EXPERIMENTAL AND CLINICAL TRANSPLANTATION

Key words : Biomarkers, Forkhead box protein P3, Regulatory T cells, Renal transplant

Kidney transplant is the standard treatment for patients with end-stage renal disease (ESRD) chosen to exclude active infection or malignancy, high risk of perioperative mortality, unsuitable anatomy for technical success, as well as noncompliance for social, financial, or mental health reasons.¹ The advent of kidney transplantation has led to dramatically important improvements in renal allograft survival and remarkably decreased morbidity and mortality rates in patients with ESRD. However, kidney transplantation continues to face several challenges, such as the adverse effects of immunosuppressive therapy, immunologic rejection, and relative shortages of available organs. Of great importance, long-term graft loss, particularly through antibody-mediated rejection, remains a major unresolved challenge of kidney transplant.^2,3

Successful long-term kidney allograft survival may be hindered by a wide range of complications, resulting from prolonged immunosuppression and suboptimal efficiency of that treatment. Of note, long?term graft survival has improved only modestly during recent decades, and immune?mediated injury remains a leading cause of graft loss.4 Data found in the literature point to some cellular and transcriptional signatures of operational tolerance in kidney transplant.⁵ In light of this, the improvement of long-term allograft survival is still a goal in kidney transplant through induction of donor-specific tolerance.

Transplant tolerance, which is characterized by decreased alloreactive effector T cells (Teffs) and increased regulatory T cells (Tregs) in grafts and associated lymphoid tissues in the periphery, has been described as graft acceptance without long-term use of immunosuppressive drugs.³ Regulatory T cells are subsets of T cells involved in the maintenance of peripheral tolerance and contribute to self-tolerance, tolerance to alloantigen, and transplant tolerance, mainly by actively suppressing the activation and expansion of reactive Teffs. The suppression of Teff activity and the function by Tregs have been demonstrated to result in allograft tolerance in several mouse models involving skin, islets, heart, and kidney allografts.^6,7 In addition to their important role in allotransplant tolerance, Tregs possess great potential for induction or promotion of tolerance while decreasing the need for immunosuppression and its associated adverse effects.⁸

The recent identification and characterization of Tregs have opened up exciting opportunities for tolerance induction, immunotherapy, and immunomodulation in transplantation.⁹ Accumulating evidence has indicated that more than 1 population of Tregs is engaged in the maintenance of peripheral tolerance.^10,11 In addition, Tregs have been implicated in the pathogenesis of diabetes, which is the leading cause of ESRD, suggesting that these cells play a role in both the pathogenesis of chronic kidney disease and the induction of transplant tolerance.² Several types of Tregs have been characterized, in which natural and inducible CD4+CD25+ Tregs are the most prominent types.¹²

Based on the above findings, in this study, we compared the expression profiles of forkhead box protein P3 (FOXP3) and interleukin 35 (IL-35) in kidney transplant recipients (KTRs) with excellent long-term graft function under immunosuppression (ELTGF) versus KTRs with acute rejection. Our goal was to find an expression pattern that might be considered prognostic for kidney transplant outcomes.

Materials and Methods

Ethics statement
All procedures performed involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Declaration of Helsinki and its later amendments or comparable ethical standards. The research protocol was approved by the Ethics Committee of Shiraz University of Medical Sciences (IR.sums.IREC.1393.7206). All participants provided written informed consent in accordance with the Declaration of Helsinki.

Patients
This case-control study included KTRs who were seen for treatment between September 2014 and March 2015. Of the 40 KTRs included in this study, there were 27 KTRs with ELTGF and 13 KTRs with acute rejection. Rejection episodes were identified by an expert nephrology team, based on approved clinical diagnostic criteria and confirmed by needle biopsy and elevated serum creatinine and blood urea nitrogen levels¹³. The standard immunosuppressive regimen for kidney transplant consisted of cyclosporine (5 mg/kg initially, then a maintenance dosage of 2 to 2.5 mg/kg; cyclosporine level was 50 to 150 ng/mL), prednisolone (120 mg/d initially, tapering to 10 mg/d), and mycophenolate mofetil (1000 mg twice daily). Table 1 shows the inclusion and exclusion criteria for each group.

