Journal: Immunology

Article Title: CD4 + NKG2D + T cells induce NKG2D down-regulation in natural killer cells in CD86-RAE-1 ε transgenic mice

doi: 10.1111/imm.12203

Figure Lengend Snippet: Generation and identification of CD86-RAE transgenic mice. The CD86-RAE-1ε transgene encompassing the CD86 promoter, the retinoic acid early transcript-1ε (RAE-1ε) coding sequence, and the bovine growth hormone polyadenylation signal (2·6-kb) (a). Founder mice identified by PCR were further screened by Western blotting. Top, RAE-1ε expression was detected in liver tissues F0-1, -12, -17, -48, -40 mice. Bottom, α-tubulin blot (b). RAE-1ε expression on spleen lymphocytes, as identified by flow cytometry. Grey area: isotype control; dotted line: control mice; solid line: CD86-RAEε mice (c). Immunofluorescence histology of the thymus, intestine, liver, lung and kidney stained with anti-I-A/I-E antibody (green) and anti-RAE-1 antibody (red) and counterstained with DAPI. Similar patterns were obtained for the mice of lines 48, 1 and 17. The photographs shown are from a line 48 mouse, and were taken at a magnification of × 200 (d). Changes in RAE-1ε expression on myeloid cells and lymphocytes in response to overnight stimulation with poly (I:C) ex vivo (50 ng/ml) (e) and in vivo (100 μg) (f). Grey area: before stimulation; red line: after stimulation. The figures in black and red show the results before and after stimulation, respectively.

Article Snippet: In brief, we coated plastic wells with recombinant mouse RAE-1 ε (2 μg; R&D Systems) by incubation at 4° overnight.

Techniques: Transgenic Assay, Sequencing, Western Blot, Expressing, Flow Cytometry, Immunofluorescence, Staining, Ex Vivo, In Vivo

Journal: Immunology

Article Title: CD4 + NKG2D + T cells induce NKG2D down-regulation in natural killer cells in CD86-RAE-1 ε transgenic mice

doi: 10.1111/imm.12203

Figure Lengend Snippet: Weak NKG2D-dependent function of natural killer (NK) cells in CD86-RAE-1ε mice. NKG2D expression on freshly isolated NK cells, gated on CD3− NK1.1+ cells (a). Transcript levels for NKG2D-L, NKG2D-S, DAP10, and DAP12 in NK cells from wild-type and transgenic mice (b). Killing activities were assessed by both CD107a staining and the lactate dehydrogenase release assay (c). After the incubation of NK cells with Ba/F3-RAE or Ba/F3 cells, CD107a expression on NK cells was assessed by flow cytometry (d). Splenocytes from transgenic mice or wild-type mice were stained with CFSE and PKH-26, respectively, mixed in equal proportions and injected intravenously into recipient mice. After 12 hr, fluorescence ratios were determined for the cells recovered from spleens, by flow cytometry (e). Interferon-γ (IFN-γ) production by NK cells from transgenic mice and wild-type mice stimulated with PMA/ionomycin, Ba/F3 or Ba/F3-RAE cells. Results are expressed as means ± SD. Asterisks indicate P < 0·05 (f).

Article Snippet: In brief, we coated plastic wells with recombinant mouse RAE-1 ε (2 μg; R&D Systems) by incubation at 4° overnight.

Techniques: Expressing, Isolation, Transgenic Assay, Staining, Release Assay, Incubation, Flow Cytometry, Injection, Fluorescence

Journal: Immunology

Article Title: CD4 + NKG2D + T cells induce NKG2D down-regulation in natural killer cells in CD86-RAE-1 ε transgenic mice

doi: 10.1111/imm.12203

Figure Lengend Snippet: The adoptive transfer of normal natural killer (NK) cells into CD86-RAE-1ε mice decreased NKG2D expression. Splenocytes from wild-type mice were stained with CFSE and intravenously injected into transgenic mice or wild-type mice. Fluorescent cells were recovered after 12 hr. Frequencies of CD3− NK1.1+ NKG2D+ cells in donor mice were analysed before and after injection, and the frequencies of spleen CD3− NK1.1+ NKG2D+ cells in recipient mice were also determined (a). B16BL6-RAE (b) or B16BL6 cells (c) (2 × 106) were injected subcutaneously into the back of the animal (n = 6). Tumour area was determined daily. CD86-RAE-1ε mice and wild-type mice (n = 5) were supplied with drinking water containing 2·5% dextran sodium sulphate (DSS). Body weight was measured daily (d). Expression levels of NKp46, NKG2A, 2B4, Ly49D, Ly49H and KLRG1 on NK cells, identified by gating on CD3− NK1.1+ cells (e). Results are expressed as means ± SD. Asterisks indicate P < 0·05. Each experiment was carried out three times.

