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j.kint.2017.08.008

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mini review
www.kidney-international.org
Novel aspects of epitope matching and practical
application in kidney transplantation
Wai H. Lim1,2, Germaine Wong3,4, Sebastiaan Heidt5 and Frans H.J. Claas5
1
Department of Renal Medicine, Sir Charles Gairdner Hospital, Perth, Western Australia, Australia; 2School of Medicine and
Pharmacology, The University of Western Australia, Perth, Western Australia, Australia; 3Sydney School of Public Health, The University of
Sydney, Sydney, New South Wales, Australia; 4Centre for Kidney Research, The Children’s Hospital at Westmead and the Centre for
Transplant and Renal Research, Westmead Hospital, Sydney, New South Wales, Australia; and 5Eurotransplant Reference Laboratory,
Department Immunohematology and Blood Transfusion, Leiden University Medical Centre, Leiden, The Netherlands
This review describes the recent developments in the
applicability and clinical significance of epitope matching
in kidney transplantation. As incremental human leukocyte
antigen (HLA) mismatches are associated with increased
risk of rejection and allograft loss, HLA-matching remains
one of the standard immunological triage tests to
determine transplant suitability. Advancements in tissuetyping techniques have led to innovative concepts of HLAmatching at the epitope level. Epitopes are configurations
of polymorphic amino acid residues that are recognized by
B cells, and antibodies reactive with these epitopes lead to
rejection and/or premature allograft loss. Even though
there is substantial advancement in the characterization
and understanding of epitopes, evidence supporting the
added clinical benefit of epitope matching over traditional
antigen matching and the ability to identify clinically
relevant immunogenic epitopes remains poorly defined.
We present an overview of the evidence surrounding the
immunogenicity of mismatched donor epitopes and the
applicability of HLA epitope matching in kidney
transplantation. A better understanding of the
immunogenicity and structural characteristics of HLA
epitopes will guide clinicians to integrate epitope matching
as an important parameter for donor selection in kidney
transplantation.
Kidney International (2017)
j.kint.2017.08.008
-, -–-;
http://dx.doi.org/10.1016/
KEYWORDS: acute rejection; chronic kidney disease; epitope; histocompatibility; kidney transplantation; tissue typin
Copyright ª 2017, International Society of Nephrology. Published by
Elsevier Inc. All rights reserved.
Correspondence: Wai H. Lim, Department of Renal Medicine, Sir Charles
Gairdner Hospital, Perth, Western Australia, Australia 6009. E-mail:
wai.lim@health.wa.gov.au
Received 26 May 2017; revised 30 July 2017; accepted 3 August 2017
Kidney International (2017) -, -–-
T
he human leukocyte antigen (HLA) system plays an
essential role in the regulation of the body’s immune
system to counteract pathogens through antigen
presentation and the recognition of “self ” and “nonself.” It
makes use of a group of cell-surface antigen-presenting
proteins classified as class I and II major histocompatibility
complex molecules. Humans have 3 class I (A, B, C) antigens
that are present on all nucleated cells and 8 class II (DPA1/B1,
DQA1/B1, DRB1, DRB3/4/5) antigens that are present only
on antigen-presenting cells and endothelial cells. The heterodimers HLA-A, -B, and -DRB1 contribute to the majority of
the polymorphisms and therefore have been the predominant
focus of HLA matching in the allocation of donor kidneys for
transplantation. Matching at the HLA-ABDR loci remains the
cornerstone of deceased donor kidney allocation because of
the association between increasing numbers of HLA-ABDR
mismatches and incremental risk of rejection and graft
loss after kidney transplantation.1 In addition, HLA mismatches predispose to the development of donor-specific
anti-HLA antibodies (DSAs), which are strongly linked to
antibody-mediated rejection (AMR) and late allograft
loss.2,3 The presence of DSAs is often an impediment
to repeat transplantation, with highly sensitized patients experiencing a longer waiting period for a suitable donor kidney
compared with unsensitized patients. HLA-DQ matching is
not routinely considered in the allocation of donor kidneys,
but it is assumed that close HLA-DR matching has reduced
the likelihood of HLA-DQ mismatches. However, a recent
registry study of 788 kidney transplant recipients showed
that in those who received zero HLA-DR mismatched
kidneys, 11% of recipients will still have any mismatches at
the HLA-DQ locus. In this study, recipients who received
HLA-DQ mismatched kidneys have a 50% greater risk
of the development of acute rejection, including AMR,
independent of mismatches at the HLA-ABDR loci.4
The evolution of our understanding of the HLA system is
closely linked to advancements in technology. Traditional
serology-based HLA typing methods can be completed
relatively quickly but are dependent on the availability of
specific cell types, viability, and appropriate antisera. The
emergence of molecular HLA typing techniques over the
past 2 decades has allowed for a more specific, flexible,
and robust means of high-resolution HLA typing. Data
1
mini review
generated via the genome project and the initiation of polymerase chain reaction techniques further refined DNAbased techniques for HLA-typing, which has led to the
development of a number of polymerase chain reaction–
based techniques currently in use.
