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• W1982704558 abstract "The cell-intrinsic mechanisms guiding naive CD8+ T cells for clonal expansion and memory generation via homeostatic proliferation (HP) are unclear. Here, we have shown that HP of naive CD8+ T cells requires IL-7-, but not IL-15-induced mTOR kinase activation. HP-induced mTOR enhances transcription factor T-bet for functional maturation and CD122 expression, which sensitizes for an IL-15-dependent memory transition by favoring transcription factor Eomesodermin over T-bet. Inhibition of mTOR blocks T-bet and CD122 expression but preserves memory in an IL-15-independent manner by promoting Eomesodermin expression. The ability of rapamycin to augment HP-induced memory was cell-intrinsic given that silencing mTOR in CD8+ T cells generated identical outcomes. Strikingly, HP-induced CD8+ T cell memory generated by IL-15-dependent or -independent mechanisms demonstrated identical tumor efficacy. These results indicate a central role for mTOR in HP-induced CD8+ T cell responses and demonstrate the importance for CD8+ memory in HP-induced tumor efficacy. The cell-intrinsic mechanisms guiding naive CD8+ T cells for clonal expansion and memory generation via homeostatic proliferation (HP) are unclear. Here, we have shown that HP of naive CD8+ T cells requires IL-7-, but not IL-15-induced mTOR kinase activation. HP-induced mTOR enhances transcription factor T-bet for functional maturation and CD122 expression, which sensitizes for an IL-15-dependent memory transition by favoring transcription factor Eomesodermin over T-bet. Inhibition of mTOR blocks T-bet and CD122 expression but preserves memory in an IL-15-independent manner by promoting Eomesodermin expression. The ability of rapamycin to augment HP-induced memory was cell-intrinsic given that silencing mTOR in CD8+ T cells generated identical outcomes. Strikingly, HP-induced CD8+ T cell memory generated by IL-15-dependent or -independent mechanisms demonstrated identical tumor efficacy. These results indicate a central role for mTOR in HP-induced CD8+ T cell responses and demonstrate the importance for CD8+ memory in HP-induced tumor efficacy. Lymphopenia-associated IL-7 induces mTOR activity in naive CD8+ T cells for HP mTOR regulates T-bet for functional maturation and IL-15-dependent CD8+ memory mTOR blockade alters transcriptional programs and produces IL-15-independent memory HP-induced CD8+ memory via both pathways produces equivalent tumor immunity The stimulation of naive CD8+ T cells with antigen, costimulation, and cytokines induce vigorous proliferation and associated differentiation that can give rise to various effector and/or memory phenotypes (Ahmed and Gray, 1996Ahmed R. Gray D. Immunological memory and protective immunity: Understanding their relation.Science. 1996; 272: 54-60Crossref PubMed Scopus (1375) Google Scholar). Alternatively, naive CD8+ T cells can undergo proliferation in the absence of antigen, in response to space (self-antigen-MHC, cytokines like interleukin-7 [IL-7], and/or IL-15) secondary to lymphopenia (genetic deficiency, infection, irradiation, or unprimed neonatal animals) (Ernst et al., 1999Ernst B. Lee D.S. Chang J.M. Sprent J. Surh C.D. The peptide ligands mediating positive selection in the thymus control T cell survival and homeostatic proliferation in the periphery.Immunity. 1999; 11: 173-181Abstract Full Text Full Text PDF PubMed Scopus (614) Google Scholar, Goldrath and Bevan, 1999Goldrath A.W. Bevan M.J. Low-affinity ligands for the TCR drive proliferation of mature CD8+ T cells in lymphopenic hosts.Immunity. 