Cell Cycling Through Cdc25A

[Cell Cycle 5:9, 907-912, 1 May 2006]; ©2006 Landes Bioscience

Cell Cycling Through Cdc25A Extra View

Transducer of Cytokine Proliferative Signals ABSTRACT A balance between survival and proliferative signals maintains a constant number of T lymphocytes that populate the mammalian immune system, a process termed “homeostasis”. Central to this process is the availability of a stromal cell product—the cytokine interleukin-7 (IL-7). We recently showed that IL-7, in addition to protecting cells from apoptosis, drives the cell cycling of lymphocytes through regulation of the stability of the phosphatase, Cdc25A, a key activator of cyclin-dependent kinases (cdks). IL-7 achieves this by controlling the activity of p38 MAP kinase (MAPK), which can phosphorylate Cdc25A, triggering its degradation. Sustained expression of Cdc25A had diverse effects: it promoted cell cycling, even in presence of cell cycle inhibitors such p27Kip1, and prevented cell shrinkage in response to cytokine deprivation. Herein we show a role for Cdc25A as a transducer of cytokine-driven proliferation and discuss novel implications for cell growth from the perspective of the requirements for maintenance of lymphocyte homeostasis.

Cycle Control and Tumorigenesis Group; Institute of Molecular and Cell Biology; Singapore

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*Correspondence to: Annette Khaled; BioMolecular Science Center; University of Central Florida; 12722 Research Parkway; Orlando, Florida 32826 USA; Tel.: 407.882.2254; Fax: 407.384.2816; Email: [email protected]

The size of the T cell pool in lymphoid organs and blood remains relatively constant through life. If the numbers are severely depleted, as a result of disease or therapy, the survivors can be triggered to replicate, a process of homeostatic proliferation, until the size of the pool returns to normal. At first, homeostatic proliferation of T cells was thought to occur in response to foreign antigen and increased available “space”,1,2 but recent findings have revealed that this spontaneous proliferation is instead largely driven by interactions with self-peptide/MHC complexes (more so for naive T cells than memory T cells) and the cytokine, interleukin-7 (IL-7).3-6 How T cells, which are normally in G0, enter into cycle during homeostatic proliferation remains poorly understood. What has clearly emerged over the recent years is the notion that availability of the cytokine, interleukin-7 (IL-7), is essential for regulating T-cell proliferation. IL-7 is a stromal cell product found in various organs including the thymus, spleen, lymph node, bone marrow and intestines.7 The production of IL-7 appears to be constitutive; hence its levels are primarily regulated by consumption after binding IL-7 receptors on lymphocytes. IL-7 acts as a limiting factor—abundant IL-7 induces survival and proliferation, while loss of IL-7 leads to apoptosis.6 Evidence for this comes from the observation that T-cell depletion, such as occurs upon HIV infection, causes a rise in circulating IL-7 levels.8,9 Severe combined immunodeficiency syndrome (SCID) occurs as a consequence of deficient IL-7 signaling.10,11 In contrast, excess IL-7 causes lymphoid hyperplasia leading to T and B cell lymphomas.12,13 For many years, IL-7 was recognized as a key survival factor required for normal development of thymocytes.14 Severe lymphopenia results from IL-7 deficiency,15 as well as from the absence of IL-7 signaling components, such as the IL-7 receptor α chain (IL-7R),16,17 the common gamma c (γc) chain,18 the kinase, JAK3,19 and the transcription factor, STAT5 (Yao, Z. et al, PNAS in press). Naïve or memory CD4 and CD8 T cells also require survival signals from IL-7,20 dying rapidly upon transfer to IL-7 deficient mice,4,5 while expanding in IL-7 transgenic mice.21 IL-7 promotes survival of thymic or peripheral T cells largely through modulation of the activity of a family of anti-apoptotic or pro-apoptotic proteins named after their founding member, BCL-2.22,23 Expression of BCL-2 was able to rescue thymic development in IL-7R deficient mice,24,25 while loss of the death proteins BAX26 or BIM27 was able to partially restore thymic numbers. These studies clearly demonstrated the pro-survival function of IL-7. The question remained unanswered, though, could IL-7 also promote proliferation? Development of γδ T cells or B cells is not restored by expression of BCL-2 in IL-7R deficient mice.24,28,29 Moreover BCL-2 deficient mice have only a modest loss of thymic cells,30 and BCL-2 expression could not overcome the thymic defect in γc-deficient mice.31

