Masashi Ohtani

Innate production of TH2 cytokines by adipose tissue-associated c-Kit+Sca-1+ lymphoid cells

Innate immune responses are important in combating various microbes during the early phases of infection. Natural killer (NK) cells are innate lymphocytes that, unlike T and B lymphocytes, do not express antigen receptors but rapidly exhibit cytotoxic activities against virus-infected cells and produce various cytokines. Here we report a new type of innate lymphocyte present in a novel lymphoid structure associated with adipose tissues in the peritoneal cavity. These cells do not express lineage (Lin) markers but do express c-Kit, Sca-1 (also known as Ly6a), IL7R and IL33R. Similar lymphoid clusters were found in both human and mouse mesentery and we term this tissue ‘FALC’ (fat-associated lymphoid cluster). FALC Lin-c-Kit+Sca-1+ cells are distinct from lymphoid progenitors and lymphoid tissue inducer cells. These cells proliferate in response to IL2 and produce large amounts of TH2 cytokines such as IL5, IL6 and IL13. IL5 and IL6 regulate B-cell antibody production and self-renewal of B1 cells. Indeed, FALC Lin-c-Kit+Sca-1+ cells support the self-renewal of B1 cells and enhance IgA production. IL5 and IL13 mediate allergic inflammation and protection against helminth infection. After helminth infection and in response to IL33, FALC Lin-c-Kit+Sca-1+ cells produce large amounts of IL13, which leads to goblet cell hyperplasia—a critical step for helminth expulsion. In mice devoid of FALC Lin-c-Kit+Sca-1+ cells, such goblet cell hyperplasia was not induced. Thus, FALC Lin-c-Kit+Sca-1+ cells are TH2-type innate lymphocytes, and we propose that these cells be called ‘natural helper cells’.

Mammalian target of rapamycin and glycogen synthase kinase 3 differentially regulate lipopolysaccharide-induced interleukin-12 production in dendritic cells

Phosphoinositide 3-kinase (PI3K) negatively regulates Toll-like receptor (TLR)-mediated interleukin-12 (IL-12) expression in dendritic cells (DCs). We show here that 2 signaling pathways downstream of PI3K, mammalian target of rapamycin (mTOR) and glycogen synthase kinase 3 (GSK3), differentially regulate the expression of IL-12 in lipopolysaccharide (LPS)-stimulated DCs. Rapamycin, an inhibitor of mTOR, enhanced IL-12 production in LPS-stimulated DCs, whereas the activation of mTOR by lentivirus-mediated transduction of a constitutively active form of Rheb suppressed the production of IL-12. The inhibition of protein secretion or deletion of IL-10 cancelled the effect of rapamycin, indicating that mTOR regulates IL-12 expression through an autocrine action of IL-10. In contrast, GSK3 positively regulates IL-12 production through an IL-10-independent pathway. Rapamycin-treated DCs enhanced Th1 induction in vitro compared with untreated DCs. LiCl, an inhibitor of GSK3, suppressed a Th1 response on Leishmania major infection in vivo. These results suggest that mTOR and GSK3 pathways regulate the Th1/Th2 balance though the regulation of IL-12 expression in DCs. The signaling pathway downstream of PI3K would be a good target to modulate the Th1/Th2 balance in immune responses in vivo.

PI3K-Akt-mTORC1-S6K1/2 Axis Controls Th17 Differentiation by Regulating Gfi1 Expression and Nuclear Translocation of RORγ

The PI3K-Akt-mTORC1 axis contributes to the activation, survival, and proliferation of CD4(+) T cells upon stimulation through TCR and CD28. Here, we demonstrate that the suppression of this axis by deletion of p85α or PI3K/mTORC1 inhibitors as well as T cell-specific deletion of raptor, an essential component of mTORC1, impairs Th17 differentiation in vitro and in vivo in a S6K1/2-dependent fashion. Inhibition of PI3K-Akt-mTORC1-S6K1 axis impairs the downregulation of Gfi1, a negative regulator of Th17 differentiation. Furthermore, we demonstrate that S6K2, a nuclear counterpart of S6K1, is induced by the PI3K-Akt-mTORC1 axis, binds RORγ, and carries RORγ to the nucleus. These results point toward a pivotal role of PI3K-Akt-mTORC1-S6K1/2 axis in Th17 differentiation.

Cutting Edge: mTORC1 in Intestinal CD11c+CD11b+ Dendritic Cells Regulates Intestinal Homeostasis by Promoting IL-10 Production

The mammalian target of rapamycin (mTOR) controls cell growth and survival through two distinct complexes called mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2). Although several reports have suggested the involvement of mTORC1 in development and function of dendritic cells (DCs), its physiological roles remain obscure. We therefore established mTORC1 signal-deficient mice lacking Raptor, an essential component of mTORC1 signal, specifically in DC lineage (referred to here as RaptorDC−/−). RaptorDC−/− mice exhibited cell expansion in specific subsets of DCs such as splenic CD8+ DCs and intestinal CD11c+CD11b+ DCs. We also found that impaired mTORC1 signal resulted in the suppression of IL-10 production along with enhanced CD86 expression in intestinal CD11c+CD11b+ DCs and that RaptorDC−/− mice were highly susceptible to dextran sodium sulfate-induced colitis. Our results uncover mTORC1-mediated anti-inflammatory programs in intestinal CD11c+CD11b+ DCs to limit the intestinal inflammation.