Role of epitope matching in kidney transplantation
Every HLA molecule is composed of a unique set of serologic
epitopes made of polymorphic amino acid residues, and it is
these structures and their conformation and position that
determine antibody accessibility, recognition, and subsequent
reactivity. Different HLA molecules can share individual
epitopes and therefore epitopes that are not present on
self-HLA molecules are considered foreign by the immune
system. HLAMatchmaker (http://www.epitopes.net) is a
computer algorithm that determines HLA compatibility between donors and recipients by assessing the 3-dimensional
molecular modeling of the epitope-paratope interfaces of
antigen-antibody complexes (Figure 1).5 The earlier version
of the program considered each HLA as a string of continuous short linear sequences of polymorphic amino acid
residues on the molecular surface called triplets,5 but the
updated eplet version considers longer and often discontinuous sequences of polymorphic amino acid in
antibody-accessible positions (Figure 2).5,6 The “theoretical”
eplets that have already been defined are listed in the
web-based HLA epitope registry (http://epregistry.ufpi.br/
terms/index). By evaluating the position and number of
foreign (i.e., nonself) and shared eplets (i.e., self, hence antibodies cannot be induced to self) between donors and recipients, HLAMatchmaker is able to calculate the number of
eplet mismatches for each donor-recipient pair. Even though
high-resolution 4-digit HLA typing is required, 4-digit alleles
can be estimated from 2-digit alleles using the catalog of
common and well-documented (CWD) HLA alleles, taking
into account the ethnic groups, the most likely allelic typing
based on haplotype frequency and the frequencies of the
paired haplotypes in the population. The common and welldocumented alleles are predominantly derived from the
Caucasian population, and therefore patients or donors who
are not Caucasian may potentially have an incorrect allele
assigned.7 Entry into the HLAMatchmaker of the extended
HLA typing at the HLA-C, -DRB3/4/5, -DP, and -DQA1B1
alleles is essential to analyze differences in amino acid residues between the potential immunizer (i.e., donor) and the
HLA alleles of the potential antibody producer (i.e., recipient). Four-digit HLA typing methods are time-consuming,
and therefore at present are not practicable in deceased
donor kidney allocation in which a rapid turnaround in HLA
typing is essential; consequently, 2-digit HLA typing methods
remain the standard typing technique for deceased donors.
However, high-resolution 4-digit molecular HLA typing
methods should be undertaken for all potential kidney
transplant candidates, particularly highly sensitized patients
in whom the correct assignment of permissible DSAs is
critical to determine transplant potential.
2
WH Lim et al.: Epitope matching in kidney transplantation
Along with advances in the typing of HLA alleles, it is
recognized that anti-HLA antibodies may bind to distinct
epitopes expressed on each serologically defined antigen. The
techniques used to detect these anti-HLA antibodies have
evolved from complement-dependent cytotoxicity assays to
more sensitive techniques including flow cytometry and
solid-phase assays (e.g., enzyme-linked immunosorbent or
Luminex assay [Luminex Corporation, Austin, TX), which
allows a more accurate assessment of a transplant candidate’s
immunologic risk pretransplantation.
Clinical significance of anti-HLA DSAs
AMR is recognized as one of the dominant causes of late
allograft loss after kidney transplantation,8 commonly preceded by the presence of DSAs, combined with complement
activation leading to subsequent vascular and/or endothelial
cell injury. Several epidemiologic studies have shown that the
presence of pretransplantation DSAs is associated with up to a
40 times increased risk of acute AMR, transplant glomerulopathy, and late allograft loss, and, therefore, transplant
eligibility in patients with preexisting DSAs should be carefully considered.2 In a probabilistic Markov model, screening
with a Luminex assay (using a DSA mean fluorescence intensity [MFI] threshold of 500 to determine suitability)
resulted in a savings of more than US$1.1 million, prevented
11 acute rejection episodes within 1 year, 6 episodes of allograft loss over 5 years, and gained 54 quality-adjusted
life-years over 20 years for every 100 kidney transplants
compared with screening with complement-dependent cytotoxicity alone.9
There is increasing evidence that the development of de
novo DSA posttransplantation, especially DSAs directed
against HLA-DQ, is associated with AMR and/or allograft
loss.2,3,10 Detection of DSAs that are capable of binding
complement (i.e., C1q-binding DSAs) may further improve
determination of kidney transplant recipients at a greater risk
of AMR and allograft loss, but it remains unclear whether
early intervention or more frequent monitoring of these recipients before the development of antibody-mediated injury
is of clinical benefit.11
Clinicians should be aware of the limitations when
interpreting the results of solid-phase assays. The generated
output of a Luminex assay is a numerical value expressed as