1999; 11: 183-190Abstract Full Text Full Text PDF PubMed Scopus (461) Google Scholar, Kieper and Jameson, 1999Kieper W.C. Jameson S.C. Homeostatic expansion and phenotypic conversion of naïve T cells in response to self peptide/MHC ligands.Proc. Natl. Acad. Sci. USA. 1999; 96: 13306-13311Crossref PubMed Scopus (283) Google Scholar, Le Campion et al., 2002Le Campion A. Bourgeois C. Lambolez F. Martin B. Léaument S. Dautigny N. Tanchot C. Pénit C. Lucas B. Naive T cells proliferate strongly in neonatal mice in response to self-peptide/self-MHC complexes.Proc. Natl. Acad. Sci. USA. 2002; 99: 4538-4543Crossref PubMed Scopus (113) Google Scholar). The antigen-independent proliferation exhibited by naive CD8+ T cells has been termed as homeostatic proliferation (HP), and it plays an important role in maintaining normal CD8+ T cell numbers (Cho et al., 2000Cho B.K. Rao V.P. Ge Q. Eisen H.N. Chen J. Homeostasis-stimulated proliferation drives naive T cells to differentiate directly into memory T cells.J. Exp. Med. 2000; 192: 549-556Crossref PubMed Scopus (423) Google Scholar, Ernst et al., 1999Ernst B. Lee D.S. Chang J.M. Sprent J. Surh C.D. The peptide ligands mediating positive selection in the thymus control T cell survival and homeostatic proliferation in the periphery.Immunity. 1999; 11: 173-181Abstract Full Text Full Text PDF PubMed Scopus (614) Google Scholar, Goldrath and Bevan, 1999Goldrath A.W. Bevan M.J. Low-affinity ligands for the TCR drive proliferation of mature CD8+ T cells in lymphopenic hosts.Immunity. 1999; 11: 183-190Abstract Full Text Full Text PDF PubMed Scopus (461) Google Scholar, Kieper and Jameson, 1999Kieper W.C. Jameson S.C. Homeostatic expansion and phenotypic conversion of naïve T cells in response to self peptide/MHC ligands.Proc. Natl. Acad. Sci. USA. 1999; 96: 13306-13311Crossref PubMed Scopus (283) Google Scholar, Murali-Krishna et al., 1999Murali-Krishna K. Lau L.L. Sambhara S. Lemonnier F. Altman J. Ahmed R. Persistence of memory CD8 T cells in MHC class I-deficient mice.Science. 1999; 286: 1377-1381Crossref PubMed Scopus (618) Google Scholar). Additionally, lymphopenia-induced proliferation of naive CD8+ T cells induces functional maturation resulting in suboptimal effector functions (IFN-γ and/or cytolytic T lymphocyte [CTL] activity) and transition to memory-like cells (Goldrath et al., 2000Goldrath A.W. Bogatzki L.Y. Bevan M.J. Naive T cells transiently acquire a memory-like phenotype during homeostasis-driven proliferation.J. Exp. Med. 2000; 192: 557-564Crossref PubMed Scopus (531) Google Scholar, Hamilton et al., 2006Hamilton S.E. Wolkers M.C. Schoenberger S.P. Jameson S.C. The generation of protective memory-like CD8+ T cells during homeostatic proliferation requires CD4+ T cells.Nat. Immunol. 2006; 7: 475-481Crossref PubMed Scopus (174) Google Scholar, Surh and Sprent, 2008Surh C.D. Sprent J. Homeostasis of naive and memory T cells.Immunity. 2008; 29: 848-862Abstract Full Text Full Text PDF PubMed Scopus (791) Google Scholar). The critical role of extrinsic cues like self-antigen-MHC class I and IL-7 in HP of CD8+ T cells has been demonstrated (Ernst et al., 1999Ernst B. Lee D.S. Chang J.M. Sprent J. Surh C.D. The peptide ligands mediating positive selection in the thymus control T cell survival and homeostatic proliferation in the periphery.Immunity. 1999; 11: 173-181Abstract Full Text Full Text PDF PubMed Scopus (614) Google Scholar, Goldrath and Bevan, 1999Goldrath A.W. Bevan M.J. Low-affinity ligands for the TCR drive proliferation of mature CD8+ T cells in lymphopenic hosts.Immunity. 