Loss of mTOR complex 1 induces developmental blockage in early T-lymphopoiesis and eradicates T-cell acute lymphoblastic leukemia cells

Significance mTOR, a kinase that senses and responds to nutrients, plays critical roles in organogenesis and tumorigenesis. Although mTOR inhibitors have been developed as immunosuppressants and anticancer drugs, it has remained controversial whether such medications contribute to cancer eradication. In addition, mTOR inhibition by chemical inhibitors is complicated because it may not produce predictable inhibition of the mTOR complexes mTORC1 and mTORC2. By using a genetic approach, our study clearly demonstrates that mTORC1, but not mTORC2, is essential for cell cycling of early T-cell progenitors. More importantly, we reveal that loss of mTORC1 efficiently eradicates T-cell acute lymphoblastic leukemia cells, but not myeloid leukemia. Thus, understanding the cell-context–dependent role of mTOR illustrates the potential importance of mTOR signals as therapeutic targets. mTOR is an evolutionarily conserved kinase that plays a critical role in sensing and responding to environmental determinants. Recent studies have shown that fine-tuning of the activity of mTOR complexes contributes to organogenesis and tumorigenesis. Although rapamycin, an allosteric mTOR inhibitor, is an effective immunosuppressant, the precise roles of mTOR complexes in early T-cell development remain unclear. Here we show that mTORC1 plays a critical role in the development of both early T-cell progenitors and leukemia. Deletion of Raptor, an essential component of mTORC1, produced defects in the earliest development of T-cell progenitors in vivo and in vitro. Deficiency of Raptor resulted in cell cycle abnormalities in early T-cell progenitors that were associated with instability of the Cyclin D2/D3-CDK6 complexes; deficiency of Rictor, an mTORC2 component, did not have the same effect, indicating that mTORC1 and -2 control T-cell development in different ways. In a model of myeloproliferative neoplasm and T-cell acute lymphoblastic leukemia (T-ALL) evoked by Kras activation, Raptor deficiency dramatically inhibited the cell cycle in oncogenic Kras-expressing T-cell progenitors, but not myeloid progenitors, and specifically prevented the development of T-ALL. Although rapamycin treatment significantly prolonged the survival of recipient mice bearing T-ALL cells, rapamycin-insensitive leukemia cells continued to propagate in vivo. In contrast, Raptor deficiency in the T-ALL model resulted in cell cycle arrest and efficient eradication of leukemia. Thus, understanding the cell-context–dependent role of mTORC1 illustrates the potential importance of mTOR signals as therapeutic targets.

Class I PI3K-mediated Akt and ERK signals play a critical role in FcεRI-induced degranulation in mast cells

Class IA and IB phosphoinositide 3-kinases (PI3Ks) have been shown to regulate mast cell functions such as proliferation, development, survival and degranulation, but the functional redundancy between these two PI3K signaling pathways in mast cells remains unclear. Here, we have generated mice deficient in both class IA regulatory subunit p85α and class IB catalytic subunit p110γ, and show that p85α(-/-)p110γ(-/-) mice exhibit a more severe defect in mast cell development than single-knockout mice. In addition, the in vivo passive cutaneous anaphylaxis reaction of p85α(-/-)p110γ(-/-) mice was nearly completely abrogated, whereas single-knockout mice exhibit just marginal reduction. Pharmacological inactivation of Akt in wild-type bone marrow-derived mast cells (BMMCs) led to partial reduction of degranulation, while over-expression of a constitutively active Akt partially restored the impaired degranulation in p85α(-/-)p110γ(-/-) BMMCs. We also found that the extracellular signal-regulated kinase (ERK) signaling pathway was activated in a PI3K-dependent manner upon FcεRI stimulation and that simultaneous inhibition of Akt and ERK resulted in nearly complete blockade of FcεRI-induced degranulation. Our data provide evidence that Akt and ERK pathways play redundant roles in FcεRI-induced degranulation.

Critical role of class IA PI3K for c-Rel expression in B lymphocytes

The fact that the Xid mutation of Btk impairs the ability of pleckstrin homology domain of Btk to bind phosphatidylinositol-(3,4,5)-trisphosphate, a product of class IA phosphoinositide-3 kinases (PI3Ks), has been considered strong evidence for the hypothesis that Btk functions downstream of PI3Ks. We demonstrate here that the Xid mutation renders the Btk protein unstable. Furthermore, class IA PI3K- and Btk-deficient mice show different phenotypes in B-cell development, collectively indicating that PI3Ks and Btk differentially function in BCR signal transduction. Nevertheless, both PI3K and Btk are required for the activation of NF-kappaB, a critical transcription factor family for B-cell development and function. We demonstrate that PI3Ks maintain the expression of NF-kappaB proteins, whereas Btk is known to be essential for IkappaB degradation and the translocation of NF-kappaB to the nucleus. The loss of PI3K activity results in marked reduction of c-Rel and to a lesser extent RelA expression. The lentivirus-mediated introduction of c-Rel corrects both developmental and proliferative defects in response to BCR stimulation in class IA PI3K-deficient B cells. These results show that the PI3K-mediated control of c-Rel expression is essential for B-cell functions.