The yeast high osmolarity glycerol (HOG) pathway activates the Hog1 MAP kinase, which coordinates adaptation to high osmolarity conditions. Here we demonstrate that the four-transmembrane (TM) domain protein Sho1 is an osmosensor in the HKR1 sub-branch of the HOG pathway. Crosslinking studies indicate that Sho1 forms planar oligomers of the dimers-of-trimers architecture by dimerizing at the TM1/TM4 interface and trimerizing at the TM2/TM3 interface. High external osmolarity induces structural changes in the Sho1 TM domains and Sho1 binding to the cytoplasmic adaptor protein Ste50, which leads to Hog1 activation. Besides its osmosensing function, the Sho1 oligomer serves as a scaffold. By binding to the TM proteins Opy2 and Hkr1 at the TM1/TM4 and TM2/TM3 interface, respectively, Sho1 forms a multi-component signalling complex that is essential for Hog1 activation. Our results illuminate how the four TM domains of Sho1 dictate the oligomer structure as well as its osmosensing and scaffolding functions.
The authors analysed the antigen-presenting ability of eosinophils purified from peritoneal exudate cells of interleukin-5 (IL-5) transgenic mice. The granulocyte-macrophage colony-stimulating factor (GM-CSF)-treated eosinophils induced proliferative responses of primed lymph node T cells and thymus T cells to staphylococcal enterotoxin B (SEB), while untreated eosinophils induced little or no response. GM-CSF-treated eosinophils also induced proliferation of ovalbumin (OVA)-primed lymph node T cells to OVA. Although untreated eosinophils expressed no MHC class II molecule on the surface the eosinophils could be induced to express major histocompatibility complex (MHC) class II molecules when treated with GM-CSF. In the present study, anti-I-Ak monoclonal antibodies (MoAbs) strongly inhibited proliferation of thymus T cells and proliferation of OVA-primed lymph node T cells in response to OVA, but weakly inhibited proliferation of primed T cells in response to SEB. Furthermore, CD80 (B7-1) and CD86 (B7-2) were expressed on the surfaces of untreated eosinophils. The expression of those two molecules on the eosinophils was increased by incubation with GM-CSF. Moreover, anti-CD80 or anti-CD86 MoAbs blocked proliferative responses of primed lymph node T cells and thymus T cells to SEB, and also blocked responses of primed lymph node T cells to OVA. Thus, CD80 and CD86 play an important role in stimulation of T cells by eosinophils.
Ca2+-regulated NFAT family members are transcription factors crucial for the expression of various cytokine genes and other immunoregulatory genes. Analyses of mice defective in one or two NFAT family members have revealed functions specific to each NFAT gene. However, the redundant functions of several family members limit the usefulness of gene disruption analysis. For example, CD4+ T cells isolated from NFATx-disrupted mice do not show any modulation in cytokine gene expression, perhaps because other family members compensate for its absence. To analyze the role of NFATx in the regulation of immunoregulatory genes in T cells, we made a gain-of-function mutant by creating transgenic mice expressing a constitutively nuclear form of NFATx in T cell lineages. In naive CD4+ T cells, NFATx up-regulated the expression of several cytokine genes and activation markers and suppressed the expression of CD154. In Th1 cells, NFATx enhanced the expression of the Th1 cytokine genes, IFN-γ and TNF-α. In contrast, NFATx suppressed Th2 cytokine genes such as IL-4 and IL-5 in Th2 cells. It has been reported that both NFAT1 and NFATx are required to maintain the homeostasis of the immune system. Our results suggest that NFATx exerts this function by inhibiting the expression of some critical immunoregulatory genes.
Mice that have recovered from a primary infection with Plasmodium chabaudi have been shown to resist a secondary infection. In the present study the authors investigated how natural killer (NK) cells were involved in this resistance. Spleen cells from P. chabaudiprimed C57BL/6 mice could transfer protection against P. chabaudi infection into naive syngeneic mice, but spleen cells from unprimed mice could not. T-enriched cells purified from primed spleen cells could also transfer such protection. Transfer of NK cells from primed spleen cells failed to protect against challenge infection. However, depletion of NK cells in host mice by injection of an anti-NK1.1 monoclonal antibody resulted in higher mortality relative to controls. The possible protective roles of NK cells in P. chabaudi infection are discussed.
We analyzed the mode of antigen presentation of an endogenous antigen localized in the cytoplasm or in the mitochondria. Pseudomonas aeruginosa PAO leucine-, isoleucine-, valine-binding protein (LIVAT-BP) encoded by the braC gene was used as a model antigen. Using mouse BALB/3T3 cells, we established two LIVAT-BP transfectants by transfection of a plasmid harboring the intact braC or braC gene fused with the mitochondrial transport signal derived from the yeast COXIV gene. One of the resulting transfectants, BC-15, expressed LIVAT-BP in the cytoplasm, while YZ-710 cells expressed LIVAT-BP in the mitochondria. The splenic effector cells derived from BALB/c mice primed with BC-15 cells exhibited cytotoxic T lymphocyte (CTL) activity against BC-15 cells, but not against YZ-710 cells, whereas splenic effector cells primed with YZ-710 cells exhibited CTL activity against YZ-710 cells, but not against BC-15 cells. Neither group of splenic effector cells showed CTL activity against parental BALB/3T3 cells. These CTL belonged to the CD8+ alphabeta T cell subset. Furthermore, we observed that the CTL activity against t BC-15 cells or YZ-710 cells was blocked with anti-H2-K(d) mAb, but not with anti-H2-D(d) or H2-L(d) mAb. The CTL against BC-15 or YZ-710 cells could kill parental BALB/3T3 cells in the presence of peptides produced by alkali lysis of the LIVAT-BP, suggesting that these CTL indeed recognized the peptide(s) derived from LIVAT-BP. We determined that the epitope for the CTL against BC-15 cells was QYGEGIATEV, corresponding to residues 162-171, and that the epitope recognized by the CTL against YZ-710 cells was GYKLIFRTI, corresponding to residues 123-131 of LIVAT-BP, respectively. Thus, we show here that epitope selection for MHC class I expression is affected by the intracellular localization of the antigenic protein.
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