From HLA Typing to anti-HLA antibody detection and beyond

24 Pages·2016·0.47 MB·English

by  Antonietta Picascia

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FromHLATypingtoanti-HLAantibodydetectionandbeyond:theroadahead
AntoniettaPicascia,VincenzoGrimaldi,ClaudioNapoli
PII: S0955-470X(16)30034-9
DOI: doi: 10.1016/j.trre.2016.07.007
Reference: YTRRE420
Toappearin: TransplantationReviews
Pleasecitethisarticleas: PicasciaAntonietta,GrimaldiVincenzo,NapoliClaudio,From
HLATypingtoanti-HLAantibodydetectionandbeyond: theroadahead,Transplantation
Reviews (2016), doi: 10.1016/j.trre.2016.07.007
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Transplantation Review, Review Article, Submission

From HLA Typing to anti-HLA antibody detection and beyond: the road ahead

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Antonietta Picasciaa,*, Vincenzo Grimaldia and Claudio Napolia
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aU.O.C. Division of Immunohematology, Transfusion Medicine and Transplant Immunology (SIMT),
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Regional Reference Laboratory of Transplant Immunology (LIT), Department of Internal Medicine and
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Specialistics, Azienda Ospedaliera Universitaria (AOU), Second University of Naples, Piazza L. Miraglia 2,
80138, Naples, Italy
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*Corresponding author:

Antonietta Picascia
U.O.C. Division of Immunohematology,
Transfusion Medicine and Transplant Immunology (SIMT)
Regional Reference Laboratory of Transplant Immunology (LIT),
Department of Internal Medicine and Specialistics
Azienda Ospedaliera Universitaria (AOU),
Second University of Naples,
Piazza Miraglia, 2
80138, Naples, Italy
Tel: [phone]
Fax: [phone]
e-mail: [email protected]
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Abstract
The complex polymorphism of the HLA genes and the need of a proper identification of anti-HLA
antibodies have led to continuously develop novel practical and feasible technologies in the field of
organ and tissue transplantation. Technologies to identify HLA moleculTes have evolved from the
serological to the molecular methods and a true innovation in the DNAP sequencing has taken place
with the development of Next Generation Sequencing. An interesIting field to explore is how the
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information resulting from the HLA-DNA sequencing can be applied in the clinical setting by
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including the alloimmunization assessment. Indeed, a good characterization of anti-HLA antibody
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at epitope level can reduce the risk of immunization. Many anti-HLA antibodies are specific for
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epitopes rather than for HLA antigens and the knowledge of unacceptable epitopes allows to reduce
the number of mismatched antigens. Furthermore, hNigh resolution HLA allele typing could help to
understand the epitopes against which antibodAies are developed. However, the improvements
should not only concern the diagnostic toMols in the pre transplantation phase, but also the
monitoring of post transplantation outcome. There is a growing interest in developing new non-

invasive biomarkers to monitor the reDjection. Currently, increasing evidence has focused on
miRNAs, epigenetic markers emergEed as regulators of molecular events that are differently
expressed in biopsies and blood Tas well as in urinary samples of transplanted recipients. The
implementation of Next GeneraPtion Sequencing and genome-wide expression analysis together with
functional assays may proviEde useful tools to evaluate the epigenetic modulation in transplantation
biology but many efforts are requested for translating in the clinical arena the results obtained in
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experimental models.
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Key words: anti-HLA antibodies, HLA antigens, next generations sequencing, epigenetics,
biomarkers
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1. Introduction
Human leukocyte antigen (HLA) genes are the most polymorphic of the human genome and at this
time, approximately 14.473 HLA and related alleles have been submitted to the IMGT/HLA
database release 3.24.0 [http://www.ebi.ac.uk/ipd/imgt/hla/stats.html]. TThe HLA allele number is
growing exponentially by providing a survival advantage with obviouPs inter-individual differences
in the immune response, but the dealing with the ambiguity of theI different HLA typing methods
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remains a critical challenge [1,2]. Most of HLA polymorphisms are located within the exons
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encoding for the peptide-binding groove by ensuring the recognition and the increasing repertoire of
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peptide motifs that can be bound and presented to CD8+ or CD4+T cells [3], by promoting the
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immune regulation through the self/non-self recognition. However, variable sequences have been
also found in the introns or exons encoding for otherN domains of molecules [4,5]. Some ambiguities
are more critical to resolve than others such as thAe polymorphism in the exons encoding the peptide
binding groove respect to those in other regionMs of the gene, and the polymorphisms that change the
protein sequence. At this regard, a list of so-called common and well documented (CWD) alleles,