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Original manuscript submitted: 03/06/06 Manuscript accepted: 03/10/06

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2Laboratory of Molecular Immunoregulation; National Cancer Institute-Frederick; Frederick, Maryland USA

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1BioMolecular Science Center; University of Central Florida; Orlando, Florida USA

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C. Kittipatarin1 W.Q. Li2 D.V. Bulavin3 S.K. Durum2 A.R. Khaled1,*

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Interleukin-7, lymphocyte, p38 MAPK, homeostasis, Cdc25A, cell cycle, p27kip1, growth arrest, proliferation, cytokine

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Previously published online as a Cell Cycle E-publication: http://www.landesbioscience.com/journals/cc/abstract.php?id=2693

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Cell Cycling Through Cdc25A

Figure 1. The strength of the IL-7 signal regulates survival versus proliferation. D1 cells were cultured with 0-50 ng/ml IL-7 for 24 hours. Survival and proliferation was assayed by BrdU incorporation and DNA content (7AAD) assessed by flow cytometry. Percent cells in G1, S, G2/M and undergoing apoptosis (Apop) was determined using Modfit software. BrdU incorporation was assayed using a commercially available kit.

Hence, expression of BCL-2 alone does not fully replace loss of the IL-7 signal. The absence of the apoptotic proteins BAX or BIM only resulted in partial rescue of thymic development and effects on peripheral T cell homeostasis remained unclear.26,27 To directly examine the effects of BCL-2 expression upon the homeostatic expansion of peripheral T cells in the absence of IL-7, we adoptively transferred BCL-2 expressing splenocytes into IL-7 deficient mice and observed that BCL-2 protected the transferred cells from death in the absence of IL-7. However, BCL-2 did not enable these T cells to proliferate in an IL-7 deficient host, though these cells would have normally replicated in a depleted host (Khaled, Durum, unpublished results). Likewise, we and others have found that cytokine-dependent cell lines that constitutively express BCL-2 or BCL-XL are protected from death upon cytokine withdrawal, but do not proliferate.32,33 Results such as these suggest that IL-7 has additional functions that go beyond conferring protection from apoptosis. Under lymphopenic conditions, in the absence of foreign antigen, naive T cells will undergo proliferation if IL-7 is present.34 Injection of mice with recombinant IL-7 commits T-cells to cycle, inducing a 4.3 fold increase in CD4 T cells and a 14.6 fold increase in CD8 T cells entering the S/G2/M phases of the cell cycle.35 These studies demonstrated that, under certain conditions, IL-7 can induce T cells to replicate, with the latter studies showing that T cells will proliferate in a nonlymphopenic environment if the amount of IL-7 is abnormally high. To determine whether there is a quantitative effect of IL-7, low doses inducing survival and high doses inducing proliferation, we evaluated an IL-7 dependent thymic cell line, D1, developed and studied in our laboratories,36 for the effects of IL-7 on growth. In the absence of IL-7, D1 cells underwent apoptosis within 48 hours. Concentrations of IL-7 ranging from 2–50 ng/ml promoted survival www.landesbioscience.com

