Role of HLA in cardiothoracic transplantation

In current clinical practice, transplant clinicians create collaborative working relationships with histocompatibility laboratory scientists to identify the risk of long‐term graft failure, which may assist in establishing strategies for treatment and surveillance. Transplant immunology research also focuses on optimizing human leukocyte antibody tissue typing and defines the most effective test for detecting the presence of donor‐specific antibodies. Although several studies have been conducted, data on pediatric heart transplant recipients are limited. Epitope load information may be utilized to identify donors with permissible human leukocyte antibody mismatches to increase transplant success. Although current guidelines do not consider human leukocyte antibody epitope‐based matching tools, these guidelines could be useful for identifying recipients at a high risk of donor‐specific antibody production, which would be appropriate for routine donor‐specific antibody screening to initiate early interventions to prevent antibody‐mediated rejection. Human leukocyte antibody matching at the epitope level offers an effective approach for identifying acceptable mismatches in sensitized patients and provides information about epitope loads. In the future, eplet matching may be used to define the best immunosuppressive therapy protocol for cardiothoracic organ transplantation. This report provides an overview of the role of human leukocyte antibodies in heart and lung transplantation.


| INTRODUCTION
][3] Donor HLA molecules are recognized by the recipient's immune system and trigger alloimmune responses after transplantation.HLA class II and class I matching are important for organ transplantation and play an important role in the complex process of graft acceptance.Achieving perfect HLA matching between donors and recipients would effectively prevent the development of de novo anti-HLA donor-specific antibodies (dnDSAs).Quantifying the degree of donor-recipient structural mismatch can potentially enhance the ability of clinicians to identify recipients at risk of graft failure secondary to HLA mismatch. 1,3| EVOLUTION OF TESTING, CHALLENGES, AND CLINICAL IMPLICATIONS 2.1 | DNA and molecular methods for HLA typing Molecular methods are routinely used in many tissuetyping laboratories to provide low-and high-resolution typing findings.4,5 The potential benefits of nextgeneration sequencing (NGS) have been fully demonstrated, and NGS technique has been used to examine the diversity of HLA characterized at the full gene level.6 Typically, when DNA is extracted from blood samples using automated systems, it is subsequently amplified using specific kits designed for multiplexed Polymerase Chain Reaction (PCR).These kits enable the simultaneous amplification of multiple gene regions, thereby facilitating comprehensive typing of the HLA-A, -B, -C, -DRB1/3/4/5, -DQA1, -DQB1, -DPA1, and -DPB1 genes.This multiplexed approach enables efficient and accurate HLA gene typing of extracted DNA samples.

| Solid phase assays for HLA antibody detection or identification
Solid-phase techniques currently represent an important tool in the study of pretransplant anti-HLA sensitization.In particular, Luminex single antigen assays enable the detection of preformed anti-HLA antibodies in patients immunized by transfusions, pregnancies, or previous transplants.The Luminex technique uses a panel of differently colored microbeads coated with purified HLA antigens.The patient's serum is incubated with these microbeads and the HLA antibodies bound to the antigens are labeled with antihuman IgG or IgM.The Luminex system detects the fluorescent emission from each bead, facilitating the identification of antibody specificity based on allelic results.Luminex assays not only indicate the presence or absence of an antibody potentially directed towards a specific HLA allele of the donor but also provide a semiquantitative strength indicator, the mean fluorescence intensity (MFI) value for each bead, however, MFI values do not always reflect antibody concentration, and low MFI values may sometimes hide the high titer of antibodies caused by a "prozone effect."This can be attributed to complement factor inhibition, antibody agglutination, or the presence of competing IgM.Laboratories have developed different strategies to avoid prozone effects, including serum dilution or pretreatment with EDTA.

