Open access peer-reviewed chapter - ONLINE FIRST

HLA and Transplantation

Written By

Penn Muluhngwi and Gizem Tumer

Submitted: January 19th, 2023 Reviewed: January 30th, 2023 Published: March 8th, 2023

DOI: 10.5772/intechopen.1001276

Human Leukocyte Antigens - Updates and Advances IntechOpen
Human Leukocyte Antigens - Updates and Advances Edited by Sevim Gönen

From the Edited Volume

Human Leukocyte Antigens - Updates and Advances [Working Title]

Dr. Sevim Gönen

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Abstract

The HLA (human leukocyte antigens) complex is positioned on chromosome 6 (6p21.3). The HLA genes respect the principles of Mendelian genetics and are co-dominantly expressed. The classical HLA genes are considered important for transplantation. HLA-A, -B, and -C are the classical HLA class I genes and are expressed in most of the somatic cells. HLA-DR, -DQ , -DP are the classical HLA Class II genes and are mainly expressed in antigen presenting cells such as B- cells, Activated T cells, Macrophages, Dendritic cells, and Thymic epithelial cells. In the presence of interferon, class II expression can be seen in other types of cells. This chapter includes the review of the structure and the function of the HLA molecule, and the most current HLA nomenclature. Subsequently evolution of HLA testing methodologies and advanced terminologies and techniques of HLA antibody evaluation that enhanced the bone marrow and solid organ transplantation is also discussed.

Keywords

  • HLA
  • nomenclature
  • HLA testing
  • transplantation

1. Introduction

Class I and Class II molecules are structurally different. The basic structure of the Class I molecule is comprised of one polypeptide chain and a β2 microglobulin chain (encoded by a gene on chromosome 15). While, the HLA class II molecule has 2 polypeptide chains. Alpha1 and alpha2 domains form the peptide binding domain for class I whereas alpha1 and beta1 domains form the peptide binding domain for class II. The peptide binding domains contain the majority of polymorphic regions of the HLA antigen [13]. An essential function of the HLA class I molecule is to present peptides that are the products of the degradation of cytosolic proteins to the cell surface where they can be identified by the CD8 + T cells. CD8 receptor binds to the alpha3 region of the class I molecule and presents peptides to CD8 + T cells. HLA class II molecules present peptides that are the products of the degradation of endocytosed proteins to the cell surface where they can be identified by the CD4+ T cells. CD4 binds to the beta2 region of the class II molecule [13].

The rejection of transplanted tissue likely begins with the immune system recognizing differences in HLA antigens. The HLA system is considered to be second in importance to the ABO antigens in determining the success of solid organ transplants, but it is considered most important in the case of hematopoietic stem cell transplants [1, 3, 4].

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2. Nomenclature

HLA nomenclature was updated in 2011 as the detection methods of HLA antigens dramatically changed since the discovery of the HLA system [1, 5, 6]. Originally, the polymorphisms of the Class I and Class II loci were solely based on serologic methods. With the rapid development and availability of molecular HLA typing methodologies, all HLA antigens are now uniformly named starting with the locus, antigenic specificity, and molecularly typed allele group. The asterisk “*” sign indicates that typing is performed by a molecular method and the colon “:” is a field separator.

Table 1 refers to molecular fields of HLA A*03:01:01:02 as an example and what they represent.

  • A*03 is low resolution typing by molecular method which represents the first field digits by DNA-based nomenclature. In most cases this refers to the allele family or the serologic equivalent to the antigen such as serologic A3.

  • A*03:01 is the allele and the 1st field (A*03) refers to a group of alleles that encode for the A3 antigen. 2nd field (:01) refers to an allele, which encodes a unique HLA protein (A*03:01).

  • Intermediate-resolution typing includes a subset of alleles with the same first field antigen name and multiple possible different alleles and for which some alleles are excluded (ambiguous results). An example of an intermediate-resolution typing is A*03:01/20/26/37 which means the HLA type could be typed as A*03:01 or A*03:20 or A*03:26 or A*03:37 but not the other A*03 alleles.

  • A group of alleles that specifies and encodes the same protein sequence for the peptide-binding region of an HLA molecule with the exclusion of non-expressed alleles on the cell-surface refers to high-resolution typing by molecular methodology. An example is A*03:26.

  • 3rd field refers to a synonymous (silent) mutation, which represents a change in the DNA sequence without a change in the encoded protein. E.g. the difference between A*03:01:01 and A*03:01:02 is a synonymous (silent) mutation.

  • The 4th field represents non-coding regions and after the 4th field are the expression modifiers (N, L, S, Q). Example includes Null alleles, which are alleles not expressed and denoted as capital N, L denotes low expression alleles, S is for secretory alleles, and questionable alleles are marked as Q.

    Additionally, not all HLA typing technologies used today always allow for an unambiguous assignment of a single HLA allele. For these instances, strategies of common reporting of ambiguous strings can be used. Adding P and G codes provides easy reporting of ambiguous alleles. “P” code is added after the 2nd field for HLA alleles containing nucleotide sequences that encode the same protein sequence for the peptide binding domains (PBD). PBD for HLA class I alleles are exons 2 and 3, for HLA class II alleles exon 2 only. https://hla.alleles.org/alleles/p_groups.html “G” code is added after the 3rd field for HLA alleles that have identical nucleotide sequences across the exons encoding the PBD [7]. https://hla.alleles.org/alleles/g_groups.html

SpeciesLocusAntigen EquivalentAlleleSilent MutationOutside exonExpression
HLAA*03:01:01:02N, L, S, Q
Locus*1st Field2nd Field3rd Field4th Field

Table 1.

