Pathway Phosphatidylinositol 3-Kinase/Akt Signaling T Cells

IL-12 Provides Proliferation and Survival Signals to Murine CD4 + T Cells Through Phosphatidylinositol 3-Kinase/Akt Signaling Pathway This information is current as of September 30, 2021. Jae Kwang Yoo, Jae Ho Cho, Seung Woo Lee and Young Chul Sung J Immunol 2002; 169:3637-3643; ; doi: 10.4049/jimmunol.169.7.3637

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2002 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

IL-12 Provides Proliferation and Survival Signals to Murine CD4؉ T Cells Through Phosphatidylinositol 3-Kinase/Akt Signaling Pathway1

Jae Kwang Yoo, Jae Ho Cho, Seung Woo Lee, and Young Chul Sung2

IL-12 is a pleiotropic cytokine that plays an important role in innate and adaptive immunity. IL-12 induces T cell proliferation and IFN-␥ secretion from activated T cells. It was also reported that IL-12 prevents apoptosis of CD4؉ T cells. However, the signaling mechanism that regulates these IL-12-induced responses is poorly understood yet. In this study, we demonstrated that IL-12 activates phosphatidylinositol 3-kinase (PI3K)/Akt pathway in murine CD4؉ T cells, and that this signaling pathway is required for IL-12-induced T cell proliferation and antiapoptotic function, but not for IFN-␥ induction. Through PI3K/Akt pathway, IL-12 up-regulates the expression of cell cycle-related molecule such as cyclin D3, and antiapoptotic molecules such as Bcl-2 and cellular Downloaded from inhibitors of apoptosis proteins-2, followed by down-regulation of active caspase-3. These results suggest that PI3K/Akt pathway is ,critical for mediating IL-12-induced CD4؉ T cell responses such as T cell proliferation and survival. The Journal of Immunology, 2002 169: 3637–3643.

nterleukin-12 is a pleiotropic cytokine that is secreted by ac- IL-12 might activate multiple signaling pathways for a variety of tivated professional APCs (1–3). IL-12 can induce Th1-type IL-12-induced T cell responses. http://www.jimmunol.org/ I cellular immune responses, T cell proliferation, and IFN-␥ Many cytokines and growth factors activate phosphatidylinosi- secretion from activated T and NK cells (1–5). In addition, it has tol 3-kinase (PI3K)/Akt signaling pathway in T cells (16). In mam- been reported that IL-12 prevents apoptotic cell death of the acti- malian cells, multiple isoforms of PI3K were found and subdivided vated human and mouse peripheral T cells (6–9). into three classes. Among them, class I PI3Ks are generally cou- IL-12R is composed of two subunits, designated ␤1 and ␤2. pled to extracellular stimuli and transmit signals to induce protein IL-12 activates the Janus family of protein tyrosine kinases (Jaks)3 synthesis, cytoskeletal changes, cell cycle regulation, and apopto- such as Jak2 and Tyk2 through IL-12R (10). These activated Jaks sis regulation (17). In the case of IL-2R signaling, it was reported phosphorylate tyrosine residues on IL-12R␤2, providing docking that PI3K/Akt pathway activated by IL-2 regulates cell cycle-re- by guest on September 30, 2021 sites for the Src homology 2 domain of STAT-4, and then STAT-4 lated molecules such as cyclin D3 and p27kip1 to induce T cell is phosphorylated and dimerized to bind to the IL-12-responsive proliferation (18). Recently, it was also shown that PI3K/Akt path- genes (11). STAT-4 appears to be an important mediator in IL-12R way activated by IL-2 family cytokines such as IL-2, IL-4, and signaling, because IL-12-induced IFN-␥ secretion and T cell pro- IL-15 promotes T cell survival through the modulation of an an- liferation are impaired in STAT-4Ϫ/Ϫ mice (5). Besides the Jak- tiapoptotic molecule such as Bcl-2 (19). However, it is not clear STAT pathway, IL-12 was also shown to activate mitogen-acti- yet whether IL-12 activates the PI3K/Akt pathway, leading to the vated protein kinase (MAPK) kinase (MKK) 6/p38 MAPK modulation of IL-12-induced T cell responses. In this study, we pathway, but not extracellular signal-regulated kinases (Erk) 1/2 or clearly demonstrated that IL-12 stimulates the PI3K/Akt signaling ϩ c-Jun N-terminal kinase pathway (12, 13). In particular, MKK pathway in activated CD4 T cells, and that this signaling pathway 6/p38 MAPK pathway is required to promote IL-12-induced is critical for IL-12-induced T cell proliferation and inhibition of IFN-␥ secretion and STAT-4 serine phosphorylation on serine 721, apoptosis. In particular, IL-12 modulates the expression of cell but not T cell proliferation (12–15). These results suggest that cycle-related molecule such as cyclin D3, and antiapoptotic molecules such as Bcl-2 and cellular inhibitors of apoptosis proteins (cIAP)-2 through PI3K/Akt pathway.

