CD4 + FOXP3+ regulatory T cells (Treg) are key players in the suppression of immune responses to both autoantigens and alloantigens.1 Suppression of alloreactivity is crucial for successful organ transplantation. The role of FOXP3+ Treg in the induction and maintenance of tolerance in organ transplantation has been demonstrated in several experimental models of transplantation2-4 and high FOXP3 expression is associated with transplant tolerance in humans.5,6 Combined with the encouraging findings in trials of Treg therapy for graft-versus-host disease in patients, the use of Treg as cellular therapy in solid organ transplantation is currently under investigation.7-9
Regulatory T cells are a heterogeneous population of cells originating in the thymus, the so-called natural (n)Treg, and induced (i)Treg, which develop in the periphery under a variety of conditions including chronic inflammation and infections.10-12 Naive, mature CD4+ T cells can be converted into iTreg via antigen stimulation in a cytokine milieu-rich IL2 and TGFβ.13 Other mechanisms include stimulation of the PDL1-PD1 pathway and environmental factors as the vitamin A metabolite all-trans retinoic acid.14,15 Both nTreg and iTreg have suppressive capacities,13,16 nevertheless their relative contribution in suppressing autoantigen- and alloantigen-specific immune responses in patients is unknown.
One of the major difficulties in studying the different Treg subsets is the lack of specific extracellular markers. Even discrimination between Treg and activated T cells is complicated because FOXP3, CD25, CD39, CTLA4, and GITR can be expressed by activated T cells as well.1 FOXP3 is currently the most reliable marker for the identification of the total Treg population as FOXP3 is essential for their suppressive function.10,17 At the epigenetic level, the Treg-specific demethylated region (TSDR) in the FOXP3 gene is demethylated in nTreg, whereas this region is methylated in other peripheral blood leukocyte subtypes cells, including iTreg and recently activated T cells and in nonhematopoietic tissues. Demethylation of the FOXP3 gene results in a stable, constitutive expression of FOXP3 and is used as a marker to identify nTreg.18-20
Classically, the secondary lymphoid organs are the sites for alloimmune regulation; however, infiltrating FOXP3+ T cells are also detected locally in transplanted allografts where they colocalize with antigen-presenting cells.21 Whether these intragraft FOXP3+ cells contribute to a favorable graft outcome22-24 or are a sign of immune activation25-27 is still under debate. The graft itself (exposure to donor-antigen) is essential for the generation of potent suppressors of the alloimmune response28,29 and antigen-specific suppressors home in the graft to exert their function.30,31 Moreover, the frequency of donor-antigen specific suppressors is higher locally in the graft compared to the peripheral blood. The ideal human transplantation model to study graft-infiltrating FOXP3+ T cells is heart transplantation because after heart transplantation, endomyocardial biopsies (EMBs) are routinely taken. This provides the unique opportunity to study intragraft FOXP3+ cells at various stages of the alloreactive immune response.
In the present study, we studied the origin of EMB-infiltrating FOXP3+ cells in humans. Because of the lack of specific iTreg markers, we were forced to study the presence of iTreg indirectly by combining the methylation status of the TSDR (identifying nTreg) with the FOXP3 messenger RNA (mRNA) expression (total Treg population). By including EMB of both patients who developed a histologically proven acute rejection (AR) episode requiring antirejection therapy (rejectors; International Society for Heart and Lung Transplantation (ISHLT) rejection grade ≥ 2R) and patients who remained free from rejection (nonrejectors), we correlated the presence of the different FOXP3+ subsets with their in vivo immunosuppressive capacities. Endomyocardial biopsies collected shortly after rejection treatment were examined as well. In addition, we focused on intragraft phenotypical and molecular features of cellular infiltrates in both 1R (mild rejection) EMB of rejectors (preceding rejection) and nonrejectors.
