EXPERIMENTAL AND CLINICAL TRANSPLANTATION

Objectives: Early prediction of graft rejection is critical for ensuring long-term graft survival and improving patient prognosis following heart transplant. Here, we investigated the association between interleukin 40, a proinflammatory cytokine, and key components of the innate (neutrophils) and adaptive (lymphocytes) immune systems. Furthermore, we evaluated the predictive value of the change in lymphocyte-to-neutrophil ratio for acute and chronic rejection and overall survival after heart transplant.
Materials and Methods: In this 2-center study, peripheral blood lymphocyte and neutrophil counts were monitored in 121 heart transplant recipients at multiple time points: pretransplant, early postoperatively, and 1, 3, 6, 9, 12, 18, and 24 months posttransplant. Change in the lymphocyte-to-neutrophil ratio was calculated for each patient. Serum interleukin 40 levels were measured in patients with and without a confirmed diagnosis of rejection. We included a control group of 28 healthy volunteers. We performed statistical analyses to determine the relationship between change in lymphocyte-to-neutrophil ratio and acute rejection, chronic rejection, and patient survival.
Results: Interleukin 40 levels and neutrophil percen-tages were significantly higher in the rejection group compared with the nonrejection and healthy control groups (P < .001). Conversely, lymphocyte percentage was significantly lower in the rejection group. Lymphocyte-to-neutrophil ratio was significantly different among study groups (P < .001). A negative correlation was identified between interleukin 40 levels and both lymphocyte percentage and lymphocyte-to-neutrophil ratio. Change in lymphocyte-to-neutrophil ratio demonstrated significant discriminatory power in predicting acute rejection (area under the curve = 0.631; P = .040), with lower rates of acute rejection observed in patients with a change in lymphocyte-to-neutrophil ratio of ≤0.412. Correlation coefficients among hematological indices were robust and consistent with clinical findings.
Conclusions: The significant correlation shown between interleukin 40 levels and lymphocyte-to-neutrophil ratio and rejection episodes in heart transplant recipients represents promising noninvasive biomar-kers for predicting posttransplant rejection.

Key words : Acute rejection, Biomarker, Cardiac allograft rejection, Inflammation, Patient survival

Despite important advancements in immunosup-pressive therapies, cardiac allograft rejection remains the leading cause of graft loss and mortality after heart transplant.¹ Current immunosuppressive protocols primarily aim to selectively inhibit lymphocyte activation and proliferation to facilitate allograft acceptance.² However, emerging evidence suggests that these treatments may not effectively reduce HLA-DQ antibodies, which can persist posttransplant.^3,4 Paradoxically, certain therapies may even trigger rejection by promoting the development of alloreactive B and T lymphocytes.⁵ Notably, Iida and colleagues demonstrated that transient lymphopenia can initiate cardiac allograft rejection.⁶ Consequently, achieving an optimal balance of lymphocyte inhibition is critical, not only to prevent the accumulation of donor-reactive cell types but also to avoid lymphopenia-induced rejection.⁶
In addition to the adaptive immune response, neutrophils play a pivotal role in the pathogenesis of cardiac allograft rejection. Research has shown that inhibiting neutrophil infiltration can significantly mitigate rejection and enhance graft survival.⁷ Given the combined importance of lymphocytes and neutrophils, hematological indices have gained clinical interest. Specifically, the change in the lymphocyte-to-neutrophil ratio (∆LNR) has emerged as a significant marker for acute rejection after heart transplant, offering predictive value even in patients who are immunologically high risk.⁸
The inflammatory milieu, driven by specific immune cell populations, is central to the develop-ment of rejection episodes. Interleukin 40 (IL-40), a recently identified B-cell-associated cytokine, has been implicated in these complex immune response mechanisms. First described in 2017 as a 27-kDa protein encoded by the C17orf99 gene, IL-40 is clas-sified as an orphan cytokine due to its lack of structural homology with recognized cytokine families.⁹
Current clinical management of posttransplant rejection relies on a multifaceted approach, including close clinical observation, therapeutic drug moni-toring of immunosuppressants, immunological screening for de novo anti-HLA antibodies, serial echocardiography, and gold standard endomy-ocardial biopsies. Despite their utility, an urgent need exists for noninvasive biomarkers to detect subclinical rejection. Both ∆LNR and IL-40 levels represent promising, less invasive diagnostic tools that could potentially reduce the frequency of invasive procedures and their asso-ciated complications.