Data collected included demographic characteristics (age, sex, height, weight, and relation to intended recipient), clinical information (creatinine, albumin, calcium, phosphorus, transaminases, electrolytes, panel reactive antibody, and blood cell count), whether the kidney was from a living or deceased donor, transplant date, and whether the patient had infection with cytomegalovirus virus or BK virus.

Isolation of peripheral blood mononuclear cells
Five milliliters of whole peripheral blood were collected in sterile EDTA tubes under standard aseptic conditions. Peripheral blood mononuclear cells were isolated from the blood samples using Ficoll reagent (Greiner Bio-One Ltd).

RNA extraction and cDNA synthesis
Peripheral blood mononuclear cells were washed twice with phosphate-buffered saline, followed by total RNA extraction using the TRIzol reagent (Invitrogen), according to the manufacturer’s recommendation. RNA concentrations were determined using NanoDrop spectrophotometer (Thermo Scientific) and adjusted at 250 ng/?L. After RNA extraction, cDNAs were synthesized using cDNA synthesis kit (Takara), according to the manufacturer’s instructions.

Quantitative real-time polymerase chain reaction
Real-time polymerase chain reaction (PCR) was used to determine the expression levels of IL-35 and FOXP3 in KTRs with ELTGF and those with acute rejection. Specific primers, as illustrated Table 2, were designed with Alelle ID 6 software to amplify IL-35 and FOXP3 genes. The human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene was used as a housekeeping gene (internal control). Real-time PCR was performed using SYBR Premix Ex Taq™ II reagent (Takara) and the ABI 7500 real-time PCR detection system (Applied Biosystems). All reaction mixtures were prepared in a final volume of 10 ?L containing 0.4 ?L of each primer, 5 ?l of Master Mix (Takara), 0.25 ?L ROX reference dye, and 0.1??g of cDNA.

The thermal cycle program consisted of an initial cycle at 95 °C for 5 minutes for denaturation and polymerase activation, followed by 45 cycles of denaturation at 95 °C for 30?seconds; annealing at 57.5 °C, 59 °C, and 60 °C (for GAPDH, IL-35, and FOXP3, respectively) for 15?seconds; and extension at 72 °C for 30?seconds. Finally, the relative expression levels of FOXP3 and IL-35, relative to the GAPDH control gene, were determined using the equation 2 ? ??CT.

Statistical analyses
We used Graph Pad Prism 5 software for Windows for statistical analysis. Data are presented as mean ± SD of at least 3 independent experiments. Statistical significance was determined using multiple comparison t tests. P < .05 was considered to be statistically significant.

Clinical information and demographic characteristics
A total of 40 participants were enrolled in the study. The mean ages of KTRs with ELTGF, consisting of 15 men (59%) and 12 women (41%), and those with acute rejection, consisting of 7 men (54%) and 6 women (46%), were 42.1 and 45.5 years, respectively. Table 3 shows demographic and clinical characteristics. Table 4 shows the causes of advanced renal failure in the 2 study groups.

FOXP3 and interleukin 35 gene expression profiles
Our results showed that FOXP3 and IL-35 had significantly different expression profiles in the 2 study groups (P < .01). As shown in Figure 1, the mean expression levels of FOXP3 and IL-35 in peripheral blood from KTRs with ELTGF were significantly higher than levels in the peripheral blood from KTRs with acute rejection.

FOXP3 and interleukin 35 mRNA gene expression profiles in transplant recipients under immunosup-pression with excellent long-term graft function of more and less than 10 years
We divided the 27 KTRs with ELTGF into 2 groups: those with graft survival of more than 10 years (n = 8) and those with graft survival of less than 10 years (n = 19). As shown in Figure 2, the expression levels of FOXP3 and IL-35 were compared between these 2 groups. We found that FOXP3 was significantly higher in patients with graft survival rate less than 10 years, whereas IL-35 was significantly higher in patients with graft survival rate more than 10 years (P < .05).