| Complement-fixing solid-phase assays
Luminex assays enable the identification and screening of complement-binding antibodies in human serum.Similar to the Luminex single antigen, the C1q technique uses a bead-based assay in which beads are coated with purified HLA molecules.Human serum and purified C1q molecules are then added to the test so that a specific antibody will bind to the HLA molecules and C1q would bind to any complement-binding antibodies.The beads are then washed, and a secondary anti-C1q antibody labeled with a fluorescent tag is added.The secondary antibody binds to any C1q-bound to the HLA antibodies present on the beads, and the level of fluorescence is measured.The amount of fluorescence is directly proportional to the level of complement-binding antibodies present in the human serum.C1q is used as a biomarker to identify patients who are at a greater risk of developing antibody-mediated rejection (AMR). 8

| Complement-dependent cytotoxic crossmatch
The complement-dependent cytotoxic crossmatch (CDC-XM) test consists in a donor/recipient cross-test performed before organ transplantation and facilitates the direct evaluation of the reactivity of a patient's serum against the T and B lymphocytes of a potential donor, usually due to the presence of anti-HLA DSA.This test enables the identification of anti-HLA complement-fixing antibodies directed against the donor using complementdependent cytotoxicity techniques.The test is performed by incubating donor T and B lymphocytes obtained from peripheral blood with serum from transplant candidates.Subsequently, a complement is added to the test, and after further incubation, a vital dye is added and allows the test results to be visualized under a fluorescence microscope.This test can predict hyperacute/accelerated AMR. 35,36In case of a positive CDC-XM result patient can be subjected to a desensitization treatment before transplantation (for example using plasmapheresis, imlifidase etc.) with the aim of reducing or removing the amount of circulating anti-HLA.Despite its poor sensitivity and specificity, CDC-XM represents a safety measure to prevent hyperacute rejection and, therefore, must be performed before proceeding with a transplant, especially in patients with HLA presensitization in all cases where flow cytometric crossmatch (FC-XM) is not performed or sera are not studied for anti-HLA.The CDC technique can also yield positive results in the presence of non-HLA antibodies (reaction with other antigens present on lymphocytes) or for nonspecific cytotoxicity of the serum, particularly B lymphocytes.In some cases, positivity of the test results from the presence of IgM-type anti-HLA antibodies.CDC-XM can achieve a positive result in the presence of DSAs with a high MFI > 10,000/15,000 (depending on whether the DSA are specific for HLA class I or II).The positivity of the test is determined based on the ability of antibodies, either by isotype or "titer," to fix complement. 35,365 | Flow cytometric crossmatch FC-XM is a technique that aids in the detection of anti-HLA antibodies against the donor.This method detects both complement-binding and non-complement-binding antibodies.Subsequently, monoclonal antibodies labeled with different fluorochromes are added to identify the donor T and B lymphocytes and antihuman immunoglobulin (IgG or IgM) labeled with a third fluorochrome (e.g., fluorescein isothiocyanate) to identify antibodies related to lymphocytes.33,34 The results are then analyzed by flow cytometry.This test provides high sensitivity and information on the isotypes of DSA(s) present in the serum tested, and enables stratification of the "immunological risk" of a transplant according to the degree of positivity (channel shift).Moreover, it correlates well with the results of antibody studies using Luminex techniques.Flow cytometric crossmatch yields a positive result in the presence of DSA with MFI > 4000/8000 (depending on whether the DSAs are specific for HLA class I or II molecules).It can also give positive results for non-anti-HLA antibodies.In fact, in some cases, the test can yield false positive results due to the presence, in the serum of the transplant candidate, of non-HLA antibodies, autoantibodies, or monoclonal antibodies administered to patients undergoing desensitizing treatments.7,33,34 3 | HLA MATCHING

