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Interplay between mTOR and STAT5 signaling modulates the balance between regulatory and effective T cells Juan Shan a,b , Li Feng a , Guixiang Sun a , Peng Chen a , Yanni Zhou a , Mengjuan Xia a , Hongsheng Li a , Youping Li a,c,∗ a Key Laboratory of Transplant Engineering and Immunology of Health Ministry of China, West China Hospital, Sichuan University, Chengdu 610041, Sichuan Province, PR China b Regenerative Medicine Research Center, West China Hospital, Sichuan University, Chengdu 610041, Sichuan Province, PR China c Chinese Cochrane Centre, Chinese Evidence-Based Medicine Centre, Chengdu 610041, Sichuan Province, PR China
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Article history: Received 17 February 2014 Accepted 21 October 2014 Available online xxx Keywords: Regulatory T cell mTOR signaling STAT5 signaling MicroRNA-155 Immune balance Rapamycin
a b s t r a c t Background: Immune response outcome, inflammation or tolerance, often depends on the balance between regulatory T cells (Tregs) and effective T cells (Teffs). Rapamycin (Rapa) has been reported to selectively expand Tregs and promote de novo generation of foxp3+ Tregs, suggesting its potential role in inducing tolerance. But the mechanism by which Rapa regulating the Treg–Teff balance is yet to be understood. Methods: Mouse CD4+ CD25− Teffs and CD4+ CD25+ Tregs are sorted by MACS. These T cell subsets were labeled with CFSE and cultured with anti-CD3/CD28 Ab ± IL-2 for 6 days. Two rounds of stimulation of 3 days each were performed. Rapa or Jak Inhibitor I was added to the culture when indicated. Cells were harvested after each round of stimulation. CFSE dilution, FOXP3, miR-155 expression and the signaling via the mTOR and STAT5 pathways were determined. And miR-155-mimic and miR-155-antagomir were transfected into purified CD4+ T cells respectively to detect miR-155 role in regulating STAT5 signaling. Results: Firstly, we confirmed that the effect of Rapa on proliferating T cells is time-dependent: it reduces both Teffs and Tregs proliferation at early stage, but selectively promotes Tregs proliferation after second round of stimulation. Then we found there is direct interaction between mTOR and STAT5 signaling, and this interaction explained the time-dependent effect of Rapa and may participate in deciding Teff–Treg balance: mTOR inhibition up-regulated the expression of phos-STAT5 in both proliferating Tregs and Teffs via miR-155. And foxp3 is the down streaming target of phos-STAT5, thus Rapa could maintain expanded Tregs function and promote de novo generation of foxp3+ Tregs. However, the phos-4E-BP1 expression pattern is different in proliferating Tregs and Teffs. 4E-BP1 is the common target of mTOR and STAT5 signaling, and plays a key role in cell proliferation. Rapa inhibits phos-4E-BP1 expression in both Tregs and Teffs at early stage of proliferation, but selectively raises its expression in Tregs after second round of stimulation. This may explains why Rapa inhibits Teffs growth, but delays Tregs proliferation. Conclusion: Together, these findings indicate that the dynamic interaction between mTOR and STAT5 signaling modulates the reciprocal differentiation of the effective and regulatory T cells, and differently affect their proliferation activity. This provides a new insight of how Treg–Teff balance is regulated. © 2014 Elsevier GmbH. All rights reserved.
Introduction
Abbreviations: Tregs, regulatory T cells; Teffs, effective T cells; Rapa, Rapamycin; miR-155, micro-RNA 155. ∗ Corresponding author at: Key Laboratory of Transplant Engineering and Immunology of Health Ministry of China, West China Hospital, Sichuan University, Chengdu 610041, Sichuan Province, PR China. Tel.: +86 028 85164032; fax: +86 028 85164034. E-mail address: [email protected] (Y. Li).
The balance between effector T cells (Teffs) and regulatory T cells (Tregs) is critical for establishing tolerance to self- and non-self-antigens. Directing T-cell differentiation toward Tregs has important clinical implications for treating autoimmune disease and enabling transplant tolerance. Rapamycin (Rapa) is an immunosuppressive drug used to prevent allograft rejection. It inhibits proliferation of many cell types, including T cells, by inhibiting the serine/threonine protein
http://dx.doi.org/10.1016/j.imbio.2014.10.020 0171-2985/© 2014 Elsevier GmbH. All rights reserved.
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kinase called mammalian target of Rapa (mTOR), the activation of which is required for protein synthesis and cell-cycle progression. However, Tregs and Teffs are not equally sensitive to Rapa: it selectively blocks proliferation of CD4+ CD25− Teffs, whereas allows CD4+ CD25+ Tregs to growth in long-term culture (Battaglia et al., 2005; Strauss et al., 2007, 2009). Moreover, Rapa can promote de novo generation of foxp3+ Tregs from naïve Teffs (Battaglia et al., 2005; Gabryˇsová et al., 2011), and appears to maintain or induce Tregs in vivo and promotes tolerance in transplant recipients when compared to calcineurin inhibitors (Ruggenenti et al., 2007; Hendrikx et al., 2009; Bestard et al., 2011; Ekong et al., 2012). These studies highlight the importance of Rapa and its target mTOR in controlling the delicate balance between Teffs and Tregs. But the mechanism by which Rapa selectively expands Tregs and promotes foxp3 expression is yet to be understood. It is worth noting that only in long-term culture can Rapa greatly promotes Tregs proliferation (Battaglia et al., 2005; Strauss et al., 2007, 2009). It reduces both Teffs and Tregs proliferation at early stage of stimulation when compared to medium cultures (KeeverTaylor et al., 2007; Basu et al., 2008; Zeiser et al., 2008; Golovina et al., 2008). This suggests that the role of mTOR signaling in proliferating Tregs is time-varying. Pim-2 is a serine/threonine-protein kinase which has considerable overlap with mTOR by sharing common downstream targets including Bad and 4E binding protein-1 (4E-bp1) (White, 2003), so it can mediate resistance to Rapa. In addition, IL-2 and STAT5 signaling is critically required for Tregs development and foxp3 expression (Antov et al., 2003), and STAT5 phosphorylation can also induce pim-2 (Mizuki et al., 2003). However, the relationship between mTOR, pim-2 and STAT5 in T cells is not fully delineated. And we thought of the interaction and differential expression pattern of these signaling molecules in Tregs and Teffs might be the mechanism by which Rapa regulating the Treg/Teff balance. In this study, we show that there is direct interaction between mTOR and STAT5 signaling in both Tregs and Teffs: mTOR inhibition weakens Teffs growth, but make Tregs shift to STAT5/pim-2 dependent proliferation, which is more beneficial to foxp3 maintain and induction. These results demonstrate that the interplay between mTOR and STAT5 signaling participated in modulating the Treg–Teff balance. Materials and methods Animal Male BALB/c (H-2d) mice weighting 25–30 g and aging 8–12 weeks were used in these experiments. They were bred and maintained under specific pathogen-free conditions in Experimental Animal Center of West China hospital (Sichuan University, Chengdu, China), and all experiments were performed in accordance with the protocols approved by the Institutional Animal Care and Use Committee.