that are identified and reported at leaDst three time, has been updated [6]. In the context of
transplantation field, the identificatioEn of CWD and null alleles, these latter sequence variants that
prevent expression of protein oTn the cell surface, is considered critical. Moreover, CWD
identification and allele prevalePnce were used to categorize G and P groups. Indeed, HLA alleles
that have identical nucleotidEe sequences across the exons encoding the peptide binding domains are
designated as G group, Cwhereas HLA alleles having nucleotide sequences that encode the same
protein sequence for the peptide binding domains are defined P group [6].
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The high allelic variability represents a barrier in the transplantation field by leading to the need of
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a proper evaluation of HLA compatibility. Indeed, the donor-recipient histocompatibility and
alloimmunization remain the crucial factors affecting success of several organ transplantation [7-
10]. A good HLA compatibility may improve graft survival and the deleterious effects of preformed
and de novo anti-HLA antibodies are well known [7,11]. The accurate identification of anti-HLA
antibodies is useful for an optimal compatibility test to avoid false positive by excluding clinically
irrelevant antibodies. Furthermore, anti-HLA antibody monitoring may be an useful tool to identify
patients with high risk of antibody-mediated rejection and graft failure in post transplantation time
[10,12,13].
The complex polymorphism of the HLA system, the need of a proper and accurate identification of
anti-HLA antibodies, together with the lack of reliable markers useful for immune monitoring in the
clinical routine, have led to continuously develop new practical, feasible and inexpensive
technologies in the transplantation field. This review focuses on the novel approaches used in the
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immunogenetic laboratories regarding both HLA typing and antibody detection as well as the recent
involvement of epigenetic tags as valuable biomarkers in organ transplantation.

2. HLA Typing from the serological to the molecular methods T
Technologies to identify HLA genes evolved from the serological toP the molecular methods and
their improvements have gone up with the increased knowledge abIout the genetic complexity and
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polymorphism of HLA system.
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Serological approach, based on the complement dependent cytotoxicity (CDC) was used for a long
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time and still today, it remains useful to identify the null alleles.
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With the development and the application of Polymerase Chain Reaction (PCR), the DNA-based
techniques have taken over in typing laboratories foNr their accuracy and reproducibility as well as
their greater sensitivity and resolution by generatiAng low and high-resolution results. DNA-methods
include PCR-sequence specific primers (SSP),M PCR-sequence-specific oligonucleotide (SSO), Real-
time PCR, Sequencing-Based Typing (SBT), and the next-generation sequencing (NGS) [14,15].

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2.1. DNA-based methods for HLAE Typing: SSP and SSO
SSP-PCR has represented an effecTtive alternative to the serological method in clinical practice. This
assay is based on the use of pPrimer designed to amplify a specific allele only if its sequence is
perfectly matched with the cEorresponding primer, so that one mismatched nucleotide between the 3’
primer and the template prevents the amplification. The discrimination between the different alleles
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takes place during the PCR process and it is determined by the presence or absence of amplicons.
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This approach is simple, fast and ideal for typing of single samples, but many separate PCR
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reactions are needed for high-resolution results. Furthermore, the high number of new alleles and
primers makes this approach disadvantageous for typing a large number of samples [16,17].
SSO-PCR assay relies on the locus-specific amplification of the HLA polymorphic regions using
primers specific for exons 2 and 3 for class I and exon 2 for class II, followed by hybridization of
amplified DNA, bound to a solid support with sequence-specific oligonucleotide probes. Usually,
oligonucleotides are composed of 20 nucleotides and the hybridization occurs in stringent
conditions. SSO probes can be immobilized on a solid support as color-coded microbeads, and
hybridized with labeled PCR products as in SSO with Luminex platform. PCR products are
biotinylated and detected by strepavidin-conjugated with phycoerythrin dye. After denaturation,
PCR products are rehybridizated to complementary DNA probes bound to microbeads. Lastly, the
Luminex flow analyzer identifies the fluorescent intensity on each bead and an interpretation
software analyzes reaction patterns and assigns the matching HLA alleles. This method, more
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adaptable to the growing number of alleles allows to test a large number of samples, even if the
execution times are longer than SSP, due to the need to several steps. The resolution level of SSO
depends on the number of probes, often 50-100 probes for an intermediate/high level resolution but,
as for SSP, the polymorphism outside the amplified region cannot bTe identify. Therefore, the
development of a 500-bead system associated with a LABScan 3D haPs allowed to reduce or solve
many ambiguities through a powerful software able to differentiate Ithe different probes and analyze
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the reaction pattern with reliability [18].
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By excluding the SSO with Luminex technology, DNA microarrays were reported as a variant of
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the SSO method, but not used routinely in clinical typing for the high costs [19].
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To date, the SSP remains the gold standard method to type the deceased organ donor for speed and
ease of interpretation. The ideal HLA typing methoNd should be accurate with cost affordable and
automatable to ensure the standardization of assaAy. However, new and more sensitive methods are
being developed due to the growing number ofM alleles and the need to resolve many ambiguities.

