Heart transplant is the best treatment option for end-stage heart failure. The major goals in solid-organ transplant are organ survivability and functionality. The effects of anti-HLA antibodies and cytokines are important for immune response. Cytokine gene polymorphisms are also effective during cytokine release. Here, we report a heart transplant recipient who was diagnosed with antibody-mediated rejection posttransplant and had an antibody response resistant to desensitization therapy. After transplant, panel reactive antibody screening and identification class I and II tests and Luminex single antigen class I and II tests were performed. Desensitization treatment included intravenous immunoglobulin, plasmapheresis, rituximab, and bortezomib. Because of these reasons, cytokine gene polymorphism tests (consistent with low, intermediate, and high expression levels for tumor necrosis factor α, transforming growth factor Β1, interleukin 6 and 10, and interferon γ) were conducted. We found polymorphic regions compatible with the high-release, proinflammatory action of tumor necrosis factor α and interleukin 6, which induced inflammation and B-cell activation, and polymorphic regions compatible with the intermediate release of the potent immunosuppressive effects of transforming growth factor Β1 and interleukin 10, suggesting that the patient may not be able to effectively suppress the activation of the immune system. The influence of cytokine gene polymorphism on the formation of a resistant antibody response in a patient, despite desensitization, contributed to the proinflammatory response in which these cytokines were involved.
Key words : Inflammatory response, Luminex single antigen, Panel reactive antibody
Heart transplant (HT) has become an accepted therapy in patients with end-stage heart failure. Despite improved immunosuppression regimens, rejection remains the most common cause of death in the first 5 years after HT.1
Presently, incompatibility of human leukocyte antigens (HLA) between the donor and the recipient remains a primary obstacle to modern solid-organ transplant procedures, including HT. HLA molecules are classified as class I (HLA-A, -B, -C) and class II (HLA-DR, -DQ, -DP), which are located in chromosome 6.2 The expression of HLA on the cell surface makes it possible to distinguish self from nonself. Although HLA class I molecules are constitutively expressed on all nucleated cells, the class II HLA molecules are expressed on human antigen-presenting cells, such as dendritic cells, B cells, and macrophages, as well as on activated T cells.3 The expression of HLA class I or II on endothelial cells of the allograft vessels may explain the rejection that occurs in the presence of donor-specific antibodies of class I or class II. Allograft damage by antibodies occurs mainly due to the activation of the complement system.The binding of antibodies to the HLA antigen triggers C1q and the complement cascade. Antibodies against major histocompatibility complex can cause direct damage to capillary endothelium and indirect damage through the recruitment of inflammatory cells with Fc receptors. As a result, cellular inflammation, thrombosis, and hemorrhage can develop, leading to dysfunction of the cardiac allograft.4,5
Both cellular and humoral immune-mediated processes that can damage the allograft are primarily directed against HLA. Antibodies against HLA can be found in patients prior to transplant after exposure to foreign HLA through pregnancy, blood transfusion, and previous transplant.1 Donor-specific anti-HLA antibodies develop in up to 50% of patients following solid-organ transplant. It is known that donor-specific anti-HLA antibodies are harmful following HT, leading to increased cellular rejection, antibody-mediated rejection, cardiac allograft vasculopathy, and mortality.6 Allosensitization levels in patients are assessed using panel reactive antibody (PRA) measurement, which also provides information on the presence of circulating antibodies. Results are expressed as a percentage of PRA and are based on the percentage of positive responses in a panel of cells from 60 to 100 individuals.7 Heart transplant patients with more than 10% of PRA are at risk for earlier and more severe rejection as well as worse survival. The potential targets for desensitization include B cells, plasma cells, antibodies, and the complement system.8,9
Sensitization refers to the condition of the recipient before transplant in which HLA antibodies are circulating in the blood or to the state in which HLA antibodies are produced immediately after transplant because of immune memory reactions. Risk factors for sensitization are blood transfusion, pregnancy, use of homografts in previous cardiac surgery, prior transplant, and use of ventricular assist devices pretransplant.10
Cytokines are essential mediators that have a regulatory role in innate and adaptive immunity. Differences in the release of cytokines that originate from polymorphic structures are also effective. Proinflammatory cytokines include tumor necrosis factor alpha (TNF-a) and interleukin 6 (IL-6), potent immunosuppressive cytokines include transforming growth factor beta-1 (TGF-b1) and interleukin 10 (IL-10), and proinflammatory cytokines include interferon-gamma (IFN-g), which are known as macrophage-stimulating cytokines.