(Fig. 1). However, we observed that at IL-7 concentrations between 2-10 ng/ml, though viability was maintained, no proliferation occurred as evidenced by a decreased number of DNA replicating cells (decreased BrdU uptake) and increased number of cells arrested in G1 (Fig. 1). Only when D1 cells were cultured in IL-7 at 50 ng/ml or greater was replication induced—almost 50% of the cells entered S/G2/M (Fig. 1). Hence, we demonstrated that two functions of IL-7—survival and proliferation—can be uncoupled based on the available amount of IL-7. That IL-7 promotes survival at low concentrations and cell division at high concentrations would be consistent with its function as a homeostatic regulator of T-cell population size. We set out to identify targets of IL-7 signaling that induced cell division, using the IL-7 dependent D1 cells to study cell cycling in response to the cytokine. Previously, we had observed that cytokine withdrawal induced the activity of a stress kinase, p38 MAP kinase (MAPK). Within a few hours of cytokine withdrawal, dependent cells displayed elevated levels of phosphorylated p38 MAPK and increased enzymatic activity37 that we correlated to apoptotic events.38,39 Others have implicated p38 MAPK in regulation of the G1 and G2/M phases of the cell cycle.40 To determine if p38 MAPK was a target of IL-7 proliferative signaling, we inhibited the activity of this kinase and observed that the D1 cells, that normally arrest in G1 upon IL-7 withdrawal, did not arrest but progressed into S phase, as evidenced by increased DNA synthesis in the absence of the cytokine.33 We observed that the same occurred with primary lymphocytes isolated from lymph nodes, indicating that growth arrest could be mediated by p38 MAPK.33 To identify the substrate of p38 MAPK that regulated cytokine-mediated proliferation, we examined various elements involved in cell cycle progression, such as the synthesis of cyclins, for