National Laboratory of DNA Medicine, Division of Molecular and Life Science, Materials and Methods Pohang University of Science and Technology, Hyoja Dong, Pohang, Korea Mice Received for publication April 17, 2002. Accepted for publication July 25, 2002. Five- to 10-wk-old C57BL/6 female mice were purchased from Japan SLC The costs of publication of this article were defrayed in part by the payment of page (Shizuoka, Japan) and used for the isolation of lymph nodes (LNs). The charges. This article must therefore be hereby marked advertisement in accordance mice were maintained in specific pathogen-free conditions and fed auto- with 18 U.S.C. Section 1734 solely to indicate this fact. claved food and water. 1 This work was supported by a National Research Laboratory grant from the Korea Institutes of Science and Technology Evaluation and Planning (2000-N-NL-01-C-202). Reagents and Abs 2 Address correspondence and reprint requests to Dr. Young Chul Sung, Division of Molecular and Life Sciences, Pohang University of Science and Technology, San 31, For MACS, anti-mouse CD11b, MHC class II, CD8, and B220 microbeads Hyoja Dong, Pohang, 790-784, Korea. E-mail address: [email protected] were obtained from Miltenyi Biotec (Auburn, CA). Purified hamster anti- mouse CD3e and CD28 NA/LE Abs were purchased from Southern Bio- 3 Abbreviations used in this paper: Jak, Janus kinase; cdk, cyclin-dependent kinase; cIAP, cellular inhibitor of apoptosis protein; Erk, extracellular signal-regulated ki- technology Associates (Birmingham, AL). For the flow cytometric analysis nase; GRR, IFN-␥ response region; LN, lymph node; MAPK, mitogen-activated pro- and cytokine ELISA, anti-mouse CD4 FITC, active caspase 3 FITC, an- tein kinase; MKK, MAPK kinase; PI, propidium iodide; PI3K, phosphatidylinositol nexin V FITC, and anti-mouse IFN-␥ capture and detection Abs were 3-kinase. purchased from BD PharMingen (San Diego, CA). Recombinant mouse

IL-2, IL-4, and IL-12 were all obtained from R&D Systems (Minneapolis, Triton X-100. The oligonucleotide sequence used was GTATTTCCCA MN) and reconstituted as described in technological notes. General re- GAAAAGGAAC. Cell lysates were incubated with GRR-bound agarose agents used were purchased from Sigma-Aldrich (St. Louis, MO), unless beads, which were then washed and subjected to SDS-PAGE. Oligo-pre- otherwise indicated. SB203580 and LY294002 were purchased from Cal- cipitated proteins were electrophoretically transferred to nitrocellulose biochem (La Jolla, CA). RPMI 1640 and FBS were purchased from Life transfer membrane and immunoblotted with anti-STAT-4 Abs (Santa Cruz Technologies (Grand Island, NY) and HyClone Laboratories (Logan, UT), Biotechnology). respectively. For immunoblot assay, rabbit Abs against p38 MAPK, Erks, Akt, Thr180/Tyr182-phosphorylated p38 MAPK, Thr202/Tyr204-phosphorylated Erks, and Ser473/Thr308-phosphorylated Akt were all purchased from New England Biolabs (Beverly, MA). Rabbit Abs against STAT-4 and cIAP-1/2 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Mouse Abs against p27kip1, Bcl-2, and cyclin D3 were also obtained from Santa Cruz Biotechnology. For IFN-␥ response region (GRR) affinity purification, streptavidin-agarose beads were obtained from Sigma-Aldrich. Preparation of murine primary CD4ϩ T lymphocytes Mouse naive CD4ϩ T cells were purified from the LNs of C57BL/6 female mice. CD4ϩ T cells were negative selected through VSϩ columns using the high gradient MACS (Miltenyi Biotec), as described previously (20). Briefly, harvested LN cells were treated with ammonium chloride lysing buffer to clear RBC. Then remaining cells were treated with anti-CD11b,

