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First Movement Practicale: Fine-tuning the Instrument of Epitope-Based Histocompatibility

First Movement Practicale: Fine-tuning the Instrument of Epitope-Based Histocompatibility. Rene Duquesnoy University of Pittsburgh Medical Center. Lecture Outline. Immunogenicity and antigenicity of epitopes CDRs and antigen-antibody interactions

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First Movement Practicale: Fine-tuning the Instrument of Epitope-Based Histocompatibility

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  1. First Movement Practicale: Fine-tuning the Instrumentof Epitope-Based Histocompatibility Rene Duquesnoy University of Pittsburgh Medical Center

  2. Lecture Outline • Immunogenicity and antigenicity of epitopes • CDRs and antigen-antibody interactions • HLAMatchmaker analysis of antibody reactivity with Luminex single allele panels • Commonly recognized class II epitopes • Epitope immunogenicity • HLAMatchmaker and platelet transfusions

  3. Identification of Structurally Defined HLA Epitopes • Empirical approach • Look for correlations between antibody reactivity patterns and the presence of distinct polymorphic amino acid residues in different sequence positions on reactive alleles • Conduct absorption/elution of sera with selected alleles • HLAMatchmaker • Epitope structure is based on three-dimensional modeling of different antigen-antibody complexes, molecular contact points between epitope and paratope and the contribution of important residues to epitope functionality • Consider the immunogenetic relationship between antibody producer and immunizer: concept of immunogenicity versus antigenicity • Determine if the antibody reactivity patterns correspond to HLAMatchmaker-predicted epitope mismatches

  4. Important Consideration Differentiate between Immunogenicity of epitopes (induction of specific antibodies)  and Antigenicity of epitopes (reactivity with antibodies)

  5. HLA Mismatch Immunogenicity • Mismatched HLA antigens have different epitope loads • HLA epitopes have different degrees of immunogenicity A better understanding of HLA immunogenicity will permit a permissible mismatch strategy for non-sensitized transplant patients

  6. Antigenicity • Reactivity of epitopes with specific antibodies • Serum screening methods • Complement-dependent lymphocytotoxicity: CDC, AHG • Antigen-binding assays: Elisa, Flow cytometry, Luminex with single antigens • Technique-dependent antibody reactivity

  7. How Do Antibodies React with Antigens?

  8. Six Complementarity-Determining Regions of the Antibody Combining Site • CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3 represent the “binding face” or “paratope” which contacts the structural epitope comprising 15-25 amino acids

  9. Crystal Structure of HLA-A1-MAGE-A1 Complex with Fab-Hyb3 Hulsmeyer et al. J. Biol. Chem., 2005. 280: 2972-2980, 2005

  10. T-Cell Receptor-HLA Complex TCR a b HLA

  11. Six Complementarity-Determining Regions of the Antibody Combining Site • CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3 represent the “binding face” or “paratope” which contacts the structural epitope comprising 15-25 amino acids • The antibody specificity is often determined by a centrally located loop (CDR-H3) that binds to the functional epitope a configuration of 3-6 amino acids in the structural epitope • Eplets may define these functional epitopes • Other CDRs serve as contact sites to stabilize binding to antigen (they play a role in the affinity of antibody)

  12. Noncovalent Forces in Antigen-Antibody Complexing

  13. The Reaction between Antibody and Antigen Depends on the Binding Force

  14. Complementarity Determining Regions

  15. Structural Basis of a HLA-B51 Mismatch “Seen” by A2,A68; B27,B44 “Seen” by A2,A68; B35,B44 Polymorphic Residues on B51

  16. B51 for A2,A68; B27,B44 B51 for A2,A68; B35,B44 ? ? How do antibodies react with structurally defined epitopes?

  17. HLAMatchmaker-Based Analysis of Human Monoclonal Antibody Reactivity Demonstrates the Importance of an Additional Contact Site for Specific Recognition of Triplet-Defined Epitopes Rene J. Duquesnoy, Arend Mulder, Medhat Askar, Marcelo Fernandez-Vina and Frans H.J. Claas Human Immunology 66: 749-761 2005