MATERIALS AND METHODS
In total, 42 patients who received a heart transplant between January 1997 and November 2010 were included (Table 1). All patients received induction therapy with antithymocyte globulin, and all patients except 5 received triple maintenance immunosuppressive therapy. Endomyocardial biopsies were sampled routinely and scored by a team of pathologists according to the ISHLT grading system.32,33 Endomyocardial biopsies scored according to the working formulation of 1990 were reclassified based on the revised working formulation of 2004. To avoid bias introduced by different pathologists,34 all included samples were scored by the same team of pathologists. According to the ISHLT definition of rejection, only patients with an EMB scored as a rejection grade ≥ 2R were considered to experience a clinically relevant AR, which was treated with antirejection therapy consisting of 1 g methylprednisolone on three consecutive days. Of these patients (rejectors; n = 28), 15 patients had multiple rejection episodes during the first year after transplantation. Here we studied EMB of the first rejection episode including grade 1R EMB taken 8 days (median; range, 3-56 days) before AR, grade 2R EMB (32 days after transplantation; range, 9-332 days) and grade 1R EMB collected 13 days (median; range, 6-70 days) after rejection treatment. Of patients with ISHLT biopsy grade 0R and 1R EMB the first year after transplantation, which were never treated with antirejection therapy (nonrejectors; n = 14), we included grade 1R EMB taken 30 days (range: 10-202) after transplantation, which is matched in time with the grade 1R EMB of the rejectors (taken 24 days [7-276] after transplantation). Detailed characteristics at the moment of 1R EMB sampling are depicted in Table 2 (1R EMB collected before rejection and 1R of nonrejectors) and Table S1 (SDC, http://links.lww.com/TP/B149) (1R EMB collected after rejection). Antibody-mediated rejection was only examined on clinical indication, and this was not the case for the included EMB. Of all patients, we studied time zero biopsies which were collected before reperfusion during the transplantation procedure. All biopsy samples were collected after informed consent and approval by the local medical ethical committee.
DNA and RNA Isolation
Endomyocardial biopsies were lysed with proteinase K for 2 to 3 hours at 56°C (QIAamp DNA mini kit; Qiagen, Venlo, The Netherlands). Total DNA and RNA was isolated using phenol chloroform extraction as previously described35 without the addition of guanidine thiocyanate to collect both DNA and RNA in the aqueous phase.
Quantitative Analysis of the Methylation Status of the TSDR in FOXP3
In total, 750 ng DNA isolated from the EMB was treated with bisulfite using the EZ DNA Methylation-Direct Kit (Zymo Research, Irvine, CA) according to the manufacturer's instructions. During bisulfite treatment, demethylated cytosines were converted into uracils, whereas methylated cytosines remain unmodified. After the bisulfite treatment, the TSDR of the FOXP3 gene was amplified in duplo (intra-assay CV: 15%) by quantitative real-time polymerase chain reaction (PCR) using the StepOnePlus Real-Time PCR System and the TaqMan Genotyping Master Mix (all Applied Biosystems, Foster City, CA). Methylation-specific and demethylation-specific amplification primers and probes were chosen as suggested by Wieczorek et al.36 The percentage of cells with a demethylated TSDR was calculated using the ratio of amplified demethylated TSDR copies and the sum of amplified methylated and demethylated TSDR copies. In female patients, the percentage of demethylated TSDR was multiplied by 2 to correct for the X-linked nature of the FOXP3 gene.
mRNA Expression Analysis
The mRNA expression levels were quantified using Taqman gene expression assays for FOXP3 (Hs00203958.m1), CD3ε (Hs00167894.m1), IL10 (Hs00174086.m1), TGFβ (Hs00171257.m1), IL2 (Hs00174114.m1), IFNγ (Hs00174143.m1), IL17A (Hs00174383.m1) and the housekeeping gene GAPDH (Hs99999905.m1). Complementary DNA synthesis was performed with random primers and quantitative real-time PCR amplification was performed using the StepOnePlus Real-Time PCR System and the TaqMan Gene Expression Master Mix (all Applied Biosystems).
