Monitoring of molecular profiling of regulatory T cell biomarkers by using noninvasive strategies to predict outcome in renal transplantation.

Background. T-cell mediated acute rejection (aTCMR) is still an issue in kidney transplantation for it is associated with chronic rejection, graft loss, and overall worse outcomes. For these reasons, a standard non-invasive molecular tool to detect is desirable to offer a simpler monitoring of kidney transplant recipients (KTRs). The purpose of our study was to examine, in peripheral blood before and after transplantation, the expression patterns of regulatory T cell (Treg)-related genes: the forkhead box P3 (FOXP3) and the two CTLA-4 isoforms (full-length and soluble) to predict aTCMR onset, de novo donor-specific antibodies (DSA) development and renal dysfunction one year after transplantation. Methods. We profiled by using a relative quantification analysis (qRT-PCR 2- ∆∆CT ) the circulating mRNA levels of these biomarkers in peripheral blood of 89 KTRs within the first post-transplant year (at baseline and 15, 60, 365 days and when possible at acute rejection) and compared the results with 24 healthy controls. mRNA drastically reduced and gradually comparison with very low by the time of aTCMR Healthy controls exhibited of kidney transplantation in the wake of “precision medicine”. While the potential of these molecules as a therapeutic target needs further investigation, we found out preliminary evidence that solCTLA-4 can qualify as a candidate non-invasive biomarker of cellular and humoral alloreactivity in clinical transplantation. the four sample collection time-points; b) no steroid-resistant rejection. Twenty-four healthy blood donors with comparable age, sex, and ethnicity volunteered as controls (all Caucasian subjects). KTRs were treated according to the local immunosuppression protocol. Induction therapy was carried out with basiliximab (Simulect®, Novartis, Basel, Switzerland) in 82 cases (92.1%), and with antithymocyte globulins (Thymoglobulin®, Sanofi, Paris, France) in 7 cases (8.9%). Maintenance therapy comprised prednisone, a calcineurin inhibitor (CNI) (either tacrolimus [Advagraf®, Astellas Pharma, Tokyo, Japan], in 75 cases (84.3%), or cyclosporine [Neoral®, Novartis] in 14 cases (15.7%), and a proliferation signal inhibitor. The latter consisted of mycophenolic acid in 77 cases (86.5%), and everolimus (Certican®, Novartis) in 12 cases (13.5%). Patients treated with everolimus were considered separately, given the effect of this drug on an increased risk of aTCMR within the first postoperative year in this analysis.

Conclusions. Peripheral blood mRNA levels of Treg-associated molecules might help shape immunosuppression, tailor monitoring and achieve a better long-term clinical course 6 of kidney transplantation in the wake of "precision medicine". While the potential of these molecules as a therapeutic target needs further investigation, we found out preliminary evidence that solCTLA-4 can qualify as a candidate non-invasive biomarker of cellular and humoral alloreactivity in clinical transplantation.

BACKGROUND
T-cell mediated acute rejection (aTCMR) is still an issue in kidney transplantation for its association with chronic rejection, graft loss, and overall worse outcomes. In addition, immunosuppressive therapy bears a risk of infection, malignancy and cardiovascular disease. For this reason, tailored immunosuppression strategies are useful to curb adverse events associated with kidney transplantation. Therefore, risk prediction and early diagnosis of aTCMR through non-invasive methods can be crucial for allograft survival and immunosuppression management (1)(2)(3)(4). For these reasons, developing a standard clinical and molecular assessment procedure offers a simpler monitoring of KTRs. The study of biomarkers of aTCMR and immune dysregulation in renal transplantation has progressively focused on regulatory T cells (Tregs). This CD4 + CD25 + FOXP3 + lymphocytic subpopulation, which spreads from the thymus as effector and memory suppressive cells, is essential in suppressing alloimmune response and maintaining tolerance in transplantation FOXP3 expression is the major determinant of Tregs phenotype and function. Another important marker recently investigated in kidney transplantation and aTCMR is CTLA-4 (CD152). CTLA-4 is implicated in self-tolerance and acts as a braking co-inhibitor of activated CD4 + and CD8 + T cell responses. Tregs represent the principal 7 cellular population expressing the CTLA-4 (5). CTLA-4 is encoded by the homonymous gene located on chromosome 2 (2p22) in two transcripts in humans: a transmembrane isoform, resulting from the translation of all 4 exons of the gene (full length CTLA-4, flCTLA-4), and a truncated isoform of exon-3, encoding the transmembrane domain (soluble CTLA-4, solCTLA-4) following alternative splicing. The inhibitory function of CTLA-4 is carried out through several different mechanisms, comprehending the cell-extrinsic action of its soluble form responsible for competition with CD28, namely the CTLA-4 counterpart, which transduces instead a proliferation signal for T cells (6)(7)(8)(9).
The purpose of our study was to develop a new non-invasive diagnostic tool, based on an accurate analysis of molecular FOXP3 and CTLA-4 mRNA expression pattern in peripheral blood, during the first post-transplant year, capable to predict aTCMR onset, de novo DSA development and renal dysfunction. For doing this, we performed three different analyses: 1) a prospective longitudinal monitoring of mRNA levels of FOXP3, flCTLA-4 and solCTLA-4 during the first post-transplant year; 2) a case-control study of KTRs compared to healthy controls for FOXP3 and 3) an evaluation of diagnostic power of the variables investigated.

