The Kinases Aurora B and Mtor Regulate the G1–S Cell Cycle Progression of T Lymphocytes

ARTICLES

The kinases aurora B and mTOR regulate the G1–S cell cycle progression of T lymphocytes

Jianxun Song1,2, Shahram Salek-Ardakani1, Takanori So1 & Michael Croft1

CD28-deﬁcient T cells arrest at the G1–S transition of the cell cycle. Here we show that this is controlled by the kinase aurora B, which exists in a complex with survivin and mammalian target of rapamycin (mTOR). Expression of aurora B in Cd28–/– T cells augmented phosphorylation of mTOR substrates, expression of cyclin A, hyperphosphorylation of retinoblastoma protein and activation of cyclin-dependent kinases 1 and 2 and promoted cell cycle progression. Interleukin 2 enhanced aurora B activity, and inactive aurora B prevented interleukin 2–induced proliferation. Moreover, expression of aurora B restored Cd28–/– T cell proliferation and promoted inﬂammation in vivo. These data identify aurora B, along with survivin and mTOR, as a regulator of the G1–S checkpoint in T cells.

T cell proliferation is important during most immune responses, Survivin, an inhibitor of apoptosis family protein, has been identi- http://www.nature.com/natureimmunology as it allows a low frequency of peptide-specific T cells to attain fied as a target of signals from OX40, a tumor necrosis factor receptor numbers sufficient to resolve infection. It has been suggested that a family costimulatory molecule17. Survivin has been described as a ‘second signal’ in addition to the signal emanating from the T cell protein that either promotes mitosis as a chromosome passenger18 or receptor (TCR) is required for passage beyond a tolerance checkpoint suppresses apoptosis during mitosis19,20. However, it has been found and avoidance of adoption of a hyporesponsive state1,2.Themolecular that survivin is expressed in all phases of the T cell cycle, including G1 basis of that checkpoint has been hotly pursued. The immuno- and S and that survivin promotes G1–S progression17. Survivin might globulin ‘superfamily’ member CD28 was initially proposed as a bind many caspases19,20. However, at least during mitosis, it is thought candidate receptor able to transmit that essential costimulatory pro- that survivin interacts with the serine-threonine kinase aurora B (also liferative signal3–5. Although other costimulatory molecules in the called AIM-1) and with INCENP, an inner centromere protein21,22. tumor necrosis factor and cytokine receptor families have subse- There are no reports of aurora B kinase expression or activity in quently been shown to also control proliferation6,7,themain T cells. As with survivin, the literature on aurora B has focused on its intracellular targets of signals emanating from those receptors and involvement in chromosome segregation23–25. Nonetheless, the cell the molecular nature of checkpoints controlling T cell division remain cycle data17 have raised the possibility that aurora B might bind

© Group 2006 Nature Publishing to be identiﬁed. survivin in early phases of the T cell cycle and that its activity might T cells stimulated in the absence of CD28 produce little interleukin regulate T cell proliferation at the tolerance checkpoint. 2 (IL-2), and IL-2 receptor (IL-2R) signals can partially overcome their Here we show that aurora B is present in all phases of the T cell proliferative defect, which is manifested as a block in progression from cycle, including G1 and S. Aurora B existed in a complex with survivin the G1 phase to the S phase of the cell cycle8–10. Phosphatidylinositol- in T cells, and its kinase activity was regulated by survivin. Notably, 3-OH kinase (PI(3)K) and protein kinase B (also called Akt) are T cells stimulated in the absence of CD28 failed to upregulate aurora B activated by CD28 and IL-2R stimulation and therefore may be and survivin and had hallmark characteristics of cells blocked at the involved in that checkpoint11,12. Rapamycin suppresses T cell prolif- G1- to S-phase transition, including low expression of cyclin A, eration and G1- to S-phase cell cycle progression10.Rapamycin, hypophosphorylation of retinoblastoma protein (Rb) and weak activ- through FK506 binding protein, inhibits the serine-threonine kinase ity of cyclin-dependent kinase 1 (Cdk1) and Cdk2 (refs. 26–28). mammalian target of rapamycin (mTOR), which is a target of Akt. In Induction of aurora B in primed T cells was not blocked by rapamycin addition, the cell cycle inhibitor p27Kip1 is a target of Akt, and some but aurora B kinase activity was impaired, suggesting that the function but not all studies have suggested that p27Kip1 can control T cell of aurora B is dependent on mTOR. In agreement with those hyporesponsiveness13–15. However, forced expression of Akt is unable observations, aurora B existed in a complex with mTOR and regulated to restore proliferation of Cd28–/– Tcells16, suggesting that additional the phosphorylation of mTOR and the mTOR substrates p70S6 molecules, acting either in isolation or in synergy with Akt or mTOR, kinase (p70S6k) and 4E-BP1. Moreover, aurora B regulated Rb phos- might control that tolerance checkpoint. phorylation, cyclin A expression and Cdk1 and Cdk2 activity in a

1Division of Molecular Immunology, La Jolla Institute for Allergy and Immunology, La Jolla, California 92037, USA. 2Institute of Immunology PLA, The Third Military Medical University, Chongqing, China. Correspondence should be addressed to M.C. ([email protected]). Received 14 April; accepted 23 October; published online 26 November 2006; doi:10.1038/ni1413

NATURE IMMUNOLOGY ADVANCE ONLINE PUBLICATION 1 ARTICLES

Day 012 3 a b c Day 1 Day 2 d 4 4 Aurora B /– 10 10 WT – –/– –/– –/– 0.1% 0.1% 80 WT Cd28–/– –/– 3 3 g) Cd28

10 10 µ 250 WT Cd28 WT Cd28 WT Cd28 WT Cd28 Control 60 Aurora B (No Ag) 102 102 40 200 Survivin 20 101 101 Incorporation 150 (pmol/min/ 0 Cyclin A 0 0

c.p.m. 10 10 01234 3 100 100 101 102 103 104 100 101 102 103 104 Time (d)

10 p-Cdk1 104 104 50 31% 30% Cdk1 p-Cdk2 e 100 103 103 0 g) 80

p-Rb µ 135 WT 102 102 60 Time (d) p21 40 1 1 p27 10 10 20 Incorporation (pmol/min/ 25 6 100 100 0 20 Cyclin B 100 101 102 103 104 100 101 102 103 104 01234 4 15 4 4 Time (d) 10 Cyclin D1 10 10 5 2 12% 6% Cdk2 3 3 IL-4 (ng/ml) IL-2 (ng/ml) 0 Cyclin E 10 10 f 80 0 Cd28–/– g)

–/– –/– 2 2 µ 60 WT WT INCENP 10 10 40 Cd28 Cd28 Bub1 101 101 20 β Aurora B -actin Incorporation 100 100 (pmol/min/ 0 1 2 3 4 0 1 2 3 4 0 10 10 10 10 10 10 10 10 10 10 01234 CFSE Time (d)

Figure 1 Defective aurora B expression in primed hyporesponsive Cd28–/– Tcells.(a) Proliferation (top) and production of IL-2 (bottom left) and IL-4 (bottom right) by primed wild-type (WT) or Cd28–/– AND TCR-transgenic CD4+ T cells restimulated in vitro with APCs and moth cytochrome c (MCC) peptide, assessed at various times (top, horizontal axis) or 24 h (bottom) after restimulation. Data are means of triplicate cultures and are representative of three experiments. (b) Immunoblot of total or phosphorylated (p-) proteins in lysates of CD4+ T cells after restimulation as in a (time, above lanes). Similar data were obtained in three experiments. (c) Aurora B expression by primed CD4+ T cells labeled with carboxyﬂuorescein diacetate succinimidyl diester (CFSE) and stimulated for 1 or 2 d with peptide, assessed by intracellular staining. Ag, antigen. Numbers in boxed areas indicate percent aurora B–positive cells. Data are representative of three experiments. (d–f) Activity of kinases aurora B (d), Cdk1 (e)andCdk2(f) immunoprecipitated from CD4+ T cell lysates after restimulation (time, horizontal axes), assessed with histone H3 (d) or histone H1 (e,f) as a substrate. Data are representative of http://www.nature.com/natureimmunology three experiments.