2.2. Real Time-PCR D
Real-time PCR has several advantageEs compared with PCR, including the increased sensitivity that
allows the rapid determination ofT specific HLA alleles and number copy of alleles without post-
PCR manipulation. In particuPlar, the TaqMan assay is designed to increase the specificity of
quantitative PCR, by takingE advantage both the 5´–3´ exonuclease activity of Taq polymerase to
cleave a dual-labeled probe during hybridization, and the fluorophore-based detection, that allows a
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quantitative measurement of the product amplificated [20]. However, this approach has only been
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applied for typing of specific HLA loci as HLA-B*27, DRB1*15, but not for routine activity [21-
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23]. A further approach uses high resolution melting (HRM) that detects small differences in PCR
melting curves [24]. A fluorescent dye as SYBR Green is included in the PCR reaction. During this
process a slow increase in the temperature of amplified DNA from around 50°C up to around 95°C
allows to reach the melting temperature. The two strands of DNA are separated so as to release the
fluorescent marker, whose intensity can be measured by small increments of temperature.
Moreover, the dye binds specifically and exclusively the double-stranded DNA by emitting
fluorescence brightly. In the initial phase of HRM, there is a high level of fluorescence due to the
double-strand DNA that decreases when the two strands melt apart; through the HRM analysis, the
shape of their curves can identify the different alleles. HRM has been applied to the typing of
specific loci as the HLADQA/DQB typing in celiac disease, HLA-B*27, and B*57:01, a strong
predictor of abacavir hypersensitivity [25-27]. A new method, LINKSEQ combines the SSP and
SYBER green principles to detect PCR products without post-amplification manipulation by taking
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advantage of the fluorescent dye and using melt curve technology to identify the amplicons. Each
characteristic amplicon will melt at the same temperature each time and specific HLA alleles can be
identified, producing a low-intermediate resolution in few hours. This approach for its ease
processing has considerable advantages for deceased donor typing in sTolid organ transplantation
[15]. P
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2.3. SBT
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SBT has become the current approach for high-resolution HLA typing and for identify new alleles
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[28,29]. SBT is based on locus- or group-specific amplification, followed by cycle-sequencing, in
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which four different dye 2’3’dideoxynucleotides, by emitting light at different wavelength, are used
as substrates and cause the stop of elongation, once Nincorporated in the growing chain. After PCR
amplification, the different DNA fragments are auAtomatically separated by capillary electrophoresis
and the nucleotides at the end of fragments Mare identified by the fluorescence emitted. Although
SBT provides a high-resolution typing, frequently ambiguous allele combinations can be obtained

for several factors among which the cis/tDrans ambiguity, due to the amplification and sequencing of
both alleles [30]. Indeed, the sequencEed region can be shared by several HLA alleles thus one pair
of common HLA alleles and seveTral less-common pairs can be obtained. If the ambiguous result
depends on an identical heterozPygote sequence, a group-specific primary amplification can be used
with each allele amplifiedE and separated [28]. Usually the HLA sequencing is restricted to
polymorphic regions of exons 2 and 3 for class I and exon 2 for class II alleles. Therefore,
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additional tests are needed to clarify ambiguities located outside of the region amplified, such as by
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including exon 4 for class I and exon 3 for class II alleles. SBT approach has enabled the
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identification of over 8.000 HLA class I and II alleles, even if only less than 10% of these alleles
have been fully sequenced and identified as CWD [31], and more than 40% have been reported just
the once [32-34]. The updated CWD catalogue using SBT observations includes 1,3% of HLA-A,-
B,-C,-DRB1, -DRB3/4/5,-DQA1,-DQB1,--DPA1 and –DPB1 alleles [6]. However, SBT is an
expensive and time-consuming method and the resolution of ambiguous allele combinations implies
many economic and laborious efforts, by combining it with SSP and SSO and by requiring many
high-resolution workflows [15].