Population
One hundred and twenty-five patients consecutively underwent kidney transplantation at the Organ Transplantation Unit of the Regional Hospital of L'Aquila, Italy during the period January 2011-September 2017. Of them, 120 (96%) received a kidney from a deceased-brain donor and five (4%) from a living donor. Written informed consent was obtained from all participants.
Investigations were carried out by the rules of the Declaration of Helsinki, and the institutional ethical committee approved the study (protocol no 0098164/2011). Out of the total KTRs, 89 met the inclusion criteria for the study consisting in: a) sufficient/pure mRNA collected at least two of the four sample collection time-points; b) no steroid-resistant rejection. Patients treated with everolimus were considered separately, given the effect of this drug on an increased risk of aTCMR within the first postoperative year in this analysis.

Outcomes
Graft function was reported as estimated glomerular filtration rate (eGFR) (10). The diagnosis of acute rejection was biopsy-proven and we considered in the analysis only KTRs with acute T-cell-mediated rejection (aTCMR), classified in the categories 3 and 4 of Banff'15 classification (11). Rejection treatment was based on three intravenous one-gram methylprednisolone boluses over three days.
Screening for HLA antibody was performed by Lambda Cell Tray T cell CDC-based Class-I PRA and Lambda Antigen Tray Mixed Class-I/II ELISA (One Lambda, Canoga Park, CA, USA). Detection of donor specific antibodies (DSA) was performed on T and B lymphocytes by cell-based assays (CDC-XM) and Luminex solid-phase assays. Graft dysfunction (moderate chronic kidney disease) was defined as an eGFR less than 60 ml/min/1,73 m 2 at one year (12). Blood samples were collected at four time points (baseline, post-KTR day 15, 60, 365) and at the time of a possible aTCMR. All patients were followed-up until two years minimum.

RNA Isolation and Gene Expression Analysis
Peripheral venous blood (3 ml) was drawn directly into Tempus Blood RNA tubes (Thermo Fisher Scientific Inc., Waltham, MA-US), according to the manufacturer's protocol, frozen and stored at -20°C until processing. Whole blood total RNA was extracted using a Tempus Spin RNA isolation (Thermo Fisher Scientific Inc.), which uses an RNA isolation aTCMR-free vs. aTCMR-positive patients) was normalized with the 2 -∆CT logarithm 10 (log2 -∆CT ) compared to the endogenous control, to correct the asymmetric distribution of the data.

Statistical Analysis
Binomial variables were reported using numbers and proportions. Numerical variables were reported using means ± standard deviations (SD), or medians and interquartile ranges (IQR), as appropriate. Gene levels distribution is shown as box-or scatter-plot representations. Results were compared using Fisher's exact test or Mann-Whitney U test/Wilcoxon. Comparison between groups and correlation between variables were examined by parametric (t test/one-way ANOVA, Pearson's correlation), and nonparametric tests (Kruskal Wallis, Friedman test for repeated measures and Spearman's test), as appropriate.
Receiver-operating characteristic (ROC) curves were generated for the prediction of shortand long-term aTCMR episodes, de novo DSA development and renal dysfunction after transplantation to define the accuracy of diagnostic test and establish the best cut-off for clinical outcomes.
The predictive ability of several variables for the risk of the acute rejection, graft dysfunction and post-transplant development of DSA in our population was assessed. All factors considered in univariable analyses were based on literature review and suggestions from the clinical team. Logistic regressions were run for simply dichotomous variables. The crude odds ratios (OR), 95% confidence interval (CI) and p value were reported for each predictor in the univariable analysis. Only statistically significant variables in the univariable analysis were entered into multiple logistic regression analysis to predict the final independent factors. The model fit was assessed by chi-square, degrees of freedom and p-value. We chose a backward conditional method to select significant independent covariates.
We used the Cox proportional hazards model for time-dependent events (graft loss, death, acute rejection, de novo anti-DSA antibody development). All the covariates with p≤0.05 were introduced into multivariable models. Hazard ratios (HRs), and 95% confidence intervals (CIs) were reported for significant variables.
The significance of statistical tests was taken at two-tailed p<0.05. Analyses were run with SPSS Statistics v. 13

