Naturally occurring CD4+CD25+ T-regulator (Treg) cells, which express the forkhead box protein 3 (Foxp3) transcription factor, are generated in the thymus and are characterized by the ability to suppress proliferation of T-effector (Teff) cells in vitro.1,2 Although the immunoregulatory ability of Treg is no longer contested, where and how this population of CD4 T cells is generated and developed remains largely unknown. The major debate centers on whether Treg are generated only in the thymus. Recent evidence suggests that Treg may also be induced in the periphery.3,4 One crucial issue is whether Treg can be induced or converted from normal peripheral CD4 T cells and if this occurs, whether this converted Tregs have suppressive ability.5,6
Rapamycin (RAPA), a macrolide antibiotic produced by Streptomyces hygroscopicus, is an immunosuppressive agent widely used for preventing acute graft rejection in patients, and has been used to induce operational tolerance in mouse models.7 RAPA binds to FK506- binding protein-12, a highly conserved cytoplasmic receptor. The FK506-binding protein-12-RAPA complex then binds to and inhibits the activities of the serine/ threonine protein kinase mammalian target of RAPA, the activation of which is essential for protein translation and cell cycle initiation in T cells.8 Unlike other commonly used immunosuppressants, RAPA does not appear to interfere with tolerance induction.9,10 Some reports demonstrated that stimulation of CD4+ T cells from spleens of ovalbumin (OVA)-T cell receptor (TCR) transgenic mice in the presence of RAPA resulted in the selective expansion of naturally occurring CD4+CD25+ Treg cells, which can suppress allograft rejection in a model of allogeneic pancreatic islet transplantation.11
There is little information on how RAPA induces Teff cells to become Treg cells. In this paper, we presented evidence that RAPA can convert naive CD4+CD25− T cells into CD4+Foxp3+ regulatory T cells in vitro. We also found that the converted regulatory T cells could inhibit immune responses and maintain antigenic specificity in vitro.
Male C57BL/6 (H-2b), DBA/2 (H-2d), and C3H (H-2k) mice were provided by Beijing Vital River Laboratory Animal Co., Ltd. (Beijing, China) and were maintained in the animal facilities of Peking University People's Hospital. Animal experiments were approved by the Local Animal Ethics Committee.
Purification of CD4+CD25+, CD4+CD25−, and CD4+CD25+CD127− T cells
Single-cell suspensions were prepared from mouse spleens and lymph nodes of C57BL/6 mice, and red blood cells (RBCs) were removed using RBC lysis buffer (eBiosource, USA). CD4+CD25+ Treg cells were isolated using CD4+CD25+ Treg isolation kits (R&D Systems Inc., USA). CD4+CD25− T cells were isolated using T-cell enrichment columns (R&D Systems Inc.) and by subsequent deletion of fluorochrome-conjugated antibodies to mouse CD25, Ter119, CD11b, CD8, B220 and NK1.1 (eBiosource) positive cells using microbeads (Miltenyi Biotec, DE) and magnetic separation columns (MSC) (Miltenyi Biotec). CD4+CD25+CD127− fractions were isolated from the mixed lymphocyte reactions (MLR) with CD127 (eBiosource) negative selection, followed by CD4 (eBiosource) and CD25 positive selection using microbeads and MSC.
Purification of mitomycin C (MMC)-treated B cells
Single-cell suspensions were prepared from the spleens of DBA/2 mice. RBCs were removed using RBC lysis buffer, then B220+ cells were selected using microbeads and MSC. Purified B220+ cells were treated with 25 μg/ml MMC (Sigma, USA) at 37°C for 30 minutes.
Purification of maturation of dendritic cells (mDC)
Bone marrow cells were aspirated and harvested from DBA/2 mice femurs and tibias. RBCs were removed using RBC lysis buffer, then GR1, Ter119 and B220 (eBiosource) positive cells were depleted using microbeads and MSC. Purified bone marrow cells were cultured with 20 ng/ml granulocyte-macrophage colony-stimulating factor and treated with lipopolysaccharide (Sigma) on day 5. mDCs were separated by positive selection of CD86 on day 6.