| HLAMatchmaker
HLA mismatching is considered, among others, a risk factor for graft rejection due to the potential presence of preformed or newly developed donor-specific anti-HLA antibodies (DSA) in solid organ transplant recipients. 9raditionally, HLA mismatching (MM) has been defined as a situation in which the recipient does not share the same donor HLA molecules.However, Duquesnoy 10 has reported the importance of investigating the HLA matching at the epitope level.It is important to note that HLA molecules are composed of epitopes, which are the regions of HLA molecules that are recognized by antibodies.Small areas of amino acids exposed on the surface of each HLA molecule are called "eplets."Eplets play dominant roles in the interaction between epitopes and antibodies.It is now well established that an immunogenetic evaluation between a donor and recipient would be more accurate if epitope MM (if verified, AbVer) is considered rather than allelic MM.Several retrospective studies have demonstrated a correlation between the number of epitope MM and the appearance of class I and/or II dnDSAs.HLAMatchmaker is an algorithm that evaluates structural compatibility between donors and recipients by comparing the polymorphic amino acid sequences of class I and II HLA molecules.HLAMatchmaker describes the polymorphic amino acids within the antibody-exposed regions of HLA allele sequences.This algorithm is based on the principle that each HLA antigen is represented by different chains of epitopes that are structurally defined as potentially immunogenic and capable of inducing the production of specific antibodies.Furthermore, patients cannot produce antibodies against epitopes present in their own HLA molecules.Initially, the program was developed to increase the chances of finding acceptable donors for hyperimmune patients.1][12] In addition to HLAMatchmaker, other effective algorithms that can be used for similar purposes are HLA epitope mismatch algorithm (HLA-EMMA) and predicted indirectly Recognizable HLA Epitope (PIRCHE).

| HLA-EMMA
HLA-EMMA is a software that simultaneously compares the HLA class I and class II amino acid sequences of the donor with those of the recipient and determines the polymorphic solvent-accessible amino acid mismatches that are likely to be accessible to B cell receptors.Analysis can be performed for many donor-recipient pairs.As proof of principle, a previously described study cohort of 191 lymphocyte immunotherapy recipients assessed using HLA-EMMA showed a higher frequency of DSA formation with a higher number of solventaccessible amino acid mismatches.Overall, HLA-EMMA can be used to simultaneously analyze the compatibility of amino acid levels between donor and recipient HLA class I and II in large cohorts to ultimately determine the most immunogenic amino acid mismatches. 13

| PIRCHE
The PIRCHE algorithm predicts T cell-related immune responses against HLA-derived peptides after transplantation, considering the indirect pathway of allorecognition.This tool uses the functional peptide-binding properties of HLA molecules to simulate the immune response and provides the number of donor-mismatched peptides that may be detected by the patient's T cells; the lower the number of presented peptides, the lower the likelihood of an immune response.PIRCHE scores can be calculated between the patient and the donor at the time of donor selection, as HLA typing does not change over time, and no additional work is required, as it is a purely bioinformatics-based approach.The PIRCHE algorithm has been developed by Eric Spierings at the Utrecht University Medical Center in 2013; it is based on the principle that an increased PIRCHE score in renal transplant patients is correlated with the immunogenicity of donor HLA-mismatched molecules. 14Furthermore, a correlation between PIRCHE scores and graft survival has been reported in patients who underwent kidney transplantation; the PIRCHE algorithm predicts T-cell epitopes that may play a role in predicting rejection. 15[18]

| CHALLENGES
13]19 HLAMatchmaker can calculate the number of eplet mismatches present in the donor but not in the recipient.The newly developed Predicted Indirectly Recognizable Epitope tool 14 may be useful for this purpose; however, it is not applicable for individual patients.In recent years, the selection of optimal donors has been established for high-resolution HLA typing, which is not always possible. 20Clinical perspectives should take into consideration the importance of performing highresolution HLA typing by immunogenetic laboratories in patients and donors in order to study the immune response from the epitope point of view and by applying the new tools such as MatchMaker.In fact, in the field of vital cardiothoracic transplantations, in clinical practice, transplant teams must often overcome the HLA barrier due to the long waiting times often required for cardiac and lung transplantations.