Characteristics and follow-up of the recipients
Clinical features of the patients (n=89) transplanted between 2011 and 2017 are reported in Table 1. Mean age at transplant was 52.5±11.5 years. All patients had a minimum follow-up of two years (mean follow-up period: 38.3±23.8 months). During the entire study period, three patients (3.4%) died: in all the cases, the cause of death was an infection.
During the same period, six graft losses (6.7%) were reported. In four cases, an immunological cause was reported, namely a drug-resistant acute rejection and a chronic active antibody-mediated rejection in two cases, respectively. In the remaining two cases, a graft thrombosis and a graft pyelonephritis were reported. Seventy-three patients   Longitudinal evaluation of FOXP3 expression levels depending on the type of maintenance immunosuppression (RT-PCR reference: healthy controls) 13 The trend of FOXP3 mRNA levels over time up to one year after transplantation on all KTRs compared to healthy controls followed that observed for CTLA-4 isoforms and, by distinguishing the KTRs for immunosuppressive regimen, we observed that there were no differences between recipients treated with everolimus and recipients on mycophenolic acid for the whole duration of the monitoring (Additional File 1).

Case-control study of FOXP3 mRNA expression between KTRs and healthy controls
FOXP3 expression profiles showed differences between KTRs and healthy controls.

Association between aTCMR onset and clinical or molecular variables
We examined several clinical variables along with the molecular targets to assess their predictive ability for the cumulative risk of short-and long-term aTCMR after transplantation. There were no significant differences between KTRs with and without aTCMR within one year after transplantation concerning FOXP3 and the flCTLA-4 isoforms (Figure 3). Only solCTLA-4 levels showed a different trend after transplantation, with significant higher transcript levels fifteen days after transplantation in cases with 14 aTCMR, compared to aTCMR-free KTRs (log=0.365, IQR=-0.   Table 3).   A cut-off value >0.461 was able to identify patients with graft dysfunction with a sensitivity of 86.0% and a specificity of 58.6%.