rapamycin-sensitive way. Our observations correlate with published To elucidate the regulation of aurora B, we initially focused on studies showing that mTOR can control those events29. Notably, survivin, which is reported to interact with aurora B in certain expression of aurora B in Cd28–/– T cells was sufficient to promote nonlymphoid cells during mitosis21,22. We stimulated naive wild- G1- to S-phase progression, T cell proliferation and inflammation in type or Cd28–/– T cells with antigen and transduced them with vivo. Our data define a previously unknown checkpoint for regulating retroviral vectors encoding green fluorescent protein (GFP) and T cell proliferation, involving aurora B kinase, survivin and mTOR. wild-type (not constitutively active) aurora B or a ‘kinase-dead’ mutant in which the aurora B ATP-binding lysine residue was replaced RESULTS with an arginine residue23,30. We then sorted and restimulated GFP+ Aurora B and survivin in Cd28–/– T cells cells. Kinase assays confirmed the functionality of the retrovirus- We used AND TCR-transgenic CD4+ cells that had been activated encoded aurora B constructs (Supplementary Fig. 1). Neither © Group 2006 Nature Publishing (primed) for 6 d in vitro with peptide and antigen-presenting cells ‘kinase-dead’ nor wild-type aurora B affected the expression of Bcl-2 (APCs). After being restimulated with peptide, Cd28–/– T cells had a family antiapoptotic molecules (Fig. 2a). Survivin can be phosphory- hyporesponsive phenotype, proliferated poorly and produced little lated, which might prevent degradation and/or might be required for IL-2 and IL-4 (Fig. 1a). We assessed many cell cycle molecules and the interaction of survivin with its binding partners31,32.Therewasless found that only a limited number were adversely affected in Cd28–/– phosphorylated survivin in wild-type T cells expressing ‘kinase-dead’ T cells. Cd28–/– T cells contained less of cyclin A and phosphorylated aurora B and more phosphorylated survivin in Cd28–/– T cells forms of Cdk1, Cdk2 and Rb, all of which are known to regulate G1- expressing wild-type aurora B, even though total survivin was not to S-phase progression (Fig. 1b). Notably, the cells contained less altered (Fig. 2a). That result was in line with data from cell-free aurora B kinase and survivin at similar time points. Wild-type and systems showing that aurora B can phosphorylate survivin33. Retro- Cd28–/– T cells had similar expression of other molecules associated viral introduction of survivin into Cd28–/– T cells had no effect on with G1 and S phases, including cyclin E and the inhibitors p27 and aurora B amounts but promoted aurora B activity (Fig. 2b). These p21. To confirm the defective expression of aurora B by Cd28–/– data also correlated with results obtained with xenopus eggs and cell- T cells, we used intracellular staining to assess aurora B expression free systems22,34. in wild-type and Cd28–/– T cells labeled with carboxyfluorescein Next we analyzed the expression of aurora B and survivin diacetate succinimidyl diester. The proportion of Cd28–/– T cells in during various phases of the cell cycle in wild-type T cells. Like each division that expressed aurora B was less than that of wild-type survivin17, aurora B not only was expressed during the G2 T cells (Fig. 1c and Supplementary Fig. 1 online). The proportion of and M phases, as reported in non–T cells, but also was present wild-type cells expressing aurora B was similar to, albeit more than, during the G1 and S phases (Fig. 2c). Moreover, we immuno- the proportion shown before to express survivin17. Similarly, kinase precipitated aurora B together with survivin, and survivin assays demonstrated lower activity of aurora B (Fig. 1d), Cdk1 with aurora B, confirming that the two molecules can exist in a (Fig. 1e) and Cdk2 (Fig. 1f)inCd28–/– T cells. complex (Fig. 2d).

2 ADVANCE ONLINE PUBLICATION NATURE IMMUNOLOGY ARTICLES

abcdWT Cd28–/– IP: survivin

–/– Total –/– WT Cd28 4 10 Sub Mig Mig–survivinMig Mig–survivin G0-G1 S Input WT Cd28 103 Survivin 102 IgH 1 –aurora B 10 Aurora B G2-M Mig Mig–KR–auroraMig B Mig G1 100 Bcl-xL 0 50 100150 200 250 Aurora B Aurora B Aurora B positive (40 kDa) Bcl-2 4 p-Survivin 10 3 Bfl-1 10 Survivin Survivin 102 β-actin 101 IP: Aurora B – Bcl-xL –/ 100 Mig 0 50 100 150 200 250 80 Mig–survivin Input WT Cd28 Bcl-2 Survivin positive 104 60 IgH g) Bfl-1 µ 103 40 102 β-actin 1

BrdU 10 Incorporation (pmol/min/ 20 Survivin 100 0 50 100 150 200 250 (16 kDa) 0 7-AAD WT Cd28–/– Aurora B

Figure 2 Aurora B interacts with survivin. Primed wild-type and Cd28–/– AND TCR-transgenic CD4+ T cells infected with retroviral vector encoding GFP alone (Mig), GFP and aurora B (Mig–aurora B), GFP and survivin (Mig–survivin) or GFP and ‘kinase-dead’ aurora B (Mig–KR–aurora B) were sorted by GFP expression, were restimulated for 3 d with APCs and MCC peptide and were lysed. (a) Immunoblot analysis of total and phosphorylated proteins (left margin). Similar data were obtained in three experiments. (b) Immunoblot analysis of proteins (top) and activity of immunoprecipitated aurora B, measured with histone H3 as a substrate (bottom). Data are representative of three experiments. (c) Flow cytometry of the expression of CD4, aurora B or survivin by primed wild-type CD4+ T cells 2 d after restimulation; cultures were pulsed with BrdU for 60 min, then stained with 7-amino-actinomycin D (7-AAD). Data represent the cell cycle status of gated CD4+ T cells (top), aurora B–positive gated CD4+ T cells (middle) and survivin–positive gated CD4+ T cells (bottom). Similar http://www.nature.com/natureimmunology data were obtained in three experiments. (d) Immunoblot analysis of proteins associated with immunoprecipitated (IP) survivin (top) and aurora B (bottom) 3 d after restimulation of wild-type CD4+ T cells. Input, immunoprecipitation with control rabbit immunoglobulin G (IgG); bottom blots, control for immunoprecipitation. Data are representative of two to four experiments.

Aurora B and cell cycle progression To determine whether aurora B contributes to the progression from To investigate whether aurora B might be active during early phases of G1 to S phase, we introduced wild-type aurora B into primed Cd28–/– the cell cycle, we restimulated primed wild-type T cells in the presence T cells. Within 2 d of stimulation, expression of cyclin A and of the S-phase inhibitor hydroxyurea or in the presence of rapamycin, phosphorylation of Rb, Cdk1 and Cdk2 were upregulated in Cd28–/– which is known to arrest T cells in late G1 phase and to suppress TCR-, T cells expressing aurora B (Fig. 3c). Expression of cyclin E, which can CD28- or IL-2-induced expression of cyclin A and activation of Cdk1 also bind to Cdk1 and Cdk2, was not altered, suggesting that cyclin A and Cdk2 (refs. 29,35). Inhibitor treatment did not alter the expres- but not cyclin E is a target of aurora B. A substantial fraction sion of transcripts encoding aurora B or survivin (Fig. 3a), suggesting of primed Cd28–/– T cells transduced with the retroviral vector that induction of aurora B and survivin in primed T cells occurs encoding aurora B progressed into the S, G2 and M phases © Group 2006 Nature Publishing independently of mTOR and of progression through the S, G2 and M (Fig. 4a). Cd28–/– T cells transduced with a retroviral vector encoding phases. Immunoblot analysis confirmed aurora B and survivin protein wild-type survivin had a similar phenotype (Fig. 4a). In contrast, induction in the presence of rapamycin and hydroxyurea (Fig. 3b, retroviral expression of ‘kinase-dead’ aurora B or dominant negative top). Treatment with mTOR inhibitors reduced the number of cells in (phosphorylation-deficient; T34A) survivin17 inhibited the progres- the S, G2 and M phases, but cells blocked in the G1 and S phases sion of wild-type T cells through the cell cycle (Fig. 4a). Those still had expression of aurora B (Fig. 3b, bottom). In contrast, phenotypes were not the result of altered production of proliferative treatment with the PI(3)K inhibitors Ly294002 or wortmannin com- cytokines (Fig. 4b) or of activation of Akt or the Erk1 and Erk2 serine- pletely suppressed the induction of aurora B and survivin mRNA and threonine kinases, which are ‘downstream’ of IL-2R and IL-4R protein (Fig. 3a,b). signaling (Fig. 3c). Next we analyzed naive T cells and found that mRNA Given that rapamycin very efficiently inhibited G1- to S-phase encoding aurora B was induced even in the presence of rapamycin progression, we investigated whether aurora B function was dependent and hydroxyurea (Supplementary Fig. 2 online). Aurora B protein on mTOR. Rapamycin inhibited the kinase activity of aurora B in expression was more strongly inhibited by rapamycin in naive primed T cells (Fig. 5a). However, some kinase activity was still than in primed T cells (Supplementary Fig. 2). We visualized apparent even in the presence of rapamycin, suggesting that aurora B fewer T cells expressing aurora B by intracellular staining than when function is partially dependent on mTOR activity. Aurora B activity the inhibitors were used with primed T cells (Supplementary Fig. 2), was reduced more in naive T cells than in primed T cells treated with an observation that was partly although not fully explained by the rapamycin (Supplementary Fig. 2), again indicating that aurora B lower proportion of naive T cells that entered the S, G2 and M phases may be differentially regulated in naive and activated T cells. In in the presence of the inhibitors (data not shown). These results primed wild-type or Cd28–/– T cells retrovirally transduced with emphasized potential differences in aurora B regulation in naive and vector encoding wild-type aurora B, rapamycin partially supp- primed T cells that remain to be investigated. ressed the phosphorylation of survivin, induction of cyclin A and