HLA nomenclature: HLA A*03:01:01:02 as an example and what each field represent.

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3. HLA typing

HLA typing is the ability to determine the HLA antigens of an individual prior to transplantation. The methodologies utilized for HLA typing have varied over the years from serological or cellular typing methods (that provided low resolution or antigen group of each locus) to the molecular methods (that utilize DNA sequence variation to provide intermediate to high resolution typing on a patient). Described below some of the HLA typing technologies previously (e.g. Complement-dependent cytotoxic assay) and currently used (including sequence specific priming; SSP, reverse Sequence specific oligonucleotide probe hybridization; rSSO, and sequence-based typing; SBT).

3.1 Complement-dependent cytotoxic (CDC) assay

The CDC assay (also known as the lymphocytotoxic or serological assay) was among the first assays used to determine HLA antigens (or type) of an individual. In this assay, individual’s lymphocytes are incubated against a well-characterized panel of HLA antisera obtained from individuals sensitized to foreign HLA molecules by pregnancy or previous transplant and upon addition of rabbit serum, serving as a source of complement, cell injury is recorded by the microscopically characterizing cell membranes that take up vital or supravital dye [2, 8]. When more than 25–50% of cells are injured by a given antiserum (antibody), that population of cells is considered to have complementary antigens. An advantage of the technique is that it can be used to discern null alleles that are identified by molecular typing. Technical limitations that limit the results include the presence of unknown/rare alleles, difficulty in finding antisera to certain antigens or particular locus, homozygosity at one or more loci, and the assays inability to distinguish alleles that can only be characterized by DNA-based methods (such as HLA-DP alleles) [8, 9]. Further, CDC assays require optimization of the assay protocol including understanding the reactivity of each antisera, acceptable room temperature (22–25°C) conditions, evaluation of complement lots, avoiding contamination during pipetting and thorough mixing of assay components [2, 8, 9].

Variations or modifications of the CDC assay were later instituted to improve on its sensitivity. These modifications mostly occurred after the incubation step of anti-sera with cells such as addition of more wash steps (Amos-modified technique), addition of antihuman globulin (AHG).

3.2 DNA-based typing methods

DNA-based typing characterizes HLA polymorphism based on DNA sequence variation. Overcoming the initial restrictions of cost and incomplete primer and probes, DNA-based typing is available for every HLA lab to offer it to the transplantation community. Here are some advantages of this technology in comparison to CDC-based HLA typing: in DNA-based typing, a relatively small amount of DNA is used to perform the assay allowing for easier evaluation pediatric populations. There is no restriction of using only viable lymphocytes, the assay has the ability of accommodating variable DNA sources such as buccal swaps, whole blood, dried blood spots, cells tissues, and hair follicles, and the expression of relevant HLA antigens on the cell surface is not a requirement as in serological typing. Reagents such as DNA polymerase, primers, and probes are reproducibly synthesized and sold as standardized reagents to meet the needs of laboratory demand as oppose to antiserum in serological assays that can be in limited supply. Further, HLA typing requirements for Hematopoietic stem cell transplants (HSCT) require a minimum allele level typing which cannot be performed by serological methods [2, 9, 10].

Because DNA-based typing methods require DNA, DNA isolation technique that consistently provides adequate DNA quality and quantity from sample sources is very important. Methods of DNA isolation include Column techniques (Anion exchange columns), Salting out (cesium chloride gradient centrifugation or phenol extraction), and the use of magnetic beads (positive selection) [11]. Assessment of DNA quantity and quality can be done by UV Spectrophotometry with “Pure” DNA having a A260/280 absorption ratio of 1.8. Ratios greater than or lower than 1.8 suggest an increased presence of RNA and protein in the isolated sample respectively. There are also a fluorochrome-based methods for quantifying DSA. This is based on the principle that the fluorochrome will selectively bind double stranded DNA can be measured with a fluorometer [12].

Multiple factors can affect DNA quality. Acid Citric Dextrose (ACD) and Ethylenediaminetetraacetic acid (EDTA) are recommended as anti-coagulants for DNA isolation from a whole blood sample. Lithium heparin tubes should be avoided as lithium inhibits PCR. For recipients of hematopoietic stem cells, using whole blood may not be the best source to obtain accurate patient HLA typing as the patient’s malignancy may include mutations at HLA loci that can lead to inaccurate results. Buccal swabs are a more suitable alternative. Samples received from patients with significant numbers of circulating cancer cells e.g. when the patient is in a leukemic blast phase, Allelic dropout can be seen at the time of HLA typing. In these situations, DNA obtained from a buccal swap is recommended. Recent blood transfusions may interfere with HLA typing as well.

Polymerase chain reaction (PCR) allowed for the development of simple, rapid, and improved characterization of allelic diversity at the HLA loci. In fact, next generation sequencing incorporates PCR. Therefore, HLA typing can be more accurate and precise (with high reproducibility and reliability). Amplified nucleotide sequences of a locus can reveal where and how alleles differ allowing for the evaluation of specific polymorphic amino acid residues in peptide binding and presentation.