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has been shown to associate with Cdc25A, and depletion of βTrCP prevented the degradation of Cdc25A.48 Cdc25A, like other SCFβTrCP substrates, contains a DSG motif (DpSGΠXpS); hence, Cdc25A turnover requires phosphorylation at Ser 82 and Ser 88. Other studies identified Ser 123 as a target of phosphorylation by the CHK1/2 kinases, induced by ionizing radiation,50 and later other sites were also identified as targets of the CHK1/2 kinases.49,51 In addition to Ser 123, Ser 75 was described as a CHK1 phosphorylation Figure 2. Cytokine withdrawal induces elevated levels of the inhibitor, p27Kip, which does not interfere with site that is also involved in regulating cell cycle progression induced by a stable Cdc25A mutant. (A) HA-tagged wild type (WT) Cdc25A or Cdc25A stability.52 Mutation of Ser S75,123A mutant (MUT) Cdc25A was expressed in IL-3 dependent murine FL5.12A pro-B cells and cells 75 or Ser 82 prevented association with incubated with or without IL-3 for 8 hours. Whole cell protein lysates were prepared and immunoblotted for the SCFβTrCP complex, suggesting expression of the inhibitor, p27kip, HA-tagged Cdc25A or as loading control, p38 MAPK, using specific antibodies. FL5.12A cells were also treated with a p38 MAPK inhibitor, PD169316. (B) HA-tagged that phosphorylation of Cdc25A at S75,123A mutant (MUT) Cdc25A was expressed in IL-7 dependent D1 thymic cells and cells incubated with these sites was critical for initiating or without IL-7 for 8 hours. Whole cell protein lysates were prepared and immunoblotted for expression of ubiquitin-mediated proteolysis.49 the inhibitor, p27kip using specific antibodies. These findings suggested a model in which Cdc25A is initially phosphoryexample the D-type Cyclins, and activity of cyclin dependent kinases lated at Ser 75, as a prerequisite for subsequent phosphorylation at (Cdks), such Cdk4 or Cdk2, and concluded that Cdk2 activity was Ser 82 by an unknown kinase, leading to recruitment of the critical for IL-7-driven cell division.33 The activity of Cdks is generally SCFβTrCP complex. Mutation of Ser 123 also restored Cdc25A regulated by phosphorylation and dephoshorylation mediated by a stability under normal conditions as well as upon exposure to ionizing number of kinases and phosphatases. Cdk2 is phosphorylated at radiation.50 Modulation of Ser 123 may regulate Cdc25A stability three main sites—Thr160 which activates the kinase and Tyr 15 and during mitosis; an adjacent site, Ser 121, when phosphorylated and Thr 14 which inhibit the kinase.41 Removal of the inhibitory phos- bound by 14-3-3 proteins, could prevent the phosphorylation of Ser phorylation is mediated by a family a dual specificity phosphatases 123, ensuring stability of Cdc25A during mitosis.53 called Cdc25. There are three known members, Cdc25A, Cd25B Previous studies of Cdc25A have shown that it is destabilized by and Cdc25C, that function at diverse phases of the cell cycle: ionizing radiation, UV irradiation or drugs that block DNA replication. Cdc25A participates in the G1-to-S transition,42 while Cdc25B and These trigger DNA damage checkpoints in which protein kinases Cdc25C regulate the G2-to-M transition.43 However, this concept like ATM and ATR are activated and subsequently phosphorylate has been recently challenged in that mice lacking both Cdc25B and and activate the effector kinases, CHK1/2.54 We asked whether the Cdc25C have normal cell cycles,44 suggesting that Cdc25A may G1 arrest induced by cytokine withdrawal could be similar to a DNA functionally compensate for the loss of the other phosphatases and damage response following ionizing radiation. To answer this, we also participate in mitotic entry in addition to S-phase progression.45 assayed for the levels of CHK1 during cytokine withdrawal and p38 MAPK was first shown to have a role regulating the activity of found that this kinase, though expressed in the presence of IL-7, the Cdc25 phosphatase family when both Cdc25B (at Ser 309 and declined in cells deprived of the cytokine.33 Furthermore, using Ser 361) and Cdc25C (at Ser 216) were identified as targets for the RNA interference to silence CHK1 expression in FL5.12A cells kinase. Mutagenesis of the putative p38 MAPK phosphorylation deprived of IL-3, we demonstrated that G1 arrest still occurred upon sites on Cdc25B or Cdc25C prevented sequestration of the phos- cytokine withdrawal.33 We cannot exclude a role for CHK-1-mediated phatases by 14-3-3, a process that normally results in activation of a phosphorylation of Cdc25A as means to control protein levels of the G2 checkpoint triggered by radiation-induced DNA damage.46 But phosphatase under normal growth conditions, but another kinase did p38 MAPK also regulate the activity of Cdc25A, making this must phosphorylate Cdc25A and target it for degradation during phosphatase a target of IL-7 signaling? To answer this, we were cytokine withdrawal-induced growth arrest. We identified that assisted by our collaborators (Bulavin DV, Fornace AJ) and observed kinase to be p38 MAPK through both our studies of the effects of that the protein levels of Cdc25A did in fact decrease dramatically p38 MAPK inhibition upon Cdc25A stability33 and experiments during IL-7 withdrawal in D1 cells (as well as IL-3 withdrawal in that showed that p38 MAPK could phosphorylate Cdc25A at Ser 75 FL5.12A cells, Fig. 2) and that inhibition of p38 MAPK prevented and Ser123.53 this decline.33 To verify that the p38 MAPK-induced breakdown of Cdc25A Recent studies have shown that Cdc25A protein is targeted for accounted for the G1 arrest following IL-7 withdrawal, we generated proteosomal degradation by two ubiquitin ligase complexes—APC/C a mutant form of Cdc25A lacking the p38 MAPK target residues. (Anaphase Promoting Complex/cyclosome) and SCF (Skp1/Cul1/ Both Ser 75 and Ser 123 were substituted by alanines and the double F-box protein).47-49 Unlike APC/C, SCF-mediated-ubquitination mutein was expressed in our cytokine dependent cell lines. We also requires recruitment of phosphorylated substrates by F-box proteins. generated cytokine-dependent cell lines expressing Cdc25A containing βTrCP, a WD-repeat containing F-box protein of the SCF complexes, single mutations at Ser 75 or Ser 123. In an earlier study, expression www.landesbioscience.com