anti-MHC class II, anti-B220, and anti-CD8 microbeads, and purified by Downloaded from negative selection using the MACS Separation System. Purified cell populations were 92% CD4ϩ T cells. The purity was assessed by flow cytometric analysis on a FACScan flow cytometer (BD Biosciences, Heidel- berg, Germany) after staining with anti-CD4 FITC Abs. In vitro activation and culture of CD4ϩ T cells Purified CD4ϩ T cells were in vitro activated for 72 h with plate-bound http://www.jimmunol.org/ anti-CD3 (300 ng/well) and anti-CD28 (100 ng/well) hamster Abs in 96- well plate. T cells were cultured in RPMI 1640 supplemented with 10% heat-inactivated FBS (HyClone Laboratories), 2 mM L-glutamine, 50 mM 2-ME, and antibiotics (50 U/ml penicillin and 50 ␮g/ml streptomycin). The Ab-activated CD4ϩ T cells were harvested and washed twice with com- plete medium and used for the following experiments. Preparation of whole cell lysates and immunoblot analysis The Ab-activated T cells (5 ϫ 106 cells/ml) were pretreated for 20 min with medium alone, 5 ␮M LY294002, or 10 ␮M SB203580 (Calbiochem). After pretreatment, T cells were incubated with medium, 20 ng/ml IL-2, 100 by guest on September 30, 2021 ng/ml IL-4, or 10 ng/ml IL-12p70 (R&D Systems) for the indicated times. Then cells were harvested and whole cell lysates were prepared, as described previously (10). To quantify the phosphorylation of Akt, p38 MAPK, and Erks, equal amounts of whole cell lysates were resolved by 10% SDS-PAGE. Then Western blots were immunoanalyzed using specific Abs against the phosphorylated form of each protein. To confirm that the same amount of proteins was loaded in each lane, primary Ab/secondary Ab complex was removed, as described previously (21). The blots were then subjected to autoradiography to confirm that the Ab signal had been removed. After this procedure, the blots were reprobed with the specific Abs against total Akt, p38 MAPK, or Erks. For cell cycle and antiapoptotic molecules, we used each indicated Ab or ␤-actin as a control, followed by goat anti-rabbit or mouse HRP-conjugated Abs. Cytokine ELISA and intracellular staining Murine IFN-␥ ELISA was performed as described in the recommended protocol (BD PharMingen). Ab-activated T cells were harvested and treated with indicated inhibitors and cytokines. At 36 h after treatment, cells were collected and washed twice with PBS. Cells were fixed and permeabilized with Cytofix/Cyto- perm solution (BD PharMingen) for 20 min at 4°C, and then washed with FIGURE 1. IL-12 activates the PI3K/Akt signaling pathway. Murine naive Perm/Wash buffer (BD PharMingen). Permeabilized cells were resus- CD4ϩ T cells were prepared and stimulated, as described in Materials and pended with the wash buffer containing FITC-conjugated rabbit anti-mouse Methods. A, Cells were harvested after in vitro stimulation, and then treated for active caspase-3 Abs (BD PharMingen), and incubated for 30 min at 4°C. 20 min with medium alone or IL-12 (10 ng/ml). Oligonucleotide affinity pu- After incubation, cells were harvested and resuspended with PBS contain- rification of STAT-4 and immunoblotting were performed as described in ing 0.5% FBS and 0.1% sodium azide. Intracellular active caspase-3 levels Materials and Methods. B, Ab-activated T cells were rested in serum-free were assessed by flow cytometry. medium for 24 h, followed by stimulation with or without IL-12 (10 ng/ml). Oligonucleotide affinity purification and immunoblotting p38 MAPK inhibitor SB203580 (10 ␮M) or PI3K-specific inhibitor LY294002 (5 ␮M) was added and incubated for 20 min before IL-12 treat- Oligonucleotide affinity purification of STAT-4 proteins was performed ment. Cells were harvested at the indicated times, and equal amounts of whole using a 5Ј-biotinylated double-stranded oligonucleotide corresponding to ␥ cell lysates were resolved by SDS-PAGE, followed by immunoblot analysis, the GRR of mouse Fc RI gene (22, 23), coupled to streptavidin-agarose. ϩ Briefly, 20 min after cytokine stimulation, whole cell lysates were prepared as described in Materials and Methods. C, Activated CD4 T cells were by lysis of cells in a lysis buffer comprised of 50 mM Tris-Cl (pH 8.0), 1% treated with each indicated cytokine for 20 min, and then cells were harvested Nonidet P-40, 15 mM NaCl, 0.1 mM EDTA, 10 mM NaF, 10% glycerol, and resolved by SDS-PAGE, followed by immunoblot analysis using a set of

