Mammalian Sin1 plays key roles in the regulation of mitogen-activated protein kinase (MAPK) and mammalian target of rapamycin (mTOR) signaling. Sin1 is an essential component of mTOR complex 2 (mTORC2). The functions of Sin1 and mTORC2 remain largely unknown in T cells. Here, we investigate Sin1 function in T cells using mice that lack Sin1 in the hematopoietic system. Sin1 deficiency blocks the mTORC2-dependent Akt phosphorylation in T cells during development and activation. Sin1-deficient T cells exhibit normal thymic cellularity and percentages of double-negative, double-positive, and single-positive CD4+ and CD8+ thymocytes. Sin1 deficiency does not impair T-cell receptor (TCR) induced growth and proliferation. Sin1 appears dispensable for in vitro CD4+ helper cell differentiation. However, Sin1 deficiency results in an increased proportion of Foxp3+ natural T-regulatory (nTreg) cells in the thymus. The TGF-β-dependent differen-tiation of CD4+ T cells in vitro is enhanced by the inhibition of mTOR but not by loss of Sin1 function. Our results reveal that Sin1 and mTORC2 are dispensable for the development and activation of T cells but play a role in nTreg-cell differentiation.
Mammalian target of rapamycin (mTOR) is a conserved serine/threonine protein kinase that regulates cell growth and metabolism []. Mammalian TOR is inhibited by rapamycin, a potent suppressor of T cell-mediated immune responses []. Rapamycin inhibits IL-2-dependent T-cell proliferation, promotes the expansion of regulatory T (Treg) cells and has recently been shown to promote the development of memory CD8+ T cells [[3-5]]. Mammalian TOR function is mediated by at least two distinct multi-protein complexes called mTOR complex 1 (mTORC1), containing mTOR, raptor, mLST8 (GβL), and PRAS40 and mTORC2, containing Rictor, Sin1, and mLST8 in addition to mTOR. Nutrients, growth factors, hormones, and energy signals activate mTORC1 to phosphorylate the translational regulators S6K and 4EBP1, leading to increased cellular protein synthesis and ribosome biogenesis []. Mammalian TORC2 regulates actin polymerization and cytoskeleton function [], controls Akt activation and specificity in a PI3K-dependent manner by phosphorylating the Akt hydrophobic motif (S473 on Akt1), and regulates the stability of Akt and conventional PKC in a PI3K-independent manner by phosphorylating their turn motif (TM) (T450 on Akt1, T638 on PKCα) [[6-8]]. Mammalian TORC2 is less sensitive to rapamycin inhibition than mTORC1; however, chronic rapamycin treatment may inhibit mTORC2. Therefore, previous studies utilizing rapamycin to study mTOR were unable to properly evaluate the contribution of mTORC2 to T-cell immunity. In addition, mTOR also possesses a rapamycin-independent mTORC1 function []. Therefore, it is unclear how mTORC1 and mTORC2 each specifically contribute to T-cell function.
Recent genetic studies have begun to elucidate the mechanism of mTOR function and regulation in T cells. Delgoffe et al. recently reported that CD4-Cre mediated T-cell specific mTOR deletion impairs T-cell proliferation and inhibits TH1, TH2, and TH17 differentiation without blocking early T-cell activation []. Mammalian TOR deficiency also greatly enhanced Treg-cell differentiation in vitro, while T cells lacking Rheb, a small GTPase that positively regulates mTORC1 function, failed to spontaneously differentiate into Treg cells upon activation suggesting that mTORC2 may play a prominent role in regulating Treg-cell differentiation []. Two recent studies from independent labs have explored the function of mTORC2 in T cells using mice that specifically lack Rictor expression in T cells [[11, 12]]. In the first study, Lee et al. show that rictor−/− T cells lack functional mTORC2 and exhibit defects in Akt and PKCθ phosphorylation as well as decreased NF-κB activity, reduced proliferation, impaired T-helper cell differentiation, and increased CD4+Foxp3+ Treg-cell differentiation [], while in the second study, Delgoffe et al. [] show that rictor−/− T cells exhibit defects in proliferation and TH2 differentiation, they do not observe deficiencies in TH1, TH17, or Treg-cell differentiation.