Materials and Methods

Study population and design
This retrospective study included patients who underwent heart transplant at Başkent University Faculty of Medicine, Ankara Hospital, and Adana Dr. Turgut Noyan Training and Research Hospital (Türkiye) between February 2005 and January 2025. Exclusion criteria included retransplant or the presence of an active infection at the time of assessment. Although 121 patients were initially screened, 24 patients were excluded due to missing 3-month LNR values or incomplete follow-up data. Consequently, 97 patients with comprehensive medical records were included in the final analysis.
The study protocol was approved by the Research Ethics Committee of Başkent University Faculty of Medicine and Health Sciences (Project no: KA20/309 and KA24/117). Informed consent was obtained from all participants. The study was conducted in strict accordance with the ethical principles of the Declaration of Helsinki.

Data collection and rejection monitoring
We derived LNRs from automated complete blood counts obtained at multiple time points: pretransplant, the immediate postoperative period, and at 1, 3, 6, 9, 12, 18, and 24 months posttransplant.
We compared LNR values and ΔLNR before and after rejection treatment, correlating with follow-up endomyocardial biopsy (EMB) results. Furthermore, we examined the association between ΔLNR and chronic rejection. We used receiver operating charac-teristic (ROC) curve analysis to determine optimal threshold values for ΔLNR in predicting acute and chronic rejection and overall patient survival.

Immunosuppressive protocol
All patients received a standardized immunosup-pressive regimen. Intraoperatively, methylpred-nisolone was administered during cardiopulmonary bypass. Presurgical induction agents for adult patients included tacrolimus (1 mg), mycophenolate mofetil (MMF) (1 g), and intravenous methyl-prednisolone (500 mg). Postoperatively, a triple maintenance regimen was initiated that included tacrolimus (0.05-0.1 mg/kg/day, twice per day), MMF (2-3 g/day, twice per day), and methylpred-nisolone (1000 mg/day for the first 3 days). From postoperative day 4, prednisolone was administered at 1 mg/kg/day for 1 week, followed by a weekly reduction of 0.2 mg/kg until a maintenance dose of 10 mg/day was reached. Pediatric dosing was adjusted to tacrolimus at 0.05 to 0.1 mg/kg/day (twice per day), MMF at 25 to 50 mg/kg/day, and pred-nisolone at 1 mg/kg/day. Acute cellular rejection episodes were managed with pulse steroid therapy followed by the standard tapering protocol.

Laboratory measurements
Hematological parameters were analyzed using the CELL-DYN Ruby Hematology Analyzer (Abbott Laboratories). The LNR was calculated as the abso-lute lymphocyte count divided by the absolute neutrophil count. Biochemical markers and immuno-suppressive drug levels were measured using the Architect i1000SR Processing Module (Abbott Laboratories). Serum IL-40 levels for both the patient and healthy control groups (n = 28) were determined with a commercial human enzyme-linked immuno-sorbent analysis (ELISA) kit (Sunred, No. 201-12-5841) and processed on an ELISA plate reader (Enzo/Byonoy).

Definition and diagnosis of rejection
Acute rejection was defined as symptomatic episodes or asymptomatic cases with an International Society for Heart and Lung Transplantation grade ≥ 2R, identified via EMB. Biopsy-negative clinical rejection was diagnosed when there was a > 25% decrease in left ventricular ejection fraction accompanied by hemodynamic instability. Chronic rejection (cardiac allograft vasculopathy) was identified by progressive intimal thickening in the coronary arteries via coronary angiography or intravascular ultrasonog-raphy. For antibody-mediated rejection, the presence of donor-specific antibodies was evaluated alongside histological findings; a mean fluorescence intensity of > 1000 was considered positive.

Statistical analyses
We used IBM SPSS Statistics version 30 (IBM Corp) software for analyses. We assessed the normality of variables by using both visual methods (histograms and Q-Q plots) and analytical tests (Kolmogorov-Smirnov and Shapiro-Wilk). We presented descriptive statistics as mean ± SD for variables with a normal distribution and as median (minimum-maximum) values for variables without a normal distribution.
To compare 2 independent groups, we used the independent sample t test (Student’s t test) for continuous variables that met the assumption of normal distribution and the Mann-Whitney U test for variables that did not. For comparison of categorical variables, we used the Pearson χ² test or, when appropriate, the Fisher exact test. We analyzed changes over time in repeated measurements (at baseline, 3, 6, 9, 12, 18, and 24 months) by using the Friedman test; when a significant overall difference was found, we made pairwise comparisons between time points (post hoc analyses) using the Bonferroni correction.
We performed ROC curve analysis to determine the discriminatory power of ΔLNR in predicting the development of acute rejection and to identify the optimal cutoff value. We calculated area under the curve (AUC) values along with 95% CIs. We estimated rejection-free survival rates using the Kaplan-Meier method based on the determined ΔLNR cutoff value and compared survival differences between groups using the log-rank test.
We examined relationships between continuous variables using Spearman correlation analysis and interpreted strength of the correlation coefficients according to the criteria proposed by Schober and colleagues (2018)¹⁰ as follows: weak (0.10-0.39), moderate (0.40-0.69), strong (0.70-0.89), and very strong (0.90-1.00). In all analyses, P < .05 was considered statistically significant.