Article Snippet: In brief, we coated plastic wells with recombinant mouse RAE-1 ε (2 μg; R&D Systems) by incubation at 4° overnight.

Techniques: Adoptive Transfer Assay, Expressing, Staining, Injection, Transgenic Assay

Journal: Immunology

Article Title: CD4 + NKG2D + T cells induce NKG2D down-regulation in natural killer cells in CD86-RAE-1 ε transgenic mice

doi: 10.1111/imm.12203

Figure Lengend Snippet: Serum from CD86-RAE-1ε mice did not induce NKG2D down-regulation. The concentration of soluble retinoic acid early transcript (RAE) in the serum was determined with a sandwich ELISA kit (a). Serum levels of antibodies against RAE-1 were determined in an indirect ELISA assay (b). NKG2D expression was assessed on freshly isolated natural killer (NK) cells, and NK cells incubated with serum from a wild-type or transgenic mouse. NK cells were isolated by gating on CD3− NK1.1+ cells (c). The experiment was carried out twice.

Article Snippet: In brief, we coated plastic wells with recombinant mouse RAE-1 ε (2 μg; R&D Systems) by incubation at 4° overnight.

Techniques: Concentration Assay, Sandwich ELISA, Indirect ELISA, Expressing, Isolation, Incubation, Transgenic Assay

Journal: Immunology

Article Title: CD4 + NKG2D + T cells induce NKG2D down-regulation in natural killer cells in CD86-RAE-1 ε transgenic mice

doi: 10.1111/imm.12203

Figure Lengend Snippet: Sustained retinoic acid early transcript (RAE) expression on dendritic cell-activated natural killer (NK) cells ex vivo. CD107a expression and interferon-γ (IFN-γ) production were assessed for freshly isolated NK cells and after stimulation with dendritic cells from a wild-type mouse or a CD86-RAE-1ε mouse, at a ratio of 1 : 2, for 12 hr or 5 days (a). NKG2D, 2B4 (b), CD69, NKp46, NKG2A (c) expression on NK cells stimulated with dendritic cells at a ratio of 1 : 3 for 12 hr, as measured by flow cytometry, with gating on CD3− NK1.1+ cells.

Article Snippet: In brief, we coated plastic wells with recombinant mouse RAE-1 ε (2 μg; R&D Systems) by incubation at 4° overnight.

Techniques: Expressing, Ex Vivo, Isolation, Flow Cytometry

Journal: Immunology

Article Title: CD4 + NKG2D + T cells induce NKG2D down-regulation in natural killer cells in CD86-RAE-1 ε transgenic mice

doi: 10.1111/imm.12203

Figure Lengend Snippet: CD4+ NKG2D+ T cells contributed to the down-regulation of NKG2D on natural killer (NK) cells. Frequencies and absolute numbers of CD4+ NKG2D+ T cells in the spleen were compared between CD86-RAE-1ε mice and wild-type mice (a). Transforming growth factor-β (TGF-β) was detected by flow cytometry, with intracellular staining and gating on CD4+ NKG2D− T cells and CD4+ NKG2D+ T cells (b). NKG2D expression on CD3− NK1.1+ cells was determined following the co-culture of normal NK cells, in a 1 : 1 ratio, with CD4+ NKG2D− T cells, CD4+ NKG2D+ T cells, or CD4+ NKG2D+ T cells in the presence of anti-TGF-β antibody, for 24 hr (c). CD4+ NKG2D+ T cells did not express Foxp3, as shown by flow cytometry (d). Asterisks indicate that P < 0·05. The experiment was carried out three times.

Article Snippet: In brief, we coated plastic wells with recombinant mouse RAE-1 ε (2 μg; R&D Systems) by incubation at 4° overnight.

Techniques: Flow Cytometry, Staining, Expressing, Co-Culture Assay

Journal:

Article Title: Porphyromonas gingivalis lipopolysaccharide induces tumor necrosis factor-α and interleukin-6 (IL-6) secretion and CCL25 gene expression in mouse primary gingival cell lines: IL-6-driven activation of CCL2

doi:

Figure Lengend Snippet: (A-C) CCL2 and TNFα levels in cell-free supernatants 24 hr after stimulation with rIL-6, rIL-6 plus sIL-6R, rIL-6 plus sIL-6R plus sgp130, sgp130, or PBS; data from 3 independent sets of experiments showing an increase in CCL2 secretion from gingival cells stimulated with rIL-6 plus sIL-6R; rIL-6 plus sIL-6R plus sgp130; or sgp130 compared to PBS control cultures. (D) Neutralization of sgp130 by anti-gp130 antibody causes an increase in CCL2 secretion. (E) A gingival cell line stained with sgp130 indicated that about one-fifth of the cells express gp130. (F) Cell supernatants used for CCL2 assays had no detectable TNFα production.