In this study, we reconstituted lethally irradiated wild-type (WT) mice with Sin1−/− fetal liver hematopoietic stem cells (HSCs) and examined the T-cell development, growth, proliferation, and CD4+ effector cell differentiation in cells obtained from these mice. We show that the loss of Sin1 in T cells disrupts mTORC2 function and blocks Akt phosphorylation at the hydrophobic motif (HM) and TM sites. Although mTORC2 function is abolished in Sin1−/− T cells, we find that Sin1 is not required for thymic T-cell development. These data reveal that Akt HM and TM phosphorylation are not required for thymic T-cell development even though Akt plays an essential role in maintaining the metabolism and viability of thymocytes undergoing TCRβ selection. Furthermore, mature T-cell growth, proliferation or CD4+ helper T-cell differentiation are unaffected by Sin1 deficiency. However, we observe that Sin1−/− thymic T cells give rise to a greater proportion of natural Treg (nTreg) cells than WT thymocytes. These data support a role for mTORC2 in the regulation of Treg-cell differentiation. We also provide evidence that Akt1 and Akt2 are not required for the mTORC2-mediated regulation of thymic Treg-cell development.
Sin1 is not required for the development of major thymic T-cell subsets
We generated chimeric mice by transplanting E12.5 fetal liver cells from Sin1+/+ or Sin1−/− embryos into lethally irradiated WT CD45.1 congenic mice []. Analysis of thymic T-cell populations in these chimeric mice revealed that Sin1-deficient HSCs gave rise to equivalent proportions of CD4/CD8 double negative (DN), CD4/CD8 double positive (DP), CD4+ single positive (SP), and CD8+ SP T cells as Sin1+/+ cells (Fig. 1A). We also derived progenitor T cells from Sin1+/+ and Sin1−/− fetal liver HSCs to further characterize the role of Sin1 in early T-cell development. Sin1+/+ or Sin1−/− fetal liver HSCs were cultured on OP9-DL1 stromal cells with IL-7 to generate stable T-cell lines that resemble CD4/CD8 double-negative thymocytes []. Phenotypic analysis of the in vitro derived Sin1+/+ and Sin1−/− T cells revealed that Sin1 is not required for the development of DN1, DN2, DN3, or DN4 T cells (Fig. 1B, top). Furthermore, analysis of these progenitor T cells revealed that Sin1 is not required for TCR beta chain expression (Fig. 1B, bottom).
To assess the effect of Sin1 on mTORC2-dependent signaling, we examined Akt S473 phosphorylation in Sin1+/+ and Sin1−/− T cells differentiated on OP9-DL1. As expected, Akt S473 phosphorylation was abolished in the Sin1-deficient T cells (Fig. 1C). We also observed that PKC hydrophobic motif phosphorylation was impaired in the Sin1−/− T cells (Fig. 1C). We have previously shown that FoxO1 expression is increased in Sin1−/− pro-B cells and FoxO1 phosphorylation is impaired in Sin1−/− fibroblasts and pro-B cells [[6, 13]]. Consistently, FoxO1 expression was increased in the Sin1−/− T cells relative to the Sin+/+ T cells (Fig. 1D). FoxO1 phosphorylation was also decreased in Sin1−/− T cells relative to Sin1+/+ T cells (Fig. 1D). These data show that Sin1 deficiency impairs mTORC2-dependent signaling in developing T cells. However, Sin1 deficiency does not significantly alter thymic T-cell development.
Next, we examined if Sin1 deficiency has any effect on peripheral T-cell populations. We observed equivalent proportions of splenic CD4+ and CD8+ T cells in Sin1+/+ and Sin1−/− chimeric mice (Fig. 2A). We also measured the proportion of cytokine producing CD4+ effector T cells in the periphery of unimmunized chimeric mice. We found that the proportion of IFN-γ, IL-4, or IL-17A expressing CD4+ T cells in the spleen of unimmunized Sin1−/− chimeric mice was comparable with that of Sin1+/+ mice (Fig. 2B). These data indicate that Sin1 may not be required for peripheral T-cell differentiation.
We have previously shown that suppression of FoxO1 and FoxO3a transcriptional activity by Akt is dependent on Sin1 and mTORC2 in MEFs and in B cells [[6, 13]]. FoxO1 is a positive regulator of L-selectin (CD62L), CD127 (IL-7 receptor alpha chain, IL-7R), and Foxp3 gene expression in T cells [[15, 16]]. Therefore, we asked if Sin1−/− T cells exhibit increased expression of these FoxO1-dependent genes. CD62L expression was increased on the splenic CD4+CD44lowCD62L+Sin1−/− T cells relative to Sin1+/+ T cells (Sin1+/+, MFI = 8520 versus Sin1−/− MFI = 17,400 (Fig. 2C) but CD127 expression was equivalent on Sin1+/+ and Sin1−/− peripheral T cells (Fig. 2D).