NATURE IMMUNOLOGY ADVANCE ONLINE PUBLICATION 3 ARTICLES

ab c

DMSO LY294002WortmanninRapamycinHydroxyurea –/– Time (d): 011111 WT Cd28 Aurora B

Survivin Mig Mig–aurora MigB Mig–aurora B 5 β-actin Aurora B 4 Total Aurora B positive Total

Birc5 3 Cyclin A 104 104 104 2 56% 3 3 103 Cyclin E expression 1 10 10 Relative Control 2 p-Rb 0 102 102 10

1 1 1 8 10 10 10 Rb 0 0 0 6 10 10 10 p-Cdk1 0 50 100 150 200 250 0 50 100 150 200 250 100 101 102 103 104 Aurkb 4 104 104 104 Cdk1 31% 3 3 expression 2 10 103 10 p-Cdk2

Relative Rapamycin 0 102 102 102 Cdk2 2 0 1 1 1 No Ag 10 10 10 p-Akt 2940 DMSO LY Rapamycin 0 0 0 Wortmannin Hydroxyurea 10 10 10 0 50 100 150 200 250 0 50 100 150 200 250 100 101 102 103 104 Akt +Ag 104 104 104 p-Erk1/p-Erk2 30% 3 3 10 103 10 Erk1/Erk2

Hydroxyurea 2 2 2 10 10 10 β-actin 101 101 101 BrdU BrdU Aurora B 100 100 100 0 50 100 150 200 250 0 50 100 150 200 250 100 101 102 103 104 7-AAD 7-AAD CD4

Figure 3 Aurora B is expressed in the G1 and S phases and regulates G1- and S-phase proteins. (a,b)RT-PCR(a) and immunoblot analysis and flow http://www.nature.com/natureimmunology cytometry (b) of primed AND TCR-transgenic wild-type CD4+ T cells restimulated with APCs and MCC peptide in the presence of DMSO (control) or various chemical inhibitors. (a) RT-PCR of cell lysates 12 h after restimulation, for expression of Birc5 (encoding survivin; top) and Aurkb (encoding aurora B; bottom). Similar data were obtained in one repeat experiment. (b) Immunoblot of cell lysates (top) and intracellular staining (bottom) 24 h after restimulation, for quantification of aurora B and survivin protein. Bottom, cell cycle status of CD4+ T cells (left) or aurora B–positive CD4+ T cells (middle), assessed by pulsing with BrdU for the final 60 min of culture and staining with 7-amino-actinomycin D; right, numbers beside boxed areas indicate percent CD4+ T cells expressing aurora B. Boxed areas are sub-G0, G0-G1, S and G2-M, as in Figure 2. Data are representative of three experiments. (c)Immunoblot analysis of protein expression by primed wild-type and Cd28–/– CD4+ AND TCR-transgenic T cells infected with retroviruses (above lanes); 2 d after restimulation with APCs and MCC peptide, GFP-sorted cells were lysed for analysis. Similar data were obtained in three experiments.

phosphorylation of Rb, Cdk1 and Cdk2; complete suppression was Erk activation was not substantially affected by aurora B expression achieved by inhibition of PI(3)K activity (Fig. 5b,c). However, (Fig. 5 and data not shown), and treatment with UO126, an inhibitor progression through S phase was suppressed by rapamycin treatment of the MEK1 serine-threonine kinase that phosphorylates Erk, did not (Fig. 5d). These data suggested that although aurora B induction in prevent induction of the expression of Aurkb mRNA (encoding aurora © Group 2006 Nature Publishing primed T cells is not dependent on mTOR, substantial aurora B B) or Birc5 mRNA (encoding survivin) in primed T cells (Supple- activity, in particular its ability to promote progression through mentary Fig. 3 online). However, treatment with UO126 suppressed S phase, is dependent on mTOR. The Erk signaling pathway has also been WT Cd28 –/– abMig Mig linked to the control of T cell proliferation. 4 4 10 4.8 10 10 103 56.5 103 70.7 29.5 6.7 Figure 4 Aurora B and survivin promote G1-S 102 3.0 102 0.2 WT Cd28–/– WT Cd28–/– 101 101 cell cycle progression but not cytokine production 6 6 –/– in Cd28 T cells. (a) Cell cycle analysis of 100 100 0 50 100 150 200 250 0 50 100 150 200 250 4 4 –/– primed wild-type or Cd28 T cells 2 d after 2 2 Mig–KR–aurora B Mig–aurora B restimulation with APCs and MCC peptide; 4 4 IL-4 (ng/ml) 0 0 10 11 10 7.1 GFP-sorted cells infected with retroviruses were 25 25 103 75.2 103 64.7 pulsed with BrdU for the ﬁnal 60 min of culture 7.3 20.8 20 20 102 0.1 102 2.2 15 15 and were stained with 7-amino-actinomycin D. 10 10 101 101 5 5 Mig–T34A–survivin encodes GFP and dominant IL-2 (ng/ml) 0 0 100 100 0 50 100 150 200 250 0 50 100 150 200 250 negative survivin. Numbers in plots are percent Mig Mig Mig Mig cells in the following phases (top to bottom): sub- Mig–T34–survivin Mig–survivin aurora B –survivin 4 4 T34 Mig–survivin + 10 10 KR–auroraMig– B G0, G1, S and G2-M. Data are gated on CD4 T 12 4.6 103 71.9 103 62.3 Mig– Mig– cells and are representative of three experiments. 8.5 21.7 (b) Enzyme-linked immunosorbent assay of the 102 0.8 102 2.3 101 101

production of IL-2 and IL-4 40 h after BrdU 100 100 restimulation as in a.Dataare 0 50 100 150 200 250 0 50 100 150 200 250 mean ± s.d. from three experiments. 7-AAD

4 ADVANCE ONLINE PUBLICATION NATURE IMMUNOLOGY ARTICLES

DMSO Interaction of aurora B with mTOR a 80 Rapamycin bc 94002 94002 ThekinasemTORbindstoseveralproteins,

g) 60 DMSO LY 2 Rapamycin DMSO LY 2 Rapamycin µ Time (d): 01 1 1 Time (d): 0 1 1 1 such as raptor (‘regulatory-associated 40 IB: p-survivin IB: p-survivin protein of mTOR’) and mLst8-GbL(‘lethal 20 Survivin Survivin with sec thirteen’). The kinase mTOR, (pmol/min/ Cyclin A Cyclin A 0 or raptor indirectly, recruits downstream Aurora B incorporation 01 234 p-Rb p-Rb Time (d) substrates such as p70S6k and 4E-BP1, Rb Rb DMSO Rapamycin which control protein translation and cell d 104 104 1.5 2.9 p-Cdk1 p-Cdk1 36,37 103 58.9 103 82.8 growth . However, it has been suggested Cdk1 Cdk1 WT 2 25.0 2 9.6 10 9.6 10 1.4 that mTOR may not directly phosphor- p-Cdk2 p-Cdk2 101 101 ylate p70S6k or 4E-BP1 and that another Cdk2 Cdk2 100 100 0 50 100 150 200 250 0 50 100 150 200 250 p-Akt p-Akt kinase might be required for full activation 4 4

–/– 10 10 4.4 9.7 Akt Akt of p70S6k and 4E-BP1 (ref. 36). Therefore, 3 78.7 3 82.9 10 10 p-Erk1/ p-Erk1/

