Results: 5

1.
Figure 1

Figure 1. CD4+ T lymphocyte gating strategy.. From: Effects of Human Respiratory Syncytial Virus, Metapneumovirus, Parainfluenza Virus 3 and Influenza Virus on CD4+ T Cell Activation by Dendritic Cells.

DDAO-labeled immature MDDC were exposed to virus or controls for 4–6 h, co-cultured for up to 1 week with autologous purified CSFE-labeled CD4+ T cells, and stimulated with PMA and ionomycin prior to staining for flow cytometry. Dead cells were excluded using a live/dead stain and MDDC were excluded based on the DDAO tracer (a). T cells were identified by gating on CD3+ cells (b). Single cells were identified using forward scatter height (FSC-H) and forward scatter area (FSC-A) to analyze cell size (c). Remaining debris was removed using FSC-A to analyze size and side scatter (SSC-A) to analyze cell complexity (d). Proliferating T cells were quantified by gating on CFSE to monitor the dilution of this tracer that occurs with cell division (e; proliferated cells are boxed). Live, proliferating, singlet CD3+ cells were analyzed for the expression of IFN-γ (f), IL-4 (g), and TNF-α (h). Boolean gating was used to quantify subsets listed at the bottom.

Cyril Le Nouën, et al. PLoS One. 2010;5(11):e15017.
2.
Figure 2

Figure 2. Proliferation of CD4+ T cells in response to autologous MDDC after exposure to SEB or purified virus.. From: Effects of Human Respiratory Syncytial Virus, Metapneumovirus, Parainfluenza Virus 3 and Influenza Virus on CD4+ T Cell Activation by Dendritic Cells.

(A) Time course of CD4+ T cell proliferation in response to MDDC that were mock-treated or treated with SEB, rHRSV, or IAV, using cells from a single donor. Days of co-culture are indicated to the left. Proliferated cells are boxed, and their percentage relative to the total live population is indicated. (B) Line graphs of the data from panel A. (C) Summary of CD4+ T cell proliferation in experiments using cells from eight donors in which MDDC were mock-treated, or treated with each of the four live or UV-inactivated viruses or SEB, with proliferation measured following 7 days of co-culture. Each donor is represented by a unique symbol. The median percent proliferation for each condition is indicated by a horizontal line. Treatments sharing the same lowercase letter do not differ significantly at P≤0.05 (Friedman test with Dunns post hoc test (see Materials and Methods).

Cyril Le Nouën, et al. PLoS One. 2010;5(11):e15017.
3.
Figure 4

Figure 4. Proliferation of CD4+ T cells during co-culture with virus-treated MDDC and SEB.. From: Effects of Human Respiratory Syncytial Virus, Metapneumovirus, Parainfluenza Virus 3 and Influenza Virus on CD4+ T Cell Activation by Dendritic Cells.

(A) Time course of CD4+ T cell proliferation in response to MDDC treated with rHRSV or IAV, or mock-treated, and co-cultured with SEB, using cells from a single donor. (B) Line graph of the data from panel A. (C) Summary of CD4+ T cell proliferation on day 4 in experiments representing cells from six donors, in which MDDC were mock-treated, or treated with each of the four live or inactivated virus, and co-cultured with SEB. Day 4 was used for comparison because the greatest differences between cultures containing mock, rHRSV, or IAV stimulated MDDC were observed on this day. For comparison, using cells from three of the six donors, mock-treated MDDC were co-cultured with autologous CD4+ T cells in the presence of SEB and IFN-β (75 IU per ml), or SEB and IL-28A (0,5 µg/ml), or SEB and IL-29 (0.5 µg/ml), or SEB and a cocktail of IFN-β, IL-28, and IL-29 (75 IU per ml, 0,5 µg/ml, 0,5 µg/ml, respectively). The box plots show the median (horizontal line) for each condition flanked by the 2nd and 3rd quartile. The outer bars show the range of values. Statistical differences are indicated by lower case letters (below the x axis). Treatment groups that share a letter are not significantly different.

Cyril Le Nouën, et al. PLoS One. 2010;5(11):e15017.
4.
Figure 5

Figure 5. Cytokine expression by proliferating CD4+ T lymphocytes co-cultured with virus-treated MDDC and SEB.. From: Effects of Human Respiratory Syncytial Virus, Metapneumovirus, Parainfluenza Virus 3 and Influenza Virus on CD4+ T Cell Activation by Dendritic Cells.

(A) Time course of cytokine production by CD4+ T cells proliferating in response to MDDC that had been mock-treated or treated with live or UV-inactivated rHRSV, or live IAV, and co-cultured in the presence of SEB, using cells from the same donor represented in Figure 4 A. The percentages of live proliferated cells positive for IFN-γ+, TNF-α+, IFN-γ+ plus TNF-α+ and IFN-γ+ plus TNF-α + plus IL-4+, are shown in the pie charts. (B) Percentage of live, proliferating CD4+ T cells positive for IFN-γ (left panel), TNF-α (middle panel), and IFN-γ plus TNF-α (right panel) after four days of co-culture with SEB and MDDC treated with each of the four live or UV-inactivated viruses or mock treatment, using cells from the same six donors shown in Figure 4 C. As a control, one co-culture for each donor containing mock-treated MDDC also contained 75 IU/ml IFN-β in addition to SEB. The box plots show the median (horizontal line) flanked by the 2nd and 3rd quartile. The outer bars show the range of values. (C) Comparison of the MFI of IFN-γ (left panel) or TNF-α (right panel) by proliferating CD4+ T cells that were positive for only the single cytokine versus those positive for both cytokines. The data are from the experiment in part B (cells from six donors). Treatments sharing the same lowercase letter do not differ significantly.

Cyril Le Nouën, et al. PLoS One. 2010;5(11):e15017.
5.
Figure 3

Figure 3. Cytokine expression by CD4+ T lymphocytes proliferating in response to MDDC treated with SEB or virus.. From: Effects of Human Respiratory Syncytial Virus, Metapneumovirus, Parainfluenza Virus 3 and Influenza Virus on CD4+ T Cell Activation by Dendritic Cells.

This is a continuation of the experiment shown in Figure 2. Expression of IFN-γ, IL-4, and/or TNF-α individually and in each double or triple combination (Boolean gating) was determined in live, proliferating, singlet CD4+ T cells. (A, B) Time course of production of IFN-γ and/or TNF-α by proliferating CD4+ T cells proliferating in response to MDDC that were mock-treated or treated with SEB, or live or UV-inactivated rHRSV or IAV, using cells from the same donor shown in Figure 2 A and B. (A) Total number of proliferating CD4+ T cells expressing IFN-γ or TNF-α ~ (B) Percentages of proliferating CD4+ T cells expressing IFN-γ and/or TNF-α, shown as pie charts. (C) Expression of IFN-γ and/or TNF-α by live, proliferated CD4+ T cells in experiments representing five of the eight donors shown in Figure 2 C. Values are expressed as percentages of total proliferated CD4+ T cells. The box plots show the median (horizontal line) flanked by the 2nd and 3rd quartile. The outer bars show the range of values. No statistical differences were observed between the treatments. (D) Comparison of the MFI of IFN-γ (left panel) or TNF-α (right panel) by proliferating CD4+ T cells that were positive for only the single cytokine versus those positive for both cytokines. The data are from the experiment in part C (cells from five donors). Statistical differences are indicated by asterisks (Friedman test with Dunns post hoc test (see Materials and Methods); * = p≤0.05.

Cyril Le Nouën, et al. PLoS One. 2010;5(11):e15017.

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