  18. Antibody Producer : A2,A68; B7,B27; Cw2,Cw7 Immunizer: A3 (pregnancy) Triplets: 62Qe,142mI,144tKr,151aHe,163dT Two examples of mAbs: OK2H12: anti-62Qe OK4F9: anti-142mI

  19. Reactivity of mAb OK2H12 with 62Qe-carrying alleles

  20. 56G is the only residue shared between HLA-A3 and 62Qe-carrying alleles that react with OK2H12

  21. Shared Polymorphic Amino Acids in the 62Qe-Defined Specificity Site Critical Second Contact Site Antibody Specificity Site

  22. Shared Polymorphic Amino Acids in the 62Qe-Defined Specificity Site Critical Second Contact Site Antibody Specificity Site Distance between 56G and 62Q is 11 Angstroms, sufficient for contact by two different CDRs

  23. Shared Polymorphic Amino Acids in the 62Qe-Defined Specificity Site Critical Second Contact Site is a Self Residue Antibody Specificity Site Distance between 56G and 62Q is 11 Angstroms, sufficient for contact by two different CDRs

  24. Immunizing HLA-A3 Triplotype:62Qe,142mI,144tKr,151aHe,163dT Two examples of mAbs: OK2H12: anti-62Qe OK4F9: anti-142mI

  25. Reactivity of mAb OK4F9 with 142mI-Carrying Alleles Lymphocytotoxicity Antigen-Binding Allele Triplets shared with HLA-A3 N %Pos Rx Rx Strength Flow Beads Elisa 142mI 62Qe, ,144tKr,151aHe,163dT A*0301 79 99% 7.6 ++ ++ 142mI 62Qe, ,144tKr A*0101 48 100% 7.4 ++ + 142mI 62Qe, ,144tKr A*1101 68 97% 7.8 ++ ++ 142mI ,151aHe A*2601 37 97% 7.5 ++ ++ 142mI A*2902 16 100% 8.0 ++ ++ 142mI 62Qe, A*3001 13 100% 8.0 ++ ++ 142mI 62Qe, A*3002 12 92% 6.5 nd nd 142mI 62Qe, A*3101 29 97% 7.6 ++ ++ 142mI A*3301 13 92% 7.3 ++ ++ 142mI A*3303 12 75% 6.3 nd ++ 142mI ,151aHe A*3401 19 100% 8.0 ++ nd 142mI ,151aHe A*3402 7 100% 8.0 nd ++ 142mI ,151aHe A*6601 8 100% 8.0 ++ ++ 142mI 62Qe, A*7401 5 100% 8.0 ++ ++ 142mI A*2301 17 0% 1.0 Neg Neg 142mI ,144tKr A*2402 78 8% 1.6 nd Neg 142mI ,144tKr A*2403 4 25% 1.7 Neg Neg 142mI ,144tKr A*2407 5 0% 1.0 nd nd 142mI ,151aHe A*2501 9 0% 1.0 Neg Neg 142mI 62Qe, A*3201 22 0% 1.0 Neg Neg A2/A28 none 67 6% 1.3 Neg Neg

  26. The sequence 79G,80T,81L,82R,83G is shared between HLA-A3 and the 142mI-carrying alleles that react with OK4F9

  27. Locations of 142mI-Defined Specificity and the 79GTLRG-Defined Critical Secondary Contact Sites on A*0301 Crtical Second Contact Site (Self Sequence) Antibody Specificity Site Distance between 142I and 82R is 13 Angstroms, Sufficient for contact by two different CDRs

  28. Residue Polymorphisms and Reactivity of Bw6-Reactive SFR8-B6 Monoclonal Antibody Lutz et al. J. Immunol. 153: 4099, 1994 Studied the effect of single amino acid substitutions by mutagenesis of HLA-B7 molecules • Antibody Specificity site: 80N, 82R, 83G • Critical contact site: 90A (about 11 Angstroms away) • Permissive substitutions in 62 positions!