Patient characteristics and demographics
The study cohort consisted of 121 patients with a mean follow-up duration of 54.31 ± 52.92 months (median of 46 mo; range, 0-184 mo). The predominant indication for transplant was end-stage heart failure (n = 120; 99.2%). The mean age of patients was 32.51 ± 18.02 years, with 88 male patients (72.7%) and 33 female patients (27.3%).
Analysis of baseline comorbidities revealed hypertension in 31 patients (25.6%), coronary artery disease in 35 (29.2%), diabetes mellitus in 19 (15.7%), hypothyroidism in 13 (10.7%), chronic kidney disease in 6 (5.0%), and pulmonary arterial hypertension in 1 (0.8%).
The most frequent etiology was dilated cardio-myopathy (n = 74; 61.1%), followed by ischemic cardiomyopathy (n = 29; 24.0%) and restrictive cardiomyopathy (n = 13; 10.7%). Preoperative hemodynamic assessment showed a mean cardiac index of 2.21 ± 0.95 L/min/m² and a pulmonary vascular resistance of 2.57 ± 2.20 Wood units. Operative data indicated a mean total ischemia time of 222.95 ± 55.82 minutes, cold ischemia time of 105.13 ± 25.06 minutes, and a cardiopulmonary bypass time of 164.62 ± 64.80 minutes.
Patients who developed and who did not develop acute rejection were compared in terms of demog-raphic and operative data. Results showed no signi-ficant differences between groups in terms of patient age (P = .868), follow-up duration (P = .914), and body surface area (p = .873).
Similarly, when surgical parameters were examined, total ischemia time (226 min [range, 120-359] in those without rejection and 237 min [range, 103-356] in those with rejection; P = .591) and cardiopulmonary bypass durations (P = .120) were similar between groups. No significant differences were observed between groups regarding cardiac index, pulmonary vascular resistance, transpulmonary gradient, and mean pulmonary artery pressure values based on the presence of rejection (P > .05).

Predictive value of change in the lymphocyte-to-neutrophil ratio for acute rejection
To evaluate the dynamic immune response and recovery in the early posttransplant period, ∆LNR was utilized rather than static, single-point absolute values.
In ROC curve analysis, to assess the ability of ∆LNR to differentiate between patients with and without acute rejection, AUC was 0.631 ± 0.063 (95% CI, 0.509-0.754; P = .040), indicating significant discri-minatory power. The optimal ∆LNR cutoff value was identified as 0.412, yielding a sensitivity of 43.8% and a specificity of 84.7% (Figure 1). Patients with a ∆LNR ≤ 0.412 exhibited a significantly lower rate of acute rejection compared with those above this threshold (84.75% vs 56.25%; P = .003) (Figure 2).

Interleukin 40 levels and correlation with hematological indices
Serum IL-40 levels were evaluated in a combined rejection group (n = 42), which included patients with cellular rejection (n = 10) and newly diagnosed humoral (antibody-mediated) rejection. These fin-dings were compared against a nonrejection group (n = 30) and a healthy control group (n = 28).
Significant differences in IL-40 levels were obser-ved across the groups (P < .001). Bonferroni-corrected pairwise comparisons revealed that IL-40 levels in the rejection group were significantly higher than those in both the healthy control group (mean difference = 336.55; P = 0.039) and nonrejection group (P < .001). No significant difference was identified between the nonrejection and healthy control groups (P = .142).
Regarding LNR, a significant difference was observed between groups (P < .001). Post hoc Bon-ferroni comparisons demonstrated that LNR was significantly lower in the rejection group compared with the nonrejection group (P < .001). The healthy control group showed significantly higher LNR compared with the rejection group (mean difference = 0.37; P = .006). The difference in LNR between the rejection and nonrejection groups approached, but did not reach, significance (P = .069) (Figure 3).
Correlation analysis showed significant, albeit weak, associations between IL-40 levels and all exa-mined hematological parameters (P < .05). Specifically, IL-40 levels were negatively correlated with the lymphocyte percentage (correlation coefficient = -0.21; P = .030) and the LNR (correlation coefficient = -0.20; P = .038). In contrast, correlation coefficients among the various hematological indices were exceptionally strong and consistent with clinical expectations (≥ 0.98; P < .001). Notably, within the healthy control group, a moderate positive correlation was identified between IL-40 levels and the LNR (correlation coefficient = 0.49; P = .008).