Multiple PCR-based typing methods have been developed and applied to clinical HLA typing. Generally, the typing methods are such that the design of primer pairs targets polymorphic sequence motifs at a locus. The amplified sequence can then be analyzed by a variety of approaches including hybridization to an oligonucleotide probe (reverse sequence specific oligonucleotide-rSSO), digestion with restriction enzymes, chain termination sequencing reactions (Sequence-Based Typing-SBT) or evaluation of the mobility pattern using gel electrophoresis (sequence-specific primer-SSP). Other approaches use the specificity of the PCR itself with the 3′ end of the primer targeting the polymorphic site [10].

3.2.1 Sequence specific oligonucleotide (SSO)

The first PCR-based approach for HLA typing utilized labeled sequence specific oligonucleotide probe to hybridize onto amplified PCR products from a sample immobilized on a nylon or nitrocellulose membrane (the dot blot method) [13]. The SSO probes only bind to complementary sequence in the amplified DNA and can distinguish single nucleotide differences. Alleles in the samples could be identified by comparing patterns of probe reactivity to a panel of probes specific for informative sequence motifs [10]. Probes were labeled with either P32 (phased out), enzymes (such as horseradish peroxidase; HRP), digoxigenin or biotin. Biotin labeled probes can be detected with streptavidin conjugated to HRP (or alkaline phosphatase; AP) and a chromogenic or chemiluminescent substrate. Note that in the SSO probe typing approach a single PCR reaction amplifies all alleles at a target locus and the amplification wherever possible is locus specific.

SSO can be cumbersome with an increase in the number of probes and multiple separate hybridizations. To circumvent this limitation, the reverse hybridization approach (reverse dot blot) was developed wherein biotinylated amplified labeled PCR products are interrogated against an array of probes immobilized onto a solid support. Currently the most used reverse SSO (rSSO) assay for testing clinical samples is the bead-based rSSOP method where primers are used to amplify HLA gene polymorphic regions on Exons 2/3/4 for Class I and Exons 2/3 for Class II. In the PCR step, the amplicons are labeled with biotin. The amplicons are then applied to a panel of probes immobilized onto different uniquely fluorescently coded beads (identifiable by the Luminex technology). Note that, as opposed to SSO, the probes used in rSSO are not labeled. Each bead is coated with a unique allele- or group-specific oligonucleotide probe. Amplicons annealed to complementary probes are detected via streptavidin-phycoerythrin (SAPE) chemistry. The HLA typing is then deduced from the pattern of probe reactivity. Multiple probes must be used to characterize alleles that are amplified at each locus [10, 14].

The large number of probes and the complex probe reactivity patterns make it necessary to use computer programs for genotype analysis, which are updated periodically to incorporate newly identified alleles. In principle, with sufficient primers and probe, PCR-SSO should be capable of distinguishing all alleles. However, ambiguities may arise from the sharing of sequences between HLA alleles, the inclusion of new alleles into the typing system and software or when a probe reactivity pattern is consistent with more than one genotype. New probes are often developed to detect the newly defined alleles. Furthermore, SSO may not be as rapid as SSP. To ensure accuracy, control such as previously typed DNA, a positive control for each locus amplified and a no template negative control (molecular biology grade water) are typically included during assay setup [14].

3.2.2 Real-time PCR sequence specific priming (rtPCR-SSP)

rtPCR-SSP is based on the specificity of the primer extension. Historically it has been variously called allele-specific amplification (ASA) and amplification refractory mutation system (ARMS). In rtPCR-SSP, sequence specific primers are designed to amplify polymorphic regions of a sequence motif starting from the 3’end of the template. Amplification of targeted polymorphic sequences can be detected as bands on a gel. If there is no amplification or no product detected, the sample is assumed to lack the targeted motif. In such a case, a positive control included with test samples is examined to demonstrate successful PCR setup and exclude the potential of PCR inhibition and absence of template control. The positive control can be an unrelated monomorphic sequence that upon amplification produces a fragment distinguishable from the targeted polymorphic sequence post-gel electrophoresis. Although relatively fast for small sample size, SSP requires several PCR reactions to generate a typing resolution equivalent to intermediate or high-resolution typing. Thus, not well suited for large-scale throughput analysis.

In contrast to endpoint analysis of the PCR amplicons on a gel, modern rtPCR methodologies quantify the DNA during the exponential phase of the process. Two main fluorescent dyes, SYBR green and Tag Man, are used to detect PCR products in real-time instruments. In the PCR step, following primer extension and polymerization, SYBR green will intercalate into double stranded DNA and emit a strong fluorescent signal. When SYBR green is unbound, it exhibits little fluorescence. TagMan is a probe-based method. Here, probes labeled with a fluorophore (TagMan) and a quencher at opposite ends anneal to complementary DNA strands after the PCR denaturation step. During the extension phase, Taq DNA polymerase’s 5’exonuclease activity cleaves the hybridized probe, releasing the reporter from the quencher and producing fluorescence that can be detected. These methods are fast, have a short turn-around time, require no post-amplification processing, and provide intermediate to high resolution typing, making them the most commonly used method for HLA typing in solid organ transplantation [10, 15].