Cd28 9.7 1.9 we investigated whether aurora B could 2 2 10 3.0 10 0.6 p-Erk2 p-Erk2 Erk1/ Erk1/ function as an integral part of such an 101 101 Erk2 Erk2 BrDU β-actin β-actin 100 100 mTOR signaling unit. Immunoprecipitation 0 50 100 150 200 250 0 50 100 150 200 250 7-AAD of aurora B resulted in precipitation of p70S6k, mTOR and 4E-BP1, and immuno- Figure 5 Aurora B kinase activity and cell cycle control depend on mTOR. (a) Aurora B kinase activity precipitation of mTOR resulted in precipita- + in primed wild-type AND TCR-transgenic CD4 T cells restimulated with APCs and MCC peptide in the tion of aurora B, p70S6k and 4E-BP1 presence of DMSO (control) or rapamycin; immunoprecipitated aurora B was assessed with histone H3 as a substrate. Data are representative of two experiments. (b–d) Analysis of protein expression (b,c) (Fig. 6a). Furthermore, in wild-type T cells and cell cycle progression (d) of GFP-sorted primed wild-type and Cd28–/– AND TCR-transgenic CD4+ transduced with a retroviral vector encod- T cells infected with Mig–aurora B and restimulated with APCs and MCC peptide in the presence of ing ‘kinase-dead’ aurora B, phosphorylation DMSO (control) or chemical inhibitors. (b,c) Immunoblot analysis of cell lysates after restimulation of p70S6k and 4E-BP1 was suppressed, (time, above lanes). (d) Cell cycle progression 2 d after restimulation analyzed by BrdU and 7-amino- whereas expression of wild-type aurora B + actinomycin D staining, with gating on CD4 cells. Numbers in plots are percent cells in the following in Cd28–/– T cells resulted in enhanced phases (top to bottom): sub-Go, G1, S and G2-M. Similar data were obtained in two experiments. phosphorylation of p70S6k and 4E-BP1 http://www.nature.com/natureimmunology (Fig. 6b). Finally, p70S6k kinase activity the expression and phosphorylation of survivin, expression of cyclin A was enhanced by retroviral expression of wild-type aurora B and phosphorylation of Rb, Cdk1 and Cdk2 in aurora B–transduced in Cd28–/– T cells and was inhibited by retroviral expression of wild-type and Cd28–/– T cells (Supplementary Fig. 3). Treatment with ‘kinase-dead’ aurora B in wild-type T cells (Fig. 6c). These UO126 also partially suppressed aurora B protein expression and kinase data indicated that aurora B directly or indirectly controls the activity activity and progression through S phase (Supplementary Fig. 3). of at least two reported mTOR substrates. Adding further com- These data suggested that PI(3)K regulates the induction of aurora B plexity, we also found that ‘kinase-dead’ aurora B suppressed and expression in primed T cells, that mTOR and MEK-Erk signaling wild-type aurora B enhanced phosphorylation of mTOR (Fig. 6b), partially regulate aurora B kinase activity and that PI(3)K, mTOR and suggesting the existence of a regulatory feedback loop between aurora MEK-Erk cooperate in promoting G1- to S-phase progression. B and mTOR.

© Group 2006 Nature Publishing WT Cd28–/– abcMig – WT Mig–KR–aurora B Input IP: Aurora B Input IP: mTOR (kDa) Mig Mig Mig–KR Mig Mig– Cd28–/– mTOR 191 aurora B aurora B Mig–aurora B IB: p-p70S6k 300 97

(Thr389) g)

p70S6k µ p70S6k 64 250 p70S6k 200 51 IgH IgH p-4E-BP1 39 Aurora B (Ser65) 150

IgL 28 IgL 4E-BP1 100 4E-BP1 19 4E-BP1 50 p-mTOR (Ser2448) Incorporation (pmol/min/ 0 01 2 3 4 mTOR Aurora B mTOR Time (d)

Figure 6 Aurora B associates with and regulates the activity of mTOR, p70S6k and 4E-BP1. (a) Immunoblot analysis of proteins interacting with immunoprecipitated aurora B (left) and mTOR (right) 24 h after restimulation of primed wild-type AND TCR-transgenic CD4+ T cells with APCs and MCC peptide, assessed with a mixture of antibodies to mTOR, p70S6k and 4E-BP1 (left) or to p70S6k, aurora B and 4E-BP1 (right). Molecular weight: mTOR, about 289 kilodaltons (kDa); p70S6k, about 70 kDa; 4E-BP1, about 15 kDa; aurora B, about 40 kDa. Input, immunoprecipitation with control rabbit immunoglobulin G; bottom blots, control for immunoprecipitation. Similar data were obtained in one repeat experiment. (b,c)Immunoblot analysis (b) and kinase activity (c) of GFP-sorted primed wild-type or Cd28–/– AND TCR-transgenic CD4+ T cells transduced with retroviruses and restimulated with APCs and MCC peptide. (b) Immunoblot analysis 1 d after restimulation. Similar data were obtained in three experiments. (c)Kinase activity of immunoprecipitated p70S6k at various times after restimulation (horizontal axis), analyzed with AKRRRLSSLRA as substrate. Data are representative of two experiments.

NATURE IMMUNOLOGY ADVANCE ONLINE PUBLICATION 5 ARTICLES

–/– Figure 7 IL-2 regulates aurora B, and aurora B is WT Cd28 a bc80 Mig Mig–KR–aurora B Cdk1 required for IL-2R-induced cell cycle progression.

Control rIL-2 Control rIL-2 60 (a) Immunoblot analysis of protein expression by –/– Aurora B primed wild-type or Cd28 AND TCR-transgenic Control rIl-2 Control rIl-2 g) 40 + µ CD4 T cells 1 d after restimulation with APCs p-Survivin p-Survivin 20 and MCC peptide in the presence (rIL-2) or Survivin Survivin absence (Control) of recombinant IL-2. Similar 0 120 Cyclin A Cyclin A Cdk2 data were obtained in three experiments. (b–d) Immunoblot analysis (b), kinase activity p-Rb p-Rb 80 (c) and cell cycle status (d) of GFP-sorted primed Rb –/– +

Rb Incorporation (pmol/min/ wild-type or Cd28 AND TCR-transgenic CD4 40 p-Cdk1 p-Cdk1 T cells transduced with retroviruses and restimulated for 1 d with APCs and MCC peptide Cdk1 Cdk1 0 l in the presence or absence of recombinant IL-2. p-Cdk2 p-Cdk2 (b) Immunoblot analysis of cell lysates. (c) Kinase

Mig + rIL-2 activity of immunoprecipitated Cdk1 and Cdk2, Cdk2 Cdk2 Mig + control determined with histone H1 as substrate. (d) Cell β-actin β-actin KR–aurora B + contro cycle status determined by BrdU and 7-amino- – Mig–KR–aurora B + rIL-2 + Mig actinomycin D staining, with gating on CD4 cells.

Mig–KR–aurora B Numbers in plots are percent cells in the following d Mig (rIL-2) (control) Mig–KR–aurora B (rIL-2) phases (top to bottom): sub-Go, G1, S and G2-M. 4 4 4 10 5.8 10 9.8 10 7.6 Data are representative of three experiments. 3 62.3 3 85.2 3 86.8 10 25.6 10 1.0 10 1.7 2 2.4 2 1.0 2 1.3 10 10 10 (Fig. 7b). IL-2-induced Cdk1 and Cdk2 kinase 101 101 101

BrdU activity and G1- to S-phase progression was 100 100 100 0 50 100150 200 250 0 50 100150 200 250 0 50 100150 200 250 7-AAD also inhibited by ‘kinase-dead’ aurora B (Fig. 7c,d). This phenotype strongly resembled the reported effects of blocking mTOR29,35. Aurora B and IL-2R signaling Next we sought to determine if aurora B was activated by IL-2R IL-2 can promote T cell division via mTOR, given the ability of signals in the absence of TCR signaling. Culture in IL-2 alone resulted http://www.nature.com/natureimmunology rapamycin to block that process29,35. To determine if aurora B is in the induction of aurora B expression and kinase activity as well as required for IL-2R signaling, we primed wild-type and Cd28–/– T the phosphorylation and expression of survivin and the phosphoryla- cells and restimulated them with antigen and IL-2. Treatment with tion of Rb, Cdk1 and Cdk2 (Supplementary Fig. 4 online). However, IL-2 upregulated the expression of aurora B, survivin and cyclin A as rapamycin treatment strongly but not completely inhibited those IL- well as the phosphorylation of Rb, Cdk1 and Cdk2 (Fig. 7a). To deter- 2R-induced events. These data suggested that TCR- and IL-2-induced mine whether aurora B kinase activity was required, we primed Cd28–/– aurora B activity is partially dependent on mTOR function. T cells and transduced them with ‘kinase-dead’ aurora B and then stimulated them with antigen and IL-2. Blockade of aurora B activity Aurora B in T cell function in vivo inhibited the phosphorylation and expression of survivin, induction of To demonstrate an essential function for aurora B in physiological cyclin A expression and phosphorylation of Rb, Cdk1 and Cdk2 settings, we used a protocol17 in which naive T cells are primed

