rar β antagonist  (Tocris)

Journal: The Journal of Experimental Medicine

Article Title: Microbiota regulate the ability of lung dendritic cells to induce IgA class-switch recombination and generate protective gastrointestinal immune responses

doi: 10.1084/jem.20150567

Figure Lengend Snippet: LDC CD103 + and LDC CD24 + mediate IgA class switching in an RA- and TGF-β–dependent manner. (A) Microarray analysis of lung APC subsets. Heat map showing the differential expression of genes in the IgA CSR pathway. Colors correspond to significant fold change expression. Red, high expression; blue, low expression. (B) qRT-PCR of mRNA isolated from flow-sorted LDC CD103 + , LDC CD24 + , LMφ CD64 + , and SpDC, respectively. The expression of Integrin β8 mRNA was normalized to actin and expressed in relative units. (C) qRT-PCR of mRNA isolated from flow-sorted LDC CD103 + , LDC CD24 + , LMφ CD64 + , and SpDC, respectively. The expression of mAldha2 was normalized to GAPDH and expressed in relative units. (D–F) Flow-sorted LDC CD103 + or LDC CD24 + were cultured with naive B cells in the absence or presence of the RAR-β inhibitor LE540, and IgA + B cells were quantified. (D) Representative flow cytometry plots. (E) Cumulative data from three different experiments with seven mice pooled per experiment. (F) Escalating doses of LE540 (25 nM, 250 nM, and 2.5 µM) were added to the DC–B cell culture on day 0. Cumulative data from two independent experiments is shown (with seven mice pooled per experiment). Statistical comparisons are made with (–) LE540 condition. (G– I) Flow-sorted LDC CD103 + or LDC CD24 + were cultured with naive B cells in the absence or presence of α-TGF-β neutralizing antibody, and IgA + B cells were quantified. (G) Representative flow cytometry plots. (H) Cumulative data from three different experiments with seven mice pooled per experiment. (I) Escalating doses of α-TGF-β neutralizing antibody (8.75, 17.5, 35, and 70 µg/ml) were added to the DC–B cell cultures on days 0 and 3. Cumulative data from two independent experiments (with seven mice pooled per experiment) is shown. Statistical comparisons are made with (–) α-TGF-β condition. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Article Snippet: For the RA inhibition assays, the DC/B cell co-culture were incubated with 3 µM of RAR-β antagonist LE540 (Wako) dissolved in DMSO at a stock concentration of 1 mM and added to cultures at final concentration of 3 µM.

Techniques: Microarray, Expressing, Quantitative RT-PCR, Isolation, Cell Culture, Flow Cytometry

Journal: The Journal of Experimental Medicine

Article Title: Microbiota regulate the ability of lung dendritic cells to induce IgA class-switch recombination and generate protective gastrointestinal immune responses

doi: 10.1084/jem.20150567

Figure Lengend Snippet: LDCs induce integrin α4β7 and CCR9 on B cells in a RA dependent manner. (A–C) Flow-sorted LDCs, SpDCs, SkDCs, and MLN DCs were cultured with α-IgM– and α-CD40–stimulated CD43 neg CD19 + B cells in a DC/B cell ratio of 2:1 for 5–7 d, and the expression of integrin α4β7 and chemokine receptor CCR9 was measured. Data from three experiments, six mice pooled per experiment, are shown. Statistical comparisons to SpDC are shown. (A) Representative flow cytometry plots. (B) Quantification of α4β7 + B cells showing cumulative data. (C) Quantification of CCR9 + B cells showing cumulative data. (D and E) Flow-sorted LDCs, MLN DCs, and SpDCs were cultured with CD43 neg CD19 + B cells in the absence or presence of RAR-β inhibitor LE540, and α4β7 and CCR9 were quantified. Cumulative data from three experiments with six mice pooled per experiment are shown. Statistical comparisons to the respective (–) LE540 condition are shown. α4β7-expressing B cells (D) and CCR9-expressing B cells (E) induced by the respective DC populations. (F and G) Flow-sorted LDCs, SpDCs, SkDCs, or MLN DCs were cultured with CD43 neg CD19 + B cells and the expression of integrin α4β7 was measured. (F) Representative flow cytometry plots. (G) Cumulative data from three experiments with six mice pooled per experiment. Statistical comparisons to SpDC are shown. (H and I) Mice were immunized with CT and the expression of α4β7 + and CCR9 + IgA + cells was examined at serial time points. (H) Representative flow plots. (I) Cumulative data from three experiments (three mice per experiment) quantifying the MFI of α4β7 + and CCR9 + on IgA + B cells. Statistical comparisons to day 0 are shown. (J and K) Flow sorted LDC CD103 + , LDC CD24 + , LMφ CD64 + , or MLN DCs were cultured with CD43 neg CD19 + B cells, and the expression of integrin α4β7 on IgA + B cells was examined. (J) Representative flow cytometry plots comparing the respective lung APC subsets with IgG 2aκ isotype control. (K) Cumulative data from three independent experiments shown (with five mice pooled per experiment). Statistical comparisons to B cell alone condition are shown. (L) Flow-sorted LDCs and SpDCs (left) or MLN DCs and SpDCs were cultured CD43 neg CD19 + CD45.2 + B cells. After culture, 5 × 10 5 LDC B cells per culture condition were labeled with green (CFSE) and red (CTr) dyes, respectively, mixed in a 1:1 ratio, and adoptively transferred into CD45.1 + mice. After 18 h, CD45.2-gated transferred cells were measured in the spleen, SILP, CLP, and lungs, and the output ratio of LDC-stimulated, CFSE-labeled B cells versus SpDC-stimulated, CTr-labeled B cells was determined. The HI was represented as the yield of CFSE-labeled cells/CTr-labeled cells divided by the input ratio of CFSE-labeled cells/CTr-labeled cells. Cumulative data from three individual experiments is shown (with five mice per experiment). Red line indicates a HI ∼1. Statistical comparisons to spleen HI are shown. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Article Snippet: For the RA inhibition assays, the DC/B cell co-culture were incubated with 3 µM of RAR-β antagonist LE540 (Wako) dissolved in DMSO at a stock concentration of 1 mM and added to cultures at final concentration of 3 µM.

Techniques: Cell Culture, Expressing, Flow Cytometry, Labeling