The transcription factor Foxp3 is the master regulator of Treg-cell development. To assess the possible role of Sin1 in Treg-cell development, we first determined the proportion of thymic Treg cells in Sin1+/+ and Sin1−/− chimeric mice. We observed that Sin1−/− thymocytes gave rise to twofold more CD25+Foxp3+ Treg cells when compared with Sin1+/+ thymocytes (4% Sin1+/+ CD4+CD25+FoxP3+ versus 10% Sin1−/− CD4+CD25+Foxp3+) (Fig. 2E), indicating that Sin1 may be a suppressor of thymic Treg-cell differentiation. The proportion of CD25+Foxp3+ T cells in the spleens of Sin1+/+ and Sin1−/− chimeric mice was not significantly different (9% Sin1+/+ CD4+CD25+Foxp3+ versus 10% Sin1−/− CD4+CD25+Foxp3+) (Fig. 2E).
To determine if the Sin1-mediated suppression of thymic Treg-cell development is cell intrinsic, we generated Sin1−/− chimeric mice containing an equivalent ratio of Sin1−/− fetal liver cells (CD45.2+) and WT cells (CD45.1+). There were two times more Sin1−/− CD25+Foxp3+ Treg cells than WT Treg cells (7% Sin1+/+ CD4+CD25+Foxp3+ versus 15% Sin1−/− CD4+CD25+Foxp3+) in the same host (Fig. 2F). These data indicate that Sin1 inhibits the development of thymic Treg-cell development in a cell intrinsic manner.
Akt is a negative regulator of Treg-cell development [] and Akt activity is directly regulated by mTORC2 [[6, 13]]. Since Sin1−/− cells lack mTORC2 function and exhibit deficiencies in Akt phosphorylation and function, we hypothesized that Akt may mediate mTORC2-dependent signals to suppress thymic Treg-cell development. To test this hypothesis, we measured the proportion of thymic Treg cells in Akt-deficient mice. We determined the proportion of CD4+Foxp3+ Treg cells in the thymus of WT, Akt1−/− or Akt2−/− mice. We found that Akt1−/− and Akt2−/− mice had an equivalent proportion of CD4+Foxp3+ T cells when compared with WT mice (Fig. 3A). In addition, we also analyzed thymic Treg-cell development in Akt1−/−Akt2−/− fetal liver cell chimeric mice (these mice die at late embryonic stage E18–19). Consistent with the previous reports [], we observed that thymocyte development was blocked at the DN to DP transition in Akt1−/−Akt2−/− chimeric mice (data not shown). However, a small number of Akt1−/−Akt2−/− thymocytes were capable of developing to the CD4+ SP stage. We measured the proportion of Foxp3+CD4+ T cells within this population of Akt1−/−Akt2−/− CD4+ SP cells and found that the proportion of Treg cells was similar to that observed in mice reconstituted with WT fetal liver cells (Fig. 3B).
Sin1 is not required for T-cell growth or proliferation
Mammalian TOR is a master regulator of cellular growth. Therefore, we asked if Sin1/mTORC2 was involved in regulating T-cell growth and proliferation. We found that the size of resting CD4+ and CD8+ T cells from lymph nodes or spleen of Sin1+/+ and Sin1−/− fetal liver chimeric mice was similar (Fig. 4A, data not shown). Next, we stimulated Sin1+/+ and Sin1−/− T cells with anti-CD3 plus anti-CD28 and assessed T-cell size change and proliferation. Sin1 deficiency did not impair the blast cell growth (size increase) following T-cell activation (Fig. 4B and C). CD4+ T cells from Sin1+/+ and Sin1−/− chimeric mice also exhibited a similar activation-induced proliferative capacity as determined by a CFSE dilution assay (Fig. 4D). Finally, we examined the proliferation and survival of Sin1+/+ and Sin1−/− CD4+ T cells activated in the presence of TGF-β. We observed that Sin1 deficiency did not impair the proliferation of in vitro differentiated CD4+Foxp3+ T cells (Fig. 4E). No difference in the proportion of live cells in the cultures of Sin1+/+ and Sin1−/− T cells was observed (Fig. 4F). These data suggest that Sin1 is not required for T-cell volume (size) growth of either resting or activated T cells and that Sin1 is not required for the proliferation and survival of activated T cells.