MFI, which does not provide a quantitative measure of the
amount of anti-HLA antibodies in the sera. These MFI
results must be interpreted in the context of positive and
negative control sera as well as the threshold associated
with a positive (flow cytometric) cross-match and, therefore, comparisons of the MFI between laboratories must be
undertaken with great caution.12 Varying antigen density
between beads, differences in the preparation of sera for
testing, the possibility of false-negative/-positive results,
shared epitopes between single-antigen beads, and the
potential of multiplicity of single-antigen beads for the
different antigens are some of the factors contributing to
the difficulty in establishing a clinically relevant MFI
Kidney International (2017) -, -–-
mini review
WH Lim et al.: Epitope matching in kidney transplantation
a
HLA Matchmaker
AMS
EMS
d
Endothelial cell damage
Donor eplets
Recipient eplets
b
Anti-donor
HLA-specific
B-cell
Antibody-verified eplets
B-cell epitope prediction
Memory B-cell
Plasma cells
ELISPOT for
HLA-specific
memory B-cell
Mismatched donor eplets
c
Donor
alloantigens
(soluble)
PIRCHE
Anti-donor
HLA-specific
B-cell
Indirect
CD4+T-cell
Donor
alloantigens
T-cell help
Figure 1 | Immunologic methods to determine structural human leukocyte antigen (HLA) compatibility and the role of B cells in the
development of donor-specific anti-HLA antibodies and antibody-mediated rejection. (a) Donor-recipient HLA compatibility can be
determined by examining and determining the number of mismatched donor epitopes or eplets (HLAMatchmaker), as well as amino acid
polymorphisms (amino acid mismatch score [AMS]) and physicochemical properties (electrostatic mismatch score [EMS]) for each mismatched
donor HLA. (b) To determine whether a mismatched donor epitope or eplet (highlighted in red) is immunogenic and antigenic, these mismatched epitopes or eplets must be verified experimentally with informative donor-specific antibodies using Luminex single-antigen bead
techniques, some of which are already recorded in the International Registry of Antibody-Defined HLA Epitopes (http://www.epregistry.com.br).
Several computational algorithms designed to predict antibody or B-cell epitopes (i.e., the part of the antigen that binds to antibodies or B cells)
may also provide additional information to assist in the discrimination of true epitopes from nonepitopes. (c) In kidney transplantation, the
epitopes from HLA class I and II alloantigens can be presented as peptides by the class II molecules of the recipient’s B cells to CD4þ helper T
cells following internalization of the targeted soluble donor alloantigens. Activation of these CD4þ T cells occurs following recognition of the
donor alloantigens in the context of self-HLA molecules provided by B cells (i.e., indirect allorecognition), which then provides signals and
cytokines to the B cells to undergo activation and differentiation and generation of donor-specific anti-HLA antibodies. The predicted indirectly
recognizable HLA epitopes (PIRCHE) computational algorithm is designed to predict indirectly recognizable HLA-derived donor peptides that
are likely to induce the production of donor-specific anti-HLA IgG antibodies. (d) B-cell immunity progresses in an ordered cascade of cellular
differentiation and development, which ultimately leads to the production of antigen-specific memory B cells, which may improve immune risk
stratification for production of donor-specific anti-HLA antibodies and antibody-mediated rejection and can be quantified in the peripheral
blood using a B-cell ELISPOT approach. This process often requires regulation by helper T cells; on re-exposure to alloantigens, these memory B
cells rapidly differentiate into plasma cells. The production of pathogenic donor-specific anti-HLA antibodies by plasma cells leads to complement activation and interacts with other effector cells, ultimately causing endothelial cell injury and manifesting clinically as antibodymediated rejection.
threshold associated with adverse allograft outcomes.12,13
Despite these limitations, the detection of preexisting
anti-HLA antibodies has enabled clinicians and HLA laboratories to more reliably define the sensitization status
(determined by calculating the panel reactive antibody or
virtual panel reactive antibody, taking into account HLA
antibody specificities and the frequency of HLA antigens in
the donor population) and allow for early identification of
potentially suitable donors for highly sensitized transplant
candidates through virtual cross-matching. A consensus
document outlining the testing and methodology, limitations, and interpretations of DSA in kidney transplantation
Kidney International (2017) -, -–-
provides clinical guidance for clinicians in the monitoring
and management of DSA in kidney transplantation.14
Epitope or eplet mismatches and kidney allograft outcomes
Several cohort studies (summarized in Table 115–23) have
shown that HLA locus–specific triplet or eplet mismatches are
associated with the development of de novo DSA, acute
rejection, and/or transplant glomerulopathy, the presence of
which is strongly associated with premature graft loss.