3.2.3 Sequence-based typing

3.2.3.1. Sanger sequencing

Sequence-based typing is used to determine the exact nucleotide sequence of a gene or a region of a gene. It is considered the gold standard for HLA allele identification. Ideally sequence-based typing should result in unambiguous “high-resolution” typing (two field resolution e.g. A*02:01), describes the actual protein expressed, distinguishes null alleles, and detects new alleles. Strategies have evolved over time from the Maxam-Gilbert or Sanger sequencing wherein HLA type was determined by manual reading of radioactive tagged amplicons separated on a slab gel. The technique was modified to the Sanger capillary method that replaces the radioactive tags with fluorescent tags and automated reading of the amplicons separated by capillary gel electrophoresis. This modification made sequencing a more robust method for HLA typing, allowing for increased throughput, and decreased cost of the test. The first step in Sanger sequencing requires amplification (by PCR) of the antigen recognition site (specifically exons 2 and 3 for Class 1 and exon 2 for class II genes) using fluorescently tagged dideoxynucleotides. The amplicons (which make up both alleles of a locus) are then sequenced and compared to an HLA reference sequence in the reference data base (https://www.ebi.ac.uk/ipd/imgt/hla/) to determine allele assignments. Infrequently, alternative genotypes are obtained (i.e. ambiguous typing) wherein it is impossible to determine the phase of two or more polymorphisms at a locus and thus the level of resolution may not meet testing requirements. To resolve this, group-specific or allele-specific can be used in the PCR step to separate alleles prior to sequencing.

3.2.3.2. Next generation sequencing (NGS)

NGS is a newer methodology for high resolution typing that sequences large pieces of DNA or entire genomes. The sequence information is read as it is synthesized during the DNA synthesis reaction. Contrary to Sanger sequencing wherein a single DNA fragment is sequenced at a time, in NGS, millions of DNA fragments are simultaneously sequenced per run hence it is known as massive parallel sequencing. There are several NGS platforms that use different chemistry and detection methods, such as monitoring pH changes or capturing fluorescent signals to identify bases (A, C, G, T) as they are added one at time to the growing nucleotide chain. The fluorescent signals correspond to fluorescent tags on each of the four bases [16]. NGS has undergone several technological advances to a 2nd generation and a 3rd generation NGS (that allows for single molecule sequencing) [17].

Routine benchwork for NGS HLA typing is cumbersome and takes 2–3 days depending on the chemistry and detection method [18]. The first step in NGS usually involves amplifying the HLA gene region by PCR using primers that flank this region. The generated amplicons range in size from a single exon to all exons. The choice of the amplicon size depends on the starting material (amplification of short DNA segments is more robust than longer segments) and the target gene size. E.g. the HLA-DRB1 gene is three times longer than an HLA class I gene and amplification of this 15 kb DRB1 gene is difficult to achieve. Hence, HLA class II amplicons usually contain only a portion of the whole gene. Amplicon are kept relatively uniform in lengths for even representation during sequencing. Amplicons are then quantified, balanced, and pooled for library construction. The first step in library preparation is fragmentation. Since the generated amplicon reads usually range in size from 1000 to 6000 base pairs, these are cut into shorter fragments (200–300 base pairs) in a process known as fragmentation. Fragmentation can either be enzymic (e.g. restriction endonucleases) or physical (e.g. acoustical shearing-sound waves). Following fragmentation, oligonucleotides (adapters) are attached to the 5′ and 3′ ends of each amplicon fragment. The adapters have sequences that allow the fragments to bind to a solid support, serve as annealing sites for PCR and sequencing primers, and identify the source of the DNA (e.g. a loci or patient identifier). Adapters are “generic” (i.e. not specific for any gene) and same adapters are used for every library created. After adapter ligation the DNA is cleaned up to select for larger fragment sizes (by gel- or bead-based methods). Quantification by fluorometric or quantitative PCR (qPCR) methods can be used to ascertain the concentration of the DNA products. Multiple libraries can be pooled together and sequenced simultaneously as multiplex sequencing. Individual “barcode” sequences are added to each DNA fragment so each read can be identified and “binned” together with reads carrying the same. The created library is then sequenced on a solid surface (flow cell or bead) in the instrument sample chamber using a combination of lasers and fluorescent dyes. Using specialized software, the resulting sequence data is then analyzed, typically by aligning sequences (reads) from the sequencing data to a reference genome and comparing the obtained sequence to known HLA alleles in a database [17, 18].

Because NGS is a high throughput complex technique, many quality control checks have to be maintained to ensure generation of accurate and reliable results. It is recommended for each run should have an internal, positive, and negative controls. Good record keeping of the parameters for each sequencing run should be documented to show the quality of the run. Some parameters that can be monitored include cluster density, quality score, depth of coverage, read depth, heterozygosity (or the presence of two different alleles at a particular locus), and maintaining external proficiency testing.

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4. Applications of HLA typing in transplantation

High resolution typing is commonly used to type recipients and unrelated donors in hematopoietic stem cell (HSCT) and cord blood (CBT) transplantation to treat leukemia, lymphoma, and other serious diseases affecting the hematopoietic system. For related donor selection high resolution typing is not a requirement but sequencing may be used to identify the best matched relative when similar alleles/antigens segregate within a family and/or the parents are unavailable.

