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Risk estimation for the development of donor specific antibodies (DSA) after transplantation.
Frans ClaasLeiden University Medical Center
Eurotransplant Reference Laboratory
Leiden, the Netherlands.
Rio de Janeiro, August 4, 2018
HLA matching is still beneficial for kidney graft survival even in the presence of efficient immunosuppression.
2
50
60
70
80
90
100
0 6 12 18 24 30 36
Follow-up (months)
% g
raft
su
rvi
0 (2268)
1 (874)
2 (2885)
3 (3532)
4 (1765)
5 (696)
6 (216)
va
lEffect of HLA-A, -B, -DR matching in Eurotransplant
3
Especially, production of donor specific HLA antibodies (DSA) after
transplantation is associated with poor graft survival.
Lachmann et al. Transplantation 2009
HLA matching prevents that a patient develops HLA antibodies
after graft rejection
4
0
20
40
60
80
100
Before 0 MM 1 MM 2 MM 3 MM 4 MM 5 MM 6 MM
T [<6% PRA] I [6-84 % PRA] HS [>84% PRA]
More HLA mismatches:
more antibody formation
Sensitization status of patients on the waiting list
After graft rejection
5
Number of different HLA proteins
HLA-A HLA-B HLA-C HLA-DR HLA-DQ HLA-DP
2747 3465 2450 2311 1079 828
HLA matching prevents the induction of DSA and will improve
graft survival but..
• Many patients will be transplanted with an HLA mismatched donor.
• However…. not every HLA mismatch leads to an antibody response and graft
rejection.
Identification of non-immunogenic HLA mismatches, which do not lead to antibody
formation in an individual patient, and use this knowledge for donor selection.
Challenge:
6
How to distinguish immunogenic and non-immunogenic HLA mismatches?
One needs a reliable parameter for immune reactivity
immunogenic non-immunogenic
In soccer, the color of the shirt is a reliable parameter
At a first glace: prediction of immunogenicity of an HLA
antigen seems a ”mission impossible”
7
All yellow amino acids configurations
are potential targets for antibodies.
Every HLA allele has many polymorphic positions
HLA antigens share antibody epitopes
Towards epitope matching in kidney allocation
Immunization by pregnancy: antibodies induced by HLA-A2 react also with HLA-B17
Polymorphic structures are often shared between different HLA alleles
44 62 65 69 77 …. 163 184 193 248
A*0101 44KM 62QE 65RNM 66RNH 77ANT 163RG 184P 193PI 248VK
A*0201 44RM 62GE 65RKV 70KAH 77VDT 163TW 184A 193AV 248VK
A*0202 44RM 62GE 65RKV 70KAH 77VDT 163TW 184A 193AV 248VK
A*0203 44RM 62GE 65RKV 70KAH 77VDT 163TW 184A 193AV 248VK
A*0205 44RM 62GE 65RKV 70KAH 77VDT 163TW 184A 193AV 248VK
A*0301 44RM 62QE 65RNV 66RNQ 77VDT 163TW 184P 193PI 248VK
A*0302 44RM 62QE 65RNV 66RNQ 77VDT 163TW 184P 193PI 248VK
A*1101 44RM 62QE 65RNV 66RNQ 77VDT 163RW 184P 193PI 248VK
A*1102 44RM 62QE 65RNV 66RNQ 77VDT 163RW 184P 193PI 248VK
A*2301 44RM 62EE 66GKH 70KAH 77ENI 163TG 184P 193PI 248VK
A*2402 44RM 62EE 66GKH 70KAH 77ENI 163TG 184P 193PI 248VK
A*2403 44RM 62EE 66GKH 70KAH 77ENI 163TW 184P 193PI 248VK
A*2501 44RM 62RN 65RNV 66RNH 77ESI 163RW 184A 193AV 248VK
A*2601 44RM 62RN 65RNV 66RNH 77ANT 163RW 184A 193AV 248VK
A*2901 44RM 62LQ 65RNV 66RNQ 77ANT 163TW 184A 193AV 248VK
A*2902 44RM 62LQ 65RNV 66RNQ 77ANT 163TW 184A 193AV 248VK
A*3001 44RM 62QE 65RNV 66RNQ 77VDT 163TW 184P 193PI 248VK
A*3002 44RM 62QE 65RNV 66RNH 77ENT 163TW 184P 193PI 248VK
A*3101 44RM 62QE 65RNV 66RNH 77VDT 163TW 184P 193AV 248VK
Duquesnoy et al. 2010
HLA antigen 1 HLA antigen 2 HLA antigen 3 HLA antigen 4
Every HLA antigen carries an unique set of epitopes but the
individual epitopes can also be present on other HLA antigens
Tambur & Claas AJT 2015
The number of foreign antibody epitopes in case of a single
HLA mismatch may differ
11
12
1. An HLA antigen has many potentially immunogenic
“epitopes” (triplets/eplets) but some of these are
shared with the patients’own HLA molecules
HLAMatchmaker algorithm predicts the immunogenicity
of an HLA alloantigen.