Nevertheless, the limitations of these studies must be carefully
considered when interpreting the clinical significance of these
results. First, it remains unclear whether the use of eplet
3
mini review
WH Lim et al.: Epitope matching in kidney transplantation
Physicochemical
concept:
• Amino acid
mismatches
• Electrostatic and
hydrophobicity
mismatches
*Amino acid
side chain
*CH3
1 2
3
5 4
Triplet
Self
Nonself
Self
Nonself/self
concept
Eplet
Figure 2 | Three-dimensional structure of a class II human leukocyte antigen (HLA) molecule (light gray denotes a subunit and dark
gray denotes b subunit) showing immunogenic epitopes (triplet vs. eplet) and the theoretical concepts of nonself/self paradigm and
physicochemical properties of importance in epitope recognition. The fundamental difference between a triplet and eplet is that the target
of antibody recognition is not restricted to short linear continuous sequences of polymorphic amino acid residues (triplet [blue]), but may
include amino acid residues in discontinuous sequences within a 3-Å radius (eplet [orange]). However, the immunogenicity and antigenicity of
an mismatched HLA molecule are often not limited to the epitope, but may involve surrounding structures. In the nonself/self theoretical
concept, it is hypothesized that an immunogenic epitope is contained within a “structural epitope,” which requires the presence of nonself (i.e.,
mismatched eplet [purple]) and self (green) amino acid residues as well as the binding site for the complementarity-determining region of the
anti-HLA antibody. In addition, physicochemical compatibility such as the actual sequence and charge of amino acid residues within the
epitope (yellow) and the presence of amino acid side chain(s) beyond the epitope (number, distribution, and polarity/charge of the amino acid
side chains, denoted by the asterisk) can lead to structural changes within the epitope and therefore determine potential differential antigenicity and immunogenicity of each epitope (i.e., not every amino acid substitution within the epitope has the same impact on antigenicity or
immunogenicity).
mismatches definitively improves the discrimination or is
associated with an additive or multiplicity effect in predicting
adverse allograft outcomes compared with broad antigen
HLA mismatches. Second, the potential “clinically significant”
threshold of eplet mismatches for adverse allograft outcome is
only established at a population level. These thresholds of
adverse effects may not be generalizable to all recipients from
other populations or at an individual level. There is increasing
recognition that eplet mismatches may be associated with
DSAs and adverse outcomes in nonkidney solid organ
transplant recipients but is beyond the scope of this review.
Are all epitope or eplet mismatches the same?
It must be recognized that the number of eplet mismatches
merely reflects the probability that 1 or more of these mismatches are capable of inducing an antibody response, but a
direct causal relationship between any predefined thresholds
of eplet mismatches and adverse allograft outcomes after
transplantation has not been established and may vary
between populations. The next step to better define the
immunologic risk associated with eplet mismatches is to
differentiate immunogenic from nonimmunogenic eplets
because not every eplet is capable of inducing an antibody
4
response and, therefore, in vitro verification of those eplets
that can bind to antibody is essential to identify potentially
immunogenic eplets. The clinical significance of immunogenic eplets is not only dependent on its ability to induce an
antibody response (i.e., immunogenicity), but the antibodies
(including complementary determining regions) must have
the capacity to interact and bind to the immunizing eplet and
the eplet paired with certain amino acid configurations to
form antigen-antibody complexes (i.e., antigenicity) that may
be capable of inducing complement activation.
Antibody-verified eplets. Identification of immunogenic
eplets is possible using monoclonal antibodies directed at
defined HLA molecules24 or using antibody absorption
and elution techniques of recombinant single HLA
antigen–expressing cell lines (termed the Terasaki epitope
[TerEps]),25 with alloantibodies verified using solid-phase
Luminex single-antigen bead assays (Figure 1). The majority
of epitopes present on uncommon or rare HLA alleles remains unverified, and future research should aim to identify
these novel alleles. There is also no consensus as to which
specific techniques best identify and confirm the presence of
antibody-verified epitopes or eplets and evidence pertaining
to the test performances and validation of these techniques as
Kidney International (2017) -, -–-
Authors
Study type
Interpretation
Limitations
Epitopes: polymorphic amino acid residues expressed on HLA molecules that determine antibody accessibility, recognition, and subsequent reactivity
Single-center study of 286 consecutive
Each HLA-DR or HLA-DQ epitope mismatch,
Wiebe et al.15
Considers epitope rather than
low immunologic risk kidney
adjusted OR of 1.06 and 1.04, respectively
eplet mismatches
transplant recipients without
(P < 0.001)
Limited ethnic diversity
pretransplantation DSA, 72%
Locus-specific epitope mismatch performed
Small number of patients in whom HLA-DR
Caucasian
better in predicting dnDSA postdnDSA (N ¼ 21) and HLA-DQ dnDSA
High-resolution HLA typing
transplantation compared with locus(N ¼ 36) had developed
