Objectives: Heart transplant remains the definitive treatment for end-stage heart failure; however, long-term graft survival is often compromised by immune-mediated injury. Antibody-mediated rejection is primarily driven by donor-specific antibodies synthesized by Blymphocytes. Interleukin 40 has been associated with B-cell-mediated autoimmune pathologies, suggesting a role in donor-specific antibody development and posttransplant outcomes. We evaluated interleukin 40 levels, B-cell phenotypic changes, and rejection in heart transplant recipients.
Materials and Methods: In this clinical cohort analysis with ex vivo cell culture, we quantified serum interleukin 40 levels (enzyme-linked immunosorbent assay) in control and recipient groups (nonrejection, active rejection [pretreatment], and postdesensitization). Ex vivo assays involved incubating donor lymphocytes with autologous serum or serum positive for donor-specific antibodies, followed by culture supernatant assays. We analyzed B-cell markers (CD19, CD20, CD21, CD45, HLA DR) by flow cytometry at baseline, 6 hours, and 24 hours across serial dilutions of donor-specific antibodies.
Results: Serum interleukin 40 levels were significantly lower in stable recipients versus control and rejection patients (P < .001). Among rejection patients, interleukin 40 levels increased significantly posttreatment (P = .039). Ex vivo exposure to serum positive for donor-specific antibodies downregulated B-cell markers, particularly CD21, consistent with a transition toward a memory B-cell phenotype. Although HLA DR expression remained stable in control and autologous serum cultures, expression significantly decreased in the presence of donor-specific antibodies at 6 and 24 hours (ie, donor-specific antibodies induced significant B-cell phenotypic alterations).
Conclusions: Interleukin 40 appears closely linked to B-cell dynamics and antibody-mediated rejection pathophysiology in heart transplant. Suppressed interleukin 40 levels observed in stable recipients contrasted with posttreatment elevations in rejection patients, highlighting interleukin 40 as a potential biomarker for monitoring desensitization efficacy and graft immune status. Further multicenter prospective studies are warranted to evaluate interleukin 40 kinetics alongside established biomarkers for clinical utility in posttransplant surveillance.

Key words : Donor-specific antibodies, Heart transplantation

Introduction

Heart transplant is recognized as the definitive treatment modality for patients with end-stage heart failure or refractory arrhythmias who are nonresponsive to conventional pharmacological and nonpharmacological interventions.1 Despite significant therapeutic advancements involving novel agents and mechanical circulatory support, long-term morbidity and mortality rates remain substantial, as many patients inevitably progress to terminal heart failure. For the past 5 decades, the 1-year survival rate following heart transplant has reached approximately 90%.2,3 Consequently, elucidation of the cellular and molecular mechanisms underlying allograft failure is critical to improve long-term graft durability and patient longevity.
The immune system drives the continuum from initial graft injury to full-scale organ rejection through both cellular and antibody-mediated rejection (AMR) pathways. Antibody-mediated rejection is characterized by the synthesis of antibodies against HLA by B cells. These antibodies are predominantly donor-specific antibodies (DSA) directed against the donor's HLA molecules and thereby play a pivotal role in the rejection process.4 Hyperacute rejection occurs when preformed DSA against donor antigens are present at the time of transplant. This phenomenon is more prevalent in sensitized patients, particularly those with a history of multiple blood transfusions, prior organ transplants, or multiparous women. Pretransplant antibodies may include DSA targeting HLA class I or class II or non-HLA antibodies directed at other donor antigens.5 In heart transplant, the presence of DSA is a critical adverse factor, as it significantly impairs graft functionality and reduces survival.6,7 These anti-HLA antibodies, primarily of the immunoglobulin G isotype, induce tissue damage by activating the classic complement pathway. Accordingly, AMR represents a formidable challenge in solid-organ transplant; for heart recipients, the incidence of AMR is estimated to be 10% to 20% after transplant.4,8 Therefore, rigorous postoperative monitoring of anti-HLA antibodies is imperative.
The presence of DSA serves as a definitive marker of alloimmune activation and is strongly correlated with diminished graft survival, acute rejection episodes, and the development of cardiac allograft vasculopathy.9 Various clinical studies have evaluated specific monitoring protocols following heart transplant. Consequently, the International Society for Heart and Lung Transplantation recommends routine DSA screening to assess the recipient's alloimmune status. Although the detection of DSA does not independently constitute a diagnosis of AMR, it serves as a robust supportive parameter.9 Notably, AMR has been identified in 50% of heart transplant recipients who have experienced rejection at an average of 7 years after transplant.10,11
Cytokines are pivotal signaling molecules that facilitate communication between immune cells and dictate the trajectory of immune responses. The recently identified cytokine known as IL-40 has been shown to modulate B lymphocyte activity. Recent research has highlighted the role of IL-40 in autoimmune pathologies driven by B-cell-derived antibodies. Given that posttransplant AMR is mediated by DSA produced by B lymphocytes, and considering the role of IL-40 as a proinflammatory cytokine affecting these cells, investigating its relationship with this rejection mechanism represents an important scientific objective.
For this study, our main hypothesis was that desensitization therapy applied after a rejection attack and immunosuppression therapy in heart transplant patients without a diagnosis of rejection attack leads to changes in IL-40 levels and that a relationship exists between B-cell phenotype and IL-40 levels.

