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Results: 4

1.
FIGURE 2

FIGURE 2. From: Type I interferon reverses human Th2 commitment and stability by suppressing GATA3.

IFN-α/β suppresses Th2 cytokine expression in committed Th2 cells. Purified human CD4+/CD45RA+ cells were activated with plate bound anti-CD3/anti-CD28 for 7 days under neutralizing conditions or with IL-4 to promote Th2 development (1° condition). Cells were then washed and restimulated for an additional 7 days with anti-IL-4, IL-4, IFN-α, or IL-4 + IFN-α (2° conditions). A, Cells were stimulated with PMA + ionomycin for 6 h, and IL-4, IL-5, and IL-13 were measured by intracellular staining. B, Cells were stimulated with PMA + ionomycin for 48 h, and IL-4 was quantified by ELISA from the culture supernatants. *, p < 0.05 compared to IL-4 (A and B, condition 3).

Jonathan P. Huber, et al. J Immunol. ;185(2):813-817.
2.
FIGURE 3

FIGURE 3. From: Type I interferon reverses human Th2 commitment and stability by suppressing GATA3.

IFN-α/β specifically inhibits GATA3 expression. A, Purified human CD4+/CD45RA+ cells were activated with plate bound anti-CD3/anti-CD28 for 72 h with IL-4, IFN-α, or IL-4 + IFN-α. mRNA was isolated from cells, and relative GATA3 mRNA was quantified by real-time PCR. *, p < 0.05 compared to IL-4 (condition 2). B, Cells were activated as in A for 6 days, and GATA3 protein was assessed by intracellular staining and flow cytometric detection. C, Purified human CD4+/CD45RA+ cells were left unstimulated or were activated with plate bound anti-CD3/anti-CD28 for 6 days in the presence of the indicated cytokines. GATA3 protein was assessed by intracellular staining and flow cytometric detection, and the data are gated on live cells and expressed as the mean fluorescence intensity of the population. *, p < 0.05 compared to IL-4 (condition 3). D, Purified human CD4+/CD45RA+ cells were activated with plate bound anti-CD3/anti-CD28 for 7 days in the presence of the indicated cytokines. mRNA was isolated from cells, and relative T-bet mRNA was quantified by real-time PCR.

Jonathan P. Huber, et al. J Immunol. ;185(2):813-817.
3.
FIGURE 1

FIGURE 1. From: Type I interferon reverses human Th2 commitment and stability by suppressing GATA3.

IFN-α/β inhibits human CD4+ Th2 development. Purified human CD4+/CD45RA+ cells were activated with plate bound anti-CD3/anti-CD28 under defined cytokine conditions. Cytokines were either neutralized with anti-cytokine antibodies (no symbol), or cytokines were added as indicated by the “+” symbol. A, Cells were restimulated with PMA + ionomycin, and IFN-γ and IL-4 expression were measured by intracellular staining. Data are gated on live events. B, Cells were restimulated with PMA + ionomycin for 24 h, and IL-4 and IL-5 were quantified by cytometric bead array from the culture supernatants. C, IL-4 expression was measured by intracellular staining of samples from multiple donors. 10 donors were assessed with conditions 1–3 and 6, while 4 of the 10 donors were also assessed with conditions 4–5. Each symbol represents a separate donor. D, Induction of CRTH2 expression was assessed by flow cytometric analysis. *, p < 0.05 compared to IL-4 and to IL-4 + IL-29 (conditions 2 and 6).

Jonathan P. Huber, et al. J Immunol. ;185(2):813-817.
4.
FIGURE 4

FIGURE 4. From: Type I interferon reverses human Th2 commitment and stability by suppressing GATA3.

IFN-α suppresses Th2 cytokines by inhibiting GATA3 protein levels in committed Th2 cells. A, Cells were activated for two consecutive weeks under 1° and 2° conditions as described in Fig. 2. GATA3 protein was measured on day 14 by intracellular staining. *, p < 0.05 compared to IL-4 (condition 3). B-D, Purified human CD4+/CD45RA+ cells were activated with plate bound anti-CD3/anti-CD28 under Th2 conditions for 1 week prior to retroviral transduction with GFPRV or GATA3-GFP. GFP+ cells were sorted on day 14 and reactivated with anti-CD3/anti-CD28 in the presence or absence with IFN-α. B, Cell culture supernatants were harvested on day 3 following the GFP sort and analyzed for IL-5 and IL-13 by ELISA. C, On day 7 following the GFP sort, cells were washed and restimulated for 48 h with anti-CD3/anti-CD28. IL-4, IL-5 and IL-13 were quantified by ELISA from the culture supernatants. D, Cells were harvested on day 5 following the GFP sort and GATA3 protein was measured by intracellular staining and flow cytometric detection. Data are gated on live cells and expressed as the mean fluorescence intensity of the population.

Jonathan P. Huber, et al. J Immunol. ;185(2):813-817.

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