specific high-resolution mismatch or locusspecific low-resolution mismatch
Optimal thresholds of 10 and 17 for epitope
mismatches at the respective HLA-DR and
HLA-DQ loci were associated with minimal
development of class II dnDSA (MFI $300)
Single-center study of 195 recipients
Single high-resolution antigen
Low-resolution typing converted to
Wiebe et al.16
monitored for medication adherence
mismatch at the HLA-DR locus resulted in as
most likely high-resolution alleles
many as 27 epitope mismatches (range, 0–
Class I epitope mismatches not
27; median, 5), whereas a single mismatch at
performed or reported
the HLA-DQ locus resulted in as many as 44
Arbitrarily created thresholds of “clinically
epitope mismatches (range, 0–44; median, 7)
relevant” HLA-DR and HLA-DQ epitope
Graft loss rates per 100 patient-years:
mismatches
nonadherent recipients with $10 HLA-DR
De novo DSA not reported
vs. <10 epitope mismatches: 5.3 (range,
Direct causal relationship between class II
3.0–9.0) vs. 1.3 (range, 0.5–3.0), nonadherent
epitope mismatches and rejection or
recipients with $17 vs. <17 HLA-DQ epitope
graft loss unclear
mismatches: 6.0 (range, 3.0–11) vs. 1.5
(range, 1.0–3.0)
(Continued on next page)
5
mini review
Triplets: Linear short sequences involving 3 contiguous polymorphic amino acid residues, which may represent residues of immunogenic epitopes
Two cohorts with 0 HLA-DR
In recipients who received 0 HLA-DR
Serologic HLA-A and -B typing used
Duquesnoy et al.17
mismatched kidney transplants:
mismatched kidney transplants, graft
1. UNOS registry (N ¼ 31,879);
survival rates of those with compatible
2. Eurotransplant registry
amino acid triplets (i.e., 0-2 triplet
(N ¼ 15,872)
mismatches) were similar to those with
0 HLA-AB antigen mismatches
2 cohorts:
Strong correlation between number of triplet
CDC assay for HLA typing and antibodies
Dankers et al.18
1. Sensitized patients after allograft
mismatches and proportion of individuals
Only HLA-A and -B examined
failure (N ¼ 146)
with antibodies
Unclear whether HLA antibodies were
2. Post-delivery pregnant women
0 triplet mismatches ¼ no antibodies
donor specific
(N ¼ 1397)
11–12 triplet mismatches: cohort 1, 94% patients
produced antibodies; cohort 2, 27% women
produced antibodies
WH Lim et al.: Epitope matching in kidney transplantation
Kidney International (2017) -, -–-
Table 1 | Studies of epitope HLA matching and clinical outcomes in kidney transplantation
Authors
Study type
Interpretation
Limitations
WH Lim et al.: Epitope matching in kidney transplantation
Kidney International (2017) -, -–-
Eplet: Longer and often discontinuous sequences of polymorphic amino acid residues within a radius of a 3-
A patch located in antibody-accessible positions, which may represent residues
of immunogenic epitopes
Single-center study of 40 recipients
HLA-DQ antibody does not differentiate
Tambur et al.19
HLA-DRB3/4/5 and HLA-DP typing
after allograft failure attributed to
between the 2 chains of the DQ molecule
not performed
rejection and in whom dnDSA to
and can recognize either or both in the
Small sample size
HLA-DQ developed
same capacity
Focuses only on those in whom de novo
High-resolution typing
Eplet (as part of the immunogenic epitope)
HLA-DQ antibody has developed
alone is not sufficient to determine the
“‘strength” of the antibody response
(therefore varying MFI between individuals
with the same mismatched eplet), but
depends on other factors such as antibody
affinity and avidity
Antibody production does not always reflect a
high frequency of nonself/self DQ chains;
challenges nonself/self theory
Single-center nested case-control study
25% increased odds of TG for every 10
Indication biopsies to establish TG
Sapir-Pichhadze
(52 with TG vs. 104 controls)
additional
HLA-DP typing not performed
et al.20
HLA-DRþDQ eplet mismatches (OR, 1.25; 95%
Adjusted for the number of HLA-A and -B
CI 1.04–1.50)
mismatches (0–2), not total class I eplet
Independent association between HLA-DR and
mismatches
HLA-DQ eplet mismatches
Possibility of inaccurate estimation of total
HLA-DR: OR for TG, 3.61 (95% CI 1.33–9.80) for
number of HLA-DR and -DQ eplet
13–27 vs. <13 eplet mismatches
mismatches
HLA-DQ: OR for TG, 3.72 (95% CI 1.33–10.43)
Assumption of class I eplet mismatches has
for 11–21
little significance in the development of TG
vs. <11 eplet mismatches)
“Threshold” of HLA-DR and -DQ eplet
mismatches not clearly defined
Registry population-cohort study
Linear relationship between broad antigen
Only HLA-A, -B, and -DRB1 considered
Do Nguyen et al.21
(Australia and New Zealand)
HLA mismatches and rejection, whereas
High-resolution typing not performed/
there was a nonlinear relationship between
available
eplet mismatches and rejection
Serologic HLA typing converted to 4-digit
Eplet mismatches similar to broad antigen HLA
typing according to CWD alleles
mismatches in predicting acute rejection:
Outcome not restricted to AMR
adjusted AUC, 0.56 (95% CI 0.54–0.58) and
0.58 (95% CI 0.56–0.61), respectively
Low immunologic risk recipients (i.e., 0–2
broad antigen HLA-ABDRB1
mismatches), $20 eplet mismatches
associated with adjusted HR of 1.85 (95% CI
1.11–3.08; P ¼ 0.02) compared with <20
mismatches
mini review
6
Table 1 | Studies of epitope HLA matching and clinical outcomes in kidney transplantation (Continued)
PIRCHE: A computational algorithm designed to predict indirectly recognizable HLA-derived donor peptides that are presented by the HLA class II molecule expressed on the recipient’s B