2.4. NGS
In the last years, a true innovation in the DNA sequencing has taken place with the development of
NGS, related to different platforms above all in the research world, but the potential feasibility in
the routine diagnostic laboratory has been assessed [35,36]. NGS technology provides a whole and
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careful characterization of polymorphic HLA genes by allowing a high-resolution genotyping with
relatively simple and cost effective protocols, so as to promote the implementation of guidelines for
clinical practice. NGS could contribute to expand the knowledge about the biological relevance of
both coding and non-coding HLA sequences in stem cell and solid orgaTn transplantation [34,37].
The greatest advantage of NGS consists in eliminating ambiguities wiPth costs comparable to SBT,
and not requiring additional tests [38]. NGS process generally Iinvolves several steps: sample
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preparation and amplification, DNA library, emulsion or solid phase PCR, normalization, sequence
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run, data acquisition and bioinformatic analysis. However, the different NGS-platforms may have
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variable processing time ranging from few hours to days. The resolution of HLA ambiguites
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depends on clonal sequencing that allows to set the phase of linked polymorphism into the amplicon
through different methods such as emulsion-PCR Nor solid phase amplification [39]. The clonal
sequencing and the library generation are comAmon to the different platforms, although with
different methods. The requisites to prepare Ma library are: oligonucleotide-adaptors to capture the
single DNA molecules on a solid-phase and a barcode sequence to identify the sample from which

the target DNA originates. For this aim,D the DNA genomic can be randomly fragmented followed
by the addition of adaptors and barcEode [39]. In the Roche/ 454 NGS-platform, the single DNA
strands linked to beads are amplifiTed in an emulsion of oil-water buffer, thus millions of copies into
a micro-droplet are obtained. APfter emulsion-PCR, the millions of copies of the same DNA strand-
bead complex are positioEned in the wells of picotiter-plates for pyrosequencing reaction.
Triphosphate nucleotides are added sequentially resulting in the release of pyrophosphate that,
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converted to ATP by sulphorase, is coupled with photon emission revealed by a CCD camera and
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correlated with the amount of nucleotides incorporated. A flowgram reporting the order of the four
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nucleotides is generated [40]. This approach that combines the clonal PCR and the pyrosequencing,
has allowed an unambiguous HLA typing and it has been an evolution in the clinical setting [41-
43].
Like to 454 system, Life Technologies/Ion Torrent platform uses the synthesis sequencing approach
in which DNA molecules linked to adaptors-barcode are amplified clonally on the surface of beads
defined Ion spheres. The nucleotides sequentially added are incorporated by causing the release of
protons with a shift of pH detected and translated in a digital signal. The incorporation of two or
more identical nucleotides in the DNA strand, results in a signal proportional to the number of
nucleotides incorporated [44].
A bridging PCR system is used in the Illumina/MiSeq System, in which the clonal sequencing is
achieved through the binding of DNA to a solid phase, defined flow-cell, through adaptor-
oligonucleotides that allow not only to capture the DNA for sequencing but they act also as primers.
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With this approach, clusters from a single DNA molecule are obtained and, all four fluorescent-
nucleotides flushed over the flow cell simultaneously are used for sequencing. Fluorescent
nucleotides, coupled to a terminator, cause a stop of polymerization so that only a nucleotide is
added at each cycle and it is monitored by a CCD camera; the next sequTencing cycle occurs after
removing the terminator and fluorescent dye [45]. Recently, HosomiPchi et al. have reviewed the
phase-defined entire HLA gene sequences, regardless whether the aIlleles are rare or new, using this
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approach [46]. Pacific Biosciences technology achieves clonal sequencing using the single molecule
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real-time sequencing (SMRT) approach. The most innovative feature is the system to detect the
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light, composed of cells with thousands of zero-mode waveguides, nanophotonic structures on
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which a DNA template/polymerase complex is immobilized. Phospholinked nucleotides are
introduced by allowing the use of high concentratiNons of labeled nucleotides, needed to support
rapid DNA sequencing by synthesis. Each nucleoAtide has a terminal phosphate fluorescently labeled
that after incorporation, results in a distinctiveM and specific pulse of fluorescence allowing the DNA
sequencing in real time [34]. Recently, Mayor et al. have evaluated the applicability of the SMRT