Materials and Methods

Cell culture experiments
Cell culture protocols were established based on standard crossmatch principles.12 Samples were obtained from the Tissue Typing and Transplantation Laboratory at the Baskent University Dr. Turgut Noyan Adana Research and Medical Center. The culture medium was prepared with 500 mL RPMI 1640 supplemented with L-glutamine (Gibco), 50 mL fetal bovine serum, and 12 mL penicillin/streptomycin (Gibco). The medium was preincubated in a 37 °C water bath for 30 minutes and used consistently across all experimental stages.
To observe the effect on B-cell surface molecule changes and IL-40 release, peripheral blood mononuclear cells obtained from a healthy volunteer patient with a known HLA tissue type were combined with a serum sample containing at least 1 DSA in a culture medium to create a rejection model. The time-dependent changes in B-cell surface markers were examined, and IL-40 levels were measured from the cell culture supernatants. The B lymphocytes were isolated using the Ficoll density gradient centrifugation method. The isolated cells were seeded into culture plates at a concentration of 106 cells/mL. Three distinct culture conditions were established, that is, medium supplemented with DSA-positive serum, medium with autologous serum (named as the DSA-negative group), and medium alone.
To assess the effect of antibody concentration, both the selected DSA-positive serum and autologous serum were tested at 1:1 and 1:2 dilutions. Each experimental set was incubated at 37 °C in a CO2 incubator for 6 hours and 24 hours. All experiments were performed in triplicate.
At the 6-hour and 24-hour marks, the contents of the flasks were collected into 15-mL Falcon tubes. After centrifugation at 3000 rpm for 5 minutes, the supernatant was separated into Eppendorf tubes and stored at -20 °C for subsequent IL-40 enzyme-linked immunosorbent assay (ELISA) analysis. The remaining cell pellets were resuspended in 650 μL of phosphate-buffered saline and gently vortexed. The B-lymphocyte surface molecule expression was then analyzed using flow cytometry.

Patient selection and study groups
Heart transplant recipients were categorized into groups as illustrated in Figure 1. The AMR group was defined based on the following criteria: clinical signs of rejection, presence of anti-HLA antibodies detected via panel reactive antibody screening, identification of at least 1 DSA (mean fluorescence intensity >1000) using the Luminex single-antigen bead assay, and biopsy-confirmed findings of the graft tissue supporting an AMR diagnosis. Serum IL-40 levels in the AMR group were measured in samples collected both before and after treatment to evaluate the correlation with therapeutic response.
The control group consisted of serum samples from healthy volunteer donors. To maintain study integrity, individuals with autoimmune diseases known to elevate IL-40 levels (eg, type 2 diabetes mellitus, rheumatoid arthritis, systemic lupus erythematosus, Sjögren syndrome) were excluded from all study cohorts.