cells to the recipient’s CD4D T cells, which in turn provides T-cell help to generate donor-specific anti-HLA IgG antibodies by B cells
Single-center study of 21 recipientIdentification of HLA class I–derived PIRCHE-II
Otten et al.23
Only HLA-DRB1 background was evaluated,
donor pairs mismatched at the HLA(that can bind to recipient’s HLA-DR
with differences in findings between actual
A and -B loci, with no
molecules) were defined as recipient’s HLA
vs. “scrambled” HLA-DRB1 background
pretransplantation sensitization
class II binding epitopes within the
Small sample size, high-resolution typing was
event and renal allografts were
mismatched and nonshared donor-derived
not available for all
HLA class I molecule
removed
Focuses only on dnDSA to define
Immunogenic HLA class I molecules (for which
immunogenicity, no reported data on AMR
dnDSA was detected) contain a significantly
and “positive” DSA not defined
greater number of PIRCHE-II,
Large distribution of the data points for
HLAMatchmaker-defined number of triplets
PIRCHE-II in recipients with identified
and eplets compared with nonimmunogenic
immunogenic HLA
HLA
Unknown whether PIRCHE-II improves the
prediction of dsDSA over triplet or eplets
Other limitations including the potential
polymorphisms of T-cell receptors, variability
of the allopeptides, the potential
contribution of noncognate T-helper cell
responses, and the inability to predict the
actual peptides generated by the
proteasome may result in the inconsistency
of T-cell recognition and responses and
therefore the clinical applicability of PIRCHE-II
WH Lim et al.: Epitope matching in kidney transplantation
Kidney International (2017) -, -–-
Physiochemical disparity between donor and recipient HLA: a novel computer program that evaluates the specificity and stability of antigen-antibody binding by assessing the differences
in the interlocus and intralocus amino acid polymorphisms in antibody-accessible regions of the HLA molecule and electrostatic, and hydrophobic interactions between the 2 HLA
molecules
Single-center study of 131 recipients
Assessment of donor HLA immunogenicity
Kosmoliaptsis
HLA-typing methods not specified
relisted after allograft failure
based on AMS, EpMS, or EMS predicts
et al.22
HLA-DP typing not performed
allosensitization after graft failure,
Association between AMS, EMS, and EpMS and
independent of conventional HLA
other outcomes such as rejection and/or
mismatches
graft loss during first graft not assessed
Donor HLA-DR and -DQ alloantigens with high
(outside scope of study)
AMS, EpMS, or EMS were more likely to
Improvement in the accuracy or discrimination
induce the production of DSA
of AMS, EMS, and EpMS over conventional
Donor HLA EMS, but not AMS or EpMS,
broad antigen mismatch not assessed
predicted the development class I DSA
AMR, antibody-mediated rejection; AMS, amino acid mismatch score; AUC, area under the curve; CDC, complement-dependent cytotoxicity; CI, confidence interval; CWD, common and well-documented; dnDSA, de novo donorspecific anti-HLA antibody; EMS, electrostatic mismatch score; EpMS, eplet mismatch score; HLA, human leukocyte antigen; HR, hazard ratio; MFI, mean fluorescent intensity; OR, odds ratio; PIRCHE-II, predicted indirectly
recognizable HLA epitopes, HLA class II-presented; TG, transplant glomerulopathy.
mini review
7
mini review
well as determination of the cost-effectiveness and practicability of the techniques are essential to inform future
practices.
There are situations in which antibody reactivity to
certain epitopes are unexplained by the current understanding of immune reactivity. Duquesnoy26 proposed an
alternative nonself-self paradigm to explain this observation, which considers that each individual has a diverse
repertoire of B cells with low-avidity Ig receptors for epitopes expressed by self-HLA eplets (Figure 2). Interaction
of self-HLA eplets with B-cell Ig receptors will not elicit
B-cell activation or antibody production, whereas the
interaction of nonself eplets with B-cell Ig receptors can
potentially lead to alloantibody responses (i.e., an antibody
response to foreign HLA requires the presence of
self–amino acid configuration in the mismatched HLA
allele). However, a recent study challenges the notion of the
importance of the nonself-self paradigm, and many of the
defined antibody-verified eplets do not satisfy this criterion.19 There is no definitive laboratory evidence to
confirm that the presence of self-HLA reactive Ig receptors
is essential for the immunogenicity of mismatched eplets,
and it is conceivable that these apparent self–amino acid
sequences on the mismatched HLA alleles are shared between donors and patients but not involved in determining
immunogenicity.
A compilation of antibody-verified class I and II eplets or
eplets paired with other residue configurations to date is
available on a website-based HLA Epitope Registry (www.
epregistry.com.br).27,28 Many of the HLA class I epitopes
have been verified using monoclonal antibodies and
adsorption/elution studies, whereas the majority of the HLA
class II epitopes remain unverified, attributed in part to the
complexities of HLA class II molecules such as HLA-DP and
HLA-DQ, which contain highly polymorphic a/b chains.
Future studies examining whether antibody-verified
epitopes/eplets improve the discrimination and accuracy of
adverse allograft outcomes compared with the total number
of epitope/eplet mismatches required to establish its clinical
significance.