1999; 11: 183-190Abstract Full Text Full Text PDF PubMed Scopus (461) Google Scholar, Tan et al., 2001Tan J.T. Dudl E. LeRoy E. Murray R. Sprent J. Weinberg K.I. Surh C.D. IL-7 is critical for homeostatic proliferation and survival of naive T cells.Proc. Natl. Acad. Sci. USA. 2001; 98: 8732-8737Crossref PubMed Scopus (734) Google Scholar). It is envisioned that weak but frequent T cell receptor (TCR) interactions with abundant self-peptide-MHC class I molecules and IL-7 under conditions of lymphopenia induces cell cycling and HP of naive CD8+ T cells (Li et al., 2006Li W.Q. Jiang Q. Aleem E. Kaldis P. Khaled A.R. Durum S.K. IL-7 promotes T cell proliferation through destabilization of p27Kip1.J. Exp. Med. 2006; 203: 573-582Crossref PubMed Scopus (66) Google Scholar, Schluns et al., 2000Schluns K.S. Kieper W.C. Jameson S.C. Lefrançois L. Interleukin-7 mediates the homeostasis of naïve and memory CD8 T cells in vivo.Nat. Immunol. 2000; 1: 426-432Crossref PubMed Scopus (1254) Google Scholar, Tan et al., 2001Tan J.T. Dudl E. LeRoy E. Murray R. Sprent J. Weinberg K.I. Surh C.D. IL-7 is critical for homeostatic proliferation and survival of naive T cells.Proc. Natl. Acad. Sci. USA. 2001; 98: 8732-8737Crossref PubMed Scopus (734) Google Scholar). Although, the other common γ-chain (γc) cytokine, IL-15, has also been implicated in HP of naive CD8+ T cells, it was unable to compensate for IL-7 deficiency in HP of naive CD8+ T cells (Tan et al., 2001Tan J.T. Dudl E. LeRoy E. Murray R. Sprent J. Weinberg K.I. Surh C.D. IL-7 is critical for homeostatic proliferation and survival of naive T cells.Proc. Natl. Acad. Sci. USA. 2001; 98: 8732-8737Crossref PubMed Scopus (734) Google Scholar). However, IL-15 is important for sustained CD8+ T cell proliferation and accumulation in a lymphopenic setting (Sandau et al., 2007Sandau M.M. Winstead C.J. Jameson S.C. IL-15 is required for sustained lymphopenia-driven proliferation and accumulation of CD8 T cells.J. Immunol. 2007; 179: 120-125PubMed Google Scholar), and the reduction in HP-induced CD8+ T cells expressing CD62L and CD44hi phenotype in IL-15Rα-deficient mice indicates the need for IL-15 in promoting CD8+ T cell memory but not initiation of HP (Kennedy et al., 2000Kennedy M.K. Glaccum M. Brown S.N. Butz E.A. Viney J.L. Embers M. Matsuki N. Charrier K. Sedger L. Willis C.R. et al.Reversible defects in natural killer and memory CD8 T cell lineages in interleukin 15-deficient mice.J. Exp. Med. 2000; 191: 771-780Crossref PubMed Scopus (1277) Google Scholar, Lodolce et al., 1998Lodolce J.P. Boone D.L. Chai S. Swain R.E. Dassopoulos T. Trettin S. Ma A. IL-15 receptor maintains lymphoid homeostasis by supporting lymphocyte homing and proliferation.Immunity. 1998; 9: 669-676Abstract Full Text Full Text PDF PubMed Scopus (1044) Google Scholar, Ramsey et al., 2008Ramsey C. Rubinstein M.P. Kim D.M. Cho J.H. Sprent J. Surh C.D. The lymphopenic environment of CD132 (common gamma-chain)-deficient hosts elicits rapid homeostatic proliferation of naive T cells via IL-15.J. Immunol. 2008; 180: 5320-5326PubMed Google Scholar). Together, the γc cytokines IL-7 and IL-15 act in concert to maintain CD8+ T cell homeostasis, but their precise mechanisms of action and collaboration are not completely understood. The binding of IL-7 to IL-7Rα on naive CD8+ T cells can initiate a signaling cascade that leads to phosphorylation of signal transducers and activators of transcription 5 (STAT5) and the kinase cascades involving phosphatidylinositol 3-kinase (PI3K)-protein kinase B (PKB, also called Akt) and mammalian target of rapamycin (mTOR) for preventing naive T cell atrophy (Plas et al., 2001Plas D.R. Talapatra S. Edinger A.L. Rathmell J.C. Thompson C.B. Akt and Bcl-xL promote growth factor-independent survival through distinct effects on mitochondrial physiology.J. Biol. Chem. 2001; 276: 12041-12048Crossref PubMed Scopus (263) Google Scholar, Rathmell et al., 2001Rathmell J.C. Farkash E.A. Gao W. Thompson C.B. IL-7 enhances the survival and maintains the size of naive T cells.J. Immunol. 