showing that it seem solve the issues of cDlinical typing even if the number of samples evaluated was
low and the analysis was limited onlyE to HLA class I [47].
Several studies have reported the Tbenefits and the feasibility of NGS approaches at different levels
on large reference panels as Pan alternative sequencing methodology in the HLA laboratories
[36,39,48-50]. The clinical Eefficacy of NGS was successfully validated, through a highly accurate
genotyping set of data compared with the reference HLA genotyping data obtained from 285
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samples typed with SSO/SSP/SBT [44]. Currently, many histocompatibility laboratories are
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defining the standards for clinical typing by the different sequencing platforms. A critical evaluation
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regards whether in the binomial NGS/immunogenetics, this last with the long cycles of
development, validation, and applicability is able to conform with a technology with high rate of
turnover.

3. Anti-HLA antibody detection: old and new challenges
Despite the significant advancements achieved in transplantation field, the induced alloimmune
response plays a pivotal role in the allograft outcome [51]. The CDC assay has been for many years
the gold standard method for anti-HLA antibody detection. CDC results are related to the
expression level of HLA molecules on the donor lymphocytes, as well as to the amount, avidity and
subclasses of immunoglobulines, as recently reported [52,53]. However, Solid Phase Assays
(SPAs), including fluorescent bead-based assay with Luminex platform, for their increased
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sensitivity and accuracy are a valid tool for the accurate detection of anti-HLA antibodies, but many
technical and interpretive issues continue to be discussed, yet [9,54].
SPAs use isolated HLA molecules as target and the anti-HLA antibodies are revealed by the
binding of fluorescent-labeled secondary antibody, thus allowing a seTmiquantitative evaluation
through the Mean Fluorescent Intensity (MFI) values. The SPAs linkedP to Luminex have developed
a multilevel analysis for antibody characterization, using three Itypes of panels with different
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composition ranging from screening to single antigen, latter using beads coated with single
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recombinant HLA molecules [55]. Today the single antigen assays (SABs) are widespread used but
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many unresolved issues remain regarding the clinical interpretation and the relevance of the anti-
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HLA antibodies detected [9]. Several factors can affect the SAB interpretation including the inter-
and intra-laboratory variability, and the lack of stanNdardization above all of MFI that reflects the
average amount of antibodies bound to a specificA bead but not their levels [9,56]. The amount and
type of antigens on the beads and their not naMtive conformation can contribute to the variability of
the results, by causing an over-estimation of the antibody strength. Moreover, the strength of anti-

HLA antibodies revealed by MFI values Ddoes not always correlates with their power harmful due to
the potential low avidity or inability tEo activate an immune response. Thus, several improvements in
the manufacturing process and in tThe antibody analysis software have helped to get more consistent
results [10,55]. P
SABs have allowed a comEprehensive characterization of the anti-HLA antibodies in sensitized
patients. The presence of preformed anti-HLA antibodies was clearly associated with antibody-
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mediated rejection occurrence. Indeed, as reported also recently by Richter et al. pretransplantation
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immunized patients with SAB-anti-HLA antibodies, had a significantly reduced graft survival,
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indicating that pretransplant sensitization against HLA antigens per se increases the risk for graft
loss, even if associated with natural anti-HLA antibodies [57] . Conversely, SAB can result in a
miss- or under-estimation of high titer anti-HLA antibodies caused by the prozone effect or by the
change in tertiary structure of HLA antigens. The prozone effect hinders the binding of
alloantibodies due to several factors including complement or IgM interference. Different methods
can overcome the prozone effect such as the EDTA or DTT treatment, addition in the sera of C1-
ihibitor and heat inactivation [55].
Furthermore, the ability of anti-HLA antibodies to bind C1q or other complement components have
driven the development of new SABs to detect C1q- or C3d-binding antibodies that could allow to
evaluate the cytotoxic potential of anti-HLA antibodies, considered as increased risk of damaging
[58-62].