ASHI

1
ASHIs New World Symphony

• A Master Piece With Five Movements

2
First Movement Perfecting The Match

• Rene Duquesnoy
• University of Pittsburgh Medical Center

3
Matching The Traditional Way

• Count the number of A, B, DR antigen mismatches
• Why do so many zero-antigen mismatches fail?
• Why are many mismatches successful?
• Determine unacceptable mismatches for highly
  sensitized patients
• Why do see so many graft failures?
• Why are many patients never transplanted?
• Crossreactive antigen matching for platelet
  transfusions of refractory thrombocytopenic
  patients
• Why are so many such transfusions unsuccessful?
• Why do some non-crossreactive mismatches work so
  well?

4
New Developments in HLA

• Complete typing for all HLA loci
• Better methods for HLA antibody identification
• HLA structure and polymorphisms
• HLA and transplant immunity

5
HLA Effect on Transplantation

• Humoral Immunity
• Complement-dependent antibodies
• Complement-independent antibodies
• Cellular Immunity
• Direct Allorecognition
• Cytotoxic CD8 T-cells
• Effector CD4 T-cells
• Regulatory T-cells
• Indirect Allorecognition
• Effector CD4 T-cells
• NK cells (KIR)
• HLA-restricted Immune Responses
• Anti-viral immunity
• Cytotoxic CD8 T-cells
• Recurrent autoimmune disease
• Effector CD4 T-cells
• Graft-versus-Host Reactivity
• Mediated by donor T-cells

6
Antibody Responses to HLA

• Class I HLA antigens
• HLA-A and HLA-B
• HLA-C
• MICA
• Class II HLA antigens
• DRB1
• DRB3, 4, 5
• DQB and DQA
• DPB and DPA

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HLA Antigens Have Multiple Epitopes

• Private epitopes and public epitopes shared
  between cross-reacting antigens (CREGs)
• Epitopes defined by polymorphic amino acid
  residues
• HLAMatchmaker
• Original version (2002) triplets, i.e. linear
  three-residue sequences
• New version (2006) eplets, i.e. patches of
  residues within a 3 Angstrom radius of a
  polymorphic residue on the molecular surface.

Duquesnoy A Structurally Based Approach to
Determine HLA Compatibility at the Humoral Immune
Level, Human immunology, 67 847-862, 2006
8
Amino Acid Polymorphisms On The Molecular Surface
Are Responsible for Epitopes That Induce Specific
Alloantibodies
How can we visualize the polymorphic residues?
9
Locations of Polymorphic Residues on HLA-A, B, C
Molecules
From Kostyu et al. Human Immunology 57, 1-18,
1997
10
Space Fill Model of HLA Class I Molecule
Top view
Side view
a1
a1
a2
peptide
b2M
a2
a3
Visualization of polymorphic residues with
three-dimensional structural modeling of HLA
molecules with Cn3D program downloadable from the
NCBI website www.ncbi.nlm.nih.gov
11
Polymorphic Residues on Class I Antigens
HLA-A2
HLA-B27
HLA-Cw3
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Topography of Polymorphic Residues on HLA-DR and
HLA-DQ Molecules
HLA-DQ
HLA-DR
?1
?1
?1
?1
?2
?2
?2
?2
13
The HLA type of the antibody producerdetermines
what structural components of an immunizing HLA
antigen can be seen as non-self
HLAMatchmaker Concept
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Structural Basis of a HLA-B51 Mismatch
Polymorphic Residues on B51
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Structural Basis of a HLA-B51 Mismatch
Seen by A2,A68 B27,B44
Polymorphic Residues on B51
16
Structural Basis of a HLA-B51 Mismatch
Seen by A2,A68 B27,B44
Seen by A2,A68 B35,B44
Polymorphic Residues on B51
17
Structural Basis of a HLA-B51 Mismatch
Seen by A2,A68 B27,B44
Seen by A2,A24 B7,B8
Seen by A2,A68 B35,B44
Polymorphic Residues on B51
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Matching at the Epitope Level Provides a Better
Assessment of HLA Compatibility than Matching at
the Antigen Level
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HLAMatchmaker Original Version
Donor HLA-A,B mismatches are defined by linear
sequences of amino acid residues (triplets) on
alloantibody-accessible sites (i.e. a-helices and
b-turns) of HLA molecules
Human Immunol. 63 339-352, 2002 63 353-363,
2002
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Histocompatibility Determination by HLAMatchmaker

• The HLA-A,B,C type of the patient represents six
  strings of self-triplets that cannot induce
  specific alloantibodies
• Each donor HLA antigen represents a string of
  triplets
• Compatibility is assessed by lining up donor
  triplet strings with patient triplet strings to
  determine differences between donor and patient
  (Microsoft Excel program)

Human Immunol. 63 339-352, 2002 63 353-363,
2002
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Notation System for Triplets and Eplets

• The number indicates the amino acid sequence
  position
• Polymorphic residues are listed with the single
  amino acid letter code (monomorphic residues are
  not listed)
• Examples 9F 12SV . 66RNV 76ES 90A etc.