Day 3 Mig + PCC Day 3 a 300 PCC b c 500 Figure 8 Aurora B is sufﬁcient to induce PBS Mig–aurora B + PCC © Group 2006 Nature Publishing –/– Mig + PBS cells 400 proliferation of Cd28 Tcellsin vivo.Naive cells + 200 WT Cd28 –/– + ) –/– + 400 200 ) 4 wild-type or Cd28 AND TCR-transgenic CD4 4 300 CD4 300 150 CD4 + 10 10 + 3 × × T cells were stimulated with APCs and MCC 3 ( ( β 200 100 200 100 β V

peptide and were transduced with retroviruses. + 100 50 100 GFP+CD4+ cells were sorted, were labeled with 0 0 GFP PKH26 in some cases and were adoptively GFP+V 0 PKH26 0 150 150 transferred into groups of ﬁve naive wild-type Day 7 Day 7 300 100 100 500 recipient mice that were subsequently challenged PCC

cells 50 50 400 cells PBS with whole pigeon cytochrome c (PCC) + 200 + ) 0 0 ) 4 protein or PBS. (a) T cell recovery in vivo 3and 4 300 CD4 CD4 +

10 BrdU 10 + 3 × × 7 d after immunization. Data are mean (± s.d.) 3 ( β ( 200 100 100 100 β + + + V

number of GFP V 3 CD4 cells in pooled spleen + b 80 80 100 and lymph nodes from ﬁve individual mice per 60 60 GFP GFP+V 0 40 40 0 group. (b) Cell division or apoptosis 3 d after 20 20 immunization, determined by the extent of 0 0 Mig /– + Mig – Annexin V PKH26 dilution (top) or incorporation of BrdU WT + Mig–aurora B Cd28 –KR–aurora B after a pulse from day 1 to day 3 (middle) + Mig–aurora B + + –/– Mig after gating on Vb3 GFP cells, and by staining WT + Mig + + Cd28 with annexin V after gating on Vb3 CD4 (GFP+ and GFP–) cells (bottom). (c) T cell Mig + PCC d 300 recovery, assessed as described in a.(d)Cell 300 300 Mig–KR–aurora B + PCC 200 Mig + PBS division or apoptosis, assessed as described 200 200 100 100 100 in b.Data(a–d) are representative of two to 0 0 0 four experiments. PKH26 BrdU Annexin V

6 ADVANCE ONLINE PUBLICATION NATURE IMMUNOLOGY ARTICLES

a Cd28 –/– + cd WT + Mig Cd28 –/– + Mig Mig–aurora B –/– –/– 4 4 4 WT + Mig Cd28 + Mig WT + Mig Cd28 + Mig 10 10 10 1,000 1,000 3 3 3 10 10 10 800 21% 800 34% 2 2 2 600 600 Mon 10 10 10 Eos γ 400 44% 400 4% 101 101 101 IFN- 38%35% 40% 200 200 Lym 100 100 100 19% 34% 0 0 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 0 0 IL-4 –/– 200 400 600 800 200 400 600 800 Cd28 + Mig–aurora B 1,000 1,000 –/– ) ) 5 Cd28 + Mig–aurora B 5 0.8 MDLN 5 1.5 BALF

b ) 5

10 10 4 1,000 × × 1.2 40 0.6 800 3 24% 30 0.9 600 cells ( cells ( + 0.4 + 2 20 0.6 400 41% 1

GFP GFP 10 + 0.2 + 0.3 200 Inflammation severity 0 0 0 17% SSC CD4 CD4 – + – + 0 0 / 0 BALF eosinophils (×10 – –/ 200 400 600 800 + /– + + Mig 1,000 – + Mig –/– – –/– –/ + Mig – + Mig WT + Mig Cd28–aurora B FSC WT + Mig Cd28 –/– –/ WT + Mig Cd28 WT + Mig Cd28 Cd28 Mig Cd28 Mig–aurora B Cd28 Mig–aurora B Cd28 Mig–aurora B

–/– –/– Figure 9 Aurora B restores the ability of Cd28 TH2 cells to promote lung inflammation. Naive T cells from wild-type and Cd28 AND mice + + were stimulated in TH2-polarizing conditions and were transduced with retroviruses; 6 d after stimulation, GFP CD4 cells were sorted and were adoptively transferred into naive wild-type recipient mice (n ¼ 4 mice per group). All mice were then challenged for 2 d with aerosolized whole pigeon cytochrome c and were analyzed 24 h later. (a) Intracellular staining analysis of IL-4 and IFN-g in primed TH2 cells restimulated for 7 h with APCs and + + + + + MCC peptide in vitro, with gating on Vb3 CD4 cells. Numbers in bottom right corners indicate percent IL-4 cells. (b)GFPCD4 cells in lung-draining lymph nodes (MDLN) and in bronchoalveolar lavage fluid (BALF). Data represent mean (± s.d.) T cell counts from four individual mice. (c) Lung sections stained with hematoxylin and eosin (mice and T cell transduction, above images). Original magnification, 200. Bottom right, quantification of lung inflammation in sections of four individual lungs in each group. (d) Forward-scatter (FSC) and side-scatter (SSC) profiles of cell populations in bronchoalveolar lavage fluid. Numbers beside outlined areas indicate percent eosinophils (Eos), monocytes (Mon) and lymphocytes (Lym). Bottom right, quantification of eosinophils; data are mean (± s.d.) total eosinophils from four mice after differential cytospin counting. Similar results were obtained http://www.nature.com/natureimmunology in one repeat experiment.

in vitro, transduced with retroviral vectors encoding GFP and then cells, although able to produce TH2 cytokines, did not promote lung adoptively transferred into wild-type recipient mice, in which their inflammation or eosinophil recruitment and did not accumulate in proliferation and function can be tracked in vivo with GFP as a large numbers in draining lymph nodes or in the lungs (Fig. 9b–d). –/– marker. In the first experiments, we stimulated T cells in neutral However, transferred Cd28 TH2 cells transduced with a retrovirus conditions. For wild-type T cells, that resulted in a primed population encoding aurora B triggered lung inflammation and eosinophilia producing mainly IL-2 but also some mixed T helper type 1 and type 2 and accumulated in substantial numbers in draining lymph cytokines, and for Cd28–/– T cells, it resulted in a primed population nodes and in the lungs (Fig. 9b–d). These results suggested that producing few cytokines (Fig. 1 and Fig. 4). aurora B is sufficient to restore T cell proliferation in vivo in the After we challenged recipient mice with antigen, the transferred absence of CD28. wild-type T cells infected with the control retrovirus proliferated extensively over 3–7 d, whereas the proliferation of transferred DISCUSSION © Group 2006 Nature Publishing Cd28–/– T cells infected with the control retrovirus was impaired The function of aurora B has been analyzed in various types of cells, (Fig. 8a,b). In contrast, transferred Cd28–/– T cells infected with a although so far not in lymphoid cells. Existing data show that aurora B retrovirus encoding aurora B underwent substantial proliferation is present in the M phase and is important during mitosis, acting with (Fig. 8a,b). We obtained similar trends in terms of the survival of binding partners survivin and INCENP as a ‘passenger protein’ to transferred T cells (Fig. 8b). Aurora B expression did not completely promote chromosome segregation and cytokinesis23,39,40. However, restore the proliferation and population expansion of Cd28–/– T cells our data here have shown that in primary T cells, aurora B was also to wild-type amounts in vivo (or in vitro; data not shown), most active earlier in the cell division process. Moreover, aurora B, survivin, probably reflecting the fact that these T cells still had impaired mTOR, p70S6k and 4E-BP1 immunoprecipitated together and cross- production of cytokines such as IL-2 that contribute additional signals regulated each other, suggesting that they form an active complex to through different kinases. Transferred wild-type T cells infected with a control cell cycle progression at the G1- to S-phase transition. retrovirus encoding the ‘kinase-dead’ aurora B mutant had defective That conclusion is in agreement with data on survivin and with the proliferation and excessive apoptosis (Fig. 8c,d). existing assumption that aurora B functions efficiently only when able Finally, to exclude the possibility of potential effects of defective IL-2 to bind survivin. Data from published studies, which we further production by Cd28–/– T cells, we used a protocol38 in which we confirmed here, show that survivin is expressed and active during primed wild-type and Cd28–/– Tcellsin vitro inthepresenceofIL-2 G1 and S phase in proliferating T cells17 and correlate with data from and added IL-4, antibody to IFN-g (anti-IFN-g) and anti-IL-12 to other published studies demonstrating an S-phase block in thymocytes 41,42 induce T helper type 2 (TH2) differentiation, which we confirmed by from mice in which survivin is deleted in the T cell lineage .Other intracellular staining (Fig. 9a). We transduced those in vitro–generated observations made with cord blood and bone marrow CD34+ stem TH2 cells with retroviruses and transferred them into wild-type cells have shown that survivin expression is induced during the G1 recipient mice, which we then subjected to antigen inhalation. In phase after exposure to growth factors43,44. The cross-regulation –/– 22,33,34 contrast to transferred wild-type TH2 cells, transferred Cd28 TH2 between aurora B and survivin shown here and in other studies