fraction using the CD25-PE mAbs and anti-PE microbeads. The positive selection fraction is CD4+ CD25+ Tregs, and the negative depletion fraction is CD4+ CD25− Teffs. Purity ranged from 92% to 98% as measured by flow cytometry. T cells culture and stimulation Purified CD4+ CD25+ Tregs and CD4+ CD25− Teffs were cultured in U-bottomed, 96-well plates and stimulated when indicated with 2 g/ml CD3 (clone 145-2C11; BD Biosciences), 2 g/ml CD28 (clone 37.51; BD Biosciences), 1000 IU/ml IL-2, at a concentration of 1 × 105 cells/well. Two rounds of stimulation of 3 days each were performed. Culture medium consisted of RPMI 1640 supplemented with 100 U/ml penicillin, 100 mg/ml streptomycin, 2 mM l-glutamine, 10 mM Hepes, 0.1 mM non-essential amino acids, 1 mM sodium pyruvate, 50 M 2-mercaptoethanol and 10% fetal calf serum. For proliferation detection, T cells (1 × 106 ) were pre-labeled with 5 M CFSE (Sigma–Aldrich, St. Louis, MO) before stimulation. After 2 or 5 days cultured T cells were harvested and analyzed by FACS or Western blot. mTOR and STAT5 signaling blockers Rapamycin (Sigma–Aldrich, St Louis, MO, USA) 100 nM was used to block the PI3K/mTOR signaling, and Jak Inhibitor I (Calbiochem, Merck KGaA, Darmstadt, Germany) 1 M was used to block the phosphorylation of STAT5. Similar volumes of DMSO vehicle were added to T-cell cultures as control groups. Flow cytometry analysis Intracellular FOXP3 staining was performed using the FOXP3 Fix/Perm Buffer set (Biolegend) according to the manufacturer’s recommendations. Western blot analysis Total cell lysates and western blot analysis were performed as previously described (Luo et al., 2012). The antibodies used were the following: anti-phospho-p70S6K, anti-phospho-S6, antiphospho-STAT5 (Tyr694), and anti-phospho-4E-BP-1 (all from Cell Signaling Technology, Beverly, MA); anti-Pim-2 (EPR6987; Epitomics, Burlingame, CA); and anti-FoxP3 (150D/E4; eBioscience, San Diego, CA). Quantitative real-time PCR Total RNA (including miRNAs) was extracted using MirVana TM miRNA Isolation Kit (Ambion, Austin, USA) according to the manufacturer’s instructions. After transcription into cDNA, the expression of miR-155 respective of the normalizer U6 was quantified using the miR-155-5p RT kit and U6-RT primer kit (Ruibo, Guangzhou, China).
Cell isolation and sorting
Nucleofection
Splenic mononuclear cells were obtained by Ficoll-Hypaque density gradient centrifugation. Cells recovered from the gradient interface were washed twice in PBS, counted, and immediately used for MACS using the CD4+ CD25+ regulatory T cell isolation kit (Miltenyi Biotec, Bergisch Gladbach, Germany). Briefly, CD4+ T cells were purified from mononuclear cells by negative selection using biotin-conjugated monoclonal antibodies (mAbs) directed against CD8a, CD11b, CD45R, CD49b, Ter-119, and anti-biotin microbeans, yielding a population of CD4+ cells with purity of 90–95%. Then, CD4+ CD25+ Tregs were positively selected from purified CD4+ T
Nucleofection was performed with Mouse T Cell Nucleofection Kit and Nucleofection device (Amaxa, Koelin, Germany) according to the manufacturer’s recommendations. Briefly, 1 × 106 sorted CD4+ T cells were resuspended in 100 ml nucleofection solution. Five microliters of GFP Vector or oligonucleotides, including miR155-mimic, mimic-control, miR-155-antagomir and antagomir control (Ruibo, Guangzhou, China), were added to the solution and mix gently. Then the mixtures were transferred to electroporation cuvettes and nucleofected using the X-01 program. Five hundred microliters pre-warmed nucleofection medium were added
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Fig. 1. Tregs activated in the presence of Rapa had a delayed kinetic of proliferation. Freshly isolated CD4+ CD25+ Tregs and CD4+ CD25− Teffs were labeled with CFSE, and were activated with anti-CD3/CD28 Ab + IL-2 in the presence or absence of Rapa. CFSE dilution was measured 2 days (A) and 5 days (B) after activation. Percentages of suppression in comparison to proliferation of naive control cells are indicated. *p < 0.05 compared with norapa group.
in the cuvettes immediately after transfection. Transfected cells were transferred to 12-well plates with 1.5 ml pre-warmed nucleofection medium and incubated in a humidified 37 ◦ C/5% CO2 incubator. Dead cells were removed with dead cell removal kit (Miltenyi Biotec, Bergisch Gladbach, Germany) after 6 h incubation. The transfection efficiency which is evaluated by monitoring GFP expression using flow cytometry 24 h after transfection is around 50%. Results
control culture. In contrast, the proliferative activity of Teffs remained significantly reduced (Fig. 1B). These results are consistent with previous data showing that Tregs activated in the presence of Rapa had a delayed kinetic of proliferation. We next examined Foxp3 expression in the expanded T cell populations. The results showed only Rapa-exposed Tregs preserved Foxp3 expression (Fig. 2A). And Rapa treatment increased the frequency of FOXP3+ cells induced in Teffs, but Foxp3 expressing cells were only observed in the divided population (Fig. 2B), suggesting the de novo generation of Foxp3 is depend on Teffs activation.