22
Potential Donor has a HLA-B8 Mismatch

• For which patient is this antigen
• a better mismatch?
• Patient 1 HLA-A2,A30 B18,B27 Cw2,Cw4
• Patient 2 HLA-A2,A31 B42,B53 Cw2,Cw7

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HLA-B8 Mismatch forHLA-A2,A30 B18,B27 Cw2,Cw4
Mismatched triplets are in underlined bold white
font
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HLA-B8 Mismatch for HLA-A2,A30 B18,B27 Cw2,Cw4
HLA-B8 has six mismatched triplets
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HLA-B8 Mismatch for HLA-A2,A31 B42,B53 Cw2,Cw7
Patient HLA
9
12
14
17
41
45
56
62
66
70
74
76
80
A2
A0201
F
sV
R
gR
A
Me
G
Ge
rKv
aHs
H
Vd
gTL
A31
A3101
T
sV
R
gR
A
Me
R
Qe
rNv
aHs
iD
Vd
gTL
B42
B4201
Y
sV
R
gR
A
Ee
G
Rn
qIy
aQa
D
Es
rNl
B53
B5301
Y
aM
R
gR
A
Te
G
Rn
qIf
tNt
Y
En
rIa
Cw2
Cw0202
Y
aV
R
sR
A
Ge
G
Re
qKy
rQa
D
Vn
rKl
Cw7
Cw0701
D
aV
R
gR
A
Ge
G
Re
qNy
rQa
aD
Vs
rNl
Donor HLA
B8
B0801
D
aM
R
gR
A
Ee
G
Rn
qIf
tNt
D
Es
rNl
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Pv
A
G
A
V
E
Cw7
T
Ew
Y
Et
Q
eP
K
Pl
A
G
A
V
Eq
B8
T
Ew
Y
Dt
E
dP
K
Pi
A
G
A
V
E
B8 is a zero-triplet mismatch for
A2,A31B42,B53Cw2,Cw7
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Examples of HLA Antigens with 0-2 Triplet
Mismatches
Nr of Mismatched Triplets Patient 1 HLA-A2,A30 B18,B27 Cw2,Cw4 Patient 2 HLA-A2,A31 B42,B53 Cw2,Cw7
Zero A69, B64, B65 A32, A74, B8, B35, B54, B55, B56, B59, Cw3, Cw6
One A68, B37, B39, B73, B74, Cw6 A69, B51, B67, B70, B71, B72, B76, B78
Two B61, B70, B71, B72, Cw1, Cw3, Cw6 A33, A68, B7, B38, B39, B46, B52, B58, B64, B65, B75, B77, B81, B82, Cw1, Cw8
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Is HLAMatchmakerClinically Useful?
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KIDNEY ALLOGRAFT SURVIVAL AND HLA CLASS I
MATCHING AT THE AMINO ACID TRIPLET LEVEL
Duquesnoy, RJ , Takemoto S, de Lange P, Doxiadis
IIN, Schreuder GMT , Persijn, G and Claas FJH
Transplantation 75884-889, 2003
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Five-Year Graft Survivals of Zero-HLA-DR
Mismatched Primary Kidney Transplants with 0-4
HLA-A,B Antigen Mismatches
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Effect of HLA-A,B Triplet Matching on Five-Year
Graft Survivals of Zero-HLA-DR Mismatched Kidney
Transplants in the UNOS Database
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Effect of HLA-A,B Triplet Mismatching on Graft
Survival of Zero-HLA-DR Mismatched Kidneys in
Eurotransplant
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HLAMatchmaker Predicts Antibody Formation to HLA
Mismatches
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Volume 77(8)             27 April 2004           
 pp 1236-1239
THE NUMBER OF AMINO ACID TRIPLET DIFFERENCES
BETWEEN PATIENT AND DONOR IS PREDICTIVE FOR THE
ANTIBODY REACTIVITY AGAINST MISMATCHED HUMAN
LEUKOCYTE ANTIGENS Dankers, Marlies K. A
Witvliet, Marian D Roelen, Dave L De Lange,
Peter Korfage, Nelleke Persijn, Guido G.
Duquesnoy, René Doxiadis, Ilias I. N Claas,
Frans H. J. Department of Immunohematology and
Blood Transfusion, Leiden University Medical
Center
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Volume 77(8)             27 April 2004           
 pp 1236-1239
THE NUMBER OF AMINO ACID TRIPLET DIFFERENCES
BETWEEN PATIENT AND DONOR IS PREDICTIVE FOR THE
ANTIBODY REACTIVITY AGAINST MISMATCHED HUMAN
LEUKOCYTE ANTIGENS Dankers, Marlies K. A
Witvliet, Marian D Roelen, Dave L De Lange,
Peter Korfage, Nelleke Persijn, Guido G.
Duquesnoy, René Doxiadis, Ilias I. N Claas,
Frans H. J. Department of Immunohematology and
Blood Transfusion, Leiden University Medical
Center
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The Epitope Load Determines the Immunogenicity
of a Mismatched HLA Antigen
37
HLA Matching at the Triplet Level

