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: Molecular Therapy

Article Title: T Cells Engineered With Chimeric Antigen Receptors Targeting NKG2D Ligands Display Lethal Toxicity in Mice

doi: 10.1038/mt.2015.119

Figure Lengend Snippet: In vitro functional profiles of NKG2D-CAR-T cells. (a,b) BALB/c or (a,c) C57BL/6 T cells were engineered with the indicated CARs. (a) NKG2D-CAR T cells were stimulated with plate-bound recombinant Rae1β-Fc fusion protein and stained for production of IFNγ and TNFα, and analyzed by flow cytometry. Data is expressed as mean frequency ± SEM normalized to background levels from three independent experiments. In vitro CAR-T cell-mediated cytotoxicity was assayed using 4T1.2 tumor cells using a 6-hour AlamarBlue assay at the indicated effector: target ratios of BALB/c (b) or C57BL/6 (c) T cells. Mean frequency of viable tumor cells ± SEM from 3–4 independent experiments of triplicate wells is presented.

Article Snippet: T cells were stimulated in round-bottom 96-well plates coated with 2,000 ng/ml recombinant mouse Rae1β-Fc chimera (R&D Systems, Minneapolis, MN) or 1,000 ng/ml recombinant human HER2-Fc chimera (R&D Systems).

Techniques: In Vitro, Functional Assay, Recombinant, Staining, Flow Cytometry, Alamar Blue Assay