Mixed lymphocyte reaction (MLR)
In the converted experiment, CD4+CD25− T cells were incubated for 6 days in 96-well round-bottom plates with MMC-treated B cells at a 1:1 ratio (1×105 T cells: 1×105 MMC-treated B cells) with RAPA (LC laboratories, USA) added to the plate at the different concentrations. CD4+CD25− T Cells were harvested after 6 days and were stained using the Foxp3 detection kit (eBiosource). Cells were analyzed for expression of Foxp3 using flow cytometry (BD Biosciences, USA).
A total of 1×105 Carboxyfluorescein succinimidyl ester (CFSE) (Invitrogen, USA) labeled naive CD4+CD25− T cells/well from C57BL/6 mice were cocultured with DBA/2 or C3H mDCs (0.25×105/well) in 96-well round-bottom plates for 6 days. 1×105 or 0.25×105 converted Treg cells were added in every well as regulatory cells. Cells were harvested after 6 days culture and analyzed for proliferation of CFSE-labeled naive CD4+CD25− T cells using flow cytometry. Data were analyzed using CellQuest software.
Real-time polymerase chain reaction (rt-PCR)
mRNA was extracted from cells using an RNeasy mini-kit (Qiagen, NED). Reverse transcription to cDNA was performed using TaqMan reverse transcription reagents (Applied Biosystems, EU). Specific message levels were quantified by rt-PCR using the ABI 7300 Sequence Detection System (Applied Biosystems). A comparative threshold cycle (CT) method was used to determine foxp3 mRNA levels (Applied Biosystems, assay ID: Mm00475156_m1). Relative expression was analyzed against endogenous murine glyceraldehyde-3- phosphate dehydrogenase (GAPDH, Applied Biosystems, assay ID: Mm99999915_g1). For each sample, the Foxp3 CT value was normalized using the formula ΔCT= CTFoxp3−CTGAPDH. The mean ΔCT was determined, and the relative foxp3 mRNA expression was calculated with 2−ΔCT.
All data analyses were performed using Statistical Package for Social Sciences software (SPSS for Windows, version 13.0). Comparisons of data were analyzed by student's t test. A P value <0.05 was considered statistically significant.
RAPA converted CD4+CD25− T cells into CD4+Foxp3+ T cells in vitro with MMC-treated B cells as antigen-presenting cells (APCs)
We determined whether RAPA can affect de novo expression of Foxp3 by peripheral T cells that were stimulated with MMC-treated DBA/2 mouse B cells as APCs. Highly purified (99% pure) CD4+CD25− T cells served as the Teff cells and were cultured with MMC-treated B cells and RAPA. We found that RAPA resulted in the conversion of 7%–8% of naive CD4+CD25− T cells into the Foxp3+ Treg phenotype after 6 days of culture. RAPA induction of Foxp3 is dose-dependent from 1 nmol/L to 100 nmol/L, with maximum conversion at a concentration of 100 nmol/L at day 6 (Figure 1).
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Figure 1. Microbeads and MSC-isolated CD4+CD25− cells from naive mice were stimulated with MMC-treated B cells alone or in the presence of increasing doses of RAPA (1 nmol/L, 10 nmol/L, 100 nmol/L, 150 nmol/L) for 6 days in vitro. Foxp3 expression was monitored by FACS. The percentages of Foxp3+ cells within the total CD4+ T cell population are indicated. One representative result from at least three is shown (A). Induction of Foxp3 by RAPA is dose-dependent from 1 nmol/L to 100 nmol/L, peaking in the concentration of 100 nmol/L at day 6 and shown as means of three independent experiments (B). Error bars represent standard deviation.