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of the Cdc25A double mutein (S75, 123A) in HeLa cells could not overcome a S-phase checkpoint induced by osmotic stress (which activated p38 MAPK) or UV-irradiation (which activated CHK1).53 In contrast, we found that, just like the effects of p38 MAPK inhibition, expression of the stable Cdc25A double mutein (S75, 123A) promoted cell cycling in lymphocytes deprived of cytokines— driving the cell cycle for two-four days beyond the time at which the cells normally died. Our findings illustrated the differences in pathways to cell cycle arrest induced by DNA damaging events in fibroblasts versus cytokine withdrawal in lymphocytes, with the latter being predominantly dependent upon Cdc25A modulation. Expression of the Cdc25A single muteins showed that mutation of S123 had no effect in relieving cell cycle arrest, whereas mutation of Ser 75 had a partial effect. Full restoration of cell cycling in the absence of a cytokine growth signal was only achieved by mutation of both Ser 75 and Ser 123, suggesting that phosphorylation of both these sites during cytokine withdrawal was critical for destabilizing Cdc25A and inducing growth arrest. Figure 3. The strength of the IL-7 signal determines whether a T cell will undergo apoptosis, Another interesting feature of the cycling growth arrest or proliferation. (A). The absence of an IL-7 signal induces the activity of p38 induced by expression of the Cdc25A double MAPK which (1) phosphorylates a pH regulating protein, the Sodium Hydrogen Exchanger (NHE), leading to intracellular alkalinization that activates the apoptotic protein, BAX, resultmutein was its ability to overcome effects of the ing in mitochondrial damage, caspase activation and cell death; and (2) active p38 MAPK inhibitor p27kip1 (Fig. 2). We found (Li et al, JEM also targets for degradation the phosphatase Cdc25A by phosphorylating two critical sites, in press), as did others,55 that IL-7 stimulation Ser 75 and Ser 123, leading to growth arrest which precedes apoptosis. (B) A minimal IL-7 kip1 downregulates p27 . Expression of the Cdc25A signal induces survival, but not growth, likely through upregulation of an anti-apoptotic prokip1 double mutein did not inhibit the rise in p27 tein like BCL-2. (C) A strong IL-7 signal, downregulates p38 MAPK activity, promoting the stafollowing IL-7 withdrawal yet still could promote bility of Cdc25A, which dephosphorylates inhibitory sites on critical kinases like Cdk2 and drives cell cycle progression resulting in proliferation. cell cycling (Fig. 2). How could expression of the stable Cdc25A double mutein circumvent the inhibitory effects of p27kip1 and drive cells through S-phase? In most that Cdc25A effectively dephosphoryates and activates Cdc2 as well reports, p27kip1 functions during the G1-to-S phase transition, and, as Cdk2, and that Cdc2 can replace Cdk2 in the G1-to-S transition although it can bind most Cdks, it is a most potent inhibitor of of cytokine-dependent lymphocytes. Others found that inhibition of Cdk2/cyclin E or cyclin A complexes.56,57 In contrast, the binding Cdc2 in wild type or Cdk2 deficient MEFs retarded entry into S-phase, of p27kip1 to Cdk4/ cyclin D-type complexes is thought to stabilize hence Cdc2 could replace Cdk2 and drive S-phase progression.62 In these complexes producing the catalytically active forms that drive the case of our cytokine-dependent cells lines, we noted that protein the G1-to-S transition.58,59 This suggested the possibility that during levels of phosphorylated Cdc2-like complexes were elevated in cells cell cycle progression, induced by stable expression of the Cdc25A expressing the Cdc25A double mutein.33 This leads to speculation double mutein, p27kip1 is sequestered by Cdk4 or Cdk6-containing that Cdc25A may play an equally significant role in the regulation complexes, preventing the inhibition of Cdk2-containing complexes. of the G2-to-M progression as it does in the G1-to-S transition. So We did observe that the protein levels of G1 cdks and cyclins whether Cdc2 or Cdk2 is active, our findings show that expression remained detectable after cytokine withdrawal and therefore could of the stable Cdc25A double mutein is a potent signal for cell cycle potentially bind and sequester p27kip1. In addition, recent findings progression that, even in the presence of an inhibitor like p27kip1, demonstrated that the phosphorylated state of p27kip1 can determine can still promote cell division in dependent lymphocytes deprived of which cdk/cyclin complexes it interacts with—for example cyclin their cytokines. Our studies on the survival and proliferative functions of IL-7 in E-type complexes interact with phosphorylated p27kip1, while cyclin D-type complexes interact with unphosphorylated forms.60 Hence, lymphocytes are summarized in a model shown in (Fig. 3). In the in the absence of IL-7 or IL-3, unphosphorylated p27kip1 may accu- absence of a ligand binding the IL-7 receptor, p38 MAPK is upregmulate but not bind and inhibit cdk2/cyclin E complexes. We also ulated and has at least two principal targets: (1) the phosphorylation observed that inhibition of p38 MAPK blocked the rise in p27kip1 and activation of the sodium hydrogen exchanger (NHE1),37 which following cytokine withdrawal (Fig. 2), indicating that the activity of leads to a rise in intracellular pH that induces the activation of BAX, p38 MAPK could have an additional role in stabilizing inhibitory leading to apoptosis, and (2) the phosphorylation and degradation of forms p27kip1. Another possibility is that p27kip1 does inhibit Cdk2- Cdc25A, leading to cell cycle arrest. p38 MAPK may also indirectly containing complexes in our Cdc25A mutant cell lines and that contribute to the stabilization of p27kip1 by mechanisms that remain cycling is mediated instead by Cdc2-containing complexes, which have to be explored. A minimal IL-7 signal prevents activation of the been shown to bind p27kip1 less efficiently.61 This would suggest apoptotic pathway, perhaps by generating the anti-apoptotic protein, www.landesbioscience.com

