Journal: The Journal of Experimental Medicine

Article Title: Nfil3/E4bp4 is required for the development and maturation of NK cells in vivo

doi: 10.1084/jem.20092176

Figure Lengend Snippet: The defect in NK cell development in Nfil3 −/− mice is NK cell intrinsic. (A) Impaired IL-15–induced in vitro generation of NK cells. Lin − BM cells from WT or Nfil3 −/− mice were cultured in medium containing mSlf, mFlt3L, and mIL-7, with or without mIL-15. NK cell expansion was assessed by flow cytometry. Results shown are representative of two independent experiments. (B) Rescue by WT Nfil3. Using GFP as a marker for transfected gene expression, NK1.1 expression was determined on gated CD3 − GFP + (green) or CD3 − GFP − (red) Nfil3 −/− fetal liver cells (10 5 ) that had been retrovirally transduced with either the WT Nfil3 gene (wt) or a gene encoding the WT Nfil3 gene lacking its DNA binding domain (deleted [del]). Transduced cells were cultured with mIL-15. Numbers indicate the percentage of NK1.1 + cells generated. (C–E) The developmental defect in Nfil3 −/− mice is NK cell intrinsic. (C) Donor-derived liver NK cell populations after BMT showing overall percentages of NK cells (top) and the percentage of CD45.1 and CD45.2 NK cells (bottom). Data shown are representative of three mice per group. (D) The ratio of CD45.2/CD45.1 cells in the various indicated lymphoid cell populations after BMT. (E) The absolute numbers of CD45.2 and CD45.1 NK cells present in spleen and liver, as calculated from the total cellularity of the lymphoid organs and the percentages of CD45.2 or CD45.1 NK1.1 + CD3 − cells as determined by flow cytometry. Results shown are the mean ± SD for three mice per group.

Article Snippet: Lin − BM cells (10 5 ) were cultured for 2 wk with 50 ng/ml mSlf, 50 ng/ml mFlt3L, and 5 ng/ml mIL-7, with or without 20 ng/ml mIL-15 (all cytokines were obtained from R&D Systems).

Techniques: In Vitro, Cell Culture, Flow Cytometry, Marker, Transfection, Expressing, Transduction, Binding Assay, Generated, Derivative Assay

Journal:

Article Title: Murine lymph node-derived stromal cells effectively support survival but induce no activation/proliferation of peripheral resting T cells in vitro

doi: 10.1046/j.1365-2567.2003.01693.x

Figure Lengend Snippet: Effects of known cytokines to support the survival of T cells. T cells isolated from lymph nodes of C57BL/6 mice were cultured with various concentrations (as indicated in the figure, per ml) of IL-4, IL-6, IL-7, and IFN-β for 24 or 48 hr, and cell viability was determined. The data of cells cultured in the stromal-conditioned medium (SCM) containing 50% of culture supernatant of the LNS cells were added in each panel to compare the effect of each cytokine with that of SCM. Three different experiments were performed, and the average values are shown.

Article Snippet: Antibodies and recombinant cytokines Recombinant murine IL-4, IL-7, IFN-α/β, and anti-mouse IL-4, anti-mouse IL-6, and anti-mouse IL-7 antibodies were purchased from R & D systems, Inc. (Minneapolis, MN).

Techniques: Isolation, Cell Culture

Journal:

Article Title: Murine lymph node-derived stromal cells effectively support survival but induce no activation/proliferation of peripheral resting T cells in vitro

doi: 10.1046/j.1365-2567.2003.01693.x

Figure Lengend Snippet: Blocking of the activity to support T-cell survival with neutralizing antibodies against various cytokines. (a) T cells were cultured with 1·0 ng/ml (IL-4, IL-6 and IL-7) or 10 U/ml (IFN-β) of cytokines with or without indicated neutralizing antibodies (5 µg/ml) for 24 or 48 hr, and cell viability was determined. T cells were also cocultured with the confluent layer of LN-derived stromal cells (+ LNS) or cultured in SCM with or without a cocktail of all antibodies (5 µg/ml each) for 24 or 48 hr, and cell viability was determined. Three experiments were performed, and the data are means ± SE. (b) IL-4, IL-6, and IL-7 levels in SCM were measured by ELISA. Three independently prepared SCM were analysed. *Results below the reliability level in the ELISA system we used (<15·6 pg/ml).

