Key words : Histocompatibility, Immunologic desensitization, MHC class II, Organ transplantation

Introduction

Kidney transplantation is the most common type of organ transplant from living donors and donors after brain death. In contrast, heart transplants are rarely performed, and the number of donors is limited. As life expectancy increases worldwide and with the persistence of risk factors such as arterial hyper-tension and coronary heart disease, the prevalence of heart failure is projected to increase by 46% from 2012 to 2030 and therefore remain the leading cause of death.¹ According to data published by the Republic of Türkiye Ministry of Health, as of November 2023, 24 449 individuals were waiting for kidney transplant and 1422 were waiting for heart transplant.² Despite strides in the field over the past 4.5 decades, substantial challenges persist that hinder the widespread application and success of heart transplant.

Although the prevalence of patients with end-stage heart failure is rising, the consistent availability of donor organs remains a constraining factor. Damage to transplanted tissue is among the most salient issues in the domain of transplantation, with the immune system of the recipient often recognizing transplanted cellular elements as foreign and eliciting an immune response.³ Human leukocyte antigens (HLAs) have been shown to play a pivotal role in this process, and the compatibility between recipient and donor HLAs directly influences the survival and functionality of the transplanted tissue or organ (graft tissue).

HLA molecules represent a family of highly polymorphic antigens that are encoded by the major histocompatibility complex (MHC). The primary function of these molecules is to present short peptides, originating from both intracellular and extracellular sources, to T lymphocytes. All nucleated cells express HLA class I on their surface. In contrast, the expression of HLA class II is exclusive to antigen-presenting cells.⁴ HLA molecules play a pivotal role in the human immune system, facilitating the recognition of “non-self” antigens and the subsequent activation of defense mechanisms.⁵ However, their polymorphism poses a major challenge in the field of transplantation, as it can compromise graft survival.

HLA sensitization, defined as the development of antibodies directed against foreign HLAs, is a common occurrence in transplant recipients. These antibodies, known as donor-specific antibodies (DSAs), can be directed against donor antigens, thereby disrupting the immune response to foreign antigens. Of note, DSAs are not exclusively anti-HLA antibodies, and evidence about antibodies directed against other donor antigens has also recently emerged.⁶ Donor-specific antibodies have been observed to develop before transplant (preformed DSAs) or appear de novo afterward (de novo DSAs).⁷

Pretransplant DSAs and de novo DSAs have been observed in 3% to 11% and in 10% to 30% of heart transplant patients, respectively. Donor-specific antibodies have been identified as a critical com-ponent of antibody-mediated rejection (AMR), a major cause of graft loss.⁸ Evidence of AMR is present in 50% of heart transplant recipients, who develop rejection an average of 7 years after transplant.⁹ Recently, rates of early allograft rejection after heart transplant have decreased. Research has focused on the effects of AMR on graft failure, which is a significant contributor to mortality and accounts for 35% to 40% of mortalities before year 5 posttransplant.¹⁰

The transmission of HLA molecules is a codo-minant process, with contributions from both the mother and father. These molecules determine the individual’s tissue group, with 6 alleles from the mother (HLA-A, -B, -C, HLA-DR, -DQ, -DP; haplotype) for HLA class I and HLA-DR, HLA-DP, and HLA-DQ for class II. In total, 12 different haplotypes are transmitted, contributing to the genetic compo-sition of the individual. The presence of these 12 HLA alleles, which can be examined for compatibility, is a critical factor in solid-organ transplants. In these transplants, the HLA-A, HLA-B, and HLA-C for HLA class I and HLA-DR, HLA-DP, and HLA-DQ for class II molecules play pivotal roles. A comprehensive understanding of HLA structures is paramount for evaluating the T-cell-mediated response and for assessing HLA antibodies produced by B cells (plasma cells) against the HLA structure before and after transplant (de novo).^11,12

Genes belonging to the HLA class II category consist of 2 chains, designated as α and β, which are both formed from 2 domains: α1, α2, β1, and β2. The transmembrane domains, α2 and β2, serve to anchor the HLA class II molecule to the cell membrane, whereas the peptide-binding groove is formed by the heterodimer α1 and β1.¹³ Of these genes, HLA-DPB1, HLA-DQA1, HLA-DQB1, and HLA-DRB1, which have been most extensively characterized, encode proteins that display processed antigenic peptides for recognition by T helper cells. Therefore, binding of peptides to the HLA molecules occurs through the interaction between the amino acid residues of the MHC groove and those of the peptides. These pockets, defined by their unique capacity to bind a particular side chain of a peptide, exhibit variations in size, shape, and function.¹⁴

The HLA tissue compatibility between the recipient and the donor and the presence of antibodies against the HLA structure in the recipient’s serum play essential roles. In this study, we investigated the response of anti-HLA antibodies to desensitization treatment and evaluated their clinical significance associated with AMR.