In HSCT donor selection, preference is given to finding HLA-identical siblings. If no related donor is identified then the search for unrelated donors with matches at HLA-A, B, C, DRB1 is recommended. In fact, matching at these loci has been shown to increase survival following transplantation [19]. Matching for HLA-DQB1 and HLA-DRB3, -DRB4, and -DRB5 alleles is also recommended but not required while for the DPB1 loci, matching is for alleles with low immunogenic potential (permissive alleles). If a patient is to receive a mismatched transplant and determined to be sensitized (having HLA antibodies), finding the best matched donor may require typing of other donor loci to exclude donors to whom donor specific antibodies are present [20, 21]. This is done in an attempt to reduce the risk of graft failure. The requirement on matching for CBT is less stringent: a > 4/6 matches at HLA-A, B, DRB1 is the traditional match requirement [20].

Other center specific criteria to guide donor selection stem cell transplantation include killer immunoglobulin-like receptor (KIR) matching [22], HLA-DQ heterodimer (DQα-DQβ) matching [23], and HLA-B leader peptide matching. These parameters have each been reported to impact graft outcome [24]. The B leader peptide is an exon 1 encoded nonamer which when presented by HLA-E molecules stabilizes cell surface expression and enhances binding to CD94/NKG2-A on NK cells. Polymorphisms in the B leader peptide impact stability to HLA-E and binding to CD94/NKG2-A [25, 26]. In unrelated donor selection with mismatched HLA-B alleles, leader matched donors is associated with lower risk of GVHD compared to leader mismatched recipient/donor pairs [24]. Functional sequence variation at position 21 of Exon 1 of the leader peptide is also of importance. While this position is invariant for HLA-A and HLA-C, it is dimorphic in HLA-B and can either code for threonine (T) or Methionine (M) at the second position of the leader peptide. HLA B-matched pairs with MM leader genotype have worse outcomes (mortality and acute GvHD) relative to HLA B-matched pairs with TT leader genotype [27]. KIR genotyping offers an additional immunogenetic criteria for assessing haploidentical donors [22]. KIR genotyping is used to provide an assessment of Natural Killer (NK) cell alloreactivity. KIRs are polymorphic receptors on NK cells that interact with MHC class I molecules. On Interaction, NK cells become competent and able to activate itself against abnormal cells. NK cells without this interaction remain inactive through inhibitory receptors, specifically self-tolerant to autologous healthy cells. In allogenic HSCT, donors NK cells become alloreactive when inhibitory KIR receptors do not recognize recipient MHC Class I antigens [28]. This missing-self alloreactivity triggers Graft vs. leukemia (GVL) without promoting GVHD [29]. Thus, NK alloreactive donors (mismatched donors) will be preferred to donors having matched KIR ligands as recipients. It was recently reported that certain HLA-DQ heterodimers have lower risk than others; specifically donor and recipients matched for DQA1*02/03/04/05 paired with a DQB1*02/03/04 have lower relapse risk than other HLA-DQ heterodimer matched or mismatched pairs [23]. This opens additional avenues to risk categorize donors.

In addition to HLA compatibility, other considerations for donor selection in HSCT include Donor age (younger donors preferred), CMV matching, Donor gender (male donors preferred), stem cell source (Bone marrow vs. peripheral Blood), ABO compatibility, donor center location and specific center practices [30].

In solid organ transplantation (SOT), high resolution typing is not commonly used. This is because the priority for donor selection is avoidance of rejection and avoidance of donor specific antibodies in sensitized patient. Nevertheless, high resolution typing in SOT may guide donor section by allowing for more accurate interpretation of virtual crossmatches, DSA identification and selection of lower risk donors to whom the recipient does not have alloantibody [31].

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5. HLA antibody testing

Microlymphocytotoxicity or CDC assay can be utilized to test serum against selected target cells to show the presence or absence of HLA antibodies. The testing is routinely done in HLA crossmatching. The assay is performed by testing serum from a potential recipient against lymphocytes from a potential donor. An enhanced variation of the CDC assay by adding antiglobulin reagent to increase the sensitivity has been used. These assays do not detect the specificity of the HLA antibodies.

5.1 Single antigen bead assay by Luminex

Current gold standard assay for HLA antibody testing is the Luminex (specialized flowcytometry) technology where each 5.6-micron polystyrene beads or microparticles are internally dyed with a unique combination of red and infrared dye. Utilizing of different intensities of the two dyes creates the bead set of 100 reactions by its unique spectral signature when excited by a laser beam. This permits multiplexing of up to 100 reactions in a single tube. Finally, the beads are coated with one recombinant HLA antigen or a haplotype of purified HLA antigen(s) (solid-phase methodology). Once the patient serum is exposed to the bead set, in the presence of an HLA antibody, the HLA antibody binds to its corresponding antigen on the bead. A fluorescent-labeled antihuman globulin (AHG) detects the antibody which produces a positive reaction and is measured by the mean fluoresce intensity (MFI) value. Flow cytometry and Luminex methods are more sensitive than CDC testing and detect the antibodies in the IgG form that may or may not fix complement. These assays are very sensitive. Therefore, the clinical significance of antibody detection by these methods should be interpreted along with a flow cytometric crossmatch prior to transplantation [13233].