Sin1 is not required for TH1, TH2, and TH17 effector T-cell differentiation in vitro
To test the function of Sin1 in effector T-cell differentiation, we purified CD4+ T cells from Sin1+/+ or Sin1−/− chimeric mice, activated these cells in vitro and differentiated these cells under TH1, TH2, or TH17 polarizing conditions. Sin1+/+ and Sin1−/− T cells cultured under TH1, TH2, or TH17 polarizing conditions gave rise to equivalent proportions of IFN-γ (30% Sin1+/+ versus 35% Sin1−/−), IL-4 (6% Sin1+/+ versus 5% Sin1−/−), or IL-17 (15% Sin1+/+ versus 14% Sin1−/−) expressing cells, respectively(Fig. 5A). We obtained same results when we cocultured Sin1−/− T cells with WT congenic T cells under the same TH polarizing conditions (data not shown) indicating that Sin1 is not required for effector T-cell differentiation into the TH1, TH2, or TH17 lineages.
Sin1 is required for mTORC2-dependent phosphorylation of Akt in T cells
To examine if Akt phosphorylation at the mTORC2 target sites S473 and T450 was defective in Sin1−/− T cells, resting Sin1+/+ or Sin1−/− CD4+ T cells were stimulated with anti-CD3 antibody and Akt S473 phosphorylation was measured. As expected, compared with unstimulated T cells, anti-CD3 stimulation induced Akt S473 phosphorylation in Sin1+/+ T but failed to induce this phosphorylation in Sin1−/− T cells (Fig. 5B). Consistent with our previous observations in Sin1−/− fibroblasts and B cells, Akt T450 phosphorylation in Sin1−/− T cells was also deficient (Fig. 5C). These data show that Sin1-deficient T cells lack mTORC2 function and show defective Akt phosphorylation at the HM and TM sites.
Sin1 and mTOR differentially regulate TGF-β-dependent Treg-cell differentiation
Our observation that Sin1 deficiency promotes thymic Treg-cell development is consistent with a current model in which mTORC2-Akt signal inhibits FoxO1 activity, which is required for Treg-cell differentiation [[10, 12]]. To test if Sin1 may also inhibit the TGF-β-dependent Treg-cell differentiation of peripheral CD4+ T cells, purified Sin1+/+ or Sin1−/− CD4+ T cells were differentiated in the presence or absence of TGF-β. Without TGF-β Sin1+/+ and Sin1−/− CD4+ T gave rise to very few numbers of Foxp3+ cells (1.4% versus 1.6%) (Fig. 6A). In the presence of TGF-β, Sin1−/− CD4+ T cells consistently gave rise to fewer Foxp3+ Treg cells when compared with Sin1+/+ CD4+ T cells (28% versus 38%, respectively) (Fig. 6A). These data are surprising since we predicted that loss of mTORC2 function would enhance Treg-cell differentiation similar to that of Sin1−/− thymocytes. Our results raise the possibility that Sin1 may have mTORC2-independent functions
that may influence TGF-β-dependent Treg-cell differentiation in the periphery.
To directly test the function of mTOR during Treg-cell differentiation, we induced Treg-cell differentiation of WT naïve CD4+ T cells with TGF-β in vitro in the presence or absence of mTOR inhibitors rapamycin or pp242 []. Rapamycin specifically inhibits mTORC1 while pp242, a specific mTOR kinase inhibitor, targets both mTORC1 and mTORC2 []. We observed that rapamycin (30 nM) did not significantly change the proportion of Treg cells generated in the presence of TGF-β (untreated = 53% versus rapamycin treated = 50%). However, pp242 treatment (100 nM) consistently resulted in an increase in the proportion of Treg cells generated in response to TGF-β (untreated = 53% versus pp242 treated = 68%) (Fig. 6B). Both rapamycin and pp242 blocked mTORC1-dependent phosphorylation of ribosomal protein S6 while only pp242 blocked mTORC2-dependent HM site phosphorylation of Akt (Fig. 6C). Overall our data support a model in which inhibition of both mTORC1 and mTORC2 is necessary to promote TGF-β-induced Treg-cell differentiation.