mTOR inhibition delays Tregs proliferation
Tregs activation requires mTOR signaling
Studies have showed Rapa inhibits both Tregs and Teffs proliferation at the early stage of stimulation, but selectively promote the expansion of Tregs in long-term cultures (Battaglia et al., 2005; Strauss et al., 2007, 2009). We first confirmed T cells in different amplification stage have varying sensitivity to Rapa. To do this, Purified CD4+ CD25+ Tregs and CD4+ CD25− Teffs were activated in vitro with anti-CD3/CD28 Ab + IL-2 in the presence or absence of 100 nM Rapa. Two rounds of stimulation of 3 days each were performed. T cell proliferation was monitored by CFSE dilution after 2 days and 5 days culture. In the first 2 days of culture, the proliferative capacity to polyclonal activation was markedly reduced in both Tregs and Teffs when exposed to Rapa (Fig. 1A). But after second round of stimulation, Tregs cultured with Rapa seems to have resumed its proliferative activity, which proliferated to a similar extent in
For there have been conflicting reports on whether or not mTOR is required in Tregs activation (Zeiser et al., 2008; Wang et al., 2011; Bensinger et al., 2004), we studied the phosphorylated STAT5, p70S6K1 and 4E-BP1 expression and cell proliferation of Tregs in response to different stimulation. P70S6K1 and 4E-BP1 are direct targets of mTOR, which phosphorylation can indicate mTOR activity. Western results showed neither anti-CD3 alone nor in combination with anti-CD28 stimulation can activate Tregs mTOR signaling (Fig. 3A). Tregs constitutively express the IL-2 receptor IL-2R␣ (CD25), so high level expression of phos-STAT5 was detected when stimulated with IL-2 alone (Fig. 3A), however, CFSE results showed Tregs were hypoproliferative to IL-2 (Fig. 3B). Only anti-CD3/CD28 Ab + IL-2 stimulation can activate Tregs, and at the same time activate their mTOR
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Fig. 2. Rapa treatment maintains Foxp3 expression in Tregs, and increases the frequency of induced Foxp3+ T cells in Teffs. Foxp3 expression of 5 days expanded CD4+ CD25+ Tregs was assessed by FACS analysis (A), CD4+ CD25− Teffs on days 2 were detected for CFSE dilution and FOXP3 expression (B). *p < 0.05 compared with norapa group.
signaling (Fig. 3A). These data suggesting Tregs require mTOR signaling to activate, STAT5 signaling alone cannot activate Tregs. In addition, we found an interesting phenomenon that although the concentration of added IL-2 is the same in each group, the expression of phos-STAT5 was higher in Tregs stimulated with IL-2 alone than these stimulated in combination with anti-CD3/CD28Ab (Fig. 3A). And STAT5 signaling is required for Tregs function and their Foxp3 expression (Antov et al., 2003; Angela et al., 2004; Passerini et al., 2007), so we detected Foxp3 expression in the
two groups and the results showed the Foxp3 expression was lower in activated Tregs, when their mTOR signaling was also activated (p < 0.05) (Fig. 3C). So it seems as mTOR activation could lower STAT5 signaling strength, and correspondently reduce the foxp3 expression, suggesting a competition relationship may exist between mTOR and STAT5 signaling, and this is related to the expression of Foxp3. Therefore, we further explore the relationship between the two pathways and its role in Tregs proliferation and Foxp3 expression.
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Fig. 3. Tregs activation requires mTOR signaling. (A) Results of Western blots comparing expression levels of phospho-P70S6K, phospho-4EBP-1 and phospho-STAT5 in CD4+ CD25+ Tregs cultured in different stimulators (anti-CD3 alone or in combination with anti-CD28, IL-2 alone or in combination with anti-CD3/CD28). (B) Proliferation activity of CD4+ CD25+ Tregs cultured in different stimulators for 72 h. One representative experiment is shown. (C) Foxp3 expression of 72 h expanded CD4+ CD25+ Tregs was assessed by FACS analysis *p < 0.05 compared with IL-2 group.
mTOR inhibition force Tregs shift to STAT5/pim-2 dependent proliferation To detect whether there is direct interplay between mTOR and STAT5 signaling, Rapa was used to block the mTOR signaling, the expression of the downstream targets of both mTOR and STAT5 signaling, including phos-STAT5, phos-S6, phos-4E-BP1, Pim-2, and Foxp3, were detected by western blot. The mTOR and STAT5 signaling cascades were showed in Fig. 6. The results showed mTOR signaling was effectively inhibited by Rapa in 2 days cultured Tregs: the expression of phos-S6 and 4E-BP1, downstream targets of mTOR, was lower in Tregs cultured with Rapa than that in control group. But we found that phos-STAT5 expression was up-regulated by Rapa, as well as its downstream target Pim-2. But the expression
of phos-4E-BP1, the common downstream target of mTOR and Pim2, was still higher in medium cultured Tregs than in Rapa-exposed ones (Fig. 4A). However, the situation changed in 5 days cultured Tregs: phos-4E-BP1 expression in Rapa-exposed Tregs exceeded that in control group (Fig. 4B), it seemed as the 4E-BP1 phosphorylated via STAT5/Pim-2 signaling is sufficient to compensate that from mTOR signaling after second round of stimulation. Both phos-S6 and 4E-BP1 play key roles in cell proliferation. These findings indicated that at the early stage of amplification, Rapa reduce Tregs proliferation by inhibiting mTOR/S6 signaling, but force Tregs shift to STAT5/pim-2 dependent proliferation, and promote its proliferation at late stage of amplification via STAT5/Pim-2/4E-BP1 signaling. This may explain why Rapa delay, but not inhibit Tregs proliferation.
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Fig. 4. mTOR inhibition force Tregs shift to STAT5/pim-2 dependent proliferation, which is more beneficial to Foxp3 expression; mTOR inhibition induces Foxp3 expression in Teffs via STAT5, but proliferating Teffs depend more on mTOR signaling than Tregs. Freshly isolated CD4+ CD25+ Tregs were stimulated with anti-CD3/CD28 + IL-2 and cultured in the presence or absence of Rapa. Jak inhibitor was added when indicated. Expression levels of phospho-S6, phospho-STAT5, phospho-4EBP-1, PIM-2 and Foxp3 in 2 days-cultured (A) and 5 days-cultured Tregs (B) were detected by Western blots. CD4+ CD25− Teffs were stimulated with anti-CD3/CD28, and cultured in the presence or absence of Rapa. IL-2 or Jak inhibitor was added to the culture when indicated. Expression levels of phospho-S6, phospho-STAT5, phospho-4EBP-1, PIM-2 and Foxp3 in Rapa-exposed or medium-cultured Teffs were compared by Western blots (C). One representative experiment is shown.
Fig. 5. mTOR inhibition up-regulates STAT5 signaling via miR-155. Freshly isolated CD4+ CD25+ Tregs and CD4+ CD25− Teffs were stimulated with anti-CD3/CD28 + IL-2 and cultured in the presence or absence of Rapa. The expression pattern of miR-155 and the effect of Rapa on it were detected by real-time RT-PCR (A). The effect of miR-155 antgomir and mimic were detected by real-time RT-PCR (B). The effect of miR-155 on phos-STAT5 was detected by western blot (C).
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Fig. 6. Possible mechanism about how mTOR modulates the balance between regulatory and effective T cells. High mTOR signaling facilitates both Tregs and Teffs proliferation, but it would reduce Foxp3 expression since reducing STAT5 activity via miR-155/SOCS1 pathway. In contrast, mTOR inhibition weakens Teffs growth, but gradually make Tregs shift to STAT5/pim-2 dependent proliferation via miR-155, which is more beneficial to foxp3 maintain and induction. Thus, the dynamic interaction between mTOR and STAT5 signaling modulates the proliferation and reciprocal differentiation of the Tregs and Teffs, thus influence the immune balance.