Isolating Treg cells from the MLR
Our method of Foxp3+ staining effectively distinguishes the Teff and the Treg populations, but results in cell inactivation. CD127− cells comprise >75% of the Foxp3+ cells in the entire population of CD4+ T cells.12,13 Therefore, we obtained CD4+CD25+CD127− T cells using microbeads and MSC from naive C57BL/6 CD4+CD25− T cells after a 6-day MLR with DBA/2 MMC-treated B cells plus 100 nmol/L RAPA. We found that there was no difference in the percentage of Foxp3 expression between naive C57BL/6 CD4+CD25+ T cells and converted Treg by flow cytometry. Foxp3 expressions were (72.84±2.38)% and (75.90±1.59)% in naive CD4+CD25+ T cells and converted Treg (CD4+CD25+CD127−) cells, respectively (P >0.05. Figure 2). To determine the mechanism of the suppression, mRNA was isolated from naive C57BL/6 CD4+CD25+ T cells and from the converted Treg cell preparations. We analyzed Foxp3 gene expression of the two groups using rt-PCR. The Foxp3 levels were not different between the two groups.
RAPA-converted Treg cells (CD4+CD25+CD127−) have suppressive ability in vitro
We further determined whether CD4+CD25+CD127− T cells from the MLR can suppress alloantigen-triggered proliferation of naive CD4+CD25− T cells. As shown in Figure 3, CFSE-labeled naive C57BL/6 CD4+CD25− T cells underwent proliferation during a 6-day co-cultivation with either allogeneic DBA/2- or C3H-derived mDCs. We compared the suppressive ability of converted Treg cells (derived from naive C57BL/6 CD4+CD25− T cells after a 6-day MLR with DBA/2 MMC-treated B cells plus 100 nmol/L RAPA) with C57BL/6 naive CD4+CD25+ T cells when they were cocultured with the same DBA/2 mDCs. Figures 3A and 3B show that the ability of converted Treg cells to suppress alloantigen-triggered proliferation of naive CD4+CD25− T cells was significantly higher than that of C57BL/6 naive CD4+CD25+ T cells. The inhibition rates were, respectively, (87.4±4.6)% and (68.1±3.9)% when CD4+CD25− T cells were cocultured with converted Treg cells or naive CD4+CD25+ T cells at a 1:1 ratio and (76.8±4.6)% and (53.5±6.1)% when CD4+CD25− T cells were cocultured with converted Treg cells or naive CD4+CD25+ T cells at a 4:1 ratio. We found that CD4+CD25+CD127− T cells obtained in the MLR with MMC-treated B cells of DBA/2 mice plus 100 nmol/L RAPA exert powerful inhibition on naive CD4+CD25− T cells stimulated with the DBA/2-derived mDCs.
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Figure 2. Foxp3+ expression was compared between the C57BL/6 converted Treg cells (CD4+CD25+CD127− T cells) and C57BL/6 naive CD4+CD25+ T cells. One representative result from at least three experiments is shown.
The suppression of converted Treg cells tended to be alloantigen specific, as shown in Figures 3B and 3C. CD4+CD25+CD127− T cells converted during DBA/2 alloantigen stimulation suppressed C3H alloantigen- triggered naive CD4+CD25− T cell proliferation in suppression assay with lower efficiency. The inhibition rate decreased from (87.4±4.6)% to (69.4±5.0)% when CD4+CD25− T cells or converted Treg cells were added to the coculture with allogeneic DBA/2-derived mDCs at a 1:1 ratio and from (76.8±4.6)% to (53.5±6.1)% when CD4+CD25− T cells or converted Treg cells were added to the coculture with allogeneic C3H-derived mDCs at a 4:1 ratio.