Determination of interleukin 40 levels
The IL-40 concentrations in culture supernatants and serum samples from both patient and control groups were quantified using a commercial human IL-40 ELISA kit (Shanghai Sunred Biological Technology, product No. 201-12-5841) according to the manufacturer's instructions. Absorbance was measured using an ELISA plate reader (Enzo Life Sciences/Byonoy). Clinical samples were obtained with informed consent under the approval of the Institutional Review Board. The assay demonstrated an analytical range of 15 to 3000 ng/mL, with a detection sensitivity of 12.825 ng/mL. To ensure methodological rigor, the intra-assay and interassay coefficients of variation were confirmed to be <8% and <10%, respectively. All samples were processed in duplicate to minimize experimental error.

Flow cytometry analysis
Cell viability was assessed using 7-aminoactinomycin D. To define the lymphocyte population isolated from peripheral blood, we used a CD45 monoclonal antibody (Beckman Coulter Life Sciences). The B-cell surface markers were identified using CD19, CD20, CD21, and HLA-DR monoclonal antibodies (Beckman Coulter Life Sciences). Changes in the expression of these surface molecules in the presence of DSA were monitored via flow cytometry at baseline, as well as after 6 hours and 24 hours of incubation. These evaluations included analyses across serial DSA dilutions to determine the dose-dependent effects on B-cell phenotypes.

Statistical analyses
Flow cytometry data. We initially considered 1-way analysis of variance to evaluate differences between study groups and dependent variables; however, the assumption of homogeneity of variances was violated (Levene test, P < .05). Due to this violation and the relatively small sample size, we used a bootstrap-enhanced approach instead of the classic 1-way analysis of variance to obtain more robust and reliable estimates.
Enzyme-linked immunosorbent assay data. For statistical analyses of serum IL-40 levels, we used SPSS software (version 25.0; IBM). We assessed the normality of data distribution with the Shapiro-Wilk test. Because data did not follow a normal distribution, we used the Kruskal-Wallis test for intergroup comparisons of quantitative variables, followed by the Dunn-Bonferroni post hoc test for pairwise comparisons. We analyzed differences between dependent (paired) measurements (eg, pretreatment and posttreatment) with the Wilcoxon signed-rank test. P < .05 was considered statistically significant.

Results

Flow cytometry findings
The baseline (0-hour) cell viability was recorded to be 97.94%. Across all experimental conditions and flasks, cell viability rates remained consistently above 95% and thereby ensured the reliability of the subsequent surface marker analyses.
Analysis of the 6-hour flow cytometry data revealed that the percentage of CD45/CD20/CD21-positive lymphocytes in the DSA-positive group was significantly lower versus baseline, the 6-hour control group, the DSA-negative group, and the DSA-negative 1:2 dilution group (P < .05). However, this lymphocyte percentage was significantly higher in the 6-hour DSA-positive group versus the 24-hour DSA-positive group and the 24-hour DSA-positive 1:2 dilution group (P < .05) (Figure 2).
In the 24-hour DSA-positive group, the percentage of CD45/CD20/CD21-positive lymphocytes was significantly lower versus all other experimental conditions. This significant reduction (P < .05) was observed versus baseline, the 6-hour control group, the 6-hour DSA-positive group, the 6-hour DSA-positive 1:2 dilution group, the 24-hour control group, the 24-hour DSA-positive 1:2 dilution group, and all DSA-negative groups (including 1:2 dilutions).
Flow cytometry analysis revealed a significant decrease in CD20/HLA-DR-positive lymphocytes within the DSA-positive group at 6 hours versus all control and DSA-negative groups (P < .05) (Figure 3). This reduction persisted at 24 hours in the DSA-positive group, which indicated a time-dependent, antibody-mediated downregulation of HLA-DR expression on B cells (P < .05).

Interleukin 40 levels in cell culture supernatants
Comparative analysis via ELISA of IL-40 concentrations in cell culture supernatants revealed no significant differences. The levels remained stable regardless of the incubation period (6 hours vs 24 hours) or the presence of DSA, which suggested that acute in vitro exposure to DSA does not immediately alter IL-40 secretion in this model.