5.1.1 Detection and identification of HLA antibodies

Solid phase method can be utilized as an enzyme immunoassay where HLA antigen is bound to a solid phase plate or flow cytometer or Luminex detects the HLA antigen on the bead. Currently, three types of Luminex systems exist for HLA antibody detection and identification. These include a pooled HLA antigen system, a phenotype panel system, and a single antigen system. The pooled antigen system is mainly used as a screening tool for the presence or absence of HLA antibody. This assay is more sensitive but less specific. Reflex testing to identify the specificity of the HLA antigen can be performed to detect either by using the phenotype bead panel or a single antigen bead (SAB) testing. These assays have increased specificity. The manufacturing of the pooled antigen, phenotype panel, and single antigen bead panel are different. The pooled antigen and phenotype panel use affinity columns to purify HLA antigens to coat the beads whereas the single antigen beads are coated by recombinant HLA antigens. A variation of SAB assay is also available to identify the antibodies that bind complement. This assay detects antibodies that bind complement (C1q or C3d). Donor specific HLA antibodies that have the capability to bind complement are associated with antibody mediated rejection and graft loss [1, 32, 33].

5.1.2 The advantages and disadvantages of antibody detection by Luminex technology

The Luminex technology provides fast, sensitive, and specific way of HLA antibody identification. These features of this assay significantly improved the clinical practice in the post-solid organ transplant setting, where the formation of new HLA antibodies to the graft can be identified quickly to determine a rejection episode or reduced graft function is reported. The speed and the specificity also aid for the utilization of apheresis to remove the antibodies to improve graft function in the post-transplantation setting. The Luminex methodology not only detects the specificity of the HLA antibody but also determines the strength and/or the avidity of the antibody which is as MFI value. Knowing the MFI value of antibodies present in the serum allows for risk stratification to the management of highly sensitized patients and proper adjustment of immunosuppressive medications [1, 32, 33].

There are some disadvantages with the Luminex technology. The assay is expensive and there are gaps in the antibody repertoire within the bead panels. If a patient has antibody where it is not represented in the bead panel, the assay provides false negative results. Also, standard Luminex kits do not provide information for the complement fixation capacity of the antibody. Since the HLA antibodies coating the single antigen bead assay are recombinant, presence of denatured antigens on beads may result in false positive results due to the cryptic epitopes becoming accessible during the manufacturing process. Inhibitory factors in patient serum may prevent antibody binding to single antigen bead (SAB) causing some beads to appear false negative. Treating the patient’s serum with and dithiothreitol (DTT), ethylenediaminetetraacetic acid (EDTA), Heat inactivation or performing serial dilutions can resolve the issue. The single antigen bead assay is a semi-quantitative assay generally indicating more fluorescence as more antibodies bind to the beads. Meaning that if all of beads are occupied with HLA antibodies, there is the possibility that an excessive amount of HLA antibodies in a patient’s serum may not be accurately detected. To ensure accurate assessment of antibody strength, titration studies are recommended. Also, if a patient has antibodies that recognize an epitope that is shared across multiple antigens, then the antibody may appear weaker than what it actually is. These shared epitope patterns or cross-reactive groups can create a false perception of compatibility, particularly when the antibody strength is not fully represented. To provide a more accurate assessment of HLA antibody strength, crossmatches can be used to performed [1, 3133].

5.2 Flow Crossmatch

Since 1969, when Patel and Terasaki first demonstrated in their landmark paper the significance of recipient antibodies against donor antigens in mediating early rejection or graft loss in kidney transplantation [34], HLA laboratories now regularly perform crossmatches between donor cells and recipient serum. In their paper, Paul and Terasaki described the CDC assay discussed in the typing and antibody sections. The CDC crossmatch (CXM) assay is in essence an in vitro “surrogate” of what is to be expected from an allograft transplanted into the patient. Isolated lymphocytes are expected to express the same surface proteins as on allograft endothelium. The CDC crossmatch (CXM) assay has now morphed into the Flow crossmatch (FXM) assay. The FXM assay is a cell-based complement-independent assay where viable donor lymphocytes (T and B cells) are incubated with recipient serum to detect the donor-specific antibodies. Instead of quantifying the level of cell death as in CXM (an indicator of incompatibility), fluorescence is measured from a DSA-dependent fluorochrome conjugated secondary anti-IgG antibody. Lymphocytes can be isolated from lymph nodes or whole blood (using magnetic beads or ficoll/percoll methods) and pronase treated to reduce non-specific interactions. T cells and B cells are distinguished with different fluorescently antibodies specific to B and T lymphocyte surface proteins and florescence in the assay detected with a flow cytometer. The assay is a semi-quantitative method that reports results in terms of median channel shifts from a baseline or in relation to a set of MESF (molecules of equivalent soluble fluorescence) beads, and is more sensitive and less subjective than the visual assessment of cell death used in CXM [35, 36].

The Flow crossmatch assay is limited by interlaboratory variability as the technique and threshold for positivity varies between labs. Interactions in the FXM assay are not only HLA-specific nor donor specific. Therapeutic antibodies, specifically humanized and chimeric antibodies, can cause false positive reactions via non-specific immunoglobulin binding to Fc receptors. Such false positive FXM results can lead to denial of an otherwise acceptable donor organ. It is therefore essential that the crossmatch test occurs alongside extensive characterization of the patient’s HLA antibody [35, 37].

Some Therapeutic interferences and consequences on Flow crossmatch:

  • Rituximab (anti-CD20): False positive B cell crossmatch.

  • Daclizumab (anti-CD25): False positive T and B cell crossmatch.