Prediction algorithms for B-cell antigenic epitopes. Continuous
and discontinuous antigenic epitopes can be predicted using
several conformational B-cell epitope prediction methods,
which evaluate aspects of physicochemical (e.g., hydrophobicity
and propensity for binding) and/or structural geometric characteristics (e.g., protrusion index) using machine-learning or
linear combination algorithms. Prediction of these B-cell epitopes based on regions protruding from the globular surface of
the protein may therefore “quantify” the probability of effective
epitope-antibody interaction (i.e., antigenicity). However, the
accuracy of B-cell epitope prediction methods for an independent set of known protein monomer structures is relatively
poor, with reported areas under the curve of between 0.57 and
0.64.29 This may be explained by the reliance predominantly on
common protein binding-site prediction methods, which are
likely to be too simplistic in predicting B-cell epitopes. The
8
WH Lim et al.: Epitope matching in kidney transplantation
potential clinical applicability of these prediction methods in
the assessment of immunologic risk cannot be recommended at
present, and future studies evaluating the association between
modified prediction methods and clinical outcomes are
required.
Determination of amino acid and electrostatic mismatches. A
novel computer program that assesses the physiochemical
disparity between donor and recipient HLA, in addition to
comparing the interlocus and intralocus amino acid polymorphisms in antibody-accessible regions of the HLA molecule, may improve identification of immunogenic epitopes.
The specificity and stability of antigen-antibody binding are
not only determined by structural compatibility, they are also
determined by the electrostatic and hydrophobic interactions
between the 2 molecules, which can be assigned electrostatic
and hydrophobicity mismatch scores, respectively. These
scores have been shown to provide additional predictive value
for class I and II alloantibody responses that is independent of
the number of amino acid mismatches (Table 1 and
Figure 2).30 The same authors also showed that comparing
amino acid polymorphisms between donor and recipient
HLA, as well as determining physicochemical properties, is
superior to the traditional approach of considering the
summation of the number of broad antigen or eplet HLA
mismatches in predicting allo-sensitization after allograft
failure and the development of locus-specific DSA.22 Future
research examining the association between this method of
assessing HLA incompatibility and hard clinical outcomes in
AMR and rejection-related allograft loss will be important.
Novel assays or methods for predicting humoral immune
responses
The majority of prediction methods for determining humoral response to alloantigens after kidney transplantation
focus on the differences in structural and/or physicochemical properties between donor and recipient HLA alleles, but
largely ignore the ability of the recipients to induce the
production of these deleterious alloantibodies. The detection of HLA-specific memory B cells, which are capable of
rapidly differentiating into antibody-secreting plasma cells
and activating alloantigen-specific T cells with appropriate
stimulation, may improve the risk stratification in predicting the production of alloantibody and adverse kidney
allograft outcomes.31 Sensitive enzyme-linked immunospot
assays (ELISPOT) are able to detect HLA class I– and
II–specific memory B cells and thereby possibly improve
prediction of the development of HLA-locus specific DSA
(Figure 1).32,33
It has long been recognized that T-cell help is essential in
the development of IgG antibodies by promoting the differentiation and proliferation of antigen-specific naïve B cells
into memory B and plasma cells, as well as undergoing
antibody class switching in response to specific antigens.
Peptides derived from HLA molecules can be presented
as peptides by self-HLA class II molecules of the B cells to
cognate helper T cells, which then provide signals and
Kidney International (2017) -, -–-
WH Lim et al.: Epitope matching in kidney transplantation
cytokines to these B cells to undergo activation and differentiation into plasma cells. The PIRCHE (predicted indirectly
recognizable HLA epitopes) computational algorithm is
designed to predict indirectly recognizable HLA-derived
donor peptides that are likely to induce the production of
donor-specific anti-HLA IgG antibodies.23 A high number of
PIRCHEs, likely to represent a higher number of epitopes that
can be presented by HLA class II molecules, correlate with
clinical alloreactivity but not with the number of eplet mismatches. However, potential polymorphisms of T-cell receptors, variability of the allopeptides, the potential
contribution of noncognate T-helper cell responses as well as
the inability to predict the actual peptides generated through
cleavage by lysosomal enzymes may result in the inconsistency of T-cell recognition and responses; therefore, the
PIRCHE prediction method may be of limited utility at an
individual level (Figure 1).34
Utility of epitope or eplet matching in the allocation of donor
kidneys for transplantation
There are 2 main benefits of using epitope matching in the
allocation of donor kidneys: (i) to avoid allocating donor
kidneys with a high load of immunogenic epitopes, therefore preventing future allosensitization with the development of anti-HLA antibodies (important in those requiring
retransplantation), and (ii) selecting a suitable donor kidney
for highly sensitized patients based on identifying acceptable versus unacceptable immunogenic epitopes (virtual
cross-match). Even though there are uncertainties with
regard to the clinical significance of immunogenic epitopes
and identification of immunogenic epitopes remains a work
in progress, clinical application of epitope matching in
kidney transplantation has already been successfully
implemented.