In cultured Teffs, phos-STAT5 expression was also higher in Rapa-exposed Teffs than that in medium culture ones. However, the phos-4E-BP1 expression was inhibited by rapa in both 2 days and 5 days cultured Teffs (Fig. 4C). It suggested that proliferating Teffs are more depend on mTOR signaling than Tregs after second round of stimulation, which makes Teffs keep sensitive to Rapa in long-term culture. These findings contribute to our understanding of how Rapa differently affects Tregs and Teffs proliferation, and selectively expands Tregs in long-term culture. mTOR inhibition promote Foxp3 expression by up-regulating STAT5 signaling STAT5 signaling has been reported relevance for Treg development and homeostasis (Antov et al., 2003), activation of STAT5 is critical for induction and maintenance of Foxp3 expression in Teffs and Tregs, respectively (Angela et al., 2004; Passerini et al., 2007). To detect whether Foxp3 expression induced by Rapa correlated its role in up-regulating STAT5 signaling, Jak inhibitor was added to block STAT5 signaling in expanded T cells. Western blot results showed that Rapa up-regulated foxp3 expression in both proliferating Tregs and Teffs when compared to medium control, but its up-regulation effect disappeared when added Jak inhibitor (Fig. 4A–C). These findings indicated that Rapa sustains Foxp3 expression via STAT5 signaling, contributing to our understanding of how Rapa affect the expression of Foxp3. mTOR inhibition up-regulate STAT5 signaling via miR-155 Crucial function of miR-155 in immune regulation was proven by Rodriguez et al. (2007) using the miR-155 knock-out mouse which showed a severe autoimmune phenotype in lung. Then miR155 was found to be able to inhibit SOCS1 expression (Lu et al., 2009; Jiang et al., 2010), which negatively regulates the activity and
function of Tregs by inhibition the expression phos-STAT5 signaling (Lu et al., 2009; Yao et al., 2012), that is by targeting SOCS1, miR155 can regulate STAT5 signaling. And our RT-PCR results showed Rapa could up-regulate miR-155 expression in both Tregs and Teffs (Fig. 5A). So we supposed Rapa may regulate STAT5 signaling via miR-155. To prove this, we performed gain-and loss-of-function analysis by transfection miR-155-mimic and miR-155-antagomir into purified CD4+ T cells (Fig. 5B). Then transfected CD4+ T cells were cultured with and without Rapa, and phos-STAT5 expression in different groups was detected. The western results showed in Rapa-cultured group, phos-STAT5 expression was reduced when miR-155 was inhibited, but phos-STAT5 expression in miR-155 over-expression CD4+ T cells was up-regulated even when cultured without Rapa (Fig. 5C). Suggesting miR-155 involved in the interaction between mTOR and STAT5 signaling.
Discussion The appropriate balance between Teffs and Tregs is essential for maintaining self-tolerance and enabling transplant tolerance. Rapamycin (Rapa) has been reported to selectively expand Tregs (Battaglia et al., 2005; Strauss et al., 2007, 2009) and promote de novo generation of foxp3+ Tregs (Battaglia et al., 2005; Gabryˇsová et al., 2011), suggesting its potential role in regulating “Tregs–Teffs” balance. In 2007, Zeiser et al. (2008) has found when compared to Teffs, Tregs reduced the usage of the mTOR signaling, but preferentially use STAT5 to proliferate. This explained why Rapa inhibits Teffs proliferation but not Tregs. However, the effect of Rapa on proliferating T cells is time-dependent: it reduces both Teffs and Tregs proliferation at early stage (Keever-Taylor et al., 2007; Basu et al., 2008; Zeiser et al., 2008; Golovina et al., 2008), but greatly promotes Tregs proliferation in long-term culture (Battaglia et al., 2005; Strauss et al., 2007, 2009). This suggests that the role of mTOR signaling in proliferating Tregs is time-varying. So, we studied the
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mechanism of the time-effect role of mTOR signaling in regulating “Tregs–Teffs” balance. Firstly, we confirmed that Tregs cells in different amplification stage have varying sensitivity to Rapa, and verified the necessity of the mTOR signaling in the activation and proliferation of Tregs: although high level expression of phos-STAT5 was detected when stimulated with IL-2, Tregs were not activated. This demonstrated that STAT5 is not sufficient to activate Tregs, they cannot only be amplified without mTOR signaling. Then we found there is direct interaction between mTOR and STAT5 signaling. mTOR inhibition up-regulated STAT5 activity and its down streaming targets including Pim-2 and Foxp3 in both proliferating Tregs and Teffs, and this contribute to our understanding of how Rapa maintain and induce Foxp3 expression in Tregs and Teffs. However, the phos-4E-BP1 expression pattern is different in Tregs and Teffs when cultured with Rapa. 4E-BP1 is the common target of mTOR and STAT5 signaling, and plays a key role in cell proliferation. Rapa inhibits phos-4E-BP1 expression in both Tregs and Teffs at early stage of proliferation, but selectively raises its expression in Tregs via STAT5 after second round of stimulation. This may explains why Rapa inhibits Teffs growth, but delays Tregs proliferation. Finally, we found miR-155 was involved in Rapa’s effect on up-regulating STAT5 signaling. Together, these findings demonstrate the strength of mTOR can regulate immune balance via modulate STAT5 signaling: High mTOR signaling facilitates both Tregs and Teffs proliferation, but it would reduce Foxp3 expression since low STAT5 activity in proliferating Tregs. In contrast, mTOR inhibition weakens Teffs growth, but gradually make Tregs shift to STAT5/pim-2 dependent proliferation via miR-155, which is more beneficial to foxp3 maintain and induction (Fig. 6). To our knowledge, this is the first study of the interaction between mTOR and STAT5 signaling in T cells. The dynamic interaction between mTOR and STAT5 signaling modulates the proliferation and reciprocal differentiation of the Tregs and Teffs, thus influence the immune balance. To regulate the activity and time-phase of mTOR and STAT5 signaling in T cells would become one of the potential therapeutic strategies to enhance the number of Tregs in vivo, and have potential therapeutic implications for the treatment of autoimmunity or induction of transplant tolerance. Conflict of interest None of the authors of this manuscript have a financial interest related to this work. Acknowledgements This work was supported by National Basic Research Program of China No. 2009CB522401, and by the Natural Science Foundation of China No. 81273255. References Angela, M., Thornton, Erin, E., Donovan, Ciriaco, A., Piccirillo, Shevach, E.M., 2004. IL2 is critically required for the in vitro activation of CD4+ CD25+ T cell suppressor function. J. Immunol. 172, 6519–6523.
