Rene Duquesnoy University of Pittsburgh Medical Center

Name: Rene Duquesnoy University of Pittsburgh Medical Center
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Description: Rene Duquesnoy University of Pittsburgh Medical Center

Rene Duquesnoy University of Pittsburgh Medical Center
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 HLAMatchmaker analysis of antibody reactivity with Luminex single allele panels Commonly recognized class II epitopes Epitope immunogenicity HLAMatchmaker and platelet transfusions

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

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

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

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

How Do Antibodies React with Antigens?

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 amino acids

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

T-Cell Receptor-HLA Complex
TCR b a HLA

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 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)

Noncovalent Forces in Antigen-Antibody Complexing
Electrostatic forces Attraction between opposite charges Hydrogen bonds Hydrogen shared between electronegative atoms (N,O) Van der Waals forces Fluctuations in electron clouds around molecules oppositely polarize neighboring atoms Hydrophobic forces Hydrophobic groups pack together to exclude water molecules

The Reaction between Antibody and Antigen Depends on the Binding Force
CDR Contact Site Reaction Too low No SpecCDR No Eplet Negative Borderline Is SpecCDR insufficient? Eplet No binding? Intermediate SpecCDR is sufficient or requires a 2nd CDR Eplet + 2nd Contact Site Binding only Strong SpecCDR + 2nd CDR + more CDR? Eplet + 2nd Contact Site + more? Binding and conformational change of Fc to expose C1q binding site

Complementarity Determining Regions

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

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

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:

Two examples of mAbs: OK2H12: anti-62Qe OK4F9: anti-142mI
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

Reactivity of mAb OK2H12 with 62Qe-carrying alleles

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

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

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

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

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

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

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

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

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!

Many Residue Substitutions Do Not Affect Reactivity With Antibody

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

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

Complement-Dependent Cytotoxic Antibody versus Non-Cytotoxic (Binding Only) Antibody

Serological Analysis of the HLA-A10 Complex
“Monospecific” antisera against the HLA-A10 splits A25 and A26 (7th Int. Workshop) Typing Serum Reaction with A25 Reaction with A26 Anti-A25 Yes No Anti-A26

Serological Analysis of the HLA-A10 Complex Duquesnoy and Schindler: Tissue Antigens 7: 65-73, 1976 Hackbarth and Duquesnoy: Transplant. Proc. 9: 43-45, 1977 Antisera against the HLA-A10 splits A25 and A26 Absorption studies: Serum Reaction with A25 Reaction with A26 Anti-A25 Cytotoxicity No Cytotoxicity Only Binding Anti-A26 Can HLAMatchmaker explain this?

Anti-A25 Serum Unshared Triplets Serum HLA-type Ab Producer Ag Jun
A1,A3;B7.B8 A25* Cytotoxic 80rIAlr, 150tAHe,156W, 183A, 193Av A26 Only Binding 150tAHe,156W,183A, 193Av * Immunizing Antigen

Anti-A26 Serum Unshared Triplets Serum HLA-type Ab producer Ag Mich
A3,A23;B7,B13 A26* Cytotoxic 76An, 90D, 150tAHe, 156W,163R,183A,193Av A25 Only Binding 90D,150tAHe,156W, 163R,183A,193Av *Immunizing Antigen

Anti-A10 Sera Serum HLA-type Ab Producer Ag Unshared Triplets Sand
A1,A24;B7,B44 A25* Cytotoxic 66rNv, 150tAHe,156W, 183A, 193Av A26 Cytotoxic 66rNv, 150tAHe,156W, 183A, 193Av Elli A1.A32;B13,B64 A26* Cytotoxic 150tAHe,156W A25 Cytotoxic 150tAHe,156W * Immunizing Antigen

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

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

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 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

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

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

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

Anti- HLA-DP Reactivity of Serum Group B Pt 43 Patient types as DPB1
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?

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

Molecular Locations of Positions 84 and 87

84DE+87AV Corresponds to an Antibody-Defined Epitope

HLAMatchmaker-Predicted Unacceptable DPB Alleles

HLAMatchmaker-Predicted Acceptable DPB Alleles

HLAMatchmaker Determination of Unacceptable DPA Alleles

Predominant Eplets Reacting with anti-HLA-DP Antibodies
* 55DE is similar to the 57DE eplet on DR11 All 55DE-reactive sera reacted also with DR11

Retransplant Candidates Have Donor-Specific Antibodies that React with Structurally Defined HLA-DR,DQ,DP Epitopes Duquesnoy et al. Transplant immunology, 18: , 2008

Three Groups of HLA-Sensitized Patients
A. Sensitizing tissue is absent Previous transplant has been removed Prior transfusion and pregnancy B. Sensitizing tissue is present Transplanted organ still in place No evidence of pre-transplant sensitization C. Combination of A and B

Incidence of Anti-HLA-DR, -DQ and -DP Antibodies in HLA Class II Sensitized Patients with or without a Transplant Present * p= ** p<0.0001

Effect of eplet mismatching and the Incidence of antibodies to donor DRB1, DRB3, DRB4 and DRB5 mismatches