The early and reliable prediction of acute rejection after heart transplantation is paramount for ensuring graft longevity and reducing long-term mortality. Although EMB remains the diagnostic gold standard, EMB is associated with significant procedural risks, including vasovagal syncope, pericardial tamponade, ventricular arrhythmias, perforation, pneumothorax, and tricuspid valve damage. Furthermore, repeated sampling from the same myocardial regions can induce fibrosis, potentially leading to inadequate pathological assessments. Given these invasive const-raints and the risk of complications, an escalating need exists for noninvasive biomarkers to optimize the posttransplant monitoring of these high-risk patients.
As noted by Martini and colleagues, biomarker research in transplant has gained significant momentum, with hematological parameters emerging as cost-effective tools for clinical decision-making. In this context, LNR and its temporal variation (ΔLNR) serve as unique indicators that reflect the immune system’s remodeling process posttransplant.¹¹ Our findings demonstrated that postoperative LNR values significantly decreased compared with preoperative levels, followed by a gradual upward trend during follow-up. Notably, acute rejection rates were signi-ficantly lower in patients with low ΔLNR values, suggesting that ΔLNR holds substantial diagnostic value in predicting acute rejection episodes. However, no significant correlation was observed between ΔLNR and long-term survival in our cohort.
Although the neutrophil-to-lymphocyte ratio (NLR) is widely utilized to assess systemic inflamma-tion in cardiovascular diseases, our study favored the LNR for specific biological and methodological reasons. The primary objective of posttransplant immunosuppressive therapy is the selective inhibition of T-lymphocyte activation and proliferation. Therefore, early shifts in lymphocyte populations more directly reflect immunological activity and the predisposition toward rejection compared with neutrophil-dominant indices. Moreover, the physiolo-gical neutrophilia and marked lymphopenia typically observed in the early postoperative phase can disp-roportionately elevate the NLR, thereby potentially weakening its predictive accuracy.
Our results align with those of Choi and colleagues, who also investigated the association between ΔLNR and acute rejection.⁸ Our present study expanded on previous literature by incorporating a longer follow-up period (1 year or more), examining the relationship between LNR and acute rejection, and including a comprehensive mortality analysis. These additions demonstrated that changes in the LNR are not limited to acute episodes but may also reflect long-term immune dynamics following heart transplant.
Another significant contribution of this study was the elucidation of the relationship between ΔLNR and IL-40, a recently identified proinflammatory cytokine. Previous research has linked IL-40, produced by B cells and other immune populations, to various autoimmune and inflammatory conditions, such as rheumatoid arthritis, diabetes, and ankylosing spondylitis.¹² Jia and colleagues proposed that IL-40 facilitates the progression of inflammatory diseases through a multicellular cooperative network involving B cells, neutrophils, and stromal cells.¹³ Furthermore, Bagiş and colleagues reported elevated IL-40 levels in the serum and pericardial fluid of heart disease patients, suggesting an association with chronic inflammation.¹⁴
Both innate and adaptive immune responses drive the acute and chronic inflammation characteristic of rejection mechanisms. Our study demonstrated a clear association between elevated IL-40 levels and LNR shifts. Interestingly, the lowest IL-40 levels were observed in transplant recipients without rejection, suggesting that effective immunosuppressive treat-ment successfully suppressed the inflammatory milieu in this group. This finding is consistent with previous data regarding patients diagnosed with antibody-mediated rejection.¹⁵
A key limitation of this study was its retrospective nature. Hematological indices and LNR values are susceptible to influence by infections, steroid regimens, and various inflammatory states. Despite the exclusion of patients with active infections, the potential impact of these confounding variables may not have been entirely eliminated.

This study highlights the significant correlation and predictive utility of IL-40 and LNR as inflammatory biomarkers in heart transplant recipients. These parameters offer valuable, noninvasive insights into the immunological status of patients during both short- and long-term follow-up, facilitating the early detection of rejection-related clinical conditions.

Volume: 24 Issue: 6 June 2026

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