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BCL-2, but still leads to growth arrest perhaps through degradation of Cdc25A and/or induction of p27kip1. While a strong IL-7 signal downregulates p38 MAPK and produces a stable form of Cdc25A that dephosphorylates Cdks leading to cell cycle progression. Cdc25A is responsive to IL-7 mediated signaling and may in fact be the critical factor that “senses” the strength of the IL-7 signal driving the homeostatic proliferation of peripheral lymphocytes. In turn, cell cycling, perhaps through induction of activity of E2F transcription factors recently shown to be required for homeostatic proliferation,63 might also provide survival signals that prevent the induction of apoptosis. We found that cytokine-deprived cells expressing the Cdc25A double mutein did not shrink in size or undergo the typical morphological changes that characterize apoptotic cells.33 This suggested to us that restoring cell cycling could be sufficient to maintain both survival and proliferation of cells grown in the absence of cytokine signals, while the converse was not true— maintaining survival, for example by overexpressing BCL-XL, will not promote proliferation. Cdc25A itself, when localized to the cytosol, has been suggested to have anti-apoptotic effects through interactions with protein kinase B (PKB, Akt),64 while others have found that Cdc25A modulates the phosphorylation of ERK, a kinase involved in relaying growth stimuli.65 Another possibility to be explored is that cycling cells are metabolically active and in this manner prevent the stress signals that trigger early apoptotic events; hence expression of Cdc25A could sustain mitochondrial energetics in the absence of cytokines. Further study of the proliferative and perhaps survival activity of Cdc25A could provide important clues about the function of IL-7 in the maintenance of peripheral lymphocytes, revealing novel insights on the mechanisms underlying disorders that result from deregulation of critical homeostatic controls. Reference List 1. Rocha B, Dautigny N, Pereira P. Peripheral T lymphocytes: expansion potential and homeostatic regulation of pool sizes and CD4/CD8 ratios in vivo. Eur J Immunol 1989; 19:905-11. 2. Lee SK, Surh CD. Role of interleukin-7 in bone and T-cell homeostasis. Immunol Rev 2005; 208:169-80. 3. Ernst B, Lee DS, Chang JM, Sprent J, Surh CD. The peptide ligands mediating positive selection in the thymus control T cell survival and homeostatic proliferation in the periphery. Immunity 1999; 11:173-81. 4. Schluns KS, Kieper WC, Jameson SC, Lefrancois L. Interleukin-7 mediates the homeostasis of naive and memory CD8 T cells in vivo. Nat Immunol 2000; 1:426-32. 5. Tan JT, Dudl E, LeRoy E, Murray R, Sprent J, Weinberg KI, Surh CD . IL-7 is critical for homeostatic proliferation and survival of naive T cells. Proc Natl Acad Sci USA 2001; 98:8732-7. 6. Khaled AR, Durum SK. Lymphocide: cytokines and the control of lymphoid homeostasis. Nat Rev Immunol 2002; 2:817-30. 