Materials and Methods

We studied 18 heart and renal transplant patients who received desensitization treatment with the diagnosis of AMR. We evaluated 60 samples from the 18 patients who were followed in our center for at least 4 years and had information available before and after desensitization (plasmapheresis, intrave-nous immu-noglobulin [IVIg], and rituximab). The Tissue Typing and Transplantation Laboratory of Başkent University Adana Dr. Turgut Noyan Research and Medical Center performed the pret-ransplant and posttransplant anti-HLA antibody tests. Serum samples of patients were tested with the panel reactive antibody screening class I and II test (One Lambda, Inc) and the single antigen class I and/or II Luminex method (One Lambda, Inc). We considered mean fluorescence intensity (MFI) ≥1000 as positive. To determine the presence of DSAs, donor HLA tissues were studied with the high-resolution methods of sequence-based typing and next-generation sequencing.

Statistical analyses
We used Statistical Package of Social Science version 25.0 (IBM Corp) for statistical analyses. We used the Shapiro-Wilk test to examine the suitability of variables for normal distribution. We summarized descriptive statistics as number and percentage for categorical variables and as mean and SD and median (minimum-maximum) for quantitative variables. We analyzed differences between de-pendent groups for categorical variables with the McNeimar test. We evaluated differences between 2 groups for conti-nuous variables with the Mann-Whitney U test because the measurements could not be matched. Significance level was determined as α = .05.

Results

The presence of anti-HLA antibodies before transplant was found to be approximately 4% in our study patients. Among patients included in the evaluation, anti-HLA antibody (MFI >10 000) was detected before transplant in 2 recipient candidates, one of whom was class I and the other was class II. In studies conducted during the subsequent monitoring of patients, MFI values of anti-HLA antibodies decreased after treatment. Furthermore, some anti-bodies were detected below 1000 MFI, which is accepted as the limit value. The patient, who was found to have been highly sensitized before transplant and was at the top of the waiting list, received desensitization treatment. The transplant was performed within 1 month of completion of her treatment. The recipient had antibodies against donor class I HLA characteristics (HLA-A*02:01), which was present pretransplant. The patient’s biopsy result 5 months after transplant was diagnosed as AMR and found to be compatible with clinical findings. The desensitization treatment (plasmapheresis and IVIg) was repeated, and DSA level was determined as MFI >6500. During follow-up, the patient’s clinical condition was determined to be stable; after 2 years, DSA MFI was <1000.

Our study results showed that class II anti-HLA antibodies present before desensitization persisted in the long-term follow-up after treatment. After desensitization treatment, anti-HLA antibodies, particularly those directed against class II HLA-DQ (DQB and DQA), were more resistant to desensitization treatment to reduce antibodies that developed posttransplant than those that targeted class I HLA and class II HLA-DR. Antibodies with MFI values of 10 000 and above did not decrease at the expected level after desensitization and even increased in some patients, whereas those with MFI <10 000 decreased. Patient’s sex and antibodies details are given in Table 1. In patients who received a second desensitization treatment, a time-dependent (approximately in 2 years) decrease in resistant antibodies was observed (Table 2).

Discussion

Immunological studies have contributed to the development of the concept of systemic immunity, which posits that immunity is not the effect of just 1 cell or a single factor but rather a systematic action involving a complex network of various cells and factors.¹⁵ HLA molecules play an important role in antigen presentation and ensure the initiation of an effective immune response. HLA class I molecules (HLA-A, HLA-B, HLA-C) are present in all nucleated cells, and HLA class II molecules (HLA-DR, HLA-DQ, HLA-DP) are expressed on antigen presenting cells.¹⁶

The primary function of MHC class II molecules located on the surface of dendritic cells is to present antigens to CD4+ T cells, thereby serving as the most critical protein in the antigen presentation process. The HLA-DQ structure exhibits distinct modi-fications and expressions compared with other HLA structures. The HLA-DQ protein is regulated by the HLA-DQA1 and HLA-DQB1 genes. Both the α and β chains are polymorphic at the HLA-DQ locus. As a result, the endoplasmic reticulum contains 2 α chains and 2 β chains, and 4 different HLA-DQ α/β com-bination pairs can be expressed on the cell surface. This phenomenon is referred to as cis- or trans-pairing of the chains.¹⁷