Article Snippet: Antibodies and recombinant cytokines Recombinant murine IL-4, IL-7, IFN-α/β, and anti-mouse IL-4, anti-mouse IL-6, and anti-mouse IL-7 antibodies were purchased from R & D systems, Inc. (Minneapolis, MN).

Techniques: Blocking Assay, Activity Assay, Cell Culture, Derivative Assay, Enzyme-linked Immunosorbent Assay

Journal:

Article Title: Murine lymph node-derived stromal cells effectively support survival but induce no activation/proliferation of peripheral resting T cells in vitro

doi: 10.1046/j.1365-2567.2003.01693.x

Figure Lengend Snippet: Cell surface markers of T cells cultured with LNS cells. Freshly isolated T cells and T cells cocultured with LNS cells, cultured in SCM or cultured with recombinant IL-4 (1 ng/ml), IL-7 (2 ng/ml), or IFN-β (100 U/ml) for 48 hr were stained with fluorescence-conjugated antibodies and analysed by flow cytometry. Numbers in each panel indicates the percentages of cells in a quadrant where the numbers are.

Article Snippet: Antibodies and recombinant cytokines Recombinant murine IL-4, IL-7, IFN-α/β, and anti-mouse IL-4, anti-mouse IL-6, and anti-mouse IL-7 antibodies were purchased from R & D systems, Inc. (Minneapolis, MN).

Techniques: Cell Culture, Isolation, Recombinant, Staining, Fluorescence, Flow Cytometry

Journal:

Article Title: Murine lymph node-derived stromal cells effectively support survival but induce no activation/proliferation of peripheral resting T cells in vitro

doi: 10.1046/j.1365-2567.2003.01693.x

Figure Lengend Snippet: DNA synthesis and cell cycle analysis of T cells cocultured with lymph node-derived stromal (LNS) cells, cultured in SCM or cultured with a cytokine. (a) T cells isolated from C57BL/6 lymph nodes were cultured in a simple medium (SM), cocultured with LNS cells, cultured in SCM or cultured with the indicated recombinant cytokine. Concentrations of cytokines were 10 ng/ml for IL-4, IL-6 and IL-7 and 100 U/ml for IFN-β. After 24 or 48 hr of culture, cells were pulsed with [3H]thymidine, and its uptake was measured as described in Materials and Methods. Three independent experiments were performed, and data are presented as means ± SE. (b) Fresh T cells, T cells cultured with IL-7 (10 ng/ml) for 48 hr, T cells cultured in SCM, and T cells cultured with LNS cells for 48 hr were prepared, and the cell cycles were analysed as described in Materials and Methods. Markers and numbers in each panel represent the areas of G0/G1, S, and G2/M phases and the percentages of cells in the area, respectively.

Article Snippet: Antibodies and recombinant cytokines Recombinant murine IL-4, IL-7, IFN-α/β, and anti-mouse IL-4, anti-mouse IL-6, and anti-mouse IL-7 antibodies were purchased from R & D systems, Inc. (Minneapolis, MN).

Techniques: DNA Synthesis, Cell Cycle Assay, Derivative Assay, Cell Culture, Isolation, Recombinant

Journal:

Article Title: Murine lymph node-derived stromal cells effectively support survival but induce no activation/proliferation of peripheral resting T cells in vitro

doi: 10.1046/j.1365-2567.2003.01693.x

Figure Lengend Snippet: Expression of Bcl family proteins in T cells cultured with LNS cells. Fresh T cells or T cells cultured with LNS cells or with a recombinant cytokine (IL-4, IL-6, and IL-7, 10 ng/ml) in the absence of supporting cells and cytokines (T alone) for 48 hr, were collected and analysed for their expression of Bcl-2 and Bcl-XL by immunoblotting as described in Materials and Methods. In the right panel, T cells were cultured with IFN-β (100 U/ml) for 24 hr with or without a neutralizing antibody and analysed for Bcl-XL expression.

Article Snippet: Antibodies and recombinant cytokines Recombinant murine IL-4, IL-7, IFN-α/β, and anti-mouse IL-4, anti-mouse IL-6, and anti-mouse IL-7 antibodies were purchased from R & D systems, Inc. (Minneapolis, MN).

Techniques: Expressing, Cell Culture, Recombinant, Western Blot