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Interplay between mTOR and STAT5 signaling modulates the balance between regulatory and effective T cells Juan Shan a,b , Li Feng a , Guixiang Sun a , Peng Chen a , Yanni Zhou a , Mengjuan Xia a , Hongsheng Li a , Youping Li a,c,∗ a Key Laboratory of Transplant Engineering and Immunology of Health Ministry of China, West China Hospital, Sichuan University, Chengdu 610041, Sichuan Province, PR China b Regenerative Medicine Research Center, West China Hospital, Sichuan University, Chengdu 610041, Sichuan Province, PR China c Chinese Cochrane Centre, Chinese Evidence-Based Medicine Centre, Chengdu 610041, Sichuan Province, PR China
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Article history: Received 17 February 2014 Accepted 21 October 2014 Available online xxx Keywords: Regulatory T cell mTOR signaling STAT5 signaling MicroRNA-155 Immune balance Rapamycin
a b s t r a c t Background: Immune response outcome, inflammation or tolerance, often depends on the balance between regulatory T cells (Tregs) and effective T cells (Teffs). Rapamycin (Rapa) has been reported to selectively expand Tregs and promote de novo generation of foxp3+ Tregs, suggesting its potential role in inducing tolerance. But the mechanism by which Rapa regulating the Treg–Teff balance is yet to be understood. Methods: Mouse CD4+ CD25− Teffs and CD4+ CD25+ Tregs are sorted by MACS. These T cell subsets were labeled with CFSE and cultured with anti-CD3/CD28 Ab ± IL-2 for 6 days. Two rounds of stimulation of 3 days each were performed. Rapa or Jak Inhibitor I was added to the culture when indicated. Cells were harvested after each round of stimulation. CFSE dilution, FOXP3, miR-155 expression and the signaling via the mTOR and STAT5 pathways were determined. And miR-155-mimic and miR-155-antagomir were transfected into purified CD4+ T cells respectively to detect miR-155 role in regulating STAT5 signaling. Results: Firstly, we confirmed that the effect of Rapa on proliferating T cells is time-dependent: it reduces both Teffs and Tregs proliferation at early stage, but selectively promotes Tregs proliferation after second round of stimulation. Then we found there is direct interaction between mTOR and STAT5 signaling, and this interaction explained the time-dependent effect of Rapa and may participate in deciding Teff–Treg balance: mTOR inhibition up-regulated the expression of phos-STAT5 in both proliferating Tregs and Teffs via miR-155. And foxp3 is the down streaming target of phos-STAT5, thus Rapa could maintain expanded Tregs function and promote de novo generation of foxp3+ Tregs. However, the phos-4E-BP1 expression pattern is different in proliferating Tregs and Teffs. 4E-BP1 is the common target of mTOR and STAT5 signaling, and plays a key role in cell proliferation. Rapa inhibits phos-4E-BP1 expression in both Tregs and Teffs at early stage of proliferation, but selectively raises its expression in Tregs after second round of stimulation. This may explains why Rapa inhibits Teffs growth, but delays Tregs proliferation. Conclusion: Together, these findings indicate that the dynamic interaction between mTOR and STAT5 signaling modulates the reciprocal differentiation of the effective and regulatory T cells, and differently affect their proliferation activity. This provides a new insight of how Treg–Teff balance is regulated. © 2014 Elsevier GmbH. All rights reserved.
Introduction
Abbreviations: Tregs, regulatory T cells; Teffs, effective T cells; Rapa, Rapamycin; miR-155, micro-RNA 155. ∗ Corresponding author at: Key Laboratory of Transplant Engineering and Immunology of Health Ministry of China, West China Hospital, Sichuan University, Chengdu 610041, Sichuan Province, PR China. Tel.: +86 028 85164032; fax: +86 028 85164034. E-mail address: [email protected] (Y. Li).
The balance between effector T cells (Teffs) and regulatory T cells (Tregs) is critical for establishing tolerance to self- and non-self-antigens. Directing T-cell differentiation toward Tregs has important clinical implications for treating autoimmune disease and enabling transplant tolerance. Rapamycin (Rapa) is an immunosuppressive drug used to prevent allograft rejection. It inhibits proliferation of many cell types, including T cells, by inhibiting the serine/threonine protein
http://dx.doi.org/10.1016/j.imbio.2014.10.020 0171-2985/© 2014 Elsevier GmbH. All rights reserved.
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kinase called mammalian target of Rapa (mTOR), the activation of which is required for protein synthesis and cell-cycle progression. However, Tregs and Teffs are not equally sensitive to Rapa: it selectively blocks proliferation of CD4+ CD25− Teffs, whereas allows CD4+ CD25+ Tregs to growth in long-term culture (Battaglia et al., 2005; Strauss et al., 2007, 2009). Moreover, Rapa can promote de novo generation of foxp3+ Tregs from naïve Teffs (Battaglia et al., 2005; Gabryˇsová et al., 2011), and appears to maintain or induce Tregs in vivo and promotes tolerance in transplant recipients when compared to calcineurin inhibitors (Ruggenenti et al., 2007; Hendrikx et al., 2009; Bestard et al., 2011; Ekong et al., 2012). These studies highlight the importance of Rapa and its target mTOR in controlling the delicate balance between Teffs and Tregs. But the mechanism by which Rapa selectively expands Tregs and promotes foxp3 expression is yet to be understood. It is worth noting that only in long-term culture can Rapa greatly promotes Tregs proliferation (Battaglia et al., 2005; Strauss et al., 2007, 2009). It reduces both Teffs and Tregs proliferation at early stage of stimulation when compared to medium cultures (KeeverTaylor et al., 2007; Basu et al., 2008; Zeiser et al., 2008; Golovina et al., 2008). This suggests that the role of mTOR signaling in proliferating Tregs is time-varying. Pim-2 is a serine/threonine-protein kinase which has considerable overlap with mTOR by sharing common downstream targets including Bad and 4E binding protein-1 (4E-bp1) (White, 2003), so it can mediate resistance to Rapa. In addition, IL-2 and STAT5 signaling is critically required for Tregs development and foxp3 expression (Antov et al., 2003), and STAT5 phosphorylation can also induce pim-2 (Mizuki et al., 2003). However, the relationship between mTOR, pim-2 and STAT5 in T cells is not fully delineated. And we thought of the interaction and differential expression pattern of these signaling molecules in Tregs and Teffs might be the mechanism by which Rapa regulating the Treg/Teff balance. In this study, we show that there is direct interaction between mTOR and STAT5 signaling in both Tregs and Teffs: mTOR inhibition weakens Teffs growth, but make Tregs shift to STAT5/pim-2 dependent proliferation, which is more beneficial to foxp3 maintain and induction. These results demonstrate that the interplay between mTOR and STAT5 signaling participated in modulating the Treg–Teff balance. Materials and methods Animal Male BALB/c (H-2d) mice weighting 25–30 g and aging 8–12 weeks were used in these experiments. They were bred and maintained under specific pathogen-free conditions in Experimental Animal Center of West China hospital (Sichuan University, Chengdu, China), and all experiments were performed in accordance with the protocols approved by the Institutional Animal Care and Use Committee.
