Figure 6.
Figure 6. Influence of DC supernatants on T-cell proliferation. (A) Purified human CD4+ T cells were stimulated with aAPCs in the presence of supernatants derived 24 hours after washout from differentially treated DCs, or DC-related soluble factors (sCD25). Proliferation was assessed by flow cytometry using CFSE-labeled CD4+ T cells; CD25 costaining was used as a control of proliferation. In PGE2-treated samples, the tryptophan concentration was adjusted (+ tryptophan, right column of dot plots) to the corresponding concentration measured in the control supernatant (left column of dot plots). On the right, soluble CD25 concentrations for the corresponding DC supernatants are shown. Black bars represent PGE2-treated DCs; mean ± standard deviation of all experiments is shown. *P < .05. Two representative experiments of 9 are shown. (B) Analysis of cell viability (left panel) and cell proliferation (right panel) of the IL2-dependent cell line CTLL-2. Supernatants from DCs matured with TNFα and αCD40 in the presence or absence of PGE2 were added to CTLL-2 cells. Where indicated, recombinant IL2 was added to DC cultures during the last 48 hours of maturation. Cell viability and cell numbers of CTLL-2 cells were assessed at 48 hours. CTLL-2 cultures exposed to PGE2-treated supernatants are represented by black bars. Mean ± standard deviation of at least 3 experiments is shown. *P < .05. (C) Proliferation of human T cells stimulated by aAPCs as described in panel A. Influence of blockade of IDO by 1-MT (1 mM) after washout during the last 24 hours of DC culture prior to collection of the supernatant (right histogram plot) was studied. One of 3 experiments is shown. (D) DC-derived soluble factors suppress the alloantigen-specific T-cell activation. Purified human CFSE-labeled allogeneic CD4+ T cells were incubated with DCs matured with or without PGE2 in the presence or absence of the corresponding DC supernatant or fresh medium. Shown here are the percentage of all dividing cells as well as the percentage of cells dividing more than once. At least 30 000 events within the positive gate were assessed for each of the conditions shown here. Reanalysis was performed at least twice for each experiment by 2 individual investigators and shown here is 1 representative experiment of 3. (White bars) Percentage of allogeneic T cells proliferating in response to maDCs in absence of PGE2; MLR was performed in fresh medium. (Dark gray bars) Percentage of allogeneic T cells proliferating in response to maDCs in presence of PGE2; MLR was performed in fresh medium. (Light gray bars) Percentage of allogeneic T cells proliferating in response to maDCs in absence of PGE2; MLR was performed in supernatants derived from maDCs. (Black bars) Percentage of allogeneic T cells proliferating in response to maDCs in presence of PGE2; MLR was performed in supernatants derived from maDCs in presence of PGE2.

Influence of DC supernatants on T-cell proliferation. (A) Purified human CD4+ T cells were stimulated with aAPCs in the presence of supernatants derived 24 hours after washout from differentially treated DCs, or DC-related soluble factors (sCD25). Proliferation was assessed by flow cytometry using CFSE-labeled CD4+ T cells; CD25 costaining was used as a control of proliferation. In PGE2-treated samples, the tryptophan concentration was adjusted (+ tryptophan, right column of dot plots) to the corresponding concentration measured in the control supernatant (left column of dot plots). On the right, soluble CD25 concentrations for the corresponding DC supernatants are shown. Black bars represent PGE2-treated DCs; mean ± standard deviation of all experiments is shown. *P < .05. Two representative experiments of 9 are shown. (B) Analysis of cell viability (left panel) and cell proliferation (right panel) of the IL2-dependent cell line CTLL-2. Supernatants from DCs matured with TNFα and αCD40 in the presence or absence of PGE2 were added to CTLL-2 cells. Where indicated, recombinant IL2 was added to DC cultures during the last 48 hours of maturation. Cell viability and cell numbers of CTLL-2 cells were assessed at 48 hours. CTLL-2 cultures exposed to PGE2-treated supernatants are represented by black bars. Mean ± standard deviation of at least 3 experiments is shown. *P < .05. (C) Proliferation of human T cells stimulated by aAPCs as described in panel A. Influence of blockade of IDO by 1-MT (1 mM) after washout during the last 24 hours of DC culture prior to collection of the supernatant (right histogram plot) was studied. One of 3 experiments is shown. (D) DC-derived soluble factors suppress the alloantigen-specific T-cell activation. Purified human CFSE-labeled allogeneic CD4+ T cells were incubated with DCs matured with or without PGE2 in the presence or absence of the corresponding DC supernatant or fresh medium. Shown here are the percentage of all dividing cells as well as the percentage of cells dividing more than once. At least 30 000 events within the positive gate were assessed for each of the conditions shown here. Reanalysis was performed at least twice for each experiment by 2 individual investigators and shown here is 1 representative experiment of 3. (White bars) Percentage of allogeneic T cells proliferating in response to maDCs in absence of PGE2; MLR was performed in fresh medium. (Dark gray bars) Percentage of allogeneic T cells proliferating in response to maDCs in presence of PGE2; MLR was performed in fresh medium. (Light gray bars) Percentage of allogeneic T cells proliferating in response to maDCs in absence of PGE2; MLR was performed in supernatants derived from maDCs. (Black bars) Percentage of allogeneic T cells proliferating in response to maDCs in presence of PGE2; MLR was performed in supernatants derived from maDCs in presence of PGE2.

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