| CLINICAL IMPLICATIONS
Although graft survival rates have improved substantially, sensitization remains a major problem in solid organ transplantations. 20Comparing cardiothoracic and kidney findings in pediatric and adults recipients, we describe in Tables 1 and 2 some studies related to new immunological tests, such as epitope and HLA allele matching.In kidney transplantation, a significant emphasis has been placed on understanding the impact of HLA mismatches.It is worth noting that children constitute a relatively small fraction of patients enrolled in renal transplantation programs.Although there is some controversy regarding the importance of HLA matching in kidney allocation, it could be useful to simultaneously analyze the effect of HLA-ABC MM on rejection, graft survival, and sensitization in the same population. 21Poor survival in living donors is less common in pediatric patients than in adults, and the key to appreciating waiting times is knowing the matchability score of the patient and understanding the local kidney-offering scheme. 22ltimately, the decision regarding the type of transplant and the degree of acceptable HLA MM depends on each patient's circumstances and must take into consideration the added complications of end-stage kidney disease and dialysis.Early transplantation planning is crucial in pediatric patients, and HLA matching can improve long-term outcomes, reduce HLA sensitization, and enhance access to retransplantation.HLA matching is fundamental for reducing the "dialysis-free period" over the lifetime of pediatric patients.Matching at the HLA-A, -B, and -DR loci has been reported to increase graft survival compared with that of nonmatched organs. 23In addition, multiple nonimmunological factors have been demonstrated to significantly affect graft and recipient outcomes.On average, recipients with matched kidneys spent less time on dialysis, which is beneficial for recipient survival, independent of the matching status.Caucasian recipients were more likely to receive a matched kidney and therefore spent less time on dialysis than recipients from other racial groups. 24Regarding heart and lung transplantation, as few studies have been conducted and the results of some reports are contradictory, the correlation between mismatched epitope loads and worse posttransplant results remains obscure.However, posttransplant immune monitoring still poses challenges in interpreting dnDSA data in the absence of an acute condition. 25,26ools based on HLA epitope matching, such as HLA-EMMA, could be used to predict long-term risks and enable personalized therapeutic trials; however, the major limitations of many studies may be related to not using high-resolution typing, and not all HLA epitope mismatches are equally immunogenic.An alternative approach to overcome these limitations is the identification of HLA epitopes.The majority of the cited studies included adult recipients, but considering graft survival, more efforts by clinicians should focus on pediatric patients in the future.Furthermore, transplant physicians face the challenge of considering the limited number of donors and time constraints in the procurement process, making it difficult to prospectively match HLA alleles in heart allocation.However, this might change with the use of ex vivo heart and lung perfusion machines.
The immunogenicity of heart transplantation differs from that of lung transplantation, and patients should be evaluated separately. 27HLA allele findings are relatively common in lung and heart transplant candidates; therefore, it was impossible to exclude all cardiothoracic transplants.Nevertheless, these patients may benefit from increased immunosuppression, which reduces the risk of dnDSA production. 28The antibody-verified HLA-DQ epitope mismatch load can be used to guide personalized posttransplant immunosuppression.The adoption of molecular matching for DQA1 and DQB1 alleles could also help minimize dnDSA formation and potentially improve transplantation outcomes. 29,30Thus, HLA mismatch may be an important risk factor for graft failure in pHTR. 30Furthermore, it could be useful to have a complete and scheduled list of recommendations for pre-and post-transplantation monitoring. 31HLA matching at the epitope level offers an effective approach for identifying acceptable mismatches in hyperimmune patients and providing information about epitope loads. 32The ability of clinicians to identify recipients at risk of graft failure is HLA allele matching, particularly at HLA-B, was associated with superior long-term graft survival in pediatric heart transplant recipients.Further risk stratification in recipients with a higher degree of allelic mismatch may be achievable.