In this study, we provide the first evidence examining the function of Sin1 in T cells. Our analysis of Sin1−/− fetal liver chimeric mice reveals that Sin1 is largely dispensable for the development of thymic T cells and peripheral CD4+ and CD8+ T-cell populations. Since Sin1 is essential for mTORC2 function, our data also indicate that mTORC2 is not required for T-cell development. Akt is the best characterized mTORC2 target and is required for T-cell development [[6, 7, 20]]. Akt1−/−Akt2−/− T cells show a profound block in thymic development at the DN to DP transition due to a dramatic increase in the rate of thymocyte cell death []. Sin1−/− T cells develop normally despite having a partial loss of Akt function due to impaired HM and TM phosphorylation. The dramatic difference in T-cell developmental phenotypes observed in Akt1−/−Akt2−/− and Sin1−/− chimeric mice indicates that the functional outcomes of Akt signaling can be subdivided into HM phosphorylation-independent signals and HM phosphorylation-dependent signals. Our data show that T-cell development is not dependent on Akt HM phosphorylation. These findings are consistent with our previously proposed model in which mTORC2-dependent Akt HM phosphorylation is required to confer Akt specificity toward a limited subset of Akt substrates []. Our data also suggest that Akt, when activated via phosphorylation of activation loop, plays a central role for DN–DP transition, most likely by controlling the survival of thymic T cells. Furthermore, our data suggest that phosphorylation of Akt at the activation loop may be sufficient to support TCR/CD3-mediated peripheral T-cell proliferation and survival.
Since mTOR is an evolutionarily conserved regulator of cellular growth and metabolism, we investigated if Sin1 deletion may affect the size of resting peripheral T cells or activated T cells and proliferation. Sin1 deficiency had little effect on resting T-cell growth and activation induced blast cell growth. Furthermore, Sin1 deficiency did not impair antigen receptor/co-receptor-dependent T-cell proliferation in vitro. These results contrast with those reported in mice bearing a T-cell-specific rictor deletion that show a modest defect in activation induced T-cell proliferation [[12, 21]]. It is possible that the differences in the in vitro T-cell stimulation conditions between our assays may account for the difference in experimental results since we stimulated our T cells in the presence of plate-bound anti-CD3 antibody plus soluble anti-CD28 in the presence of exogenous IL-2.
FoxO1 is an mTORC2-dependent Akt substrate that has been shown to play a key role in regulating T-cell development, homeostasis, and effector cell differentiation [[16, 22]]. FoxO1 is required for proper expression of the genes that encode L-selectin (CD62L), interleukin 7 receptor alpha chain (CD127), and Foxp3 [[15, 16, 22]]. We have previously shown that Sin1 deficiency results in decreased FoxO1 phosphorylation at the Akt target sites, leading to increased FoxO1 transcriptional activity [[6, 13]]. Consistently, we observed an increased proportion of Foxp3 expressing nTreg cells in the thymus and an increased expression of CD62L expression on naive peripheral CD4+ T cells in Sin1−/− chimeric mice. Surprisingly, Sin1 deficiency did not affect IL-7R expression on resting peripheral T cells. We have previously shown that in developing progenitor B cells, the mTORC2-Akt-FoxO1 signaling negatively regulates IL-7R expression []. IL-7R expression is suppressed in antigen activated T cells. It is possible that the loss of mTORC2 function has no effect on IL-7R expression in resting T cells because these cells normally have a very low level of Akt signaling. Mammalian TORC2 may play a more important role in suppressing IL-7R expression after T-cell activation since TCR signaling strongly induces the Akt signaling pathway. Alternatively, it is possible that another kinase may phosphorylate and regulate FoxO1 activity in place of Akt in Sin1−/− T cells. The serum and glucocorticoid-dependent kinases (SGKs) may also phosphorylate FoxO proteins and negatively regulate FoxO transcriptional activity []. This may explain why we did not observe a complete loss of FoxO1 phosphorylation in Sin1−/− T cells. SGK1 has been shown to be positively regulated by both mTORC1 and mTORC2-dependent mechanisms [[24, 25]]. Since mTORC1 activity is not inhibited by Sin1 deficiency it is possible that SGK1 may play an important role in the regulation of FoxO1 in Sin1−/− T cells. Interestingly, like our previous observation in pro-B cells [], we observed a significant increase in FoxO1 expression in Sin1−/− T cells. These data raise the possibility that Sin1 may regulate FoxO1 expression, although the exact mechanism through which this regulation occurs is currently unclear.