1mMNa3VO3, 1 mM PMSF, 1 mM DTT, protease inhibitors, and 0.1% Abs. Data are representative of two independent experiments. The Journal of Immunology 3639

Results IL-12 induces CD4ϩ T cell proliferation via PI3K/Akt IL-12 directly activates the PI3K/Akt signaling pathway pathway-dependent modulation of cyclin D3 molecules To investigate which signaling pathways are activated by IL-12, IL-12 has been known as a cytokine that induces the proliferation we examined the activation of STAT-4 molecule and several ki- of activated CD4ϩ T cells (3, 5). In addition, it has also been nases such as PI3K/Akt, p38 MAPK, and Erk1/2 by immunoblot reported that the PI3K/Akt pathway is an important cellular sig- analysis. As expected, IL-12 activated STAT-4 and induced their naling pathway that regulates multiple cellular responses, one of binding to the GRR DNA (Fig. 1A). When STAT-4 is activated by which is to induce cellular proliferation through the modulation of IL-12, it is translocated to the nucleus and regulates the transcrip- cell cycle-related molecules such as cyclin D3 and cyclin-depen- tion of the IL-12-responsive genes through the binding of con- dent kinase (cdk) inhibitor, p27kip1 (17, 18). As shown in Fig. 2A, served DNA sequences, GRR (23, 24). In addition, the treatment the proliferation of CD4ϩ T cells was enhanced by IL-12 treatment of IL-12 induced the phosphorylation of p38 MAPK, which was compared with the cells treated with medium alone. This IL-12 ␮ significantly inhibited by 10 M p38 MAPK inhibitor, SB203580. effect was completely inhibited by LY294002, but not by However, Erk1/2 were not activated by the treatment of IL-12. SB203580, suggesting that PI3K/Akt, but not p38 MAPK pathway These results agree well with the previous reports that IL-12 stim- ϩ is critical for IL-12-induced CD4 T cell proliferation. ulates MKK6/p38 MAPK, but not Erk1/2 signaling pathway in It has been well known that both up-regulation of cyclin D mol- activated CD4ϩ T cells (12, 13). Particularly, IL-12 induced Akt ecules and down-regulation of cdk inhibitors such as p27kip1 or phosphorylation, which was significantly blocked by 3.5 IC (5 50 WAF1/cip1 ␮ p21 are needed and sufficient to induce the cell cycle pro- M) of PI3K-specific inhibitor, LY294002 (25) (Fig. 1B). As re- Downloaded from gression through the G /S phase boundary in many cell types (26). ported previously (16, 19), IL-2 family cytokines such as IL-2 and 1 IL-4 also induced Akt phosphorylations, which appeared to be less Particularly, it was reported that PI3K/Akt pathway is involved in than those by IL-12 (Fig. 1C). Therefore, we demonstrated that, in IL-2-induced T cell proliferation through the regulation of both kip1 addition to STAT-4 and p38 MAPK, IL-12 directly activates the cyclin D3 and p27 expression (18). Interestingly, the treatment PI3K/Akt signaling pathway in murine CD4ϩ T cells. of IL-12 up-regulated the expression of cyclin D3, but down-regulated p27kip1, compared with medium alone (Fig. 2B). In addition, IL-12-induced up-regulation of cyclin D3 was significantly inhib- http://www.jimmunol.org/ ited by the treatment of LY294002, but not by SB203580. How- ever, the modulation of p27kip1 was not affected by both LY294002 and SB203580, which suggests that another signaling pathway except both PI3K/Akt and p38 MAPK pathways might be involved in IL-12-induced down-regulation of p27kip1. Taken together, these results demonstrated that IL-12 induces CD4ϩ T cell proliferation through the modulation of cell cycle-related mole-

cules such as cyclin D3 and p27kip1, and that PI3K/Akt pathway by guest on September 30, 2021 activated by IL-12 is associated with the regulation of cyclin D3, but not with that of p27kip1.