fraction using the CD25-PE mAbs and anti-PE microbeads. The positive selection fraction is CD4+ CD25+ Tregs, and the negative depletion fraction is CD4+ CD25− Teffs. Purity ranged from 92% to 98% as measured by flow cytometry. T cells culture and stimulation Purified CD4+ CD25+ Tregs and CD4+ CD25− Teffs were cultured in U-bottomed, 96-well plates and stimulated when indicated with 2 g/ml CD3 (clone 145-2C11; BD Biosciences), 2 g/ml CD28 (clone 37.51; BD Biosciences), 1000 IU/ml IL-2, at a concentration of 1 × 105 cells/well. Two rounds of stimulation of 3 days each were performed. Culture medium consisted of RPMI 1640 supplemented with 100 U/ml penicillin, 100 mg/ml streptomycin, 2 mM l-glutamine, 10 mM Hepes, 0.1 mM non-essential amino acids, 1 mM sodium pyruvate, 50 M 2-mercaptoethanol and 10% fetal calf serum. For proliferation detection, T cells (1 × 106 ) were pre-labeled with 5 M CFSE (Sigma–Aldrich, St. Louis, MO) before stimulation. After 2 or 5 days cultured T cells were harvested and analyzed by FACS or Western blot. mTOR and STAT5 signaling blockers Rapamycin (Sigma–Aldrich, St Louis, MO, USA) 100 nM was used to block the PI3K/mTOR signaling, and Jak Inhibitor I (Calbiochem, Merck KGaA, Darmstadt, Germany) 1 M was used to block the phosphorylation of STAT5. Similar volumes of DMSO vehicle were added to T-cell cultures as control groups. Flow cytometry analysis Intracellular FOXP3 staining was performed using the FOXP3 Fix/Perm Buffer set (Biolegend) according to the manufacturer’s recommendations. Western blot analysis Total cell lysates and western blot analysis were performed as previously described (Luo et al., 2012). The antibodies used were the following: anti-phospho-p70S6K, anti-phospho-S6, antiphospho-STAT5 (Tyr694), and anti-phospho-4E-BP-1 (all from Cell Signaling Technology, Beverly, MA); anti-Pim-2 (EPR6987; Epitomics, Burlingame, CA); and anti-FoxP3 (150D/E4; eBioscience, San Diego, CA). Quantitative real-time PCR Total RNA (including miRNAs) was extracted using MirVana TM miRNA Isolation Kit (Ambion, Austin, USA) according to the manufacturer’s instructions. After transcription into cDNA, the expression of miR-155 respective of the normalizer U6 was quantified using the miR-155-5p RT kit and U6-RT primer kit (Ruibo, Guangzhou, China).
Cell isolation and sorting
Nucleofection
Splenic mononuclear cells were obtained by Ficoll-Hypaque density gradient centrifugation. Cells recovered from the gradient interface were washed twice in PBS, counted, and immediately used for MACS using the CD4+ CD25+ regulatory T cell isolation kit (Miltenyi Biotec, Bergisch Gladbach, Germany). Briefly, CD4+ T cells were purified from mononuclear cells by negative selection using biotin-conjugated monoclonal antibodies (mAbs) directed against CD8a, CD11b, CD45R, CD49b, Ter-119, and anti-biotin microbeans, yielding a population of CD4+ cells with purity of 90–95%. Then, CD4+ CD25+ Tregs were positively selected from purified CD4+ T
Nucleofection was performed with Mouse T Cell Nucleofection Kit and Nucleofection device (Amaxa, Koelin, Germany) according to the manufacturer’s recommendations. Briefly, 1 × 106 sorted CD4+ T cells were resuspended in 100 ml nucleofection solution. Five microliters of GFP Vector or oligonucleotides, including miR155-mimic, mimic-control, miR-155-antagomir and antagomir control (Ruibo, Guangzhou, China), were added to the solution and mix gently. Then the mixtures were transferred to electroporation cuvettes and nucleofected using the X-01 program. Five hundred microliters pre-warmed nucleofection medium were added
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Fig. 1. Tregs activated in the presence of Rapa had a delayed kinetic of proliferation. Freshly isolated CD4+ CD25+ Tregs and CD4+ CD25− Teffs were labeled with CFSE, and were activated with anti-CD3/CD28 Ab + IL-2 in the presence or absence of Rapa. CFSE dilution was measured 2 days (A) and 5 days (B) after activation. Percentages of suppression in comparison to proliferation of naive control cells are indicated. *p < 0.05 compared with norapa group.
in the cuvettes immediately after transfection. Transfected cells were transferred to 12-well plates with 1.5 ml pre-warmed nucleofection medium and incubated in a humidified 37 ◦ C/5% CO2 incubator. Dead cells were removed with dead cell removal kit (Miltenyi Biotec, Bergisch Gladbach, Germany) after 6 h incubation. The transfection efficiency which is evaluated by monitoring GFP expression using flow cytometry 24 h after transfection is around 50%. Results
control culture. In contrast, the proliferative activity of Teffs remained significantly reduced (Fig. 1B). These results are consistent with previous data showing that Tregs activated in the presence of Rapa had a delayed kinetic of proliferation. We next examined Foxp3 expression in the expanded T cell populations. The results showed only Rapa-exposed Tregs preserved Foxp3 expression (Fig. 2A). And Rapa treatment increased the frequency of FOXP3+ cells induced in Teffs, but Foxp3 expressing cells were only observed in the divided population (Fig. 2B), suggesting the de novo generation of Foxp3 is depend on Teffs activation.
mTOR inhibition delays Tregs proliferation
Tregs activation requires mTOR signaling
Studies have showed Rapa inhibits both Tregs and Teffs proliferation at the early stage of stimulation, but selectively promote the expansion of Tregs in long-term cultures (Battaglia et al., 2005; Strauss et al., 2007, 2009). We first confirmed T cells in different amplification stage have varying sensitivity to Rapa. To do this, Purified CD4+ CD25+ Tregs and CD4+ CD25− Teffs were activated in vitro with anti-CD3/CD28 Ab + IL-2 in the presence or absence of 100 nM Rapa. Two rounds of stimulation of 3 days each were performed. T cell proliferation was monitored by CFSE dilution after 2 days and 5 days culture. In the first 2 days of culture, the proliferative capacity to polyclonal activation was markedly reduced in both Tregs and Teffs when exposed to Rapa (Fig. 1A). But after second round of stimulation, Tregs cultured with Rapa seems to have resumed its proliferative activity, which proliferated to a similar extent in
For there have been conflicting reports on whether or not mTOR is required in Tregs activation (Zeiser et al., 2008; Wang et al., 2011; Bensinger et al., 2004), we studied the phosphorylated STAT5, p70S6K1 and 4E-BP1 expression and cell proliferation of Tregs in response to different stimulation. P70S6K1 and 4E-BP1 are direct targets of mTOR, which phosphorylation can indicate mTOR activity. Western results showed neither anti-CD3 alone nor in combination with anti-CD28 stimulation can activate Tregs mTOR signaling (Fig. 3A). Tregs constitutively express the IL-2 receptor IL-2R␣ (CD25), so high level expression of phos-STAT5 was detected when stimulated with IL-2 alone (Fig. 3A), however, CFSE results showed Tregs were hypoproliferative to IL-2 (Fig. 3B). Only anti-CD3/CD28 Ab + IL-2 stimulation can activate Tregs, and at the same time activate their mTOR
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Fig. 2. Rapa treatment maintains Foxp3 expression in Tregs, and increases the frequency of induced Foxp3+ T cells in Teffs. Foxp3 expression of 5 days expanded CD4+ CD25+ Tregs was assessed by FACS analysis (A), CD4+ CD25− Teffs on days 2 were detected for CFSE dilution and FOXP3 expression (B). *p < 0.05 compared with norapa group.