We have also determined if Akt mediates the Sin1–mTORC2 signals to regulate the development of thymic nTreg cells by examining the nTreg-cell development in Akt1−/−, Akt2−/−, and Akt1−/−Akt2−/− mice. We had previously used a similar experimental approach to identity Akt2 as the specific mediator of mTORC2-dependent FoxO1 regulation in B cells []. Disruption of Akt1, Akt2, or both Akt1 and Akt2 did not alter the proportion of CD4+ thymic nTreg cells when compared with WT mice. Therefore, it is possible that either Akt3 is the principle mediator of mTORC2-dependent FoxO1 regulation or, alternatively, FoxO1 may be inhibited by other mTORC2-dependent AGC kinases such as SGKs.
We also explored the function of Sin1 in CD4+ T-helper cell differentiation. We did not observe any deficiency in the ability of Sin1−/− CD4+ T cells to differentiate into TH1, TH2, or TH17 effector cells. These data also differ from the results reported in rictor−/− T cells from two different groups [[12, 21]]. Lee et al.  reported that Rictor-deficient CD4+ T cells show impaired TH1 and TH2 differentiation while Delgoffe et al.  only observed a deficiency in TH2 differentiation in rictor−/− T cells. Lee et al. also report that PKC phosphorylation is deficient in rictor−/− T cells and that ectopic expression of PKCθ rescues TH2 differentiation in rictor−/− T cells. Interestingly, we observe that PKC–HM phosphorylation is deficient in Sin1−/− T cells, however, we failed to observe a deficiency in TH2 differentiation in Sin1−/− T cells. It is possible that the disparity between our data and those observed in rictor−/− T cells could be partially due to differences in the in vitro experimental conditions used to induce TH cell differentiation in the three studies. Alternatively, it is possible that Rictor may also influence TH-cell differentiation through a mechanism that is independent of mTORC2. Analysis of the roles Rictor and Sin1 in the context of a physiologic T-cell immune response should resolve these issues.
Our observation that Sin1 deficiency in T cells results in an increased proportion of thymic Treg cells is consistent with previous studies linking mTOR and FoxO transcription factors to regulatory T-cell differentiation. Surprisingly, however, we observed that peripheral Sin1−/− CD4+ T cells gave rise to fewer Foxp3+ cells when stimulated in the presence of TGF-β. The unexpected finding that Sin1−/− T cells had slightly decreased TGF-β-dependent Treg-cell differentiation suggests that Sin1 may regulate Treg-cell development independent of mTORC2 function. It is possible that Sin1 may regulate TGF-β-dependent Treg-cell differentiation through the MAPK signaling pathway []. In this regard, we have recently shown that deletion of MEKK2/3, which bind to and are negatively regulated by Sin1, augments TGF-β-dependent Treg-cell differentiation []. Future investigations into the role of Sin1–MAPK signaling in T cells will help elucidate the mechanism underlying this phenotype.
Materials and methods
Sin1−/‒ mice and Akt1−/−, Akt2−/−, and Akt1−/−Akt2−/− mice were described previously [[6, 13]]. CD45.1+ congenic mice were purchased from The Jackson Laboratory and used as recipients for the fetal liver hematopoietic cell transfers. Mice receiving fetal liver cell transplants were irradiated with 700–900 cGy prior to cell transfer. 0.5–1 × 106 total fetal liver cells were suspended in sterile 1 × PBS and injected via the tail vein. Successful donor cell engraftment was verified by the presence of CD45.2+ peripheral blood mononuclear cells. All mice were housed in the animal facilities at Yale University and all animal procedures were approved by the Yale IACU Committee.
OP9-DL1/progenitor T-cell cultures
Mouse fetal liver hematopoietic cells were obtained from embryonic day 11.5–12.5 Sin1+/+ and Sin1−/− littermate embryos. Fetal liver cells were cultured on confluent OP9-DL1 bone marrow stromal cells in RPMI1640 medium supplemented with 10% fetal bovine serum, 100 U/mL penicillin, 100 μg/mL streptomycin, 5 μg/mL gentamicin, 50 μM β-mercaptoethanol, and 10 ng/mL mouse IL-7 (Constem, CT). Stable T-cell lines were grown at 37°C in an atmosphere containing 5% CO2.