7. Jiang Q, Li WQ, Aiello FB, Mazzucchelli R, Asefa B, Khaled AR, Durum SK. Cell biology of IL-7, a key lymphotrophin. Cytokine Growth Factor Rev 2005; 16:513-33. 8. Fry TJ, Connick E, Falloon J, Lederman MM, Liewehr DJ, Spritzler J, Steinberg SM, Wood LV, Yarchoan R, Zuckerman J, Landay A, Mackall CL. A potential role for interleukin-7 in T-cell homeostasis. Blood 2001; 97:2983-90. 9. Napolitano LA, Grant RM, Deeks SG, Schmidt D, De Rosa SC, Herzenberg LA, Herndier BG, Andersson J, McCune JM. Increased production of IL-7 accompanies HIV-1-mediated T-cell depletion: implications for T-cell homeostasis. Nat Med 2001; 7:73-9. 10. Puel A, Ziegler SF, Buckley RH, Leonard WJ. Defective IL7R expression in T(-)B(+)NK(+) severe combined immunodeficiency. Nat Genet 1998; 20:394-7. 11. Giliani S, Mori L, de Saint BG, Le Deist F, Rodriguez-Perez C, Forino C, Mazzolari E, Dupuis S, Elhasid R, Kessel A, Galambrun C, Gil J, Fischer A, Etzioni A, Notarangelo LD. Interleukin-7 receptor alpha (IL-7Ralpha) deficiency: cellular and molecular bases. Analysis of clinical, immunological, and molecular features in 16 novel patients. Immunol Rev 2005; 203:110-26. 12. Fisher AG, Burdet C, LeMeur M, Haasner D, Gerber P, Ceredig R. Lymphoproliferative disorders in an IL-7 transgenic mouse line. Leukemia 1993; 7 Suppl 2:S66-8. 13. Rich BE, Campos-Torres J, Tepper RI, Moreadith RW, Leder P. Cutaneous lymphoproliferation and lymphomas in interleukin 7 transgenic mice. J Exp Med 1993; 177:305-16. 14. von Freeden-Jeffry U, Moore TA, Zlotnik A, Murray R. IL-7 knockout mice and the generation of lymphocytes. In: Durum SK, Muegge K, eds. Cytokine knockouts. Totowa, NJ: Humana Press Inc.; 1998:21-36. 15. von-Freeden-Jeffry U, Vieira P, Lucian LA, McNeil T, Burdach SE, Murray R. Lymphopenia in interleukin (IL)-7 gene-deleted mice identifies IL-7 as a nonredundant cytokine. J Exp Med 1995; 181:1519-26.


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Bcl-2 rescues T lymphopoiesis in interleukin-7 receptor-deficient mice. Cell 1997; 89:1033-41. 26. Khaled AR, Li WQ, Huang J, Fry TJ, Khaled AS, Mackall CL, Muegge K, Young HA, Durum SK. Bax deficiency partially corrects IL-7 receptor alpha deficiency. Immunity 2002; 17:561-73. 27. Pellegrini M, Bouillet P, Robati M, Belz GT, Davey GM, Strasser A. Loss of Bim increases T cell production and function in interleukin 7 receptor-deficient mice. J Exp Med 2004; 200:1189-95. 28. Maraskovsky E, Peschon JJ, McKenna H, Teepe M, Strasser A. Overexpression of Bcl-2 does not rescue impaired B lymphopoiesis in IL-7 receptor-deficient mice but can enhance survival of mature B cells. Int Immunol 1998; 10:1367-75. 29. Kondo M, Akashi K, Domen J, Sugamura K, Weissman IL. Bcl-2 rescues T lymphopoiesis, but not B or NK cell development, in common gamma chain-deficient mice. Immunity 1997; 7:155-62. 30. Matsuzaki Y, Nakayama K, Tomita T, Isoda M, Loh DY, Nakauchi H. 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