Several studies have demonstrated a correlation between HLA-DR and HLA-DQ gene polymorphisms and an increased propensity for the development of autoimmune, infectious, and malignant diseases.^18-21 The role of HLA-DQ in celiac disease has been especially well clarified. After the intake of gluten-containing foods, enzymes can modify food proteins to induce an autoimmune response in a restricted manner, causing intestinal epithelial inflammation.²² This disease is closely associated with HLA-DQ2 and HLA-DQ8; gluten-modified gliadin peptides bind to HLA-DQ2 and HLA-DQ8 and then activate autoimmune responses in T cells through antigen presentation.²³ A study on DSAs after kidney transplant demonstrated that endothelial cells stimu-lated with HLA-DR and HLA-DQ antibodies exhi-bited increased proinflammatory cytokine secretion and a decrease in regulatory T cell expansion, and HLA-DQ antibodies strongly promoted proinflam-matory responses.²⁴ Increased frequency and levels of de novo DSAs associated with AMR have suggested that HLA-DQ has greater immunogenicity.²⁵ Our study results showed that HLA-DQ antibodies were detected as a larger group compared with other loci, which is consistent with the results of this study. In our study, we evaluated anti-HLA antibody and changes in patients who underwent heart and renal transplant and received desensitization before and after treatment. When the results were evaluated, HLA-DQ antibodies, especially those with MFI >10 000, showed no response to treatment, with significant differences in treatment response from other antibodies.

HLA mismatch can be recognized by macrop-hages, B cells, and dendritic cells. B cells can recognize HLA mismatch, engulf the peptide, process it, and present the identifying epitopes of the donor’s HLA mismatch to specific CD4 lymphocytes with MHC II restriction.²⁵ The interaction between specific B and T cells leads to the preparation of specific naive B cells with germinal center formation in regional lymph nodes and activation and induction of anti-HLA antibody development.

Some of the antigen-recognizing B cells differen-tiate into memory B cells or short-lived plasmablasts and long-lived plasma cells. Memory B cells are derived from B cells that are less mutated and have lower affinity receptors than cells that will develop into plasma cells. The repertoire of plasma cells and memory B cells is not the same. The repertoire of plasma cells and the antibodies they produce are up to 100 times more restricted than the repertoire of memory B cells.^26,27 These differences between memory B cells and plasma cells suggest that treat-ment aimed at blocking DSA production may not stop the production of memory B cells.

The heterogeneity of the anti-HLA antibody repertoire, which includes antibodies that bind to specific features on HLA molecules, are therefore highly donor specific. Cross-reactive alloantibodies may be donor reactive but not donor specific, and some may bind to more than one HLA molecule. Consequently, the breadth of circulating antibodies with HLA reactivity may not be a direct output of the plasma cell repertoire. Sensitized individuals have intact long-lived plasma cells that continuously secrete anti-HLA antibodies and resting memory B cells that mount a rapid anamnestic antibody response and are capable of secreting large amounts of antibody when re-exposed to the same antigen.

The role of HLA mismatch and antibodies to mismatched epitopes in the mechanism of rejection is important. Treatments, including desensitization protocols, are used to prevent the harmful effects of existing or subsequently developed antibodies. In some patients, desensitization protocols have been shown to be unsuccessful in reducing anti-HLA antibody levels. First-line desensitization agents that are commonly used include plasmapheresis and IVIg. Plasmapheresis has been observed to remove antibodies along with circulating plasma proteins. However, it is important to note that the effectiveness of this approach is limited.^28-30 High-dose IVIg is utilized for the purpose of desensitization.³¹ The mechanism of action of high-dose IVIg involves the inhibition of FcRn, thereby curtailing the rate of antibody recycling and reducing the antibody half-life. In addition, this treatment can neutralize the effector functions of pathogenic antibodies, inhibit complement activation, and activate immune inhi-bitory signaling via FcƴRIIb receptors on immune cells.³² Intravenous immunoglobulin can also help prevent opportunistic infections in patients under-going desensitization.³³ However, activation of memory B cells and pathogenic antibody-producing donor reactive plasma cells cannot be completely prevented by the combination of plasmapheresis and IVIg.

One hypothesis to explain this is that there is no effect on plasma cells, especially memory plasma cells. With rituximab, this may seem obvious because it is an anti-CD20 monoclonal antibody, and this CD20 marker is no longer expressed by plasma cells. Rituximab, as well as IVIg and antithymocyte globulin, do not induce plasma cell apoptosis or block antibody production.³⁴ In 2 patients in our study, class I DSA positivity (MFI >10 000) decreased with repeated treatments within 2 years (to MFI <2000). However, desensitization protocols do not work the same in every patient and do not ensure effective removal of all DSAs. The long-term outcomes of patients with persistent DSAs are much worse compared with nonsensitized patients.³⁵

In our study, we found that class II antibodies developed more in patients awaiting transplant and in transplant recipients compared with other loci.We showed that class II HLA-DQ antibodies are especially at the forefront and respond to desensi-tization therapy more resistant than class I antibodies. However, in heart transplants, decisions on transplant are made primarily with HLA-A, HLA-B, and HLA-DR tissue compatibility. Our study results showed that the identification of the class II HLA-DQ tissue group and anti-HLA antibodies against it played an important role in reducing the need for desensiti-zation therapy and maintaining graft tissue functionality.

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