2001; 167: 6869-6876PubMed Google Scholar, Wofford et al., 2008Wofford J.A. Wieman H.L. Jacobs S.R. Zhao Y. Rathmell J.C. IL-7 promotes Glut1 trafficking and glucose uptake via STAT5-mediated activation of Akt to support T-cell survival.Blood. 2008; 111: 2101-2111Crossref PubMed Scopus (243) Google Scholar). The ability of IL-7 to induce these pathways suggests a role for IL-7 in regulating CD8+ T cell survival and growth during lymphopenia-induced HP. The fact that activated CD8+ T cells upregulate CD122 (IL-2 receptor β chain), the signaling component of the IL-15R complex, provides an explanation for the reliance on IL-15 for memory CD8+ T cell generation (Intlekofer et al., 2005Intlekofer A.M. Takemoto N. Wherry E.J. Longworth S.A. Northrup J.T. Palanivel V.R. Mullen A.C. Gasink C.R. Kaech S.M. Miller J.D. et al.Effector and memory CD8+ T cell fate coupled by T-bet and eomesodermin.Nat. Immunol. 2005; 6: 1236-1244Crossref PubMed Scopus (817) Google Scholar, Judge et al., 2002Judge A.D. Zhang X. Fujii H. Surh C.D. Sprent J. Interleukin 15 controls both proliferation and survival of a subset of memory-phenotype CD8(+) T cells.J. Exp. Med. 2002; 196: 935-946Crossref PubMed Scopus (278) Google Scholar, Ku et al., 2000Ku C.C. Murakami M. Sakamoto A. Kappler J. Marrack P. Control of homeostasis of CD8+ memory T cells by opposing cytokines.Science. 2000; 288: 675-678Crossref PubMed Scopus (693) Google Scholar). However, CD122 is a direct gene target of the transcription factor T-bet, whose expression has been inversely correlated with CD8+ T cell memory generation (Intlekofer et al., 2005Intlekofer A.M. Takemoto N. Wherry E.J. Longworth S.A. Northrup J.T. Palanivel V.R. Mullen A.C. Gasink C.R. Kaech S.M. Miller J.D. et al.Effector and memory CD8+ T cell fate coupled by T-bet and eomesodermin.Nat. Immunol. 2005; 6: 1236-1244Crossref PubMed Scopus (817) Google Scholar, Joshi et al., 2007Joshi N.S. Cui W. Chandele A. Lee H.K. Urso D.R. Hagman J. Gapin L. Kaech S.M. Inflammation directs memory precursor and short-lived effector CD8(+) T cell fates via the graded expression of T-bet transcription factor.Immunity. 2007; 27: 281-295Abstract Full Text Full Text PDF PubMed Scopus (1131) Google Scholar, Matsuda et al., 2007Matsuda J.L. George T.C. Hagman J. Gapin L. Temporal dissection of T-bet functions.J. Immunol. 2007; 178: 3457-3465PubMed Google Scholar). It is reasonable to suggest that IL-15 rescues effector cells for memory; yet this notion has not been formally tested, particularly in HP-induced generation of CD8+ T cell memory. Although the antigen stimulated and HP-induced CD8+ T cells seem to undergo proliferation, clonal expansion, and functional maturation for memory (Haluszczak et al., 2009Haluszczak C. Akue A.D. Hamilton S.E. Johnson L.D. Pujanauski L. Teodorovic L. Jameson S.C. Kedl R.M. The antigen-specific CD8+ T cell repertoire in unimmunized mice includes memory phenotype cells bearing markers of homeostatic expansion.J. Exp. Med. 2009; 206: 435-448Crossref PubMed Scopus (221) Google Scholar, Hamilton et al., 2006Hamilton S.E. Wolkers M.C. Schoenberger S.P. Jameson S.C. The generation of protective memory-like CD8+ T cells during homeostatic proliferation requires CD4+ T cells.Nat. Immunol. 2006; 7: 475-481Crossref PubMed Scopus (174) Google Scholar, Suzuki et al., 2008Suzuki T. Ogawa S. Tanabe K. Tahara H. Abe R. Kishimoto H. Induction of antitumor immune response by homeostatic proliferation and CD28 signaling.J. Immunol. 