  • Alemtuzumab (anti-CD25): False positive T and B cell crossmatch.

  • Antithymocyte globulin (multiple targets): may or may not cause false positive reactions.

  • IVIG (pooled human Ig): False positive T and B cell crossmatch at high concentrations.

5.3 Luminex crossmatch

In the Luminex crossmatch, isolated donor lymphocytes are coated onto class I and Class II specific beads. Captured monoclonals bind donor HLA antigens onto specific beads. HLA antigen-captured beads are then incubated with recipient sera. The presence of donor specific antibodies is detected with a Luminex analyzer after the addition of PE conjugated anti-human IgG antibody. The advantage of the Luminex crossmatch is that there is no requirement of viable lymphocytes, isolated donor cells can be frozen for future use and up to 91 assays can be performed at a time. Unfortunately, the ability for the Luminex crossmatch to detect DSA to HLA-DQ and DP is questionable [38].

5.4 Virtual crossmatch (VXM)

A virtual crossmatch (VXM) is the use of a patient’s HLA-antibody profile to access compatibility with HLA antigens of a donor prior to a physical crossmatch and/or transplantation. It is not a physical test. Transplant programs have become increasingly reliant on VXMs to improve cold ischemic time and potentially reduce delayed graft function without increase risk of rejection [39] and offer the ability to evaluate low titer antibodies. The accuracy of a VXM is determined by the information used to perform the VXM. Factors such the ability of detect HLA antibodies in patient serum, the resolution level of Donor HLA typing and resolution of ambiguities, and the frequency and timeliness of HLA antibody testing [31, 40]. Having a more recently tested serum sample when performing improves the predictability of a VXM.

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6. Applications of HLA antibody testing and Crossmatching in the clinical setting of solid organ transplantation

The heterogeneity in MHC region affords the immune system the ability to target diverse non-self-antigens (and pathogens). Development of anti-HLA antibodies occurs by three main mechanisms (listed in decreasing order sensitizing potential); prior transplantation, pregnancy, and blood transfusion. Patients having anti-HLA antibodies are described as sensitized. The presence of prior anti-HLA antibodies can result in rapid rejection of a transplanted organ or tissues; especially if there are preexisting donor specific anti-HLA antibodies (DSA). Detection and avoidance of anti-HLA antibodies is critical for successful transplantation. Further, well matched donor/recipient pairs have improved graft survival and limit sensitization in patients who may need retransplantion in future [41].

For patients not sensitized at transplantation, there is still the risk of developing de novo DSA or T- cell mediated rejection after transplantation. The likelihood of these events increases with the degree of mismatched antigens (i.e. antigens present in the donor but not in the recipient). The acceptable degree of matching and HLA typing resolution necessary for transplantation depends on the type of transplant (solid organ vs. stem cell) [41].

6.1 Allorecognition in transplantation

Allorecognition develops when the host T cells identify non-self-antigens and elicit for allograft rejection. There are two defined pathways for allorecognition. The “direct pathway” is involved with the presentation of the intact (non-processed) donor MHC peptide to host T cells while the presentation of processed peptides of the donor MHC molecules to host antigen presenting cells is referred as “indirect pathway.” This second pathway requires T cell activation with co-stimulatory signals in addition to antigen recognition. In the alloimmune response, T Helper 1 (Th1) cells play a role in acute and chronic cellular rejection in solid organ transplantation and humoral antibody mediated rejection (AMR) is driven by Th2 cells. Once helper T cells activate the cytotoxic T cells, acute and chronic cellular rejection can be triggered [42].

6.2 HLA antibody production and graft survival

Individuals may generate anti-HLA antibodies when exposed to foreign HLA molecules through sensitizing events. Both pre-formed HLA antibodies and de novo donor specific antibodies are known to increase risk of rejection and premature allograft failure [43].

There are four major clinical types of rejection seen in kidney allografts where HLA antibodies are implicated: 1) hyperacute, 2) humoral/ antibody-mediated, 3) acute cellular, and 4) chronic [44].

Hyperacute rejection happens within minutes up to 24 hours after transplantation. Pre-formed donor specific HLA antibodies initiate a coagulopathy by binding HLA antigens expressed on the endothelium of the glomeruli and the graft microvasculature. This form of rejection has been virtually eliminated by the advanced technology of accurate HLA typing, timely identification of HLA antibodies, and enhanced techniques of crossmatching prior to transplantation. The absence of preformed HLA antibodies decreases the risk of hyperacute rejection and early humoral rejections. Currently no effective treatment is available for hyperacute rejection and is not reversible.

Humoral/antibody-mediated rejection often takes place within one [1] to three [3] months after transplantation. The patients may present with a rapid rise in the serum creatinine with an initial good baseline renal function. Pathology consists of the infiltration of endothelialitis, thrombosis, and C4d deposition in the peritubular capillaries and glomeruli. Development of de novo donor specific anti-HLA antibodies, and possibly non-HLA antibodies play a role in the pathogenesis. Apheresis, IVIG, and appropriate immunosuppressant therapy have been successful in the treatment of this form of rejection.