• Reduces antibody formation
• Improves kidney transplant survival
• Increases the availability of suitably matched
  donors

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The Highly Sensitized Transplant Candidate

• Match or Treat?

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The Highly Sensitized Transplant Candidate

• Match or Treat and Match?

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Transplant Strategy For Highly Sensitized
Candidates

• Sensitization history
• Laboratory assessment of sensitized state
• Serum antibody reactivity and specificity
• Identification of acceptable mismatches
• Assess transplantability of patient (what is the
  chance of finding a suitably matched donor?)

42
HLAMatchmaker for the Highly Sensitized Patient
43
ExamplePatient HLA-A3,11B18,62 (PRA96)
HLAMatchmaker Results
44
Lymphocytotoxicity Screen Patient
HLA-A3,11B18,62 (PRA96)

1. 48/50 panel cells gave consistently positive
   reactions with patients serum
2. Two negative cells A3,A26B62,- and
   A11,-B18,51
3. A26 and B51 have unshared triplets but none of
   them are apparently recognized by patients
   antibodies

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Patient 3 HLA-A3,11B18,62 (Contd)
4. Eight mismatched triplets on A26 and five
mismatched triplets on B51 are not recognized by
patients antibodies they are acceptable triplet
mismatches 5. Enter A26 and B51 as negative
antigens in HLAMatchmaker 6. The program will
identify additional HLA antigens with triplets
that either matched or acceptable
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Patient 3 HLA-A3,11B18,62 (PRA96)
HLAMatchmaker Results after Serum Analysis
Neg Ag
Neg Ag
Zero-trp mm
After A26 and B51 had been entered as negative
antigens, HLAMatchmaker identified eight HLA
antigens as acceptable/zero triplet mismatches
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Serum Screening May Identify Acceptable Antigen
Mismatches and Increase the Probability of
Finding a Suitable Donor for a Highly Sensitized
Patient
49
Chances of Finding a Zero or Acceptable HLA-A,B
Mismatch for a High PRA Patient (30 patients)
Eurotransplant Patients, Leiden, The Netherlands
50
Eurotransplants Acceptable Mismatch Program Now
Includes HLAMatchmaker

• Validation of HLAMatchmaker by direct
  cross-matches
• HLA-A mismatch
• Expected Observed
• negative negative
• 0 triplet 18 18
• 1 triplet 25 25
• HLA-B mismatch
• Expected Observed
• negative negative
• 0 triplet 54 54
• 1 triplet 133 131

AM are Acceptable Mismatches 5-85 and gt85 PRA
are conventional ET-KAS cases
Claas et al. The Acceptable Mismatch Program as
a Fast Tool to Transplant Highly Sensitized
Patients Awaiting a Post-Mortal Kidney Short
Waiting Time and Excellent Graft Outcome.
Transplantation 78190-193, 2004
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Shortcomings of Current HLAMatchmaker Algorithm

• Limited to linear three-residue sequences (i.e.
  triplets)
• Does not always consider conformational
  influences of nearby polymorphic residues
• Triplets do not identify all corresponding
  serologically defined determinants