2008; 180: 4596-4605Crossref PubMed Scopus (11) Google Scholar), several distinctions have been noted. The early activation markers typically associated with antigen-stimulated CD8+ T cells are not observed with HP-induced stimulation, and the HP-induced functional maturation is weaker and transient. In addition, HP-induced CD8+ T cells display higher CD44 and CD122, and also retain expression of CD62L (Goldrath et al., 2000Goldrath A.W. Bogatzki L.Y. Bevan M.J. Naive T cells transiently acquire a memory-like phenotype during homeostasis-driven proliferation.J. Exp. Med. 2000; 192: 557-564Crossref PubMed Scopus (531) Google Scholar, Murali-Krishna et al., 1999Murali-Krishna K. Lau L.L. Sambhara S. Lemonnier F. Altman J. Ahmed R. Persistence of memory CD8 T cells in MHC class I-deficient mice.Science. 1999; 286: 1377-1381Crossref PubMed Scopus (618) Google Scholar). The HP-induced memory cells display weaker ability to mount antigen-recall responses and are therefore considered to be less potent than antigen-induced memory cells (Cheung et al., 2009Cheung K.P. Yang E. Goldrath A.W. Memory-like CD8+ T cells generated during homeostatic proliferation defer to antigen-experienced memory cells.J. Immunol. 2009; 183: 3364-3372Crossref PubMed Scopus (30) Google Scholar). Nevertheless, HP-induced CD8+ T cell responses have been shown to impart considerable efficacy against infectious and tumor challenge (Dummer et al., 2002Dummer W. Niethammer A.G. Baccala R. Lawson B.R. Wagner N. Reisfeld R.A. Theofilopoulos A.N. T cell homeostatic proliferation elicits effective antitumor autoimmunity.J. Clin. Invest. 2002; 110: 185-192Crossref PubMed Scopus (274) Google Scholar, Hamilton et al., 2006Hamilton S.E. Wolkers M.C. Schoenberger S.P. Jameson S.C. The generation of protective memory-like CD8+ T cells during homeostatic proliferation requires CD4+ T cells.Nat. Immunol. 2006; 7: 475-481Crossref PubMed Scopus (174) Google Scholar). Recent evidence demonstrates an essential role for mTOR in memory CD8+ T cell differentiation. Rapamycin treatment of lymphocytic choriomeningitis virus (LCMV)-infected mice was shown to increase the quantity and quality of memory CD8+ T cells in a T cell-intrinsic manner (Araki et al., 2009Araki K. Turner A.P. Shaffer V.O. Gangappa S. Keller S.A. Bachmann M.F. Larsen C.P. Ahmed R. mTOR regulates memory CD8 T-cell differentiation.Nature. 2009; 460: 108-112Crossref PubMed Scopus (1006) Google Scholar). We have further demonstrated the ability of mTOR to instruct effector versus memory cell fate of antigen-specific CD8+ T cells by regulating the expression of transcription factors T-bet and Eomesodermin (Eomes) (Rao et al., 2010Rao R.R. Li Q. Odunsi K. Shrikant P.A. The mTOR kinase determines effector versus memory CD8+ T cell fate by regulating the expression of transcription factors T-bet and Eomesodermin.Immunity. 2010; 32: 67-78Abstract Full Text Full Text PDF PubMed Scopus (442) Google Scholar). However, its role in integrating self-MHC and cytokine signals (IL-7 and/or IL-15) to regulate lymphopenia-induced homeostatic proliferation, functional maturation, and memory function has not been reported. The increasing use of bone marrow transplantation (BMT), radiation, and chemotherapy behooves us to better understand the cell-intrinsic mechanisms that regulate HP-induced CD8+ T cell clonal expansion and memory. In this study, we present the evidence for a central role of mTOR in bridging IL-7 and IL-15 mediated HP-induced CD8+ clonal expansion, memory functions, and tumor efficacy. This understanding will enable new approaches to harness homeostatic proliferation for immunity and restrict self-reactivity. The homeostatic cytokines IL-7 and IL-15 play a crucial role in lymphopenia-induced HP that restores CD8+ T cell numbers and promotes memory generation (Surh and Sprent, 2008Surh C.D. Sprent J. Homeostasis of naive and memory T cells.Immunity. 2008; 29: 848-862Abstract Full Text Full Text PDF PubMed Scopus (791) Google Scholar, Takada and Jameson, 2009Takada K. Jameson S.C. Naive T cell homeostasis: From awareness of space to a sense of place.Nat. Rev. Immunol. 