Yohsuke Harada

Convergence of Itch-induced ubiquitination with MEKK1-JNK signaling in Th2 tolerance and airway inflammation

The immune system is capable of mounting robust responses against invading pathogens but refrains from attacking self. Many studies have focused on tolerance induction of Th1 cells, whose failure results in development of autoimmune diseases. However, the molecular mechanisms governing tolerance induction in Th2 cells and its relation to allergic responses remain unclear. Here we used both in vivo and in vitro protocols to demonstrate that Th2 cells either containing a mitogen and extracellular kinase kinase 1 (MEKK1) mutant or lacking JNK1 or the E3 ubiquitin ligase Itch cannot be tolerized. In a mouse allergic model, injection of high-dose tolerizing antigen failed to block the development of airway inflammation in Itch-/- mice. This study suggests that MEKK1-JNK signaling regulates Itch E3 ligase-mediated tolerogenic process in Th2 cells. These findings have therapeutic implications for allergic diseases.

The 3′ Enhancer CNS2 Is a Critical Regulator of Interleukin-4-Mediated Humoral Immunity in Follicular Helper T Cells

A main role for interleukin-4 (IL-4) is in humoral immunity, and follicular helper CD4(+) T (Tfh) cells may be an intrinsic IL-4 source. Here we demonstrate that conserved noncoding sequence 2 (CNS2) is an essential enhancer element for IL-4 expression in Tfh cells but not in Th2 cells. Mice with a CNS2 deletion had a reduction in IgG1 and IgE production and in IL-4 expression in Tfh cells. Tracking of CNS2 activity via a GFP reporter mouse demonstrated that CNS2-active cells expressed several markers of Tfh cells: CXCR5, PD-1, and ICOS; the transcriptional master regulator Bcl6; and the cytokines IL-21 and IL-4. These CNS2-active cells were mainly localized in B cell follicles and germinal centers. The GFP(+) Tfh cells were derived from GFP(-) naive T cells after in vivo systemic immunization. These results indicate that CNS2 is an essential enhancer element required for IL-4 expression in Tfh cells controlling humoral immunity.

Mechanisms of NKT cell anergy induction involve Cbl-b-promoted monoubiquitination of CARMA1.

Repeated injection of α-galactosylceramide, an agonistic ligand for natural killer T (NKT) cells, results in long-term unresponsiveness or anergy, which severely limits its clinical application. However, the molecular mechanisms leading to NKT anergy induction remain unclear. We show here that the decreased IFN-γ production and failed tumor rejection observed in anergized NKT cells are rescued by Cbl-b deficiency. Cbl-b E3 ligase activity is critical for the anergy induction, as revealed by the similarity between Cbl-b−/− and its RING finger mutant NKT cells. Cbl-b binds and promotes monoubiquitination to CARMA1, a critical signaling molecule in NFκB activation. Ubiquitin conjugation to CARMA1 disrupts its complex formation with Bcl10 without affecting its protein stability. In addition, CARMA1−/− NKT cells are defective in IFN-γ production. The study identifies an important signaling pathway linking Cbl-b-induced monoubiquitination to NFκB activation in NKT cell anergy induction, which may help design approaches for human cancer therapy.

Grb2 and Gads exhibit different interactions with CD28 and play distinct roles in CD28-mediated costimulation.

Although both CD28 and ICOS bind PI3K and provide stimulatory signal for T cell activation, unlike CD28, ICOS does not costimulate IL-2 secretion. CD28 binds both PI3K and Grb2, whereas ICOS binds only PI3K. We have generated an ICOS mutant, which can bind Grb2 by replacement of its PI3K binding motif YMFM with the CD28 YMNM motif, and shown that it induces significant activation of the IL-2 promoter. However, this mutant ICOS was insufficient to activate the NF-κB pathway. In this study, we show that Gads, but not Grb2, is essential for CD28-mediated NF-κB activation, and its binding to CD28 requires the whole CD28 cytoplasmic domain in addition to the YMNM motif. Mutagenesis experiments have indicated that mutations in the N-terminal and/or C-terminal PXXP motif(s) of CD28 significantly reduce their association with Gads, whereas their associations with Grb2 are maintained. They induced strong activity of the NFAT/AP-1 reporter comparable with the CD28 wild type, but weak activity of the NF-κB reporter. Grb2- and Gads-dominant-negative mutants had a strong effect on NFAT/AP-1 reporter, but only Gads-dominant-negative significantly inhibited NF-κB reporter. Our data suggest that, in addition to the PI3K binding motif, the PXXP motif in the CD28 cytoplasmic domain may also define a functional difference between the CD28- and ICOS-mediated costimulatory signals by binding to Gads.

CD28 stimulation triggers NF-kappaB activation through the CARMA1-PKCtheta-Grb2/Gads axis.

