1. Epitope analysis with MatchIt software Ivan Balazs, Ph.D. HLA Epitopes in Clinical Transplantation Pittsburgh, PA June 15-16, 2011

2. Epitope Analysis with single antigens Why do it: –Rationalize/explain serum reactivity patterns –Distinguish between Donor vs de novo antibodies –Facilitate assignment of permissible and non-permissible antigens Information needed: –Patient HLA typing (4 digit res.) –Donor HLA typing (4 digit res.) –Pre-Transplant Ab sensitization events –Post-Transplant Ab results –List of functional epitopes

3. Epitope Analysis with single antigens. Confounding events (life is not easy!!!): Unknown number and source of sensitizing event –Transfusions (blood or components) –Pregnancies & aborted pregnancies Sensitization to multiple Ag(s) and similar or very different levels of Abs There may be epitopes not accounted for (e.g. effect of internal aa changes on Ag conformation and antibody affinity) Unfortunately not every case lends itself to this type of analysis.

4. Confounding events (continued) Sample analysis requires time and money. Mixture of Ab and presence of multiple IgG subtypes (Adsorption/elution with single Ags; Subtype specific Ab) Interference by IgM (inactivate or eliminate) High titer serum (signal detector saturation) –Repeat assay at 1:4, 1:16 dilution to bring within range Therefore, not every case can be analyzed

5. MatchIt Software antibody analysis MatchIt software for Ab was designed as a LIMS system for the analysis of serological results obtained with Lifecodes products MatchIt software can also be used for epitope assignment HLAMatchmaker software for epitope sharing, permissible/non-permissible Ag assignments

6. MatchIt software for antibody analysis Epitope assignment It utilizes the list of epitopes proposed by Duquesnoy in HLAMatchmaker and publications: –M. Marrari & R. J. Duquesnoy. Correlations between Terasaki’s HLA class II epitopes and HLAMatchmaker-defined eplets on HLA-DR and -DQ antigens. Tissue Antigens (2009): 70: 134 –R. J. Duquesnoy & M. Marrari. Correlations between Terasaki’s HLA class I epitopes and HLAMatchmaker-defined eplets on HLA-A, -B and -C antigens. Tissue Antigens (2009): 70: 117 Customizable list of epitopes and specificities i.e. import/export list) Automated assignment of epitopes by tail analysis. Possibility to perform manual assignments

7. Epitope distribution in Class I Ag

8. Automated Tail Analysis of epitopes

9. Maual epitope Analysis Donor HLA Type A2,A11,B7,B40

10. Examples of Class I epitopes Donor: A*24:02

11. Examples of Class I epitopes MM Donor: A*02:05

12. Examples of Class I epitopes MM Donor: A*35:01

13. Examples of Class II epitopes MM Donor: DQA*03:02/B*03:03

14. Examples of Class II epitopes MM Donor: DQA*05:01/B*03:01

15. Examples of Class II epitopes MM Donor: DRB3*01:01 DQB*03:01

16. There is still a lot to be learned To help interpret the patterns of antibody reactivity we need more experimental results for: –Empirical confirmation of immunogenic epitopes (modeling is just the start) –Estimating relative frequency/immunogenicity of different epitopes (not all mismatched Ag are equally immunogenic) (F. Claas et al.) –What is the effect of the other patient’s Ags on the immunogenicity of the mismatched antigens (shared CDR?)

17. 16th International HLA and Immunogenetics Workshop Liverpool, UK 28th May - 3rd June 2012 Project: Development of an HLA Epitope Database Rene Duquesnoy University of Pittsburgh

18. 16th International HLA and Immunogenetics Workshop Committee: Rene Duquesnoy (Pittsburgh, PA) Chair, Domenico Adorno (L’Aquila, Italy), Ivan Balazs (Stamford, CT, USA), Frans Claas (Leiden, The Netherlands), Nadim El-Awar (Los Angeles, CA, USA), Marcelo Fernandez-Vina (Houston, TX, USA), Nils Lachmann (Berlin, Germany), Derek Middleton (Liverpool, UK), Arend Mulder (Leiden, The Netherlands), Craig Taylor (Cambridge, UK), Robert Vaughan (London, UK) and Cristina von Glehn (Curitiba, Brazil) This committee has the following tasks: Determine criteria for the experimental verification of epitopes reacting with informative antibodies. Address the question of clinically relevant versus irrelevant epitopes. Develop a notation system for Class I and Class II epitope repertoires Verify experimentally defined epitopes submitted by workshop participants Develop a website based HLA epitope database Please note that this project addresses only antibody-reactive epitopes, not cellular epitopes defined by T-cells.

19. Acknowledgements Gen-Probe Transplant Diagnostics, Inc. Bryan Ray Alexandra Storm Justin Mostecki Jayasree Hariharan Lioudmila Zaslavskaia