2009; 9: 823-832Crossref PubMed Scopus (258) Google Scholar). To explore potential contributions of these cytokines in regulating HP-induced CD8+ T cell proliferation, expansion, and functional maturation, we monitored carboxy fluorescein diacetate succinimidyl ester (CFSE)-labeled naive OT-1 cells after adoptive transfer into irradiated B6, B6 Il15−/− or B6 mice treated with anti-IL-7Rα. Consistent with previous findings, we demonstrated that IL-7 blockade considerably reduced lymphopenia induced OT-1 proliferation and clonal expansion on day 5 (Figure 1A ). Interestingly, IL-15-deficient (B6 Il15−/−) recipients showed no reduction in OT-1 proliferation or expansion (Figure 1B). Furthermore, the OT-1 cells transferred to recipients with IL-7 blockade failed to undergo functional maturation (IFN-γ and granzyme-B production) (Figure 1A), but no difference in OT-1 functional maturation was observed in Il15−/− recipients (Figure 1B). To confirm our in vivo observations and demonstrate the sufficiency of IL-7 to induce proliferation (CFSE), clonal expansion, and functional maturation of naive CD8+ T cells, we tested the addition of IL-7 to OT-1 cells in an in vitro system. As shown in Figure 1C, IL-7 was sufficient for inducing OT-1 proliferation and functional maturation by day 5 (Figure 1C and data not shown). In addition, IL-7 but not IL-15 at the concentration of 10 ng/ml induced cell proliferation and IFN-γ production, which was almost identical for TCR transgenic (Tg) CD8+ T cells (OT-1) or polyclonal CD8+ T cells from B6 mice (Figure S1A available online). These results establish an essential role for IL-7 in the initiation of lymphopenia-induced naive CD8+ T cell proliferation for functional maturation. In addition, the extent of lymphopenia caused by radiation dosage regulated CD8+ T cell clonal expansion and functional maturation given that CFSE-labeled naive OT-1 Thy1.1+ cells that were transferred into mice irradiated with 0, 175, and 700 rad showed dose-dependent increases in CFSE dilution, cell number, and functional maturation (IFN-γ and in vivo CTL) (Figure S1B). The energy-sensitive kinase mTOR has been shown to regulate memory generation in antigen-stimulated CD8+ T cells (Araki et al., 2009Araki K. Turner A.P. Shaffer V.O. Gangappa S. Keller S.A. Bachmann M.F. Larsen C.P. Ahmed R. mTOR regulates memory CD8 T-cell differentiation.Nature. 2009; 460: 108-112Crossref PubMed Scopus (1006) Google Scholar, Rao et al., 2010Rao R.R. Li Q. Odunsi K. Shrikant P.A. The mTOR kinase determines effector versus memory CD8+ T cell fate by regulating the expression of transcription factors T-bet and Eomesodermin.Immunity. 2010; 32: 67-78Abstract Full Text Full Text PDF PubMed Scopus (442) Google Scholar). Because HP-induced functional maturation results in CD8+ T cell memory, we explored the potential role for mTOR kinase in lymphopenia-associated CD8+ T cell response by testing the ability of HP to induce mTOR activity, by evaluating the phosphorylation of its downstream target, ribosomal protein S6 (pS6), as a functional readout of mTOR activity (Burnett et al., 1998Burnett P.E. Barrow R.K. Cohen N.A. Snyder S.H. Sabatini D.M. RAFT1 phosphorylation of the translational regulators p70 S6 kinase and 4E-BP1.Proc. Natl. Acad. Sci. USA. 1998; 95: 1432-1437Crossref PubMed Scopus (894) Google Scholar). As shown in Figure 2A , HP dramatically induced mTOR activity (pS6) in OT-1 cells, which was blocked by anti-IL-7Rα treatment (Figure 2B). In agreement, the addition of IL-7 but not IL-15 to naive OT-1 cells in vitro induced a dose-dependent (1 or 10 ng/ml) increase of mTOR phosphorylation (mTORp) and pS6 (Figures S2A and S2B). These results demonstrate that HP induces an IL-7-dependent increase in mTOR activity, which may