CD28 stimulation contributes to activation of the IL-2 promoter by up-regulating the activity of several transcription factors, including nuclear factor kappaB (NF-kappaB)/Rel family members. However, the signal-transducing cascades linking the CD28 molecule and activation of NF-kappaB remain unclear. Protein kinase C (PKC) , CARMA1 and Bcl10 have recently been reported to integrate TCR-mediated NF-kappaB activation. However, since the data in these studies were drawn from experiments in which T cells were usually stimulated with both TCR and CD28, the relative contributions of TCR- and CD28-mediated signals to initiation of the NF-kappaB pathway remain elusive. To examine the role of these molecules in NF-kappaB activation through CD28-mediated stimulation, Bcl10 was over-expressed in Jurkat cells and their NF-kappaB activation by CD28- or TCR-cross-linking was evaluated. We found that CD28 stimulation alone can induce NF-kappaB activation in Bcl10-over-expressing Jurkat cells, whereas TCR stimulation alone has only little effect. In addition, we found that Bcl10-induced NF-kappaB activation through CD28-mediated stimulation could be blocked by the dominant-negative form of PKC or CARMA1. Furthermore, genetic studies revealed that Grb2/Gads binding, but not phosphatidylinositol 3-kinase binding, is important in CD28-mediated NF-kappaB activation. These findings indicate that the PKC-CARMA1-Bcl10 signaling pathway participates in the CD28 co-stimulatory signal independently of the TCR-signaling pathway, which leads us to propose that the activation of the NF-kappaB-signaling pathway via PKC-CARMA1-Bcl10 may be markedly dependent on CD28 stimulation rather than TCR stimulation.

CD28 stimulation triggers NF-κB activation through the CARMA1–PKCθ–Grb2/Gads axis

CD28 stimulation contributes to activation of the IL-2 promoter by up-regulating the activity of several transcription factors, including nuclear factor kappaB (NF-kappaB)/Rel family members. However, the signal-transducing cascades linking the CD28 molecule and activation of NF-kappaB remain unclear. Protein kinase C (PKC) , CARMA1 and Bcl10 have recently been reported to integrate TCR-mediated NF-kappaB activation. However, since the data in these studies were drawn from experiments in which T cells were usually stimulated with both TCR and CD28, the relative contributions of TCR- and CD28-mediated signals to initiation of the NF-kappaB pathway remain elusive. To examine the role of these molecules in NF-kappaB activation through CD28-mediated stimulation, Bcl10 was over-expressed in Jurkat cells and their NF-kappaB activation by CD28- or TCR-cross-linking was evaluated. We found that CD28 stimulation alone can induce NF-kappaB activation in Bcl10-over-expressing Jurkat cells, whereas TCR stimulation alone has only little effect. In addition, we found that Bcl10-induced NF-kappaB activation through CD28-mediated stimulation could be blocked by the dominant-negative form of PKC or CARMA1. Furthermore, genetic studies revealed that Grb2/Gads binding, but not phosphatidylinositol 3-kinase binding, is important in CD28-mediated NF-kappaB activation. These findings indicate that the PKC-CARMA1-Bcl10 signaling pathway participates in the CD28 co-stimulatory signal independently of the TCR-signaling pathway, which leads us to propose that the activation of the NF-kappaB-signaling pathway via PKC-CARMA1-Bcl10 may be markedly dependent on CD28 stimulation rather than TCR stimulation.

Immune Regulation by Ubiquitin Conjugation

It becomes increasingly evident that protein ubiquitination is closely linked to many aspects of the immune system and the E3 ubiquitin ligases play an important role in those processes via specific targeting of protein substrates. Gene database searching has indicated that there are several hundred E3 ubiquitin ligases, with the majority being RING finger-containing E3 ligases. It is conceivable that there will be very complicated interplay among the E3 ligases. Several E3 ligases may target the same substrate, or otherwise, one E3 ligase may target different substrates under diverse physiological conditions. In addition, the list of de-ubiquiting enzymes involved in immune regulation will increase in the near future. It has to be mentioned that biochemical and molecular approaches are still the main tools for identification of a substrate for a particular E3 ubiquitin ligase. The physiological relevance of such findings has to be correlated with genetic investigations. In any case, the field of research on protein ubiquitination in immune modulation will continue to be a hot topic in the years to come.

CD28 stimulation triggers NF- B activation through the CARMA1-PKC -Grb2/Gads axis

CD28 stimulation contributes to activation of the IL-2 promoter by up-regulating the activity of several transcription factors, including nuclear factor kappaB (NF-kappaB)/Rel family members. However, the signal-transducing cascades linking the CD28 molecule and activation of NF-kappaB remain unclear. Protein kinase C (PKC) , CARMA1 and Bcl10 have recently been reported to integrate TCR-mediated NF-kappaB activation. However, since the data in these studies were drawn from experiments in which T cells were usually stimulated with both TCR and CD28, the relative contributions of TCR- and CD28-mediated signals to initiation of the NF-kappaB pathway remain elusive. To examine the role of these molecules in NF-kappaB activation through CD28-mediated stimulation, Bcl10 was over-expressed in Jurkat cells and their NF-kappaB activation by CD28- or TCR-cross-linking was evaluated. We found that CD28 stimulation alone can induce NF-kappaB activation in Bcl10-over-expressing Jurkat cells, whereas TCR stimulation alone has only little effect. In addition, we found that Bcl10-induced NF-kappaB activation through CD28-mediated stimulation could be blocked by the dominant-negative form of PKC or CARMA1. Furthermore, genetic studies revealed that Grb2/Gads binding, but not phosphatidylinositol 3-kinase binding, is important in CD28-mediated NF-kappaB activation. These findings indicate that the PKC-CARMA1-Bcl10 signaling pathway participates in the CD28 co-stimulatory signal independently of the TCR-signaling pathway, which leads us to propose that the activation of the NF-kappaB-signaling pathway via PKC-CARMA1-Bcl10 may be markedly dependent on CD28 stimulation rather than TCR stimulation.