Eplet mismatching and the incidence of donor-specific anti-HLA-DQ antibodies

Frequencies of antibodies to donor DQB eplet mismatches

Frequencies of antibodies to donor DQA eplet mismatches

The Antibody Response to an HLA Mismatch is Restricted to a Small Number of Epitopes

SERUM ANALYSIS AFTER TRANSPLANT NEPHRECTOMY REVEALS RESTRICTED ANTIBODY SPECIFICITY PATTERNS AGAINST STRUCTURALLY DEFINED HLA CLASS I MISMATCHES Adeyi et al. Transplant Immunology, 14: 53-62, 2005

Adeyi et al. Transplant Immunology, 14: 53-62, 2005
Thirty Patients With Rejected Kidney Transplants Underwent Allograft Nephrectomy 100% 80% PRA 60% 40% 20% 0% Before Tx Pre-AlloNx Post-AlloNx Adeyi et al. Transplant Immunology, 14: 53-62, 2005

Donor Epitopes: A3: 62Qe,66rNv,70aQs,76Vd,80gTl,144tKr,149aAh,151aHe,163dT A31: 56R,62Qe,66rNv,74iD,76Vd,80gTl,193Av B55: 131S B63: 45Ma,66rNm,70aSa,74Y,131S Adeyi et al. Transplant Immunology, 14: 53-62, 2005

Donor Epitopes (serum-reactive epitopes are shown in underlined bold font) A3: 62Qe,66rNv,70aQs,76Vd,80gTl,144tKr,149aAh,151aHe,163dT A31: 56R,62Qe,66rNv,74iD,76Vd,80gTl,193Av B55: 131S B63: 45Ma,66rNm,70aSa,74Y,131S Adeyi et al. Transplant Immunology, 14: 53-62, 2005

Donor Epitopes (serum-reactive epitopes are shown in underlined bold font) A3: 62Qe,66rNv,70aQs,76Vd,80gTl,144tKr,149aAh,151aHe,163dT A31: 56R,62Qe,66rNv,74iD,76Vd,80gTl,193Av B55: 131S B63: 45Ma,66rNm,70aSa,74Y,131S Unacceptable epitopes identify unacceptable antigens e.g. 45Ma is present on B13, B46, B57,B62, B63. B75, B76, B76 Adeyi et al. Transplant Immunology, 14: 53-62, 2005

Donor Epitopes: B13: 41T,45Ma,76En,80rTa,82aLr,144tQl,163E 82aLr is expressed by all Bw4-associated HLA-B antigens + A23, A24, A25 and A32 144tQl is unique for HLA-B13 After six months: the anti-76En and anti-82aLr antibodies have disappeared, Only anti-144tQl antibodies are detected Adeyi et al. Transplant Immunology, 14: 53-62, 2005

Relative Immunogenicity of Eplets
How often do mismatched eplets induce specific antibodies? 14th International HLA Workshop Project Analysis of donor-specific antibodies in patients whose rejected kidney transplants had been removed

Preliminary Results: Eplet Immunogenicity in 62 Cases
Duquesnoy, RJ and Claas FHJ: Progress Report of 14th International Histocompatibility Workshop Project on the Structural Basis of HLA Compatibility, Tissue Antigens, 69 (Suppl. 1): 1-5, 2007

15th International Workshop Project on Epitope Immunogenicity
Analyze post-allograft nephrectomy sera for antibodies against donor class I and class II epitopes Serum screening with single alleles (Luminex) and by CDC So far, 40 laboratories worldwide will contribute informative cases For more information go to

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

Use of HLAMatchmaker in Platelet Transfusions

Department of Transfusion Medicine, National Institutes of Health
HLAMatchmaker-Driven Analysis of Responses to HLA Typed Platelet Transfusions in Alloimmunized Thrombocytopenic Patients Ashok Nambiar, Rene J. Duquesnoy, Sharon Adams, Yingdong Zhao, Jaime Oblitas, Susan Leitman, David Stroncek and Francesco Marincola Department of Transfusion Medicine, National Institutes of Health Blood 107: , 2006

Structural epitope matching for HLA-alloimmunized
T R A N S F U S I O N P R A C T I C E Structural epitope matching for HLA-alloimmunized thrombocytopenic patients: a new strategy to provide more effective platelet transfusion support? Rene J. Duquesnoy Transfusion, 48: , February 2008

Alloimmunization-Induced Refractoriness to Random Donor Platelet Transfusions
Antibody reactivity with HLA Class I antigens Platelet-specific antigens Other antigens (MICA?, HLA class II?, blood groups) Refractory patients Hematological defects (e.g. aplastic anemia) Malignancies (e.g. leukemia) Highly sensitized liver transplant candidates

Treatment of HLA Alloimmunization-Induced Refractoriness
Platelet transfusions from HLA matched donors (Yankee et al. N Eng J Med 281:1208, 1969) Platelet transfusions from donors mismatched for cross-reacting HLA antigens (Duquesnoy et al. Amer J Hematol 2: 219, 1977)

Serological Cross-Reactivity Between HLA Antigens
HLA antigens carry “private” and “public” epitopes Cross-Reacting Groups (CREGs) of HLA antigens share the same public epitope Rodey and Fuller: Crit Rev Immunol 7:229, 1987

Duquesnoy et al. American Journal of Hematology. 2:219-226, 1977

Responses to Platelet Transfusions with Different HLA Match Grades
Duquesnoy et al. American Journal of Hematology. 2: , 1977

Limitations of the Cross-Reactivity Based HLA Match Grade System
Many BX matches are unacceptable epitope mismatches A and BU match groups may have incompatible epitopes revealed by 4-digit DNA typing CREG matching considers only HLA-A and HLA-B; how important is HLA-C?