52
New Eplet Version of HLAMatchmaker

• Duquesnoy, RJ A Structurally Based Approach to
  Determine HLA Compatibility at the Humoral Immune
  Level, Human immunology, 67 847-862, 2006
• Duquesnoy, RJ and Askar, M HLAMatchmaker A
  Molecularly Based Algorithm for
  Histocompatibility Determination. V. Eplet
  Matching for HLA-DR, HLA-DQ and HLA-DP, Human
  Immunology, 68 12-25, 2007

53
New HLAMatchmaker Version

• Based on molecular structures of
  protein-antibodies complexes and identification
  of contact residues with the specificity-determini
  ng CDRs such as CDR-H3
• Considers all surface-exposed polymorphic
  residues and residues within a 3.0-3.5 Angstrom
  radius
• These polymorphic residues clusters will be
  referred to as eplets rather than triplets
  because eplets may include nonlinear sequences
  with more than three residues
• Requirement All well-defined serological
  determinants should have corresponding eplets

54
Polymorphic Residue Positions in 3.0 Angstrom
Patches of HLA Class I Antigens
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HLA-ABC Eplets

• Provisional repertoire 203 eplets, 125 on top,
  76 on side and 32 in less accessible positions
• Many eplets are equivalent to triplets
• Excellent correlations between eplets and
  serologically defined private and public
  determinants

57
Class II Eplet Version of HLAMatchmaker

• Duquesnoy, RJ and Askar, M HLAMatchmaker A
  Molecularly Based Algorithm for
  Histocompatibility Determination. V. Eplet
  Matching for HLA-DR, HLA-DQ and HLA-DP, Human
  Immunology, 68 12-25, 2007

58
New Class II Version Considers DR, DQ and DP

• DRB1 and DRB3/4/5
• Patients can make antibodies to DR51, DR52 and
  DR53
• DQA1 and DQB1
• Patients can make antibodies to DQB and DQA
  epitopes

59
Topography of Polymorphic Residues on HLA-DR and
HLA-DQ Molecules
HLA-DQ
HLA-DR
?1
?1
?1
?1
?2
?2
?2
?2
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Class II Version Considers DR, DQ and DP

• DRB1 and DRB3/4/5
• Patients can make antibodies to DR51, DR52 and
  DR53
• DQA1 and DQB1
• Patients can make antibodies to DQB and DQA
  epitopes
• DPA1 and DPB1
• Clinical relevance of anti-DP antibodies in
  transplantation

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3.0 Angstrom Patches on DRB1,3,4,5
Polymorphic positions are underlined
62
DRB1,3,4,5 Contd
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Polymorphic Residues in 3 Angstrom DRB Patches
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DQA
DPB
DQB
DPA
Polymorphic Residues in 3 Angstrom DQ and DP
Patches
65
Determination of the Polymorphic Residue
Composition of Each Patch

• HLA Patch Generator a Microsoft Excel Macro
  developed by Grzegorz Dudek (Medigen Molecular
  Diagnostics, Warszawa , Poland)

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Eplet Numbers on HLA Class II Molecules
Locus Eplets
DRB 149
DQB 74
DQA 58
DPB 45
DPA 19
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Serologically Defined DRB Antigens and
Corresponding Unique Eplets
68
Serologically Defined DQB Antigens and
Corresponding Unique Eplets
69
DQA Alleles and Unique Corresponding Eplets
70
How Important is DRB1 Matching in Kidney
Transplantation?
71
HLA Class II Matching

• DRB1 is standard
• DRB3/4/5
• Antibodies to DR51, DR52 and DR53
• DQB1 and DQA1
• Relevance of DQB matching in transplantation
• Patients make antibodies to DQB and DQA epitopes
• DPA1 and DPB1
• Relevance of DP matching
• Anti-DP antibodies in transplantation

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A DR Antigen Mismatch Represents an Extra Epitope
Load

• DRB1 DRB3/4/5 DQBDQADPBDPA

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Conventional DR Compatibility
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Epitope-Based DR Compatibility
All are common DR-DQ haplotypes
75
Epitope-Based DR/DQ Compatibility for DR7,DR18
Patient
76
Epitope-Based DR Compatibility for DR7,DR18
Patient
Are DR8, DR9 and DR17 more desirable mismatches?
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Epitope-based HLA Class II Compatibility

• Epitope compatibility information requires
  high-resolution DRB, DQ and DP types
• It is possible to identify DR mismatches with
  relatively low epitope loads
• Such mismatches may reduce anti-class II antibody
  responses and perhaps benefit transplant outcome

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To be ContinuedFirst Movement
Practicale,Fine-tuning the Instrument