signaling (Fig. 3A). These data suggesting Tregs require mTOR signaling to activate, STAT5 signaling alone cannot activate Tregs. In addition, we found an interesting phenomenon that although the concentration of added IL-2 is the same in each group, the expression of phos-STAT5 was higher in Tregs stimulated with IL-2 alone than these stimulated in combination with anti-CD3/CD28Ab (Fig. 3A). And STAT5 signaling is required for Tregs function and their Foxp3 expression (Antov et al., 2003; Angela et al., 2004; Passerini et al., 2007), so we detected Foxp3 expression in the
two groups and the results showed the Foxp3 expression was lower in activated Tregs, when their mTOR signaling was also activated (p < 0.05) (Fig. 3C). So it seems as mTOR activation could lower STAT5 signaling strength, and correspondently reduce the foxp3 expression, suggesting a competition relationship may exist between mTOR and STAT5 signaling, and this is related to the expression of Foxp3. Therefore, we further explore the relationship between the two pathways and its role in Tregs proliferation and Foxp3 expression.
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Fig. 3. Tregs activation requires mTOR signaling. (A) Results of Western blots comparing expression levels of phospho-P70S6K, phospho-4EBP-1 and phospho-STAT5 in CD4+ CD25+ Tregs cultured in different stimulators (anti-CD3 alone or in combination with anti-CD28, IL-2 alone or in combination with anti-CD3/CD28). (B) Proliferation activity of CD4+ CD25+ Tregs cultured in different stimulators for 72 h. One representative experiment is shown. (C) Foxp3 expression of 72 h expanded CD4+ CD25+ Tregs was assessed by FACS analysis *p < 0.05 compared with IL-2 group.
mTOR inhibition force Tregs shift to STAT5/pim-2 dependent proliferation To detect whether there is direct interplay between mTOR and STAT5 signaling, Rapa was used to block the mTOR signaling, the expression of the downstream targets of both mTOR and STAT5 signaling, including phos-STAT5, phos-S6, phos-4E-BP1, Pim-2, and Foxp3, were detected by western blot. The mTOR and STAT5 signaling cascades were showed in Fig. 6. The results showed mTOR signaling was effectively inhibited by Rapa in 2 days cultured Tregs: the expression of phos-S6 and 4E-BP1, downstream targets of mTOR, was lower in Tregs cultured with Rapa than that in control group. But we found that phos-STAT5 expression was up-regulated by Rapa, as well as its downstream target Pim-2. But the expression
of phos-4E-BP1, the common downstream target of mTOR and Pim2, was still higher in medium cultured Tregs than in Rapa-exposed ones (Fig. 4A). However, the situation changed in 5 days cultured Tregs: phos-4E-BP1 expression in Rapa-exposed Tregs exceeded that in control group (Fig. 4B), it seemed as the 4E-BP1 phosphorylated via STAT5/Pim-2 signaling is sufficient to compensate that from mTOR signaling after second round of stimulation. Both phos-S6 and 4E-BP1 play key roles in cell proliferation. These findings indicated that at the early stage of amplification, Rapa reduce Tregs proliferation by inhibiting mTOR/S6 signaling, but force Tregs shift to STAT5/pim-2 dependent proliferation, and promote its proliferation at late stage of amplification via STAT5/Pim-2/4E-BP1 signaling. This may explain why Rapa delay, but not inhibit Tregs proliferation.
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Fig. 4. mTOR inhibition force Tregs shift to STAT5/pim-2 dependent proliferation, which is more beneficial to Foxp3 expression; mTOR inhibition induces Foxp3 expression in Teffs via STAT5, but proliferating Teffs depend more on mTOR signaling than Tregs. Freshly isolated CD4+ CD25+ Tregs were stimulated with anti-CD3/CD28 + IL-2 and cultured in the presence or absence of Rapa. Jak inhibitor was added when indicated. Expression levels of phospho-S6, phospho-STAT5, phospho-4EBP-1, PIM-2 and Foxp3 in 2 days-cultured (A) and 5 days-cultured Tregs (B) were detected by Western blots. CD4+ CD25− Teffs were stimulated with anti-CD3/CD28, and cultured in the presence or absence of Rapa. IL-2 or Jak inhibitor was added to the culture when indicated. Expression levels of phospho-S6, phospho-STAT5, phospho-4EBP-1, PIM-2 and Foxp3 in Rapa-exposed or medium-cultured Teffs were compared by Western blots (C). One representative experiment is shown.
Fig. 5. mTOR inhibition up-regulates STAT5 signaling via miR-155. Freshly isolated CD4+ CD25+ Tregs and CD4+ CD25− Teffs were stimulated with anti-CD3/CD28 + IL-2 and cultured in the presence or absence of Rapa. The expression pattern of miR-155 and the effect of Rapa on it were detected by real-time RT-PCR (A). The effect of miR-155 antgomir and mimic were detected by real-time RT-PCR (B). The effect of miR-155 on phos-STAT5 was detected by western blot (C).
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Fig. 6. Possible mechanism about how mTOR modulates the balance between regulatory and effective T cells. High mTOR signaling facilitates both Tregs and Teffs proliferation, but it would reduce Foxp3 expression since reducing STAT5 activity via miR-155/SOCS1 pathway. In contrast, mTOR inhibition weakens Teffs growth, but gradually make Tregs shift to STAT5/pim-2 dependent proliferation via miR-155, which is more beneficial to foxp3 maintain and induction. Thus, the dynamic interaction between mTOR and STAT5 signaling modulates the proliferation and reciprocal differentiation of the Tregs and Teffs, thus influence the immune balance.