play a potential role in regulating CD8+ T cell homeostasis under conditions of lymphopenia. We next inquired whether rapamycin, a specific inhibitor of mTOR complex-1, could block HP-induced CD8+ T cell responses. Administration of rapamycin from day 0–7 (750 μg/kg/day) blocked lymphopenia-induced mTORp and pS6 in OT-1 cells (Figure 2C) and concomitantly decreased proliferation and clonal expansion (Figures 2D and 2E). The relatively high dose of rapamycin (750 μg/kg/day) has been previously used in vivo to block Graft-versus-host diseases (GVHDs) (Blazar et al., 1998Blazar B.R. Taylor P.A. Panoskaltsis-Mortari A. Vallera D.A. Rapamycin inhibits the generation of graft-versus-host disease- and graft-versus-leukemia-causing T cells by interfering with the production of Th1 or Th1 cytotoxic cytokines.J. Immunol. 1998; 160: 5355-5365PubMed Google Scholar) and was found to be optimum in a titration study for blocking mTOR in OT-1 cells in vivo (data not shown). Notably, the lymphopenia induced IL-7-dependent increases in OT-1 functional maturation; IFN-γ, granzyme-B production, and in vivo CTL activity was reduced upon mTOR inhibition (Figures 2F and 2G). This was further corroborated by the loss of IL-7 induced mTOR activity, proliferation, clonal expansion, and functional maturation of OT-1 cells upon rapamycin treatment in vitro (Figures S2C and S2D). Interestingly, the IL-7-induced mTOR activity (pS6) was PI3K dependent (Figure S2E), supporting the previously noted observation that IL-7-induced CD8+ T cell responses are PI3K dependent. These observations indicate an essential role for IL-7 induced mTOR for regulating HP-induced CD8+ T cell expansion and functional maturation. The acquisition of type I effector functions in antigen-experienced CD8+ T cells is governed by the master regulatory transcription factor T-bet (Szabo et al., 2000Szabo S.J. Kim S.T. Costa G.L. Zhang X. Fathman C.G. Glimcher L.H. A novel transcription factor, T-bet, directs Th1 lineage commitment.Cell. 2000; 100: 655-669Abstract Full Text Full Text PDF PubMed Scopus (2554) Google Scholar). To test the notion that T-bet plays a role in IL-7-dependent HP-induced CD8+ T cell functional maturation, we transferred CFSE-labeled naive OT-1 cells into irradiated recipients and evaluated the ability of HP to induce T-bet expression in OT-1 cells on day 5 after transfer. As predicted, HP induced T-bet expression, which corresponded with the extent of OT-1 cell proliferation (Figure 3A ). Moreover, the increase in T-bet was accompanied by an increase in CD122 expression (Figure 3A), which required IL-7 in vivo, given that anti-IL-7Rα abrogated the increase in T-bet and CD122 expression (Figure 3B). These results demonstrate an essential role for T-bet in HP-induced CD8+ T cell responses and CD122 expression. To characterize the role of mTOR-T-bet-CD122 pathway in HP-induced CD8+ T cell functional maturation, we evaluated T-bet and CD122 expression on OT-1 cells in rapamycin-treated recipients. Clearly, rapamycin treatment blocked mTOR induced T-bet and subsequent CD122 expression (Figure 3C), which was confirmed by our in vitro observations (Figure S3A). By in vivo monitoring, we compared HP-induced CD8+ clonal expansion, functional maturation, and CD122 expression by WT and Tbx21−/− (T-bet−/−) OT-1 Thy1.1+ cells on day 5. The T-bet deficiency reduced CD122 expression (Figure 3D), but not proliferation (Figure 3E) or OT-1 cell numbers (Figure 3F). Notably in the absence of T-bet, HP-induced functional maturation was diminished as reflected by IFN-γ production and in vivo CTL activity (Figure 3F). Surprisingly, the Tbx21−/− OT-1 cells showed similar cell surface phenotype (day 5; CD44, CD62L, IL-7Rα, IL-15Rα, and KLRG1) except for reduced CD122 expression (Figures S3B and S3D). These results identify a previously unappreciated role for T-bet in HP-induced CD8+ T cell functional maturation and CD122 expression and suggest a potential role for mTOR kinase in bridging IL-7 and IL-15 mediated CD8+ T cell homeostasis. The fact that T-bet is essential for CD122 expression (Figure 3D) and IL-15 is important for CD8+ T cell accumulation during HP (Sandau et al., 2007Sandau M.M. Winstead C.J. Jameson S.C. IL-15 is required for sustained lymphopenia-driven proliferation and accumulation of CD8 T cells.J. Immunol. 