Patientselection and antibody monitoring • 400 recipients first renal transplantin the absence of anti-HLA antibodies [DSA or NDSA] on the day of their [sample taken on the day of transplantation, irrespective of historical/current sera]; • Antibody monitoring post-transplantation: • by solid phase assays (Flow or Luminex – kindly provided by One Lambda), Class I and II anti-HLA; DSA or NON-DSA • At 0, 1, 3 months and every 3 months thereafter • To identify and characterize the de novo appearance of anti-HLA antibodies in the absence of humoral rejection or deterioration of graft function (Stable function de novo DSA/NDSA) • Immunosuppression selected by the center (Tacro-MMF-steroids in more than 65% patients)

5. 01/46/MP: anti-HLA Follow-up (FlowPRA) Tx M13 Class I Class II

6. Patients selection and Enrollment Exclusion of 14.7% selected patients [false negative due to: increased sensitivity of method use or new immunizing event since last pre-tx sample?]

7. Demography of enrolled patients Number of patients 344 Age (years) 52 ±11,5 Male vs Female 246 vs 98(72%vs 28%) Patients with previous pregnancies 52/98 Patients with previous transfusions 76 Mean Mismatch number 3.5 ±1.2

8. Enrollment and Follow-up • First patient enrollment: 2007 • Last patientenrollment: August 2013 • Median of enrollment time: 26 months (0-82 months) • Follow-up period: • 360 patients: ≥ 12 months • 252 patients: ≥ 24 months

9. de novo appearance of anti-HLA antibodies during follow-up in patients with stable graft function Stable function de novo DSA

10. de novo appearance of anti-HLA antibodies during follow-up in patients with stable graft function Stable function de novo DSA

11. de novo appearance of anti-HLA antibodies during follow-up in patients with stable graft function Stable function de novo DSA

12. de novo appearance of anti-HLA antibodies during follow-up in patients with stable graft function Stable function de novo DSA

13. de novo appearance of anti-HLA antibodies during follow-up Stable function de novo DSA/NDSA

14. de novo appearance of anti-HLA antibodies during follow-up Stable function de novo DSA/NDSA

15. de novo appearance of anti-HLA antibodies during follow-up Stable function de novo DSA

16. Seroconversion (DSA+NDSA) and antibody specificity: Class I vs Class II 63 % patients develop de novo Class I antibodies 50 % patients develop de novo Class II antibodies

17. Seroconversion and antibody specificity: DSA vs NDSA [typing for Loci C, DP, DQ is complete] 55% patients develop DSA 95% patients develop NDSA (45% isolated NDSA)

18. DSA specificity: Class I vs Class II Patients developing de novo DSA • 25 % anti-class I • 50 % anti-class II • 25 % anti class I+ II • 11 % • 78 % • 11 % • 30 % • 64 % • 6? % • 75% patients develop anti-HLA class II DSA • Anti-DQ are the most commonly observed DSA

19. Trend of de novo DSA during a 2 year follow-up in patients with stable graft function • Usually against a very limited number of HLA targets (DQ!) • MFI does not increase with time • DSA may disappear spontaneously • NO epitope expansion

20. Trend of de novo NDSA [iDSA] during a 2 year follow-up in patients with stable graft function • Usually against many HLA targets • Most NDSA disappear spontaneously • NO epitope expansion • No evidence of conversion NDSA→DSA]

21. Considerations at this stage of the study • No statistically significant correlation has been found between those withde novo antibodies and those without: • Demographics • Presence of immunising events • Average mismatch number (4.1±1.25 vs 3.6±1.0; p=0.051) • Acute rejection episodes • Patients with de novo DSA/NDSA retain an excellent graft function: • Mean Creatine at Tx: 138.7±67.6 µmol/l • Mean Creat. 6 months post seroconversion: 125.2±35 µmol/ • Mean Creat. 12 months post seroconversion: 124±41 µmol/l

22. Conclusions • high percentage of patients with stable renal function develop an early de novo anti-HLA antibody response (27.8% at 2yrs) • These de novo anti-HLA antibodies are anti-Class I or class II antibodies; they are DSA in 55% cases. • DSA: • At this stage of the study, we have no evidence as yet that de novo anti-HLA antibodies are associated with ill-effects • Routine monitoring of DSA are likely to become part of the standard follow upprovided to transplanted patients • Usually to very a limited number of HLA targets; • may disappear spontaneously; • NOepitope expansion; • anti-DQ are the most commonly observed DSA