BTS 2014
The study indicated that regular HLA antibody screening within the first year after transplant was recommended exclusively for cardiothoracic transplant patients at an elevated risk level, exceeding the standard risk.
Sullivan PM et al J Heart Lung Transplant (2015) 34: 950-957 In a comprehensive analysis of a substantial pediatric heart recipient cohort (4851 individuals) with a higher frequency of HLA class I and class II mismatches, conducted using HLAMatchmaker, the researchers examined how genotypic and epitope-level HLA mismatching could aid in identifying recipients at a heightened risk.The findings revealed that such mismatching could serve as a valuable tool for identifying individuals who may require intensified post-transplant surveillance and management.
In a cohort of cardiothoracic transplant recipients, it was demonstrated that the highrisk epitope HLA-DQA105 + DQB102/DQB1*03:01 was significantly associated with a 4.7-fold increase in the likelihood of producing donor-specific antibodies (dnDSA).
Emilio Osorio-Jaramillo et al.Transplant International (2020) 33:1078-108 In a retrospective analysis of 1167 patients who underwent primary cardiac transplantation, an epitope MisMatch Algorithm was employed to calculate the number of amino acid differences in antibody-verified HLA eplets, known as amino acid mismatch load (AAMM), between the donor and recipient.The study revealed that patients with a higher HLA-DR AAMM load experienced inferior 1-year graft survival outcomes.In a retrospective study involving 77 patients, it was observed that a higher number of HLA Class II DPB eplet mismatches correlated with an increased risk of graft loss.While significant correlations were found between these mismatches and posttransplant outcomes such as CAV, acute cellular rejection, and acute mediated rejection, the primary causes of graft loss were predominantly cardiac in nature.These included primary graft dysfunction, rejection, and accelerated CAV.
potentially defined by the quantification of the degree of donor-recipient eplet mismatching.In the future, epitope-based matching using PIRCHE and HLA-EMMA may be a better tool for predicting dnDSA development. 15,16By using HLAMatchmaker, PIRCHE, or HLA-EMMA, some authors have found that heart and lung transplant patients with higher HLA-DR and/or HLA-DQ epitope mismatch loads showed an enhanced risk of poor transplant outcomes compared with other patients 20,21,32

| CONCLUSIONS
As we have extensively discussed throughout the paper, most studies have been performed in the field of kidney transplantation.However, it is essential that future studies, be conducted regarding pediatric cardiothoracic transplantation that assess whether HLA epitope-based matching tools such as HLA-EMMA, can be used to predict graft failure.Eplet matching in adult and pediatric cardiothoracic transplant recipients may serve as a helpful tool for identifying recipients with a higher risk of graft failure and might be useful for defining the most aggressive immunosuppressive protocol.

AUTHOR CONTRIBUTIONS
Giorgia Grutter designed the overview research and wrote paper.Antonio Giuseppe Bianculli, Paola Giustiniani, and Marco Andreani participated to describe the newer immunological tests.Antonio Amodeo, Estela Azeka, and Francesca Giovanna Iodice participated in the final revision of the overview.

T A B L E 1
Studies on the utility and efficacy of new immunological tests for heart and lung transplantation in pediatric and adult populations.Butts RJ et al.Circ Heart Fail.(2014) 7:605-11.
Cardoso B et al.J Heart Lung Transplant (2022) 41:1470-1477 37,38lty the studies byMangiola et al. and  Ellison et al.in the pediatric heart cohort, which explore the impact of various algorithms like Matchmaker, PIRCHE II, and EMMA on the development of de-novo DSA and AMR, are important additions that should be included to provide a more well-rounded perspective on Studies on the utility and efficacy of new immunological tests for kidney transplantation in pediatric and adults recipients., the study highlights the importance of considering various molecular mismatches when assessing transplant outcomes.This holistic approach may help identify high-risk patients and guide personalized immunosuppressive strategies.Mangiola and Ellison colleagues' study delves into the impact of Matchmaker and PIRCHE II algorithms on the development of de-novo DSA and AMR.The research adds depth to our understanding of the role of molecular mismatch in the context of heart transplantation and antibody-mediated immune responses.By focusing on specific algorithms, the study provides insights into the predictive power of these tools in identifying transplant recipients at risk of developing complications like DSA and AMR.The findings may have practical implications for clinicians, as they suggest that certain molecular mismatches could serve as early warning signs, allowing for proactive interventions to prevent or manage rejection episodes.37,38Insummary, both studies by Mangiola et al. and Ellison et al. contribute significantly to the field of transplant immunology by emphasizing the importance of molecular mismatch algorithms in predicting immunological responses and clinical outcomes in pHTR.Their work underscores the need for a multidimensional approach to assess the immunological risk and tailor interventions accordingly, potentially improving the long-term success of organ transplantation in this patient population.These findings may also have broader implications for the field of transplantation, suggesting avenues for further research and potential clinical applications in other organ transplant contexts.