2. Patient will not make antibodies to epitopes
present on the own HLA antigens and therefore:
3. Polymorphism of an HLA mismatch is considered in
the context of the HLA type of the potential antibody
producer.
Principles of HLAMatchmaker:
Duquesnoy, Human Immunol. 2002
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Consequence: immunogenicity of a specific HLA mismatch is
different for individual patients.
Number of foreign "epitopes” on the same HLA-B51 mismatch for:
Patient A Patient B Patient C Patient D Patients E
many quiet some few few no
The number of foreign epitopes on an HLA mismatch predicts
antibody production after renal allograft rejection
Towards epitope matching in kidney allocation Dankers et al., Transplantation 2005
Antibody detection in CDC
2
The number of triplet and eplet mismatches of an HLA
mismatch predicts also antibody reactivity in luminex..
Kosmoliaptsis et al. 2008
Antibody reactivity in highly sensitized renal transplant candidates.
16
Amino acid differences Eplet differences
Different parameters are now available to predict the
degree of sensitization after graft rejection
Kosmoliaptsis et al. AJT, 2016
Degree of sensitization
vPRA
85-100%
51-84%
10-50%
0-10%
17 Wiebe et al. JASN, 2017
Eplet load is more predictive for DSA free graft survival
than HLA antigen matching
It is not all about B cell epitopes.T cell help is crucial for the production of IgG antibodies
Tambur & Claas AJT 2015
IgG DSA
B cell epitope
T cell epitope
The number of T cell epitopes and B cell epitopes independently affect
the induction of Donor‐Specific HLA Antibodies and graft survival.
Lachmann et al. AJT, 2017
B cell epitopes:
Number of eplets
T cell epitopes:
Predictable number of
foreign peptides
derived from the
mismatched HLA, that
is able to bind to HLA
class II of the recipient.
(PIRCHE II)
Not all DSA, which develop after transplantation, are due to a
primary immune response.
Low baseline DSA resulting in AHR
Fast increase of post-transplant DSA can be explained by activity of memory B cells
Burns et al., AJT 2008
Burns et al. AJT, 2008
Karahan et al. Transplantation 2015
DR
B1*0
7:0
1
DR
B1*0
9:0
2
DR
B1*0
9:0
1
DR
B1*1
0:0
1
DR
B1*1
4:0
1
DR
B1*1
4:5
4
0
5 0 0 0
1 0 0 0 0
1 5 0 0 0
MFI
s e rum
su p e rn a ta n t
* *
Supernatant of activated memory B cells may contain antibody
specificities not detectable in the serum.
HLA-ELISPOT allows for quantification of the HLA class I- and class II-specific
memory B cells in the peripheral blood following polyclonal activation of B cells.
Peripheral
blood B cells
HLA-specific
ELISPOT
6 days
Polyclonal
activation
HLA-specific memory B cell ELISPOT
1) Pre-culture phase 2) Visualization phase
Visualization is based on reactivity
with synthetic HLA monomers
23
HLA-specific memory B cell ELISPOT assays.
Human Immunology 2015, 76: 129-136
Kidney International 2015: 874-887
Is it possible to replace HLA monomers by donor celllysate containing all donor HLA molecules?
anti-human IgG Fc
Cells producing (anti-donor) Abs
DONOR CELL LYSATE
beta-2 microglobulin-HRP
TMB substrate
HLA class I specific ELISPOT
Antibody producing hybridoma cells can be detected by
HLA class I-specific ELISPOT
PBMC
lysate
Cell lysate : HLA-A2, A30, B7, B13
Total IgG
1000 c/w 500 c/w 250 c/w
Hybridoma (VTM1F11) : anti-HLA-B7 producer
Karahan et al. AJT, 2017
PBMC lysate: HLA-A2, A30, B7, B13
All donor specificities are represented in the lysate.
Quantification of Donor‐Specific Memory B Cells in the Peripheral Blood of an
HLA‐Immunized Individual is also feasible
Karahan et al. AJT, 2017
Quantification of Donor‐Specific Memory B Cells in the
Peripheral Blood of HLA‐Immunized Individuals
Karahan et al. AJT, 2017
Conclusions
• Production of (de novo) donor specific antibodies after
transplantation is associated with poor outcome.
• HLA epitope matching is a better strategy to prevent the
induction of DSA than classical HLA antigen matching.
• The donor specific memory B cell crossmatch may be an
additional tool for risk stratification in patients with a history of
sensitization i.e. pregnancy, blood transfusion, previous
transplant or after desensitization.
29
Sebastiaan Heidt
Gonca Karahan
Dave Roelen
Cynthia Kramer
Geert Haasnoot
Arend Mulder
Acknowledgements:
Rico Buchli (Pure Protein)
Rene Duquesnoy (Pittsburgh)