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Figure 3. C57BL/6 naive CD4+CD25+ T cells suppress CFSE-labeled C57BL/6 CD4+ CD25− T-cell proliferation triggered by the mature DBA/2 DCs. The horizontal bars gate the generation of the dividing cells and the numbers refer to the percentages of these cells. The percentages of these M1 cells may reflect the inhibition rate (A). C57BL/6 converted Treg cells (derived from naive C57BL/6 CD4+CD25− T cells after a 6-day MLR with DBA/2 MMC–treated B cells plus 100 nmol/L RAPA) suppress CFSE-labeled C57BL/6 CD4+CD25− T cell proliferation triggered by the mature DBA/2 DCs (B). C57BL/6 converted Treg cells suppress CFSE-labeled C57BL/6 CD4+CD25− T-cell proliferation triggered by third party alloantigens (mature C3H DCs) at lower efficacy (C). The experiment was shown as means of 3 independent experiments. Error bars represent standard deviation. The rates of inhibition were (68.1±3.93)%, (87.4±4.55)%, (69.4±4.97)% at a 1:1 ratio (Teff:Treg), and were (54.8±4.2)%, (76.8±4.6)%, (53.5±6.1)% at a 4:1 ratio (Teff:Treg), and were (40.07±0.96)%, (40.07±0.96)%, (44.92±7.65)% at a 1:0 ratio (Teff:Treg). Statistical analyses was significant difference between the A, C and B (P <0.05), however no significant difference between the A and C (P >0.05) (D).
RAPA is widely used to effectively prevent transplant rejection. This drug is a potent and reasonably well-tolerated immunosuppressive agent, but its effects on graft-destructive Teff and graft-protective Treg cells are drastically different. Some studies demonstrated that RAPA efficacy is not due to selective expansion of naturally-occurring Treg, but induce suppressor functions in conventional CD4+ T cells upon TCR-mediated stimulation.14 RAPA may reverse the effects of AKT as a strong repressor after converting into the Treg phenotype in vitro and in vivo.15 In several other models, the ability of maturation-resistant, RAPA-conditioned DCs are markedly impaired in Foxp3− T cell allostimulatory capacity which favors the stimulation of murine alloantigen-specific CD4+CD25+Foxp3+ Treg cells.16 In all of the above mentioned studies, the researchers focus mainly on the effects of RAPA on Treg cells. However, only a few studies have examined the effects of RAPA on Teff cells.
The current study focuses on the role of RAPA during the conversion of Teff cells to Treg cells. Our experiments show that 100 nmol/L RAPA promotes conversion of about 7% of CD4+CD25− cells stimulated with MMC-treated B cells, with conversion peaking after 6 days of culture and acquisition of alloantigen-specificity. But when the concentration of RAPA exceeds 100 nmol/L, the number of converted CD4+CD25− T cells decreased. RAPA markedly enhanced the percentages of CD4+CD25+ Treg cells at a low dose, but it significantly reduced the cell numbers of all thymocyte subsets at a high dose.17 These results suggest an additional mechanism by which RAPA induces tolerance.
T-cell costimulation by APC, anti-CD3 and anti-CD28 plays important roles in the activation, proliferation and conversion of T cells. In addition to TCR recognition and binding to antigen-MHC, these constimulatory interactions provide a second activation signal. In the absence of costimulatory signals, T-cell responsiveness is impaired.18 Recent studies showed that treatment with transforming growth factor (TGF)-β and RAPA resulted in the conversion of naive CD4+Foxp3− T cells into the Foxp3+ Treg phenotype when stimulated with plate-bound anti-CD3 and anti-CD28.19 However, the Foxp3+ Treg cells were not alloantigen-specific. A previously-published report suggests that dendritic cells (DCs) partially abrogate the regulatory activity of Treg cells from the peripheral blood.20 B cells are seldom considered as professional APCs, and the majority of previous work has focused primarily on their role as terminal effectors as antibody producers.21-23 However, several reports suggest that B cell-induced Treg cells are crucial for resolving experimental autoimmune encephalomyelitis; only after reconstitution of mice with wild type B cells, did Treg numbers normalize and experimental autoimmune encephalomyelitis (EAE) symptoms disappear.24 Other studies suggest that B cells are able to directly block the proliferation of Treg cells.25,26 Therefore, in our experiments during the suppression assay, we used mDCs instead of MMC-treated B cells as APC. B cells are very poor APCs compared with DC, and although B cells have antigen-presenting function in MLR, B cells can also inhibit Treg cell proliferation.27-29 Thus, we selected mDCs as APCs to provide costimulation for CD4+CD25+CD127− T cells.