Distribution of FOXP3 and interleukin 35 expression according to patient age
As shown in Figure 3, expression levels of FOXP3 and IL-35 in patients in the 35- to 50-year age range were greater than levels shown in the other age groups.

In the present study, we compared the expression profile of FOXP3 and IL-35 in the peripheral blood of KTRs with ELTGF versus KTRs with acute rejection. Our results showed significantly different expression profiles of FOXP3 and IL-35 in the 2 study groups.

Despite the development of successful immuno-suppression protocols and remarkable improvements in short-term graft survival rates, chronic graft loss has remained a major hurdle in clinical transplantation. Importantly, none of the currently used immunosuppressive strategies has culminated in true graft tolerance, and the toxicity and complication profiles of the standard immunosuppressive agents can lead to significant patient morbidity and mortality.¹⁴ In this regard, the induction and maintenance of allograft tolerance are the most appropriate options after transplant.

Immune tolerance is an active process characterized by a central and peripheral component. Although central tolerance is a thymic-dependent process that involves the deletion of autoreactive clones through the induction of apoptosis, peripheral tolerance can be subdivided into at least 3 major categories: clonal deletion, anergy, and suppression. Immunologic self-tolerance in the periphery is achieved by the negative regulation exerted on the immune response by a variety of cells of which the best-characterized populations are Tregs.¹⁵ Regulatory T cells have been shown to play important roles in balancing the effector arm of the immune system and in preventing autoimmunity, facilitating graft tolerance after organ transplant, and inhibiting the development of antitumor immunity.^16-18 These cells may not only suppress innate and adaptive immune responses but they can also block Teffs at any stage of their activation, proliferation, differentiation, and effector functions.3 In addition, Tregs, induced in vitro or in vivo or expanded ex vivo after alloantigen stimulation, have been shown to promote tolerance to the allograft.^8,19 Most importantly, Treg migration to the graft is required to prevent graft rejection. Increased Tregs have been demonstrated to be involved in the development of immune tolerance²⁰ after solid-organ transplant, as documented in kidney transplant,^21,22 liver transplant,^23,24 and heart transplant.^25,26 In animal models of transplant, Tregs were present in tolerant allografts and were shown to migrate to the allograft tissue.²⁷

A well-studied regulator of Tregs at the molecular level is the transcription factor FOXP3, the expression of which is critical for Treg development and function. FOXP3 is a forkhead-winged helix transcription factor gene involved in immune function, and its expression has been used to define this T-cell subset.²⁸ FOXP3-positive Tregs constitute 5% to 10% of peripheral CD4+ T cells in both mice and humans and are critical for maintaining immune homeostasis.¹⁵ Evidence has supported the notion that CD4+CD25+FOXP3+ Tregs play a fundamental role in the establishment and maintenance of operational tolerance to renal allografts.^27,29 In transplantation, FOXP3+ T cells play a role in the suppression of donor-activated Teffs and in tolerance induction. Although FOXP3 expression has been clinically investigated in graft samples and peripheral blood, the significance of altered levels or cell numbers in patients is not well understood.

Our results showed that FOXP3 expression was significantly increased in KTRs with ELTGF compared with KTRs with acute rejection. There are contradictory results on the clinical and prognostic significance of FOXP3-positive cell infiltrates in renal allograft recipients with acute rejection.³⁰ In contrast to our results, FOXP3 mRNA expression in endomyocardial biopsies taken during acute cellular rejection after cardiac transplant was higher than those without histologically proven rejection. When FOXP3 gene expression in the peripheral blood was analyzed, no association with rejection or nonresponsiveness was found.²⁸ Similarly, higher FOXP3 mRNA levels in the urine of KTRs with an acute rejection episode were observed. In that study, Muthukumar and colleagues reported that KTRs with an acute rejection episode expressed high levels of FOXP3 mRNA in the urine and that lower levels of FOXP3 were associated with a poorer response to antirejection therapy, postulating that this could be a future noninvasive marker for level of renal graft function.³¹ Bunnag and colleagues reported that FOXP3 expression in human kidney biopsies was linked to rejection and was not correlated with a favorable outcome.³² Results of FOXP3 analysis from graft biopsy cores have also demonstrated a higher FOXP3 expression in allografts with acute rejection compared with stable renal allografts or with those displaying antibody-mediated rejection.³³ However, a simple correlation with the existence of FOXP3-positive cells is not always a reliable predictor of favorable kidney graft outcome. For example, Ashton-Chess and colleagues reported that expression of FOXP3 in blood and in the graft could not be distinguished between patients with and without rejection.³⁴

