Longitudinal monitoring of mRNA levels of regulatory T cell biomarkers by using non-invasive strategies to predict outcome in renal transplantation

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 [1‐4]. 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 cellular population expressing the CTLA-4 [5]. CTLA-4 is encoded by the homonymous gene located on chromosome 2 (2q33) 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 [6‐9].

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.

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, 12‐17]. 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 [18]. 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 [19]. The profiling of FOXP3 and CTLA-4 isoforms gene expression at baseline and during the first year after kidney transplantation might clarify patterns of immunological activation, for these molecules are strongly implicated in tolerance of transplanted organs. Most of the evidence concerning these candidate biomarkers comes from cross-sectional or case-control studies and the validation of these molecules as immunological biomarkers in longitudinal studies still requires proof of accuracy. The improvement of long-term graft survival is still an important goal in kidney transplantation and the induction of donor-specific tolerance represents the holy grail of solid-organ transplantation.

In our longitudinal prospective and case-control study, we examined in peripheral blood the role played by the FOXP3 and CTLA-4 transcripts as possible biomarkers of clinical outcomes, such as aTCMR, de novo DSA development and renal dysfunction. The analysis was carried out on mRNA transcripts from peripheral blood samples from KTRs monitored up to one year after transplantation, easily available with relatively non-invasive techniques, compared to tissue biomarkers obtained through biopsies. The choice of FOXP3 and CTLA-4 as candidate biomarkers of the immune response to KTR was supported by the evidence of their wide inter-individual variability, both in healthy subjects and in patients on renal replacement therapy (RRT) (Fig. 3, legend).

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 [20, 21]. 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 [22, 23].

We studied the expression of these molecules over a period spanning from before transplantation up to 1 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 [24, 25]. 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 6 months after transplantation [12]. 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 15 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 [26]. 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. 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 [27, 28].

Through the analysis of immunological, demographic and clinical parameters, we detected an association between aTCMR within the first year after transplantation and solCTLA-4 mRNA levels 15 days after transplantation, use of everolimus, and immunosuppression switch, while basiliximab as induction therapy was negatively correlated. Despite the relatively small number of acute rejection cases and, overall, the small sample size of the entire cohort should represent a note of caution for any definitive interpretation, we hypotesize a putative role of early post-transplant levels of solCTLA-4 on the susceptibility to acute rejection, through an influence on T-cell activation. This observation was already reported in autoimmune thyroid diseases, where solCTLA-4 would indirectly act as an enhancer of autoimmune response in the presence of activated intra-thyroid T lymphocytes [29]. In our experience, solCTLA-4 acted as a time-dependent four-fold risk factor for aTCMR and showed a good accuracy of prediction at ROC curve both in the entire KTRs group but mainly in patients treated only with mycophenolate acid. After transplantation, solCTLA-4 could interfere with flCTLA-4 function, stimulate T reactivity and prevent the transduction of inhibitory signals. These data support the role of solCTLA-4 in acute rejection demonstrated at DNA level in one previous genetic study by our group [30], where we 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 [31‐35]. There is growing experimental and clinical evidence that soluble isoforms play an important role in establishing and maintaining peripheral tolerance [36]. Despite the initial observation that solCTLA-4 is mainly produced by resting T cells [37], recent studies have clarified that solCTLA-4 release can rise during antigenic responses, and that this phenomenon modulates immune responses [26, 38‐40].

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 [26, 41].

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 [42‐47].

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 [16, 48]. 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 patients showed gradual expression increment up to 1 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 1 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 when 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 1 year post-transplantation.

This 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 [49, 50]. It is known that ESRD patients harbour a heterogeneous population of CD3⁺CD8⁺CD28⁻ cells with immunomodulatory but also cytotoxic characteristics to a greater extent than healthy subjects do, and which expands after transplantation [51].

The study presents some limitations. First, a relative heterogeneity exists in terms of immunosuppressive therapy adopted in the cohort. Such a phenomenon should be connected with the long period of enrollment. However, this study represents a monocenter experience, therefore limiting the potential biases derived from different managements and clinical approaches. Overall, the decision to use different immunosuppressive regimens derived from several aspects, like specific clinical necessities (using mTOR inhibitors in patients with previous history of cancer), specific immunological background (pre-transplant PRA value), or protocols of research. In all the cases, the best immunosuppressive regimens were used following the available literature.

As previously reported, another limit to consider is the sample size of the event of interest, namely the aTCMR. Unfortunately, this is the principal limit of studies focused on investigating relatively uncommon events, like aTCMR is. In this observational study we enrolled 125 consecutive kidney transplant patients, reporting 11 (8.8%) and 16 (12.8%) aTCMR cases during the first year and duting the overall period of the study. From a clinical point of view, the aTCMR frequency is in line with previous clinical experiences. We understand that the study’s small sample size can unfortunately impair our ability to construct solid diagnostic models. However, on the other side we should underline that the Treg markers studied in the present analysis have been mainly investigated in cross-sectional or case-control studies, and that their impact in longitudinal studies still requires proof of accuracy. Therefore, we think that, although limited by the numbers, our experience should be of relevance.

The molecular study of clinically relevant Treg markers in peripheral blood of KTRs suggested 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, a protection towards de novo DSA response. Furthermore, FOXP3 monitoring could be useful as indicator of acute rejection for the effect of Treg plasticity, reflective of a change from Treg into Teff cells during inflammation for the pro-apoptotic role of FOXP3, and contribute with solCTLA-4 to increased risk of graft dysfunction at one year post-transplantation.

While the potential of the soluble CTLA-4 isoform as a therapeutic target will need a wider case history and a confirmation of its role, verifying the levels of the protein in the serum or plasma, the dosage of the blood levels of these two molecules, and mainly of the solCTLA-4, could put forward as a candidate non-invasive biomarker of cellular and humoral alloreactivity in clinical transplantation. mRNA levels of 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”.