NATURE IMMUNOLOGY ADVANCE ONLINE PUBLICATION 7 ARTICLES

may explain why overexpression of survivin can compensate for low exact relationship between aurora B and mTOR and other mTOR- expression of aurora B and why introduction of either aurora B or binding proteins such as raptor, p70S6k and 4E-BP1? Aurora B mRNA survivin into T cells results in apparently similar phenotypes in terms and protein were induced to some extent in the presence of rapamycin, of G1- to S-phase progression. but LY294002 and wortmannin completely prevented aurora B up- Primed hyporesponsive Cd28–/– T cells produced little IL-2 and regulation, suggesting that PI(3)K rather than mTOR is involved in failed to undergo many signaling events associated with late G1 or promoting aurora B expression in primed T cells. However, once early S phase of the cell cycle. In Cd28–/– T cells, expression of cyclin A, induced, as well as apparently forming complexes with aurora B, a molecule required for the initiation of DNA replication at the onset mTOR and its substrates were found to be intimately involved in the of S phase, was not efficiently upregulated. Cdk1 and Cdk2, kinases action of aurora B. Thus, rapamycin blocked a substantial proportion that are activated by binding to cyclin A as well as to other cyclins such of aurora B kinase activity and aurora B affected the phoshorylation of as cyclin E, were not efficiently activated. Finally, hyperphosphoryla- Ser2448 of mTOR, a site thought to be required for full mTOR tion of Rb, an event required for entry into and progression through activity36. Data on control of mTOR or by mTOR have shown S phase, which is regulated in part by the activity of several cyclin- multiple potential direct or indirect binding partners, many of dependent kinases, including cyclin A–activated Cdk2, was not which are capable of cross-regulating each other36,37,47, such as induced26–28,45. Retroviral expression of aurora B in Cd28–/– T cells p70S6k, whose activity is dependent on mTOR but that can also resulted in upregulation of the expression of cyclin A and activity of lead to the phosphorylation of Ser2448 of mTOR48. Our data now add Cdk1 and Cdk2 and in hyperphosphorylation of Rb, whereas retroviral another kinase to that putative signaling complex and feedback loop expression of ‘kinase-dead’ aurora B in wild-type T cells resulted in and thus further complicate the task of identifying primary versus suppression of the ability of IL-2 to trigger all of those signaling events. secondary targets of aurora B. For example, we have demonstrated that Thus, several molecules critical for the passage of T cells through the aurora B regulated p70S6k phosphorylation and activity, thus raising G1- to S-phase transition were affected by aurora B activity. the issues of whether p70S6k is a chief target and whether the observed Whether the effect of aurora B on cyclin A, Cdk1, Cdk2 and Rb is effect of aurora B on mTOR phosphorylation represents feedback direct or indirect is not clear and needs further investigation. One through p70S6k. Furthermore, both p70S6k and 4E-BP1 immunopre- possibility is that Rb is the main target of aurora B. Survivin has been cipitated together with aurora B, raising the issue of whether aurora reported to bind directly to Cdk4 and to trigger the activation of B directly binds to those proteins or to mTOR. Alternatively, aurora B cyclin E–Cdk2 complexes and subsequent Rb phosphorylation43,46. might use raptor as a scaffold or adaptor to move into close proximity Although we cannot rule that out as a mechanism for aurora B of mTOR or p70S6k and 4E-BP1 (ref. 36), an idea supported by http://www.nature.com/natureimmunology function, we did not find any changes in cyclin E quantities in the studies showing physical interaction of raptor with p70S6k and absence of CD28 or after expression of aurora B, and we failed to 4E-BP1 (ref. 49). Future studies should shed light on this previously detect any cyclin-dependent kinases in aurora B or survivin immuno- unknown and most likely complex aspect of aurora B biology. precipitates (data not shown). Phosphorylation of Rb during late G1 In summary, we have provided evidence that aurora B kinase phase is required for entry into S phase and can contribute to the controls antigen-induced T cell division at the transition of cells upregulation of cyclin A through the release of E2F transcription from G1 to S of the cell cycle. Loss of aurora B activity accounted factors26,27. However, a feedback loop in which cyclin A–induced at least in part for the hyporesponsiveness of T cells that did not Cdk2 activity contributes to Rb hyperphosphorylation can also receive signals through CD28 and/or IL-2R, and aurora B worked operate45. Therefore, any effect of aurora B on Rb could be secondary cooperatively with many other kinases, including mTOR and MEK1, to an effect of aurora B on cyclin A. at that checkpoint. The exact targets of aurora B activity during the Inhibition of aurora B or mTOR activity suppressed the induction early phases of the cell cycle, the direct binding partners of aurora B of cyclin A and activation of Cdk1 and Cdk2; those observations and the molecular nature of the aurora B–mTOR cooperation remain correlate with data showing that mTOR activity is required for the to be investigated. © Group 2006 Nature Publishing passage of T cells through the G1- to S-phase transition15,29,35. Although to our knowledge a direct connection between cyclin A METHODS and mTOR has not been shown other than through rapamycin Mice and reagents. AND and AND Cd28–/– TCR-transgenic mice, expres- treatment, it is likely that upregulation of cyclin A is a consequence sing a TCR composed of variable (Vb3andVa11) chains that bind to a peptide of the activation of mTOR and of aurora B. The kinase mTOR from moth or pigeon cytochrome c, were bred onto a B10.BR background as 17 phosphorylates and activates p70S6k and phosphorylates and inacti- described . B10.BR mice were purchased from Jackson Laboratories. All vates 4E-BP1, which blocks the translation-initiation factor eIF4E. experiments were in compliance with the regulations of the La Jolla Institute Animal Care committee in accordance with guidelines of the Association for Those mTOR-mediated events are essential for initiation of the 36 the Assessment and Accreditation of Laboratory Animal Care. Reagents were as translation of certain proteins , one of which could be cyclin A. described (Supplementary Methods online). Here we have shown a direct connection between aurora B and mTOR. Aurora B and mTOR coimmunoprecipitated together with Purification of T cells and APCs. Naive CD4+ T cells were purified from spleen p70S6k and 4E-BP1. Rapamycin strongly inhibited aurora B kinase and lymph nodes by nylon wool depletion, followed by antibody and comple- activity and suppressed aurora B–mediated cell cycle progression. ment treatment17. APCs were purified from the spleens of syngeneic nontrans- Finally, forced expression of aurora B or inhibition of aurora B activity genic mice by depletion of T cells with antibody and complement17.APCswere led to alterations in the phosphorylation of p70S6k and 4E-BP1. treated for 30 min at 37 1Cwithmitomycinc(100mg/ml). Those data therefore collectively suggest that cyclin A might be a T cell cultures. Cells were cultured in 48-well plates containing 1 ml RPMI principal target of aurora B action. 1640 medium (Irvine Scientific) plus 10% FCS (Omega Scientific). Naive CD4+ As for the biology of aurora B and its relationship with mTOR, T cells were plated at a density of 5 105 cells per ml with 2 106 APCs per ml several questions remain. First, do our findings showing cooperation and 0.5–1 mM T102S peptide. For analysis of secondary responsiveness, 5 105 between mTOR and aurora B extend to other cell types, including recovered T cells were recultured with 2 106 APCs per ml and 0.5 mM nonlymphoid cells, or are they specific for T cells? Second, what is the peptide. LY294002 (5 mM), wortmannin (10 nM), hydroxyurea (10 mg/ml),