FOXP3 Expression by Immunohistochemical Analysis
Paraffin-embedded sections of 4 μm thickness were deparaffinized, rehydrated, and incubated with 1.5% H2O2 diluted in phosphate-buffered saline for 30 minutes to block the endogenous peroxidase activity. Antigen retrieval was achieved with microwave treatment in citrate buffer (0.01 M, pH 6.0), and sections were blocked for 30 minutes with 10% normal human serum and 10% normal horse serum in TengT (10 mM Tris, 5 mM EDTA, 0.15 M NaCl, 0.25% gelatin, 0.05% Tween-20, pH 8.0). After incubation overnight at 4°C with the primary antibody FOXP3 (hFOXP3; mouse clone 236A/E7, eBioscience, San Diego, CA; 1:50), the immunoreactivity was visualized with a biotinylated horse-antimouse secondary antibody combined with the Vectastain ABC Elite Kit (Vector Laboratories, Burlingame, CA). 3,3′-Diaminobenzidine (Sigma-Aldrich, Zwijndrecht, The Netherlands) was used as chromogen, and sections were counterstained with hematoxylin (Vector Laboratories). Negative control experiments were performed by omitting the incubation with the primary antibody.
The clinical characteristics of rejectors and nonrejectors were compared with an unpaired t test for continuous variables and a χ2 test or Fisher exact test for discrete variables using Graphpad Prism 5.1. To identify differences between groups the Mann-Whitney U test and Kruskal-Wallis test were used as appropriate. P less than 0.05 for 2 sides was considered statistically significant.
The clinical characteristics age, sex, primary disease, cold ischemia time, number of HLA mismatches, and the immunosuppressive regimen were not significantly different between the rejectors and nonrejectors (Table 1).
nTreg Infiltrate the Cardiac Allograft Before Rejection
In almost all time zero biopsies, cells with a demethylated TSDR, which represent nTreg, were undetectable (Figure 1A). In 1R EMB of nonrejectors, a low percentage of cells with a demethylated TSDR was observed, implying the presence of few nTreg. In contrast, 1R EMB taken before the rejection biopsy showed a significant higher frequency of infiltrating nTreg (P = 0.002) compared to the 1R EMB of nonrejectors. Only 1 of the 19 1R EMB of rejectors showed a percentage of nTreg less than 0.06%, whereas this was the case for 11 of 20 1R EMB of the nonrejectors (Figure 1B). Detailed characteristics, including primary disease, timing of histology, number of rejections the first year after transplantation, and trough levels of immunosuppressive medication at the moment of biopsy sampling are depicted in Table 2, though could not explain the relative range in the percentage of nTreg observed in the 1R EMB of both the rejectors and nonrejectors. Analysis of 2 1R EMB of the same patient (nonrejectors) demonstrated already variation in the percentage of nTreg (Table 2). Both the rejection EMB (scored as a 2R) and the 1R EMB collected after rejection displayed a significantly higher percentage of nTreg compared to 1R EMB of nonrejectors (P < 0.0001 and P = 0.0005; Figure 1A), whereas there was no significant difference (P = 0.6) between the different EMB sampling points of the rejectors. The relative high percentage of nTreg in the 1R EMB collected after rejection did not correlate with the number of rejection episodes in the first year after transplantation (Figure 1C). Detailed characteristics of the 1R EMB sampling after rejection is depicted in Table S1 (SDC, http://links.lww.com/TP/B149).