DISCUSSION
The study of urine and peripheral blood biomarkers of aTCMR and immune dysregulation in kidney transplantation is crucial because non-invasive methods are potentially game changers in clinical practice. Tregs have studied in this context for their suppressive capacity and for the prediction of better long-term graft outcomes (2,(13)(14)(15)(16)(17)(18). The role of FOXP3-positive infiltrates in renal allograft biopsies in prolonging organ survival has not yet been clarified, although long-lived grafts demonstrate a substantial presence of FOXP3 + Tregs, suggesting their beneficial effect on survival, through a regulatory function (19). The activity of Tregs modulated by the transcription factor FOXP3 is dependent on the expression of a complex group of proteins, such as CTLA-4 (20). The profiling of CTLA-4 is a target gene of FOXP3, which functions as the main Treg regulator, and whose expression levels are critical for the suppressive function of Tregs (21)(22). CTLA-4 would have a dual function: for conventional Teffs, it would act as a receptor for inhibitory signals, while, on Tregs, as an immune response suppression mediator (23)(24).
We studied the expression of these molecules over a period spanning from before transplantation up to one year after transplantation, and we observed that pre-transplant levels of CTLA-4 and FOXP3 were significantly reduced compared to healthy subjects. Accordingly, recent studies and a meta-analysis have suggested that end-stage renal disease (ESRD) and RRT influence immunity by lowering CD4 + lymphocytes and Tregs (25)(26). After transplantation, we documented a robust decrease in both CTLA-4 and FOXP3 expression compared to baseline levels, probably due a combined effect of immunosuppressive treatment and immune response. Our findings are in line with recent results obtained by flow cytometry regarding Tregs six months after transplantation (13).
The authors observed a decrease after transplantation in the percentage of both natural thymic Tregs and activated Tregs in peripheral blood. Furthermore, a higher percentage of activated Tregs before transplantation was also a predictive biomarker of long-term graft survival. The fall in biomarkers levels during the first fifteen days after transplantation might be a consequence of induction therapy. It is indeed known that these drugs can cause a profound decrease of T and NK cells. In 2015, Krepsova et al. observed a reduction in gene expression of some tolerance-associated transcripts in patients treated also with basiliximab (27). In the early post-transplant period, CNI-based maintenance therapy could also interfere with FOXP3 transcription by inhibiting IL-2 release.
In our analysis, we observed that the expression of FOXP3 in peripheral blood was lower by the time of aTCMR than in all the other time points. Perhaps, this was a consequence of a recall in the graft of FOXP3 + lymphocytes, both Tregs and activated CD4 + T cells.
Another mechanism is possibly the Treg plasticity, by which a loss of FOXP3 expression would reflect a change from Treg into Teff cells during inflammation for the pro-apoptotic role of FOXP3 (28)(29). showed an increased frequency of solCTLA-4 CT60 A/A genotype in the 3' untranslated region in patients experiencing acute rejection. This genotype predisposes to higher release of solCTLA-4 and it has been also emphasised in graft-versus-host and autoimmune diseases (32)(33)(34)(35)(36). There is growing experimental and clinical evidence that soluble isoforms play an important role in establishing and maintaining peripheral tolerance (37). Despite the initial observation that solCTLA-4 is mainly produced by resting T cells (38), recent studies have clarified that solCTLA-4 release can rise during antigenic responses, and that this phenomenon modulates immune responses (27,(39)(40)(41).
In addition, the protective influence of basiliximab compared with rATG has already been observed by other groups that evidenced, in the early post-transplant period, a higher ratio CD4 + FOXP3 + Tregs to effector T cells and an inferior incidence of rejection in KTRs treated with basiliximab (27,42).
Interestingly, we found out a negative correlation between the expression of baseline solCTLA-4 and de novo DSA production after transplantation. This association could be interpreted by considering the control that Tregs exert, through the CTLA-4 co-receptor, on the expansion of follicular T cells and the humoral immunity. Memory B cells are important in alloreactivity in kidney transplantation and B cells are involved in aTCMR as well as in chronic antibody-mediated rejection with DSA produced by B cells (43)(44)(45)(46)(47)(48).
FOXP3 is involved in the regulation of Tregs development and function, and it is considered a biomarker of kidney allograft tolerance. Previous studies, based on urinary FOXP3 mRNA of KTRs, showed higher levels in patients experiencing acute rejection compared to patients with chronic rejection or subjects with stable function (17,49). In our study, FOXP3 mRNA levels in peripheral blood were not associated with aTCMR at regression analysis but, at the time of rejection, we recorded the lowest level of transcription of the entire follow-up. However, median expression levels in aTCMR-positive 20 patients showed gradual expression increment up to one year after transplantation, while on the contrary in negative-aTCMR KTRs they first decreased until 60 days after transplantation and then increased.
In addition, in recipients with impaired graft function one year after transplantation a higher expression of pre-transplant FOXP3 and day-15 solCTLA-4 emerged. This finding suggests that these molecules might work as prognostic biomarkers, whose prediction power is increased considering donor age. Older donors (>53 years) with day-15 solCTLA-4 and baseline FOXP3 mRNA log>0.461 have an increased risk of graft dysfunction at one year post-transplantation. The association between day-15 solCTLA-4 and renal dysfunction might originate from the influence of Tregs on memory CD8 + CD28 -Teff, which has been recently implied in allograft dysfunction (50)(51). It is known that ESRD patients harbour a heterogeneous population of CD3 + CD8 + CD28cells with immunomodulatory but also cytotoxic characteristics to a greater extent than healthy subjects do, and which expands after transplantation (52). However, the correlation between impaired graft function one year after transplantation and day-15 solCTLA-4 should be explained also with other mechanisms. As an example, the solCTLA-4 increase observed in the patients with rejection should explain this connection: in fact, rejected grafts typically end in late impaired functions.

CONCLUSIONS
The expression of FOXP3 and CTLA-4 isoforms in the peripheral blood of KTRs, before or early after transplantation, suggests a dual immunological role for the solCTLA-4 molecule, which might predict a susceptibility to cellular acute rejection and/or graft dysfunction, but, on the other hand, of protection towards de novo DSA response.
While the potential of these molecules as a therapeutic target needs further investigation, we found out preliminary evidence that solCTLA-4 can qualify as a candidate non-invasive biomarker of cellular and humoral alloreactivity in clinical transplantation. mRNA levels of 21 Treg-associated genes in peripheral blood might help shape immunosuppression, tailor monitoring and achieve a better long-term clinical course of kidney transplantation in the wake of "precision medicine".