Acceptable HLA mismatches are mismatched HLA at the
broad antigen level that comprises structurally and functionally compatible eplets, which are unlikely to be of clinical
significance. Since the introduction of the Eurotransplant
Acceptable Mismatch program, the transplant waiting time
among the highly sensitized patients with multiple preexisting
anti-HLA antibodies has decreased by at least 50% while
achieving comparable short- and long-term allograft survivals
in nonsensitized patients.35 B-cell epitope analysis using
HLAMatchmaker to identify additional class I antigens that
lack antibody epitopes, which are likely to represent acceptable mismatches, has further improved the transplant potential of these highly sensitized patients. Further refinement
of the definition of unacceptable antigens, which are
identified by the presence of specific antibodies using
complement-dependent cytotoxicity assays, as well as
single-antigen bead testing in the presence of a known previous immunizing event will further assist in the selection of
an immunologically compatible donor kidney. Using modeled
data from a simulated alternative acceptable mismatch allocation model in Western Australia, we have shown that
incorporating acceptable HLA mismatch into the current
Kidney International (2017) -, -–-
mini review
allocation model improved transplant potential up to 15% in
highly sensitized kidney transplant recipients, achieving an
overall lifetime gain of 0.034 quality-adjusted life-years and
cost savings of >$4000 per recipient.36 In a novel pediatric
kidney transplant allocation program in Australia (N ¼ 19),
the exclusion of unacceptable HLA mismatch defined as a
threshold of at least 10 and 30 eplet mismatches at the class I
and II loci, respectively, was associated with the timely access
to transplantation and low rates of de novo DSA at 12-month
follow-up.37 However, long-term follow-up of this program is
required to determine whether the outcomes of these
recipients have improved.
With the ever-growing improvement in identifying and
mapping of functional HLA epitopes or eplets, clinicians
and transplant immunologists must attempt to decipher the
available data and establish a clinical framework for incorporating epitope matching in organ allocation and assessment of immunologic risk. Even though identifying and
calculating the number of eplet mismatches for every
donor-recipient pair should be considered in the initial
assessment of immunologic risk, it is unrealistic to use all
other available methods or techniques for every potential
kidney transplant candidate. Clinicians and immunologists
must try to individualize immunologic risk testing rather
than using a “one-size fits all” approach by selecting the
most appropriate assessment tool for subgroups of potential
kidney transplant candidates. For example, highly sensitized
patients and patients who are likely to require repeat
transplantation (e.g., pediatric kidney transplant candidates)
should be considered for extended immunologic testing
(e.g., identifying nonimmunogenic eplets) to ensure the
maximum survival benefit from the first kidney transplantation and minimize the risk of allosensitivity
posttransplantation. Notwithstanding the success of the
Eurotransplant Acceptable Mismatch program, there are
several gaps in the literature that must be addressed before
potential widespread uptake of epitope or eplet matching in
kidney transplantation: (i) establishing the association between immunogenic eplet mismatches and clinical outcome,
which could assist in the decision of whether to consider
immunogenic eplet mismatches rather than the total number of eplet mismatches in allocation and immunologic risk
assessment; (ii) modeling the benefits and costs of incorporating an alternative eplet-based allocating algorithm,
thereby determining the effect on the transplant potential,
benefit/cost, and projected survival of the entire transplant
cohort; (iii) modeling the benefits and costs of incorporating HLA-DQ matching into the allocating algorithm,
given that HLA-DQ DSA is the predominant de novo DSA
occurring posttransplantation; and (iv) establishing
high-resolution HLA typing for non-Caucasian ethnic
groups with incorporation of the alleles into the HLAMatchmaker, including indigenous patients and ethnic
minorities in whom there is likely to be a high prevalence
of novel HLA alleles, which is essential in the accurate
identification and calculation of immunogenic mismatched
9
mini review
eplets. The availability of these data is essential before clinicians can determine whether epitope matching can be of
benefit in the ethnic minority population groups.
Conclusion
There is little doubt that epitope matching will become one of
the standard triage tests for future kidney transplant allocation and assessment of immunologic risk. Even though the
advantage and potential clinical application of epitope
matching in kidney transplantation are relatively well established, there remains many unanswered questions, particularly with respect to the characterization of clinically relevant
epitopes. The upcoming 17th International Workshop will aid
in, at least partially, answering these questions. Future direction and challenges will aim to better refine the understanding of immunogenic epitopes as well as help clinicians
interpret and select from the myriad of immunologic tests
designed to predict allosensitization and adverse clinical
outcomes after kidney transplantation. The answers to these
important research questions will then assist clinicians and
policy makers in considering appropriate integration of
epitope matching in kidney transplantation.
DISCLOSURE
WH Lim et al.: Epitope matching in kidney transplantation
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
All the authors declared no competing interests.
20.
ACKNOWLEDGMENTS
WHL is supported by a clinical research fellowship from the University
of Western Australia (Raine Foundation) and the Health Department
of Western Australia. The authors acknowledge Anita Dening (graphic
designer, Audiovisual Department at Sir Charles Gairdner Hospital,
Perth, Australia), who helped with Figure 2.
21.
22.
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