Other reports11 have shown that the CD4 and CD25 phenotype is essential for Treg cell function. CD4+CD25+ regulatory T cells have emerged as a unique subgroup of suppressor T cells that maintain peripheral immune tolerance. The extrathymic de novo generation of Treg cells is of considerable interest. Identification of Foxp3+ cells suggests that there may be a larger population of Treg cells in human peripheral blood than previously anticipated, although this is controversial due to unanticipated expression in several activated CD25− T cell populations.30,31 Foxp3 expression cannot be used to purify cells, and no cell surface markers can be used to isolate these and other T cell subsets to examine Treg cell activity. Activated CD4+ T cells expressing CD25 under neutral TCR stimulation conditions show no suppressive ability.5,6 For this reason, isolation of Treg cells cannot depend solely on CD25 as a marker. Using a Foxp3−/GFP fusion protein knock-in mouse, Fontenot et al reported that induction of Foxp3 expression in CD4+GFP− T cells may be obtained through fluorescence activated cell sorting.9 It has been recently reported that CD127 expression is down-regulated on CD4+ Treg cells, and is inversely correlated with Foxp3 expression.12,13 It was shown that 94% of CD4+CD25+CD127− T cells express Foxp3, suggesting that better cell surface markers for the identification of CD4+Foxp3+ T cells are CD127 and CD25.12 Similar results were observed in mice and humans.32 CD4+ T cells isolated from Foxp3-GFP knock-in mice were stained for CD127, and the vast majority of CD4+Foxp3+ T cells were CD127−.31,33 Others found that natural Treg cells constitutively expressed high amounts of folate receptor 4 (FR4). This claim will be substantiated in next experiment.34
Among many unsolved questions regarding Treg cells, arguably of most important, is to find ways to promote their suppressive activity or to increase their numbers for their potential clinical application. Since successful application of adoptive cell therapy requires a large number of Treg, and the frequency of alloantigen specific Treg in naive hosts is extremely low, attention has been turned to clonal expansion or induction of Treg.35 It has been shown that expanded Tregs retain their regulatory activity.36 So, it is important to study the production and suppression of induced Treg cells.
In our experiments, CD4+CD25− Teff cells stimulated with MMC-treated B cells and RAPA for 6 days were isolated using CD4, CD25 and CD127 markers to obtain the converted Treg cells (CD4+CD25+CD127− T cells). We observed 70%–75% CD4+Foxp3+ T cells in the CD4+CD25+CD127− T cell subset. We examined their ability to suppress allogeneic naive CD4+CD25− and to respond to allogeneic APCs,37 and we showed that CD4+CD25+CD127− cells are Treg cells. Our results demonstrate that these converted Tregs cells express the Foxp3+ gene and suppress allogeneic naive CD4+CD25− T cells. The CD4+CD25+CD127+ population cannot do so, despite some expression of Foxp3+. In addition, these converted Treg cells retained antigen specificity because they were cultivated with allogeneic B cells as APCs. Antigen specificity of converted Treg cells may render them more potent regulators of secondary immune responses in vitro. Whether the induction of allogeneic- specific Treg can cause skewing of the TCR repertoire of the Treg population towards specific antigens, or if an enhancement of the suppressive capacity on a per cell basis might play a role, requires further elucidation.38
In summary, our findings provide evidence that RAPA induces T regulatory cell conversion from T effector cells and this type of cell displays more potent regulatory ability in vitro; meanwhile our results uncover an additional mechanism for tolerance induction by RAPA.
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