Several clinical and genetic factors affect the pharmacokinetics of desensitizing drugs.11 Genetic polymorphisms of IL-3 and CTLA4 have been shown to influence the dose requirements of tacrolimus (FK506). Tacrolimus, which was approved in 1994 as an immunosuppressive therapy for solid-organ transplant, is a calcineurin inhibitor.12 Single nucleotide polymorphisms in IL-1b, IL-2, IL-6, IL-6 receptor, IL-10, TGF-b, and TNF have been shown to influence increases or decreases in protein production and to affect the function of these pro- and anti-inflammatory mediators in vitro.13 Thus, the genetic polymorphisms affecting innate immunity may increase risk of organ rejection. However, genetic polymorphisms have not yet been extensively studied in solid-organ transplant, particularly with regard to HT.
A 39-year-old male patient presented for HT at the Ankara Baskent University Hospital in 2011. Immunological follow-up posttransplant was conducted at the Baskent University Adana Dr. Turgut Noyan Research and Medical Center Tissue Typing Laboratory since 2016. Panel reactive antibody tests for screening and identification of class I and II and Luminex single antigen (LSA) (One Lambda, Inc) tests for class I and II were performed with the Luminex method according to the manufacturer’s protocol with the patient’s posttransplant serum sample. The cutoff for a positive reaction was set at a mean fluorescence intensity (MFI) ≥1000.
In 2016 after transplant, the patient was diagnosed with antibody-mediated rejection. The patient then received desensitization treatment containing intravenous immunoglobulin, plasmapheresis, rituximab, and bortezomib. Results of PRA screening, identification, and LSA tests performed during the treatment process showed that the patient was resistant to treatment.
Cytokine gene polymorphism studies were performed to investigate the cause of treatment resistance. Isolated DNA samples were obtained with the use of a commercially available cytokine gene polymorphism kit (Cytokine Genotyping Tray, One Lambda, Inc), which allowed the determination of compatible polymorphism sites with low, intermediate, and high cytokine release for TGF-β1, TNF-α, IL-6, IL-10, and IFN-γ used during the polymerase chain reaction-based sequence-specific priming procedure.
After the last desensitization treatment, LSA class I of 6% (MFI: 6.473-1.145) and LSA class II of 26% (MFI: 18.564-1.383) were found. Table 1 displays the desensitization treatments given to the patient and the results of PRA screening class I and II and LSA class I and II tests. Table 2 presents cytokine gene polymorphism results.
Polymorphic regions compatible with the high-release proinflammatory action of TNF-a and IL-6 can induce inflammation and B-cell activation. Our finding of polymorphic regions compatible with the intermediate release of the potent immunosuppressive effects of TGF-b1 and IL-10 suggested that the patient was not able to effectively suppress activation of the immune system. The influence of cytokine gene polymorphisms on the formation of a resistant antibody response in a patient, despite desensitization, may also contribute to the proinflammatory response in which these cytokines are involved. According to the meta-analysis results fromYongcharoen and colleagues in 2013, individuals carrying the minor A allele of the TNF-α -308 polymorphism may be at greater risk of developing graft rejection, whereas individuals carrying the minor C alleles for the TGF-b1 -C10 and TGF-b1 -C25 polymorphisms may be less prone to developing graft rejection after HT.14 These results were in agreement with our findings.
Presently, sensitization remains a barrier for HT recipients and for potential HT donors. Despite reasonable justification, data on current therapies on desensitization and the outcomes are limited. Future research on desensitization therapy and its outcomes should therefore continue.
Volume : 20
Issue : 3
Pages : 113 - 116
DOI : 10.6002/ect.MESOT2021.P49
From the 1Department of Immunology, Baskent University, Ankara, Turkey; the 2Tissue Typing and Transplantation Laboratory, Baskent University, Adana, Turkey; the 3Department of Medical Biology, Baskent University, Ankara, Turkey; and the 4Cardiovascular Surgery Department, Baskent University, Ankara, Turkey
Acknowledgements: The authors have not received any funding or grants in support of the presented research or for the preparation of this work and have no declarations of potential conflicts of interest.
Corresponding author: Bilkay Bastürk, Baskent University Faculty of Medicine, Department of Immunology, Dr. Turgut Noyan Reseach and Medical Center, Adana, Turkey
Phone: +90 322 327 2727
Table 1. Desensitization Treatment, Panel Reactive Antibody Screening, and Luminex Single Antigen Results
Table 2. Cytokine Gene Polymorphism Results