8 ADVANCE ONLINE PUBLICATION NATURE IMMUNOLOGY ARTICLES

U0126 (10 mM) or rapamycin (100 nM) in dimethylsulfoxide (DMSO) or PBS RNA was extracted with TRIzol (Invitrogen) and was reverse-transcribed with was added at start of culture. oligo(dT)12–18 (SuperScript II; Invitrogen). Primers were as described (Supple- mentary Methods). Retroviral transduction. The cDNA encoding wild-type or ‘kinase-dead’ aurora B23 was subcloned into the Mig (mouse stem cell virus–internal ribosomal entry Note: Supplementary information is available on the Nature Immunology website. site–GFP) retroviral expression vector. Mig vectors expressing survivin and dominant negative (T34A) survivin were constructed as described17 with cDNA ACKNOWLEDGMENTS that was a gift (Acknowledgments)31. Retroviral transduction was done as We thank M. Tatsuka (Research Institute for Radiation Biology and Medicine, described after 2–3 d of primary T cell culture17 (Supplementary Methods). Hiroshima University) for aurora B cDNA, and A. Song, W. Duan, X. Tang and Y. Adams for technical assistance. The cDNA to construct Mig vectors Adoptive transfer. Primed T cells cultured with antigen for 6 d and retrovirally expressing survivin and dominant negative survivin was provided by D. Altieri transduced on days 2 and 3 were sorted based on GFP and CD4 expression and (University of Massachusetts Medical School). Supported by the US National Institutes of Health (AI50498 and AI49453 to M.C.) and the Universitywide in some cases were labeled with the dye PKH26. Then, 3.5 106 cells were AIDS Research Program (J.S.). This is manuscript 764 from the La Jolla injected intravenously into naive wild-type mice, which were challenged 1 d Institute for Allergy and Immunology. later by intraperitoneal injection of 100 mg whole pigeon cytochrome c in PBS or injection of PBS alone. Numbers of T cells were calculated based on total cell AUTHOR CONTRIBUTIONS + + numbers in spleen and lymph nodes along with percent GFP Vb3 cells J.S., S.S.-A., T.S. and M.C. designed the research and analyzed the data; visualized by ﬂow cytometry. J.S., S.S.-A. and T.S. did the research; and M.C. wrote the manuscript.

Lung inflammation. Naive T cells (1.5 106 cells/ml) were cultured with COMPETING INTERESTS STATEMENT plate-bound anti–CD3 (5 mg/ml), soluble anti-CD28 (5 mg/ml), IL-2 (5 ng/ml), The authors declare that they have no competing financial interests. IL-4 (20 ng/ml), anti-IFN-g (10 mg/ml) and anti-IL-12 (10 mg/ml) for Published online at http://www.nature.com/natureimmunology/ the induction of TH2 cell differentiation. Retrovirally transduced T cells were sorted based on GFP expression and 3 106 cells were injected intravenously Reprints and permissions information is available online at http://npg.nature.com/ into naive B10.BR mice. Lung inflammation was assessed as described38 reprintsandpermissions/ (Supplementary Methods).

Aurora B and survivin staining and cell cycle analysis. Aurora B and survivin 1. Lafferty, K.J. & Cunningham, A.J. A new analysis of allogeneic interactions. Aust. J. intracellular staining and cell cycle analysis was done as described17 (Supple- Exp. Biol. Med. Sci. 53, 27–42 (1975). 2. Jenkins, M.K. & Schwartz, R.H. Antigen presentation by chemically modiﬁed spleno- mentary Methods). cytes induces antigen-speciﬁc T cell unresponsiveness in vitro and in vivo. J. Exp. Med.

http://www.nature.com/natureimmunology 165, 302–319 (1987). Cytokine secretion, proliferation, cell division and apoptosis. Cytokines were 3. June, C.H., Ledbetter, J.A., Gillespie, M.M., Lindsten, T. & Thompson, C.B. T-cell measured by enzyme-linked immunosorbent assay as described17. Proliferation proliferation involving the CD28 pathway is associated with cyclosporine-resistant was assessed in triplicate cultures by measurement of the incorporation of interleukin 2 gene expression. Mol. Cell. Biol. 7, 4472–4481 (1987). [3H]thymidine (1 mCi/well; ICN Pharmaceuticals) during the last 12 h of 4. Linsley, P.S. et al. Binding of the B cell activation antigen B7 to CD28 costimulates T cell proliferation and interleukin 2 mRNA accumulation. J. Exp. Med. 173, 721–730 culture. Cell division was assessed in vitro by dilution of carboxyﬂuorescein (1991). diacetate succinimidyl diester or in vivo by incorporation of 5-bromodeoxyur- 5. Harding, F.A., McArthur, J.G., Gross, J.A., Raulet, D.H. & Allison, J.P. CD28-mediated idine (BrdU) given intraperitoneally 48 h before analysis or by labeling of signalling co-stimulates murine T cells and prevents induction of anergy in T-cell T cells with PKH26 before adoptive transfer in vivo. Apoptosis was assessed by clones. Nature 356, 607–609 (1992). 6. Sharpe, A.H. & Freeman, G.J. The B7–CD28 superfamily. Nat. Rev. Immunol. 2, staining with phycoerythrin-conjugated annexin V (BD Pharmingen). 116–126 (2002). 7. Croft, M. Co-stimulatory members of the TNFR family: keys to effective T-cell Immunoprecipitation and immunoblot. Live CD4+ cells were recovered by immunity? Nat. Rev. Immunol. 3, 609–620 (2003). Ficoll treatment and by positive selection with anti-CD4 microbeads (130-049- 8. Beverly, B., Kang, S.M., Lenardo, M.J. & Schwartz, R.H. Reversal of in vitro T cell clonal 201; Miltenyi Biotec). Cells were lysed for 30 min in ice-cold radioimmuno- anergy by IL-2 stimulation. Int. Immunol. 4, 661–671 (1992). 9. Boussiotis, V.A. et al. Prevention of T cell anergy by signaling through the gamma c precipitation lysis buffer. Insoluble material was removed and lysates were used chain of the IL-2 receptor. Science 266, 1039–1042 (1994).

© Group 2006 Nature Publishing for immunoblot analysis or were immunoprecipitated overnight with primary 10. Powell, J.D., Lerner, C.G. & Schwartz, R.H. Inhibition of cell cycle progression by antibodies followed by incubation for 2 h at 4 1C with protein G agarose beads. rapamycin induces T cell clonal anergy even in the presence of costimulation. Washed immunoprecipitates were boiled in SDS sample buffer. Protein content J. Immunol. 162, 2775–2784 (1999). 11. Prasad, K.V. et al. T-cell antigen CD28 interacts with the lipid kinase phosphatidyli- was determined with the Bio-Rad protein assay kit (Bio-Rad). Equal amounts nositol 3-kinase by a cytoplasmic Tyr(P)-Met-Xaa-Met motif. Proc. Natl. Acad. Sci. USA of protein (30–50 mg) were separated by SDS-PAGE (4–12% NuPage Bis-Tris 91, 2834–2838 (1994). precasting gels), were transferred onto polyvinyldiﬂuoride membranes (Invi- 12. Ahmed, N.N., Grimes, H.L., Bellacosa, A., Chan, T.O. & Tsichlis, P.N. Transduction of trogen) and were analyzed by immunoblot. All immunoblots were developed interleukin-2 antiapoptotic and proliferative signals via Akt protein kinase. Proc. Natl. Acad. Sci. USA 94, 3627–3632 (1997). with the ECL immunodetection system (Amersham Pharmacia Biotech). 13. Nourse, J. et al. Interleukin-2-mediated elimination of the p27Kip1 cyclin- dependent kinase inhibitor prevented by rapamycin. Nature 372,570–573 In vitro kinase assays. For assay of aurora B kinase activity, cell lysates were (1994). immunoprecipitated with anti–aurora B by the Catch and Release v2.0 14. Boussiotis, V.A. et al. p27kip1 functions as an anergy factor inhibiting interleukin 2 reversible immunoprecipitation system (17-500A; Upstate). Immunoprecipi- transcription and clonal expansion of alloreactive human and mouse helper T lymphocytes. Nat. Med. 6, 290–297 (2000). tates were resuspended in kinase reaction buffer (ADBI assay dilution buffer I; 15. Powell, J.D., Bruniquel, D. & Schwartz, R.H. TCR engagement in the absence of cell 32 20-108; Upstate) with [ P]ATP in magnesium-ATP ‘cocktail’ (20-113; cycle progression leads to T cell anergy independent of p27Kip1. Eur. J. Immunol. 31, Upstate), with histone H3 as substrate, and were incubated for 10 min at 3737–3746 (2001). 30 1C. Kinase reactions were transferred onto P81 phosphocellulose squares 16. Kane, L.P., Andres, P.G., Howland, K.C., Abbas, A.K. & Weiss, A. Akt provides the CD28 costimulatory signal for up-regulation of IL-2 and IFN-g but not TH2 cytokines. (20-134; Upstate) and phosphorylated histone H3 was quantiﬁed with a Nat. Immunol. 2, 37–44 (2001). scintillation counter. For assay of the activity of Cdk1, Cdk2 and p70S6k, the 17. Song, J., So, T., Cheng, M., Tang, X. & Croft, M. Sustained survivin expression from same procedure was used, except lysates were immunoprecipitated with anti- OX40 costimulatory signals drives T cell clonal expansion. Immunity 22, 621–631 Cdk1, anti-Cdk2 or anti-p70S6k, and histone H1 or AKRRRLSSLRA was used (2005). 18. Li, F. et al. Control of apoptosis and mitotic spindle checkpoint by survivin. Nature 396, as the kinase substrate for Cdk1 and Cdk2 or for p70S6k, respectively. 580–584 (1998). 19. Tamm, I. et al. IAP-family protein survivin inhibits caspase activity and apoptosis Real-time RT-PCR. Sybr Green I dye and an ABI GeneAmp 5700 sequence induced by Fas (CD95), Bax, caspases, and anticancer drugs. Cancer Res. 58,5315– BioDetector (Applied Biosystems) were used for quantitative RT-PCR. Total 5320 (1998).