Although IL-10 and transforming growth factor beta (TGF-?) are the most commonly studied immunosuppressive cytokines, the recently identified IL-35 has been shown to have potent suppressive function in vitro and in vivo. Interleukin 35 has been identified as the newest member of the IL-12 family of cytokines, including IL-23 and IL-27, which is distinct from its siblings in several ways. Similar to that of regulatory cytokines TGF-? and IL-10, IL-35 is one of the major components of the suppressive repertoire.³⁵ Interleukin 35 can directly suppress Teff proliferation in vitro in an antigen-presenting cell-free culture.36 Furthermore, not only does IL-35 have the ability to directly suppress Teff responses, it can also expand regulatory responses by propagating infectious tolerance and generating a potent population of IL-35 expressing inducible Tregs.¹⁶ Numerous studies have concentrated on the functions of IL-35 in autoimmune and inflammatory diseases, such as psoriasis,³⁷ type 1 diabetes,³⁸ arthritis,³⁹ asthma,^40,41 and leukemia.⁴² However, the role of IL-35 in solid-organ transplantation is poorly understood. Our results showed that IL-35 expression was significantly increased in KTRs with ELTGF compared with KTRs with acute rejection. Ma and colleagues demonstrated the role of IL-35 in conversion of human and murine CD4+CD25- T cells into IL-35-induced Tregs (iTr35 cells).⁴³ In their study, rhIL-35 was shown to induce EBI3 and P35 expression in CD4+CD25- T cells (including Th17 cells) and relative Tregs were shown to induce a further increase in IL-35 levels.⁴³

Our findings indicated that expression levels of FOXP3 and IL-35 in the 35- to 50-year age range were greater than levels in the other age ranges. In their study, Ashton-Chess and colleagues34 showed variability between FOXP3 expression and patient age and the time that samples were taken posttransplant. When FOXP3 mRNA expression using RT-PCR was analyzed in over 80 renal transplant biopsies, higher mRNA levels were shown to be associated with rejection, younger donor age, and longer posttransplant period, with FOXP3 expression not correlated with a favorable graft outcome.³² Intragraft FOXP3 increased within the first year after liver transplant but was not reflected by changes in blood samples. Data from protocol biopsies in recipients with episodes of subclinical cellular rejection showed a correlation between low FOXP3/CD3 ratio and poor graft function up to 5 years posttransplant.^44,45 An important point is that our statistical calculations did not show a good correlation between the expression level of IL-35 and FOXP3 and the age of individuals. On the other hand, our distribution data indicated that an age range from 35 to 50 years was associated with risk of graft rejection, whereas FOXP3 and IL-35 expression levels in patients in other age ranges were less correlated than those shown in patients from 35 to 50 years old. We also found that FOXP3 was significantly higher in KTRs with ELTGF with graft survival of less than 10 years and IL-35 was significantly higher in KTRs with graft survival of more than 10 years. These data suggest that the occurrence of FOXP3 mRNA reflects time-dependent entry of Tregs into sites of chronic inflammation.

Findings from our study suggested that expression levels of FOXP3 and IL-35 may have the potential to be used as prognostic biomarkers for kidney transplant outcomes. However, further comprehensive studies are needed. The outlined predictive power of the Treg population needs to be investigated further to be confirmed as an immune-monitoring strategy that can help to achieve better long-term kidney allograft outcomes.

Volume: 19 Issue: 11 November 2021

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