Total Treg Population Before Rejection is Comparable to the Total Treg Population in Nonrejectors
After transplantation, the expression of FOXP3 significantly increased in both EMB of nonrejectors and rejectors (Figure 2A; P < 0.0001). FOXP3 mRNA expression levels in the 1R EMB of nonrejectors were comparable (P = 0.3) to the FOXP3 mRNA expression levels in the 1R EMB taken before rejection. There was no significant difference (P = 0.6) in the FOXP3 mRNA expression between the different EMB sampling points of the rejectors. The FOXP3 mRNA expression levels in the 2R EMB were significantly increased compared to the expression in the 1R EMB of nonrejectors (P = 0.03). Relative low expression of FOXP3 after rejection (1R EMB sampled after AR) is associated with the development of multiple rejection episodes the first year after transplantation (Figure 2B).
Characterization of 1R EMB Taken Before Rejection and 1R EMB of Nonrejectors
FOXP3+ cells were identified with immunohistochemical staining in only 8 1R EMB of our study population (4 rejectors and 4 nonrejectors) as the other material has all been used to set the diagnosis. The number of FOXP3+ cells in the 1R EMB of nonrejectors varied from 0 to 2 per slide, whereas the 1R EMB taken before rejection displayed 2 to 12 positive cells per slide (Figure 3A-B). Next, we analyzed the mRNA expression of CD3, IL10, TGFβ, IL2, IFNγ and IL17A in 1R EMB of rejectors and nonrejectors. The expression of CD3 significantly increased after transplantation (Figure 3C; P < 0.005), though comparing the 1R EMB taken before rejection with the 1R EMB of nonrejectors demonstrated no significant differences. The expression of the Treg-associated cytokines IL10 and TGFβ increased after transplantation, but there was no significant difference in expression comparing the 1R EMB (Figure 3D-E). The expression of the proinflammatory cytokines IL2, IFNγ and IL17A was not detectable in several EMB, though the EMB in which the expression was detectable demonstrated that the expression was not significantly different in the 1R EMB of rejectors compared to the 1R EMB of nonrejectors (Figure 3F-H). We did not observe a correlation between the expression levels of CD3, IL10, TGFβ, IL2, IFNγ, and IL17A and the number of AR episodes. Moreover, the ratio FOXP3/CD3 was not significantly different in the 1R EMB of rejectors compared to 1R EMB of nonrejectors, and this ratio did not correlate with subsequent rejections.
Based on the demethylation status of the FOXP3 gene, we demonstrate here that nTreg infiltrate the cardiac allograft before a histomorphological-proven AR. Moreover, 1R EMB of nonrejectors show a low percentage of nTreg, whereas the total Treg population, as measured by the FOXP3 mRNA expression levels, was comparable to 1R EMB taken before rejection. Because nonrejectors do not develop an AR necessitating antirejection therapy, it is tempting to speculate that these FOXP3+ cells represents cells with a immune regulatory function (iTreg) and not FOXP3+-activated T cells.
Intragraft FOXP3+ T cells are associated with graft acceptance and better graft survival in both mice and humans.23,37,38 Adoptive transfer experiments have demonstrated the immunosuppressive capacities of intragraft FOXP3+ T cells39; nevertheless, FOXP3 expression has also been associated with rejection,26,40 and we previously reported the highest FOXP3 mRNA expression in the rejection cardiac biopsies.25 Most likely, these conflicting data are explained by the fact that FOXP3 is not exclusively expressed by a single cell type, but is expressed by nTreg, iTreg, and also activated T cells. Demethylation of the TSDR in the FOXP3 gene identifies nTreg,18-20 though both iTreg and activated FOXP3+ T cells are highly methylated at the TSDR. The lack of specific (extracellular) markers to identify iTreg in humans hinders subsequent functional experiments to prove that the observed intragraft FOXP3+ cells with a methylated TSDR in the 1R EMB of nonrejectors are indeed iTreg with immunosuppressive capacities and antigen specificities. Nevertheless, because these patients do not develop a clinically relevant rejection, it is probable that at least part of these FOXP3+ cells represents iTreg.