  29. Many Residue Substitutions Do Not Affect Reactivity With Antibody

  30. Permissive Residue Substitutions that Do Not Inhibit Reactivity of the 62Qe-Specific mAb

  31. Locations of Permissive Polymorphic Residues for 62Qe-Specific mAb Reactivity 56G 62Qe

  32. Complement-Dependent Cytotoxic AntibodyversusNon-Cytotoxic (Binding Only) Antibody

  33. Serological Analysis of the HLA-A10 Complex • “Monospecific” antisera against the HLA-A10 splits A25 and A26 (7th Int. Workshop)

  34. Serological Analysis of the HLA-A10 ComplexDuquesnoy and Schindler: Tissue Antigens 7: 65-73, 1976Hackbarth and Duquesnoy: Transplant. Proc. 9: 43-45, 1977 • Antisera against the HLA-A10 splits A25 and A26 • Absorption studies: Can HLAMatchmaker explain this?

  35. Anti-A25 Serum * Immunizing Antigen

  36. Anti-A26 Serum *Immunizing Antigen

  37. Anti-A10 Sera * Immunizing Antigen

  38. Structural Basis of Cytotoxic Antibody Reactivity against A25 and A26 Anti-A26 Anti-A25 Critical Second Contact Site :76An Critical Second Contact Site :82aLr Antibody Specificity Site: 150tAhe Antibody Specificity Site: 150tAhe Immunizing A26 antigen Immunizing A25 antigen The Critical Second Contact Site is a Non-Self Sequence and is Necessary for Complement Binding

  39. Emerging Concepts • The HLA antibody specificity site consists of a cluster of few polymorphic amino acids on the molecular surface • This site would be contacted by CDR-H3 • Antibody reactivity may require a second contact site which is about 7-15 Angstroms away from the antibody specificity site • This site would be contacted by a different CDR

  40. Interpretations of Negative Reactions of a Serum with HLA Antibodies The mismatched HLA antigen has a different epitope not recognized by patient antibody: Acceptable mismatches are identified through sharing of epitopes not recognized by patient’s antibodies

  41. Interpretations of Negative Reactions of a Serum with HLA Antibodies The mismatched HLA antigen has a different epitope not recognized by patient antibody: Acceptable mismatches are identified through sharing of epitopes not recognized by patient’s antibodies The mismatched HLA antigen carries the antibody specificity site but lacks the Critical Contact Site (CCS) for antibody binding: Acceptable mismatches are more difficult to identify

  42. Interpretations of Negative Reactions of a Serum with HLA Antibodies The mismatched HLA antigen has a different epitope not recognized by patient antibody: Acceptable mismatches are identified through sharing of epitopes not recognized by patient’s antibodies The mismatched HLA antigen carries the antibody specificity site but lacks the Critical Contact Site (CCS) for antibody binding: Acceptable mismatches are more difficult to identify Hidden polymorphisms may affect the conformation of surface residues in the antibody specificity site and/or critical second contact site

  43. HLAMatchmaker Analysis of Antibody Reactivity Patterns with Luminex Single HLA Alleles Example: Screening for antibodies against HLA-DP

  44. Anti- HLA-DP Reactivity of Serum Group B Pt 43, Patient types as DPB1*0201,-

  45. Anti- HLA-DP Reactivity of Serum Group B Pt 43 Patient types as DPB1*0201,- Questions: Do the antibodies react with DPB or DPA or both ? What are the antibody specificities ? Which DP antigens are acceptable mismatches ? Which DP antigens are unacceptable?

  46. HLAMatchmaker Analysis of Anti- HLA-DP Reactivity of Serum Group B Pt 43 (Step 1)

  47. HLAMatchmaker Analysis of Anti- HLA-DP Reactivity of Serum Group B Pt 43 (Step 2)

  48. HLAMatchmaker Analysis of Anti- HLA-DP Reactivity of Serum Group B Pt 43 (Step 3)

  49. HLAMatchmaker Analysis of Anti- HLA-DP Reactivity of Serum Group B Pt 43 (Step 4)

  50. Molecular Locations of Positions 84 and 87

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