Serum interleukin 40 levels across clinical groups
Statistical analysis of clinical samples demonstrated significant variations in serum IL-40 concentrations (as measured by ELISA) across the study cohorts. In comparison of groups, serum IL-40 levels in heart transplant recipients without rejection were significantly lower versus both the healthy control group and patients diagnosed with AMR (P < .001) (Figure 4, Figure 5). Effect of treatment analysis showed that, in the AMR group, serum IL-40 levels showed a significant increase following desensitization therapy (P = .039) (Figure 6). In rejection versus stability analysis, overall, patients with active AMR exhibited significantly higher serum IL-40 concentrations versus stable, nonrejection transplant recipients (P < .001) (Figure 4 , Figure 5).
Statistical analysis revealed that serum IL-40 levels in heart transplant recipients without rejection episodes were significantly lower versus those of both the healthy control group and patients in the rejection group following treatment (P < .001). Notably, a significant elevation in serum IL-40 concentrations was observed in the rejection group following desensitization therapy (P = .039).

Discussion

The primary finding of our study is that IL-40 levels are significantly elevated in heart transplant recipients who develop rejection versus nonrejection recipients and healthy control patients. Prior studies have shown that IL-40 correlates with active inflammation and disease severity (eg, Disease Activity Score 28 and the Systemic Lupus Erythematosus Disease Activity Index) in autoimmune diseases such as rheumatoid arthritis, systemic lupus erythematosus, and primary Sjögren syndrome.13-15 Elevated IL-40 during rejection is consistent with these previously published reports and likely reflects the intense inflammatory response and tissue injury associated with rejection. Because B cells are the main source of IL-40 in primary Sjögren syndrome and because IL-40 can induce interferon and IL-17 release from T cells,14 a similar B-cell/T-cell axis may drive the immune processes underlying heart transplant rejection.
Notably, transplant recipients on standard immunosuppression therapy who did not experience rejection had lower IL-40 levels versus healthy control patients. This suggests that corticosteroids and other immunosuppressants strongly suppress IL-40 production, indicating that IL-40 may be a sensitive marker of therapeutic response.
In vitro data showed that DSA alone did not significantly increase IL-40 levels in culture supernatants over time. This implies that the IL-40 increase observed in vivo is not directly triggered by the initial antibody-antigen interaction but rather results from the ensuing complex inflammatory cascade and cellular infiltration.
B lymphocytes have been shown to be central to adaptive immunity and differentiate into antibody-secreting plasma cells. Although CD19 is expressed throughout B-cell development, it is lost at the terminal plasma cell stage and is critical for B-cell receptor (BCR) signaling together with CD21. It has been established that CD21 (CR2) also functions as a complement receptor that binds opsonized immune complexes to augment signaling.16 Although CR2/BCR coligation enhances B-cell responses in mice, it can inhibit activation markers, cytokine production, and proliferation in human B cells.17,18 Our ex vivo data showed that DSA-positive serum reduced B-cell surface markers, particularly CD21, consistent with a shift toward a memory B-cell phenotype. We observed decreases in CD19/CD20/CD21-positive populations in all cultures relative to baseline, with a more pronounced reduction in the presence of DSA, mirroring the trend in total CD45-positive lymphocytes.
The class II molecule HLA-DR is essential for antigen presentation and is highly expressed on B cells and activated endothelial cells. Malignant cells downregulate HLA-DR to evade immune detection,19-21 and reduced monocytic HLA-DR expression is a marker of increased mortality in sepsis.22 In our study, HLA-DR expression on B cells remained stable in control and autologous serum cultures but decreased significantly at 6 hours and 24 hours only in the presence of DSA. This downregulation partially recovered at lower DSA concentrations (1:2 dilution), indicating that DSA exposure alters B-cell phenotype in a concentration-dependent manner. The relative decrease in CD45-positive lymphocytes in high-DSA conditions likely reflects antibody-mediated cytotoxicity affecting cell survival.

Conclusions

Our data indicated that IL-40 is associated with B-cell responses and the dynamics of AMR after heart transplant. The low IL-40 levels in clinically stable, nonrejection recipients and the posttreatment IL-40 increase in rejection patients support IL-40 as a potential biomarker for monitoring desensitization therapy and graft immune status. Multicenter studies should evaluate IL-40 kinetics alongside established biomarkers and clinical outcomes to determine its usefulness in posttransplant surveillance.

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