Lymphocyte staining and flow cytometry
Cells were washed with FACS buffer (1% FBS in 1× PBS with 0.1% NaN3), incubated with indicated antibodies on ice for 30 min, then washed two more times with FACS buffer, and fixed in 1% paraformaldehyde in PBS before being analyzed with a LSRII flow cytometer (BD Biosciences). For intracellular cytokine staining, cells were stimulated with phorbol 12-myristate 13-acetate (PMA, Sigma) (50 ng/mL) + ionomycin (Sigma) (500 ng/mL) for 6 h in the presence of Golgi-stop (BD Bioscience) for the last 4 h. Cells were first surface stained, fixed/permeablized with a Cytofix/Cytoperm kit (BD Bioscience), and stained with antibodies against indicated cytokines. Intracellular FoxP3 and T-bet staining were carried out according to manufacturer's instruction (EBioscience). For costaining Foxp3 with GFP, cells were fixed by cytofix buffer (BD Bioscience), permeablized by ice-cold methanol and stained with indicated antibodies in the 1× Perm/Wash buffer (BD Bioscience).
Cell purification and culture
Splenocytes and lymph node cells were first stained with anti-CD4 biotin and CD4+ cells were magnetically purified using a Biotin-selection kit (Stem cell). Purified CD4+ T cells were stimulated with plate bound anti-CD3 (3 μg/mL; 2C11) and soluble anti-CD28 (2 μg/mL; 37N) in T-cell medium (RPMI 1640, 10% FBS, 1× antibiotics, 1× nonessential amino acid, and 50 μM β-mercaptoethanol). When indicated, recombinant (r) cytokines were added into the culture: TH1: anti-IL-4 (5 μg/mL; 11B11) and IL-12 (10 ng/mL; PeproTech); iTreg-cell: anti-IL-4 (5 μg/mL, 11B11), anti-IFN-γ (5 μg/mL; R46A2), rhIL-2 (100 U/mL, PeproTech), and indicated concentration of rhTGF-β (Peprotech); TH17: anti-IL4 (5 μg/mL), anti-IFN-γ (5 μg/mL), IL-6 (20 ng/mL, Peprotech), and indicated concentration of rhTGF-β (Peprotech). When indicated, the following inhibitors were used in this study: Rapamycin (LC laboratories); pp242 [].
T-cell stimulation and immunoblotting
Naive CD4+ T cells were activated with anti-CD3/anti-CD28 antibodies in the presence of IL-2 (50 U/mL) for 4 days. Activated cells were then split into fresh culture medium with IL-2 (100 U/mL) and expanded for an additional 4 days. Cultured T cells were rested in T-cell medium without IL-2 overnight and stimulated with either plate bound anti-CD3 (5 μg/mL) + anti-CD28 (2 μg/mL) for various time points. Stimulated T cells were washed with ice-cold PBS and lysed with RIPA buffer plus freshly added protease inhibitors and phosphatase inhibitors. Total cell lysates were used for immunoblot analysis. To detect S6 and Akt S473 phosphorylation following TCR stimulation, CD4+ T cells were first stained with anti-CD3 (5 μg/mL) for 30 min on ice. After wash, T cells were cross-linked with anti-Hamster IgG for 3 min, fixed with Phosflow fix buffer I (BD Bioscience), and stained with anti-pS6 S235/236 or anti-pAkt S473 (Cell Signaling) in Phosflow perm/wash buffer (BD Bioscience) for 30 min at room temperature followed by Alex-fluor 647-conjugated anti-Rabbit IgG (Cell Signaling) in Phosflow perm/wash buffer for 15 min at room temperature.
Purified CD4+ T cells were labeled with CFSE (3 nM) at 37°C for 10 min. CFSE-labeled cells were stimulated with plate bound anti-CD3 and anti-CD28 as described [[28, 29]].
We thank Drs. A. Di Lorenzo and W. Sessa (Yale University) for the Akt1 and Akt2 knockout mice, K.M. Shokat (UCSF) for providing the pp242. This work is supported in part by grants AI063348 (NIH) and PR093728 (DOD) (to B. Su). A.S. Lazorchak is a Leukemia and Lymphoma Society fellow, and X. Chang was a recipient of Gershon and Trudeau Fellowship from Yale University.
Conflict of interest
The authors declare no financial or commercial conflict of interest.