The in vivo generation of FOXP3+ iTreg under both inflammatory and non-inflammatory conditions is now being accepted11; however, an important remaining question is whether they are just byproducts or indeed capable of regulating the immune response. Recently Haribhai et al41 demonstrated that nTregs alone are not capable to fully prevent the development of autoimmune disease in FOXP3-deficient mice, whereas the combination of nTreg and in vivo–generated iTreg is. This essential and additional role of iTreg in the regulation of immune tolerance might be explained by the difference in antigen specificity as nTreg are selected by self-antigens, whereas iTreg are induced locally at the site of inflammation.41,42 Translation of these findings into the transplantation setting would imply that iTregs are induced locally by alloantigen and could regulate the alloresponse more specifically compared to nTreg.
The graft itself, so exposure to donor-antigen, is essential for the generation of potent suppressors of the alloresponse.28,29 Studies in a humanized mouse model of skin transplantation as well as studies in a heart transplantation model in mice demonstrated that donor-antigen–specific Tregs more effectively suppress the alloresponse than polyclonal Treg.43,44 Also in humans, the generation of donor-specific Treg after kidney transplantation has been reported.45 All these studies demonstrate that donor antigen specificity is crucial for suppression of the antidonor-directed immune response, but the origin and relative contribution of different Treg subsets are unclear. In line with the findings of Haribhai et al41 in autoimmunity, Brennan et al44 demonstrated that allospecific nTreg alone ineffectively controlled cardiac rejection in mice, despite infusion of large numbers. Based on our findings in the 1R EMB of nonrejectors, we speculate that donor-antigen specific FOXP3+ iTreg are generated locally after heart transplantation which contribute to the effective suppression of the alloresponse. In other cases, in the rejectors, donor-antigen–specific iTregs are either not generated or not capable of dampening the alloresponse leading to more antidonor-directed immunoreactivity and eventually a rejection episode necessitating antirejection therapy. In line with the finding of Muthukumar et al46 that high FOXP3 mRNA levels in urinary samples during rejection in human kidney transplant patients is associated with successful reversal of rejection, we observed that higher levels of FOXP3 mRNA in EMB collected after rejection did not result in a next rejection episode in the first year after transplantation. Moreover, we demonstrate that relative high levels of nTreg during rejection could not prevent the development of a next rejection episode, which confirms our speculation that iTregs are involved in the effective regulation of the alloimmune response after transplantation.
The infiltration of nTreg in EMB of rejectors before rejection probably reflects immunoreactivity as infiltrating Treg follow a similar migratory pattern as alloreactive effector T cells.47 Even though these patients do develop a rejection episode necessitating antirejection therapy, there is no reason to assume that these nTreg have lost their immunosuppressive capacities. More likely, these nTreg are outnumbered and overruled by alloreactive T cells.48,49 The fact that the alloresponse is effectively dampened in the 1R EMB of nonrejectors cannot be explained by differences in the expression of the classical cytokines like IL2, IFNγ, IL17A, IL10, and TGFβ. Future experiments using a genome-wide approach (e.g., mRNA expression levels or at the epigenetic level studying DNA methylation) might be useful to determine the molecular and phenotypical differences in the cellular infiltrates to explain the suppressive mechanisms in the 1R EMB of nonrejectors.
Our results indicate that nTreg infiltrate the graft before rejection, suggesting that nTregs are unable to successfully impede or suppress the alloresponse. Possibly, this can be used for prediction of clinically relevant rejections, giving opportunities for timely intensifying the immunosuppressive maintenance therapy. Moreover, our results suggest that iTreg contribute to the suppression of the alloresponse. Because of the lack of specific iTreg markers, we were unable to ascertain the immunosuppressive capacities in vitro of the observed intragraft FOXP3+ cells with a methylated TSDR in the 1R EMB of nonrejectors. Still our data serve as the first indication that iTreg might play a possible role in the regulation of alloreactivity after solid organ transplantation.
The authors thank D. Lindenbergh-Kortleve for the technical assistance with the FOXP3 immunohistochemical staining.
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