Many BX Matches Have Incompatible Epitopes
Cross-Reacting Matches and Bw4/Bw6 mismatches Eplet differences between Cross-Reacting Antigens Effect of BW4/6 incompatibility on responses to BX matched platelet transfusions Patients 1-8: lower increments Patients 9-21: comparable increments McElligott et al Blood 59: 971, 1982

Eplet Differences Between Cross-Reacting Antigens Example: A2 CREG

Many BX matches are unacceptable epitope mismatches A and BU match groups may have incompatible epitopes revealed by 4-digit DNA typing CREG matching considers only HLA-A and HLA-B; how important is HLA-C?

Eplet Differences Between 4-Digit Alleles

Many BX matches are unacceptable epitope mismatches A and BU match groups may have incompatible epitopes revealed by 4-digit DNA typing CREG matching considers only HLA-A and HLA-B How important is HLA-C? Not Important (Duquesnoy et al Transplant. Proc. 9: 1827, 1977) Low expression on platelets (Mueller-Eckhart et al Tissue Antigens 16: 91, 1980) Important for some patients (Saito et al Transfusion 42: 302, 2002)

HLA-C Eplets in Positions 1-193

Example of Luminex Screen for Antibodies Specific for HLA-C (1)
What is the cut-off point to distinguish between positive and negative reactions?

Antibody react with Cw*0102 of donor Reactivity with donor’s Cw*0701? What HLA-C epitopes are recognized? Do HLAMatchmaker analysis What are the mismatched eplets?

These are the eplets on HLA-C alleles of donor and the panel Cw*1203 has no mismatched eplets for this patient One or more eplets on donor’s Cw*0102 must react with antibody Any reactivity with 65QNR of donor’s Cw*0701?

No reactivity with 65QNR of donor’s Cw*0701 No reactivity with the eplets of the negative alleles Cw*0202, Cw*0401, Cw*0501, Cw*1502, Cw*1701 and Cw*1801 Record these negative alleles in the HLAMatchmaker program

The 77TVS eplet of donor’s Cw*0102 is present on all reactive alleles 77TVS is an unacceptable mismatch

Proposed HLA Epitope-Based Matching Protocol (steps 1 and 2)
Perform HLA-A, B, C typing of patients and donors by DNA methods at the high-resolution (4-digit allele) level. Screen patient sera with HLA typed panel Complement-dependent methods: direct and/or antiglobulin-augmented lymphocytotoxicity Antigen-binding assays such as Luminex, Flow Cytometry and ELISA preferably with single HLA class I alleles HLAMatchmaker-based analysis of serum reactivity pattern to identify acceptable mismatches

Proposed HLA Epitope-Based Matching Protocol (step 3)
3. Conduct a platelet donor search Establish a computerized platelet donor registry that incorporates an HLAMatchmaker-based search engine Enter the HLA type of the patient and the non-reactive mismatched alleles in this database and the computer will generate a list of donors with matches and acceptable mismatches at the eplet level No need for platelet cross-match testing for HLA incompatibility

Proposed HLA Epitope-Based Matching Protocol (step 4)
4. Evaluate the outcome of the platelet transfusion, if increment is low then: Determine whether serum reactivity patterns have improperly been interpreted in terms of HLA mismatch acceptability Look for antibodies against platelet-specific antigens and blood groups, or autoimmune phenomena and drug reactions Consider clinical conditions such as coagulopathy, infection and hepatosplenomegaly

Prevention or Delay of HLA Alloimmunization
HLAMatchmaker-based selection of apheresis platelets with minimal numbers of mismatched eplets From existing inventories of stored platelets Do a computer search for compatible platelet donor Avoid immunogenic eplets Leukoreduction of platelet preparations prior to transfusion

Conclusions The serological cross-reacting antigen matching system introduced in 1977 should be replaced by a system that incorporates modern concepts of epitope reactivity with antibody. The proposed HLA epitope matching protocol is expected to benefit platelet transfusion outcome and increase the number of compatible donors for refractory patients.

http://www.HLAMatchmaker.net Introductory articles and tutorials
New Website Introductory articles and tutorials HLAMatchmaker related publications From UPMC From other institutions Free downloads of all programs Class I and class II matching Antibody analysis Other programs Contacts and communications

If you would like to discuss HLAMatchmaker in Pittsburgh, come visit me in my office…

on the Allegheny River

Rene Duquesnoy University of Pittsburgh Medical Center

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