FIGURE 2. IL-12 regulates the expression of cyclin D3 to induce T cell proliferation through PI3K/Akt signaling pathway. A, Seventy-two hours after Ab stimulation, cells (1 ϫ 105 cells/sample) were harvested and stimulated with medium alone or IL-12 (10 ng/ml) for the indicated times. PI3K-speciﬁc inhibitor LY294002 (5 ␮M) or p38 MAPK-speciﬁc inhibitor SB203580 (10 ␮M) was added and incubated for 20 min before IL-12 treatment. T cell proliferation was measured through the counting of total FIGURE 3. The PI3K/Akt signaling pathway is not involved in IL-12- cell numbers at the indicated times. Data are representative of three inde- induced IFN-␥ secretion from CD4ϩ T cells. Ab-stimulated T cells were pendent experiments with similar results. B, Ab-activated T cells were preincubated with each indicated inhibitor, as described in Fig. 2A, and incubated with LY294002 (5 ␮M) or SB203580 (10 ␮M) for 20 min, and then treated with medium alone or IL-12 (10 ng/ml) for 24 h. Cell culture then stimulated with medium alone or IL-12 (10 ng/ml) for 20 h. Total cell supernatants were harvested and tested for murine IFN-␥ production by lysates were resolved by SDS-PAGE, followed by immunoblot with each cytokine ELISA. Data are shown as the mean Ϯ SD of triplicate cultures indicated Ab. Data are representative of two independent results. and representative of two independent experiments. 3640 PI3K/Akt PATHWAY IN IL-12-INDUCED T CELL RESPONSES

The PI3K/Akt pathway via IL-12 is not required for not signiﬁcantly inhibit the IL-12-induced IFN-␥ production, in- IL-12-induced IFN-␥ production dicating that PI3K/Akt pathway is not required for IL-12-induced ␥ ϩ One of the important properties of IL-12 is its ability to induce the IFN- production from activated CD4 T cells. production of IFN-␥ from activated T cells (3). As expected, IL-12 induced large amounts of IFN-␥ production from activated CD4ϩ IL-12 suppresses the apoptosis of activated CD4ϩ T cells by up- T cells, which was inhibited by the treatment of SB203580 (Fig. regulation of antiapoptotic molecules through PI3K/Akt pathway 3). This result is consistent with previous reports that p38 MAPK pathway is required for IL-12-induced IFN-␥ production from ac- Because IL-12 has been shown to prevent the apoptosis of acti- tivated T cells (14, 15). In contrast, the treatment of LY294002 did vated CD4ϩ T cells in various conditions (3, 6–9), we tested Downloaded from http://www.jimmunol.org/ by guest on September 30, 2021

FIGURE 4. PI3K/Akt, but not p38 MAPK pathway is critical for the survival of CD4ϩ T cells by IL-12 in passive cell death. A, 72 h after in vitro stimulation, Ab-activated CD4ϩ T cells were harvested and treated with medium alone or each indicated concentration of IL-12 (0.1, 1, 10 ng/ml). At the indicated times, cells were harvested and incubated with annexin V FITC in a buffer containing PI and analyzed by flow cytometry. Annexin V- and PI-double-negative cells were regarded as viable cells, annexin V-positive but PI-negative cells as apoptosis-undergoing cells, and annexin V- and PI-double-positive cells as dead cells. The sum of percentages of both apoptosis-undergoing and dead cell population is presented in each FACS profile. The result shown here is representative of more than five independent experiments with similar results. B and C, Ab-activated CD4ϩ T cells were harvested and incubated for 20 min with indicated concentrations of LY294002 (B) or SB203580 (C). After preincubation, cells were treated with IL-12 (10 ng/ml) for additional 48 h and then cells were assayed for cell death by flow cytometry, as described in A. The percentage of live cells before IL-12 treatment was regarded as 100%. The percentage of relative live cells of each sample was calculated and shown in histogram. Data are representative of five independent experiments. The Journal of Immunology 3641

FIGURE 5. IL-12 up-regulates the expression of Bcl-2 and cIAP-2 molecules, but down-regulates active caspase-3 through PI3K/Akt pathway. Ab-activated CD4ϩ T cells were incubated with LY294002 (5 ␮M) or SB203580 (10 ␮M) for 20 min and then treated with medium alone or IL-12 (10 ng/ml) for additional 36 h.