2007; 179: 120-125PubMed Google Scholar), suggests a pivotal role for mTOR in integrating IL-7 and IL-15 signals to regulate HP-induced CD8+ T cell memory. To test this possibility, we adoptively transferred naive wild-type (WT) or Tbx21−/− OT-1 Thy1.1+ cells into intact or irradiated B6 recipients and monitored their persistence (day 40), and the ability to mount antigen recall-responses (day 43), as a measure of memory function. In comparison to Tbx21−/− OT-1 cells, the WT OT-1 cells underwent significant clonal expansion, persisted up to day 40 (Figure 4A ), and produced a considerable antigen-recall response; clonal expansion and effector functions, after re-challenge (Figure 4B). In contrast, the Tbx21−/− OT-1 cells failed to persist in irradiated B6 recipients (Figure 4A) and subsequently produced poor antigen-recall responses (Figure 4B). These results demonstrate an essential requirement for T-bet expression in HP-induced memory CD8+ T cell responses and identify an important role for T-bet in CD8+ T cell persistence. To determine whether T-bet mediates HP-induced CD8+ T cell memory via IL-15, we transferred naive WT or Tbx21−/− OT-1 cells into irradiated WT or Il15−/− hosts and assessed their persistence and antigen-recall responses at day 40 and 43. Consistent with an earlier report demonstrating the belated requirement for IL-15 in HP-induced CD8+ T cell responses (Sandau et al., 2007Sandau M.M. Winstead C.J. Jameson S.C. IL-15 is required for sustained lymphopenia-driven proliferation and accumulation of CD8 T cells.J. Immunol. 2007; 179: 120-125PubMed Google Scholar), WT OT-1 cells transferred into Il15−/− hosts failed to persist up to day 40 and produce antigen-recall responses (Figures 4A and 4B). Moreover, the absence of T-bet in OT-1 cells and IL-15 in recipients further abrogated the HP-induced CD8+ T cell response (in comparison to WT recipients) (Figures 4A and 4B). These results demonstrate the requirement of HP-IL-7-mTOR-induced T-bet expression for CD8+ T cell memory via the cell-extrinsic factor IL-15. These observations are further corroborated by our in vitro experiments, in which IL-7 conditioned OT-1 cells underwent a dose-dependent proliferation in response to IL-15 and this response to IL-15 was T-bet dependent (Figure S4A). In addition, the cell recovery of WT OT-1 cells in Il15−/− hosts was equivalent to the cell recovery of Tbx21−/− OT-1 cells in WT hosts, reiterating the interdependency of cell intrinsic regulation of CD122 expression by T-bet and cell extrinsic requirement for IL-15 in programming HP-induced CD8+ T cell persistence for memory. To confirm whether T-bet expression was involved with the previously noted role for STAT5 in IL-15 mediated CD8+ T cell homeostasis (Kelly et al., 2003Kelly J. Spolski R. Imada K. Bollenbacher J. Lee S. Leonard W.J. A role for Stat5 in CD8+ T cell homeostasis.J. Immunol. 2003; 170: 210-217PubMed Google Scholar), we evaluated STAT5 phosphorylation (pSTA" @default.
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• W1982704558 title "A Central Role for mTOR Kinase in Homeostatic Proliferation Induced CD8+ T Cell Memory and Tumor Immunity" @default.
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