Acute cellular rejection usually presents within the first year after transplantation and more frequently within the first half of the year. The initiation and severity of the rejection depends on the type of immunosuppression used and patient compliance as well as the degree of matching between the recipient and the donor. A gradual rise in the serum creatinine with high blood pressure without significant decrease in urine output is the typical clinical presentation. This form of rejection usually has a favorable response to therapy. Pathogenesis involves the direct, and subsequently indirect, pathway of stimulations. With the availability of novel potent immunosuppressant drugs, the frequency of acute cellular rejection has significantly decreased. Degree of compatibility has an impact on the frequency of this type rejection. HLA identical living donor transplants, and 6- antigen-matched (HLA-A, -B, -DRB1) deceased donor transplants have a decreased chance of cellular rejection.

Chronic rejection may present months to years after transplantation. It occurs as an insidious rise in serum creatinine, proteinuria, and hypertension. This form of rejection does not have a favorable response to increased immunosuppression therapy, and despite treatment renal function continues to decline. Ultimately leads to a transplant glomerulopathy.

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7. Advanced topics in HLA antibody analysis

Anti-HLA antibodies bind to immunogenic motifs, known as B cell epitopes and these epitopes can be shared across different HLA molecules within the same loci or across multiple loci. Each HLA molecule has multiple epitopes that can bind anti-HLA antibodies at once if these epitopes are spatially separated on the molecule. Advanced molecular HLA typing, now allows for better analysis of serologically defined HLA typing. This approach ultimately led to the recognition of HLA epitopes and the potential use for matching in transplantation [45].

7.1 Paratope: Epitope interactions

Complementarity-determining regions (CDR) determine the specific reactivity and affinity of an antibody. Paratope is the binding site of an antibody. B cell epitope is located within the CDRs of the antibody and is composed of 15–22 amino acid residues. Functional epitope is centrally located within the epitope and is made up of 2–5 amino acid residues within a 3-Å radius where the specificity of binding with the antibody occurs. The presence of at least one non-self-amino acid residue with the functional epitope allows for eliciting an immune response [45].

7.2 Defining epitopes

HLA B cell epitopes have been defined with two main methods: [1] Terasaki’s serological epitopes (TerEps) that are identified by analysis of cross-reactivity patterns of anti-HLA antibodies using antibody absorption/elution technique, and [2] Rene Duquesnoy’s HLAMatchmaker which defines theoretical epitopes based on differences in HLA amino acid sequences and modeling of molecular structure [45].

7.3 HLAMatchmaker

Exposure to foreign HLA antigens triggers HLA antibody production. The HLA antibodies bind to polymorphic amino acid residues known as epitopes on the antigens rather than binding to the whole HLA molecule. Each HLA molecule possesses many sites or epitopes for an antibody to bind. These epitopes may be private for only one HLA antigen or they may be shared across more than one HLA antigen (public) [46].

HLAMatchmaker (http://www.epitopes.net/) is an algorithm that analyzes HLA alleles structurally, considering these antigens as strings of unique molecular conformations that can be recognized by HLA antibodies critical in transplantation. This algorithm is based on experimentally antibody-verified epitopes described by polymorphic amino acids referred to as eplets.

Epitope repertoire was initially described based on serological cross-reactivity between HLA antigens and antibody specificities against private or public binding sites. This algorithm allows for defining the structural basis of HLA epitopes by looking at three-dimensional molecular structures and amino acid sequence differences between HLA antigens. In this model, HLA epitopes are determined by the polymorphic amino acid residues on the surface of the HLA molecule [47].

Most current version of HLAMatchmaker considers that antigenic proteins have functional epitopes where the amino acid residues are separated from each other by about 3 Ångstroms and at least one of amino acid residue is non-self. The current definition of an “eplet” is the areas of polymorphic residues within a radius of 3.0–3.5 Ångstroms. An epitope is described by the complete antigen–antibody interface (15 Ångstroms) composed of amino acids necessary for specificity and those that affect only affinity but not specificity. Utilizing HLAMatchmaker allows recipient and donor pair evaluations of humoral alloimmune response at the epitope level rather than antigen level humoral alloimmune response [46, 47].

An eplet can serve as a biomarker. A recent study from 2019 demonstrates that quantifying the number of single molecule eplet mismatches between individual class II HLA molecules (HLA-DR and HLA-DQ) may represent a precise, reproducible prognostic biomarker that can be utilized to modify immunosuppression based on individual patient risk [48].

7.4 Clinical applications of epitopes

Epitope-based matching provides a powerful matching tool to predict clinical outcomes. This tool can be used for decision-making at the time of organ allocation and dose adjustments of immunosuppression. In one study a strong linear correlation has been observed between DSA formation and the number of class I triplet mismatches in rejected post-transplant kidney grafts and also postpartum females [49].

A Canadian group in their kidney graft cohort demonstrated the association between class II epitope mismatch and DSA formation. Additionally, they found that the HLA-DR and DQ loci epitope mismatches were an independent risk factor for DSA formation [50].

The same group further characterized the epitope mismatches by defining an optimal threshold for each locus. HLA-DR (10 mismatched epitopes) and HLA-DQ (17 mismatched epitopes) were associated with a lower risk of developing DSA. On a separate cohort they subsequently investigated the synergistic impact of HLA epitope mismatch and patient non-compliance on graft survival. They concluded that increased number of epitope mismatches and patients with poor compliance had significant impact on graft loss when compared to low number of epitope mismatches [51].

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Written By

Penn Muluhngwi and Gizem Tumer

Submitted: January 19th, 2023 Reviewed: January 30th, 2023 Published: March 8th, 2023