NATURE IMMUNOLOGY ADVANCE ONLINE PUBLICATION 9 ARTICLES

20. Li, F. et al. Pleiotropic cell-division defects and apoptosis induced by interference with 35. Morice, W.G., Brunn, G.J., Wiederrecht, G., Siekierka, J.J. & Abraham, R.T. Rapamycin- survivin function. Nat. Cell Biol. 1, 461–466 (1999). induced inhibition of p34cdc2 kinase activation is associated with G1/S-phase growth 21. Wheatley, S.P., Carvalho, A., Vagnarelli, P. & Earnshaw, W.C. INCENP is required for arrest in T lymphocytes. J. Biol. Chem. 268, 3734–3738 (1993). proper targeting of Survivin to the centromeres and the anaphase spindle during 36. Inoki, K., Ouyang, H., Li, Y. & Guan, K.L. Signaling by target of rapamycin proteins in mitosis. Curr. Biol. 11, 886–890 (2001). cell growth control. Microbiol. Mol. Biol. Rev. 69, 79–100 (2005). 22. Bolton, M.A. et al. Aurora B kinase exists in a complex with survivin and INCENP and 37. Martin, D.E. & Hall, M.N. The expanding TOR signaling network. Curr. Opin. Cell Biol. its kinase activity is stimulated by survivin binding and phosphorylation. Mol. Biol. Cell 17, 158–166 (2005). 13, 3064–3077 (2002). 38. Salek-Ardakani, S. et al. OX40 (CD134) controls memory T helper 2 cells that drive 23. Terada, Y. et al. AIM-1: a mammalian midbody-associated protein required for lung inflammation. J. Exp. Med. 198, 315–324 (2003). cytokinesis. EMBO J. 17, 667–676 (1998). 39. Carmena, M. & Earnshaw, W.C. The cellular geography of aurora kinases. Nat. Rev. Mol. 24. Murata-Hori, M. & Wang, Y.L. The kinase activity of aurora B is required for kinetochore- Cell Biol. 4, 842–854 (2003). microtubule interactions during mitosis. Curr. Biol. 12, 894–899 (2002). 40. Ke, Y.W., Dou, Z., Zhang, J. & Yao, X.B. Function and regulation of Aurora/Ipl1p kinase 25. Kallio, M.J., McCleland, M.L., Stukenberg, P.T. & Gorbsky, G.J. Inhibition of aurora B family in cell division. Cell Res. 13, 69–81 (2003). kinase blocks chromosome segregation, overrides the spindle checkpoint, and perturbs 41. Xing, Z., Conway, E.M., Kang, C. & Winoto, A. Essential role of survivin, an inhibitor of microtubule dynamics in mitosis. Curr. Biol. 12, 900–905 (2002). apoptosis protein, in T cell development, maturation, and homeostasis. J. Exp. Med. 26. Hunter, T. & Pines, J. Cyclins and cancer. II: cyclin D and CDK inhibitors come of age. 199, 69–80 (2004). Cell 79, 573–582 (1994). 42. Okada, H. et al. Survivin loss in thymocytes triggers p53-mediated growth arrest and 27. Ekholm, S.V. & Reed, S.I. Regulation of G1 cyclin-dependent kinases in the mamma- p53-independent cell death. J. Exp. Med. 199, 399–410 (2004). lian cell cycle. Curr. Opin. Cell Biol. 12, 676–684 (2000). 43. Suzuki, A. et al. Survivin initiates cell cycle entry by the competitive interaction with 28. Yam, C.H., Fung, T.K. & Poon, R.Y. Cyclin A in cell cycle control and cancer. Cell. Mol. Cdk4/p16INK4a and Cdk2/cyclin E complex activation. Oncogene 19, 3225–3234 Life Sci. 59, 1317–1326 (2002). (2000). 29. Morice, W.G., Wiederrecht, G., Brunn, G.J., Siekierka, J.J. & Abraham, R.T. Rapamycin 44. Fukuda, S. & Pelus, L.M. Regulation of the inhibitor-of-apoptosis family member inhibition of interleukin-2-dependent p33cdk2 and p34cdc2 kinase activation in T survivin in normal cord blood and bone marrow CD34+ cells by hematopoietic growth lymphocytes. J. Biol. Chem. 268, 22737–22745 (1993). factors: implication of survivin expression in normal hematopoiesis. Blood 98,2091– 30. Murata-Hori, M., Tatsuka, M. & Wang, Y.L. Probing the dynamics and functions of 2100 (2001). aurora B kinase in living cells during mitosis and cytokinesis. Mol. Biol. Cell 13, 1099– 45. Shen, W.H. et al. Tumor necrosis factor alpha inhibits cyclin A expression and 1108 (2002). retinoblastoma hyperphosphorylation triggered by insulin-like growth factor-I induction 31. O’Connor, D.S. et al. Regulation of apoptosis at cell division by p34cdc2 phosphoryla- of new E2F–1 synthesis. J. Biol. Chem. 279, 7438–7446 (2004). tion of survivin. Proc. Natl. Acad. Sci. USA 97, 13103–13107 (2000). 46. Suzuki, A. et al. Survivin initiates procaspase 3/p21 complex formation as a result of 32. Wall, N.R., O’Connor, D.S., Plescia, J., Pommier, Y. & Altieri, D.C. Suppression of interaction with Cdk4 to resist Fas-mediated cell death. Oncogene 19, 1346–1353 survivin phosphorylation on Thr34 by flavopiridol enhances tumor cell apoptosis. (2000). Cancer Res. 63, 230–235 (2003). 47. Jacinto, E. & Hall, M.N. Tor signalling in bugs, brain and brawn. Nat. Rev. Mol. Cell 33. Wheatley, S.P., Henzing, A.J., Dodson, H., Khaled, W. & Earnshaw, W.C. Aurora-B Biol. 4, 117–126 (2003). phosphorylation in vitro identifies a residue of survivin that is essential for its 48. Chiang, G.G. & Abraham, R.T. Phosphorylation of mammalian target of rapamycin localization and binding to inner centromere protein (INCENP) in vivo. J. Biol. (mTOR) at Ser-2448 is mediated by p70S6 kinase. J. Biol. Chem. 280,25485– Chem. 279, 5655–5660 (2004). 25490 (2005). 34. Chen, J. et al. Survivin enhances Aurora-B kinase activity and localizes Aurora-B in 49. Hara, K. et al. Raptor, a binding partner of target of rapamycin (TOR), mediates TOR http://www.nature.com/natureimmunology human cells. J. Biol. Chem. 278, 486–490 (2003). action. Cell 110, 177–189 (2002). © Group 2006 Nature Publishing

10 ADVANCE ONLINE PUBLICATION NATURE IMMUNOLOGY