In cultured Teffs, phos-STAT5 expression was also higher in Rapa-exposed Teffs than that in medium culture ones. However, the phos-4E-BP1 expression was inhibited by rapa in both 2 days and 5 days cultured Teffs (Fig. 4C). It suggested that proliferating Teffs are more depend on mTOR signaling than Tregs after second round of stimulation, which makes Teffs keep sensitive to Rapa in long-term culture. These findings contribute to our understanding of how Rapa differently affects Tregs and Teffs proliferation, and selectively expands Tregs in long-term culture. mTOR inhibition promote Foxp3 expression by up-regulating STAT5 signaling STAT5 signaling has been reported relevance for Treg development and homeostasis (Antov et al., 2003), activation of STAT5 is critical for induction and maintenance of Foxp3 expression in Teffs and Tregs, respectively (Angela et al., 2004; Passerini et al., 2007). To detect whether Foxp3 expression induced by Rapa correlated its role in up-regulating STAT5 signaling, Jak inhibitor was added to block STAT5 signaling in expanded T cells. Western blot results showed that Rapa up-regulated foxp3 expression in both proliferating Tregs and Teffs when compared to medium control, but its up-regulation effect disappeared when added Jak inhibitor (Fig. 4A–C). These findings indicated that Rapa sustains Foxp3 expression via STAT5 signaling, contributing to our understanding of how Rapa affect the expression of Foxp3. mTOR inhibition up-regulate STAT5 signaling via miR-155 Crucial function of miR-155 in immune regulation was proven by Rodriguez et al. (2007) using the miR-155 knock-out mouse which showed a severe autoimmune phenotype in lung. Then miR155 was found to be able to inhibit SOCS1 expression (Lu et al., 2009; Jiang et al., 2010), which negatively regulates the activity and
function of Tregs by inhibition the expression phos-STAT5 signaling (Lu et al., 2009; Yao et al., 2012), that is by targeting SOCS1, miR155 can regulate STAT5 signaling. And our RT-PCR results showed Rapa could up-regulate miR-155 expression in both Tregs and Teffs (Fig. 5A). So we supposed Rapa may regulate STAT5 signaling via miR-155. To prove this, we performed gain-and loss-of-function analysis by transfection miR-155-mimic and miR-155-antagomir into purified CD4+ T cells (Fig. 5B). Then transfected CD4+ T cells were cultured with and without Rapa, and phos-STAT5 expression in different groups was detected. The western results showed in Rapa-cultured group, phos-STAT5 expression was reduced when miR-155 was inhibited, but phos-STAT5 expression in miR-155 over-expression CD4+ T cells was up-regulated even when cultured without Rapa (Fig. 5C). Suggesting miR-155 involved in the interaction between mTOR and STAT5 signaling.
Discussion The appropriate balance between Teffs and Tregs is essential for maintaining self-tolerance and enabling transplant tolerance. Rapamycin (Rapa) has been reported to selectively expand Tregs (Battaglia et al., 2005; Strauss et al., 2007, 2009) and promote de novo generation of foxp3+ Tregs (Battaglia et al., 2005; Gabryˇsová et al., 2011), suggesting its potential role in regulating “Tregs–Teffs” balance. In 2007, Zeiser et al. (2008) has found when compared to Teffs, Tregs reduced the usage of the mTOR signaling, but preferentially use STAT5 to proliferate. This explained why Rapa inhibits Teffs proliferation but not Tregs. However, the effect of Rapa on proliferating T cells is time-dependent: it reduces both Teffs and Tregs proliferation at early stage (Keever-Taylor et al., 2007; Basu et al., 2008; Zeiser et al., 2008; Golovina et al., 2008), but greatly promotes Tregs proliferation in long-term culture (Battaglia et al., 2005; Strauss et al., 2007, 2009). This suggests that the role of mTOR signaling in proliferating Tregs is time-varying. So, we studied the
Please cite this article in press as: Shan, J., et al., Interplay between mTOR and STAT5 signaling modulates the balance between regulatory and effective T cells. Immunobiology (2014), http://dx.doi.org/10.1016/j.imbio.2014.10.020
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mechanism of the time-effect role of mTOR signaling in regulating “Tregs–Teffs” balance. Firstly, we confirmed that Tregs cells in different amplification stage have varying sensitivity to Rapa, and verified the necessity of the mTOR signaling in the activation and proliferation of Tregs: although high level expression of phos-STAT5 was detected when stimulated with IL-2, Tregs were not activated. This demonstrated that STAT5 is not sufficient to activate Tregs, they cannot only be amplified without mTOR signaling. Then we found there is direct interaction between mTOR and STAT5 signaling. mTOR inhibition up-regulated STAT5 activity and its down streaming targets including Pim-2 and Foxp3 in both proliferating Tregs and Teffs, and this contribute to our understanding of how Rapa maintain and induce Foxp3 expression in Tregs and Teffs. However, the phos-4E-BP1 expression pattern is different in Tregs and Teffs when cultured with Rapa. 4E-BP1 is the common target of mTOR and STAT5 signaling, and plays a key role in cell proliferation. Rapa inhibits phos-4E-BP1 expression in both Tregs and Teffs at early stage of proliferation, but selectively raises its expression in Tregs via STAT5 after second round of stimulation. This may explains why Rapa inhibits Teffs growth, but delays Tregs proliferation. Finally, we found miR-155 was involved in Rapa’s effect on up-regulating STAT5 signaling. Together, these findings demonstrate the strength of mTOR can regulate immune balance via modulate STAT5 signaling: High mTOR signaling facilitates both Tregs and Teffs proliferation, but it would reduce Foxp3 expression since low STAT5 activity in proliferating Tregs. In contrast, mTOR inhibition weakens Teffs growth, but gradually make Tregs shift to STAT5/pim-2 dependent proliferation via miR-155, which is more beneficial to foxp3 maintain and induction (Fig. 6). To our knowledge, this is the first study of the interaction between mTOR and STAT5 signaling in T cells. The dynamic interaction between mTOR and STAT5 signaling modulates the proliferation and reciprocal differentiation of the Tregs and Teffs, thus influence the immune balance. To regulate the activity and time-phase of mTOR and STAT5 signaling in T cells would become one of the potential therapeutic strategies to enhance the number of Tregs in vivo, and have potential therapeutic implications for the treatment of autoimmunity or induction of transplant tolerance. Conflict of interest None of the authors of this manuscript have a financial interest related to this work. Acknowledgements This work was supported by National Basic Research Program of China No. 2009CB522401, and by the Natural Science Foundation of China No. 81273255. References Angela, M., Thornton, Erin, E., Donovan, Ciriaco, A., Piccirillo, Shevach, E.M., 2004. IL2 is critically required for the in vitro activation of CD4+ CD25+ T cell suppressor function. J. Immunol. 172, 6519–6523.
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Please cite this article in press as: Shan, J., et al., Interplay between mTOR and STAT5 signaling modulates the balance between regulatory and effective T cells. Immunobiology (2014), http://dx.doi.org/10.1016/j.imbio.2014.10.020