A, Total cell lysates were resolved by SDS-PAGE, and Downloaded from followed by immunoblot with each indicated Ab. Data are representative of two independent experiments. B, Intracellular staining for the detection of active caspase-3 was performed as described in Materials and Methods. Active caspase-3-positive populations were distinguished from negative populations by using iso- type control Ab. Each percentage of the active caspase- http://www.jimmunol.org/ 3-positive and caspase-3–negative population is presented in histograms. Data are representative of three independent experiments. by guest on September 30, 2021

whether PI3K/Akt pathway is involved in antiapoptotic effects of affected by the treatment of IL-12. These results suggest that IL-12 IL-12 in passive cell death. up-regulates the expression of Bcl-2 and cIAP-2 molecules Apoptotic T cell death was measured through flow cytometric through PI3K/Akt pathway in activated CD4ϩ T cells. analysis stained with both annexin V and propidium iodide (PI). Bcl-2 and cIAP-2 are known as antiapoptotic molecules that ϩ Preactivated CD4 T cells showed spontaneous apoptosis over promote cell survival through inhibition of the processing and ac- time, but the addition of IL-12 significantly reduced apoptosis in a tivity of caspase molecules (16, 29). As expected, IL-12 decreased dose-dependent manner (Fig. 4A), which is consistent with previ- the level of active caspase-3 from 70 to 58% (Fig. 5B). This result ous reports (7, 27). As shown in Fig. 4, B and C, LY294002 correlates with a recent report that IL-12 prevents apoptosis of inhibited the antiapoptotic effect of IL-12 in a dose-dependent human CD4ϩ T cells costimulated with ICAM-1 through inhibi- manner, but SB203580 did not, suggesting that IL-12 induces tion of the processing of caspase-3/9 (9). Moreover, the inhibitory ϩ CD4 T cell survival through PI3K/Akt pathway, but not through effect of IL-12 was completely blocked by the treatment of p38 MAPK pathway. LY294002, but not by SB203580, suggesting that IL-12 induces The PI3K/Akt pathway gives survival signals to many cell types CD4ϩ T cell survival in passive cell death through up-regulation of by regulating the expression of antiapoptotic molecules such as Bcl-2 and cIAP-2, followed by the down-regulation of active Bcl-2 and cIAP-1/2, or by controlling the activity of proapoptotic caspase-3 via PI3K/Akt pathway. molecules (16). Particularly, it has been reported that PI3K/Akt pathway activated by cytokines inhibits the T cell apoptosis Discussion through induction of Bcl-2 expression (19, 28). As expected, IL-12 It has been known that multiple cytokines and growth factors such enhanced the expression of Bcl-2 and cIAP-2 molecules, which as IL-2, IL-4, and insulin-like growth factors activate PI3K/Akt was inhibited by the treatment of LY294002, but not by SB203580 signaling pathway (16). In this study, we demonstrated that like (Fig. 5A). However, the expression of cIAP-1 appeared not to be IL-2 family cytokines, IL-12 activates the PI3K/Akt signaling 3642 PI3K/Akt PATHWAY IN IL-12-INDUCED T CELL RESPONSES pathway in murine CD4ϩ T cells, which is essential for IL-12- Acknowledgments induced T cell proliferation and survival. On the contrary, We thank Sang Chun Lee for devoted animal care and Chang Geun Lee Veronica et al. (24) recently reported that IL-12 could not activate and Kwan Hyuck Baek for technical assistance. PI3K/Akt pathway in human PBL T cells. This discrepancy might be due to the difference of origin of cells that were harvested, or the difference of experimental conditions in which T cells were References 1. Kobayashi, M., L. Fitz, M. Ryan, R. M. Hewick, S. C. Clark, S. Chan, R. Loudon, prestimulated. F. Sherman, B. Perussia, and G. Trinchieri. 1989. Identification and purification The PI3K/Akt signaling pathway is associated with cellular pro- of natural killer cell stimulatory factor (NKSF), a cytokine with multiple biologic liferation in T cells. It was reported that IL-2 induces T cell pro- effects on human lymphocytes. J. Exp. Med. 170:827. kip1 2. Wolf, S. F., P. A. Temple, M. Kobayashi, D. Young, M. Dicig, L. Lowe, liferation by modulation of both cyclin D3 and p27 through R. Dzialo, L. Fitz, C. Ferenz, and R. M. Hewick. 1991. Cloning of cDNA for PI3K/Akt pathway (18). 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