Article Snippet: Reagents used in this study included ultrapure P. gingivalis LPS (Invitrogen; San Diego; CA) and E. coli LPS (Sigma); recombinant IL-6 (rIL-6) (<0.01 ng endotoxin per μg cytokine (eBioscience; San Diego, CA); sIL-6R (<0.0 EU endotoxin per 1 μg cytokine receptor) (R&D Systems; Minneapolis, MN); two preparations of sgp130: recombinant human sgp130 (<1.0 EU endotoxin per 1 μg receptor) (R&D Systems), and recombinant mouse sgp130/Fc chimera (<1.0 ng endotoxin per 1 μg receptor) (R&D systems).

Techniques: Control, Neutralization, Staining

Journal:

Article Title: Porphyromonas gingivalis lipopolysaccharide induces tumor necrosis factor-α and interleukin-6 (IL-6) secretion and CCL25 gene expression in mouse primary gingival cell lines: IL-6-driven activation of CCL2

doi:

Figure Lengend Snippet: Stimulatory effects on CCL2 secretion from gingival cell lines

Article Snippet: Reagents used in this study included ultrapure P. gingivalis LPS (Invitrogen; San Diego; CA) and E. coli LPS (Sigma); recombinant IL-6 (rIL-6) (<0.01 ng endotoxin per μg cytokine (eBioscience; San Diego, CA); sIL-6R (<0.0 EU endotoxin per 1 μg cytokine receptor) (R&D Systems; Minneapolis, MN); two preparations of sgp130: recombinant human sgp130 (<1.0 EU endotoxin per 1 μg receptor) (R&D Systems), and recombinant mouse sgp130/Fc chimera (<1.0 ng endotoxin per 1 μg receptor) (R&D systems).

Techniques:

Journal: mBio

Article Title: The GPI sidechain of Toxoplasma gondii inhibits parasite pathogenesis

doi: 10.1128/mbio.00527-24

Figure Lengend Snippet: Enhanced CD36 binding and slight macrophage tropism 3 hours after infection. Mice (C57BL/6J) were injected i.p. with 10 6 parasites of CEP, CEP Δ pigj, or CEP Δpigj + PIGJ and PECs were analyzed 3 hours later by flow cytometry. ( A ) Gating strategy for panels B–G. ( B ) Total GFP+ signal in the peritoneal exudate; GFP frequency of total events. ( C ) GFP+ events were separated by size using FSC and SSC parameters to determine extracellular or “non-cell-associated” and intracellular or “cell-associated” GFP+ parasites. ( D ) Frequency of PI+ (non-viable) cells of the indicated GFP+ category. ( E ) Frequency of cell-associated GFP+ events that were GR-1 hi Cd11b + neutrophils. ( F ) Frequency of cell-associated GFP+ events that were MHCII hi F4/80 lo “BMDM,” or ( G ) MHCII lo F4/80 hi “yolk sack-derived macrophages” (YSM). Plotted are seven total experiments; each dot represents a single mouse (mice; n = 12 CEP, n = 12 CEP Δ pigj , n = 8 CEP Δ pigj + PIGJ ). Statistics performed were one-way ANOVAs with multiple comparisons and a Tukey correction; none were significant, and the values were indicated. ( H ) In vitro differentiated BMDMs were plated on coverslips overnight and infected with 100 and 300 GFP+ parasites per well, and parasites were allowed 20 minutes to invade cells before being fixed and stained for fluorescence microscopy. No permeabilization was used, and SAG1 staining was performed. Intracellular parasites were not stained with SAG1 but were GFP+, while extracellular parasites are SAG1+GFP+ . The fraction of intracellular parasites is plotted, dots represent independent experiments; n = 3 experiments. ( I ) Parasites were incubated for 1 hour with recombinant CD36-Fc and CD36 binding was measured with anti-human-IgG-Daylight-550 via flow cytometry. Representative histogram and ( J ) average + SD MFI of CD36 binding from four experiments is plotted with each experiment represented as a dot, and an unpaired t-test is shown, ** P < 0.01. For I and J, also plotted are IgG Fc staining controls of the CEP strain (Fc control).

Article Snippet: Parasites (10 7 ) were incubated with 0.5 µg recombinant human IgG Fc (R&D Systems # 110-HG) or recombinant CD36-Fc (R&D Systems, #2519 CD) in 50 µL binding buffer (0.14 M NaCl, 2.5 mM CaCl 2 , 0.01 M HEPES [pH 7.4], 3% BSA) for 1 hour at 15°C.

Techniques: Binding Assay, Infection, Injection, Flow Cytometry, Derivative Assay, In Vitro, Staining, Fluorescence, Microscopy, Incubation, Recombinant, Control