Figure 6
Figure 6. Induction of Treg cells after cocultivation with FV-infected DCs. (A) Naive TCR transgenic CD4+ T cells from DO11.10 mice were incubated with DC in a 3D collagen gel. After 24 hours, gels were digested and T cells were analyzed for the intracellular expression of the Treg cell marker Foxp3 by flow cytometry. T cells were mixed with uninfected (□) or FV-infected DCs (■), which were loaded with OVA (+Ova) or not loaded with the OVA peptide (−Ova). T cells prior to coculture served as negative control (▩). The percentage of positively stained cells in the total CD4+ T cells is shown. Cumulative data from 7 independent experiments are shown. The error bars represent standard errors of the means. Differences between cultures with FV-infected versus uninfected DCs were statistically significant (P = .006). (B) Naive TCR transgenic CD4+ T cells from DO11.10 mice were incubated with FV-infected or uninfected DCs for 3 days. After culture, T cells were isolated and added to a polyconal CD4+ T-cell proliferation assay. Left panel: as negative control, 2 × 105 nonstimulated CD4+ T cells were labeled with CFSE and analyzed by flow cytometry 3 days later. Middle panel: 2 × 105 naive CD4+ T cells were stimulated with antibodies against CD3 and CD28 and labeled with CFSE 3 days prior to analysis. At the same time, 2 × 105 total CD4+ T cells that were stimulated previously by uninfected DCs were added. However, adding nonstimulated CD4+ T cells to the culture had no influence on the proliferation of the anti-CD3, anti-CD28–stimulated T cells (data not shown). The MFI of 3 independent tests was 1155 ± 21.6. Right panel: 2 × 105 naive CD4+ T cells were stimulated with antibodies against CD3 and CD28 and labeled with CFSE 3 days prior to analysis. At the same time, a total of 2 × 105 CD4+ T cells containing about 5 × 104 Foxp3+ cells that had been stimulated previously by FV-infected DCs were added. The MFI of 3 independent tests was 1842 ± 51.7. M1 shows the percentage of nonproliferating cells in each culture. Representative data for 3 independent experiments with similar results are shown. The difference in the MFI between the groups labeled “uninfected DC” and “infected DC” was statistically significant (P < .001).

Induction of Treg cells after cocultivation with FV-infected DCs. (A) Naive TCR transgenic CD4+ T cells from DO11.10 mice were incubated with DC in a 3D collagen gel. After 24 hours, gels were digested and T cells were analyzed for the intracellular expression of the Treg cell marker Foxp3 by flow cytometry. T cells were mixed with uninfected (□) or FV-infected DCs (■), which were loaded with OVA (+Ova) or not loaded with the OVA peptide (−Ova). T cells prior to coculture served as negative control (▩). The percentage of positively stained cells in the total CD4+ T cells is shown. Cumulative data from 7 independent experiments are shown. The error bars represent standard errors of the means. Differences between cultures with FV-infected versus uninfected DCs were statistically significant (P = .006). (B) Naive TCR transgenic CD4+ T cells from DO11.10 mice were incubated with FV-infected or uninfected DCs for 3 days. After culture, T cells were isolated and added to a polyconal CD4+ T-cell proliferation assay. Left panel: as negative control, 2 × 105 nonstimulated CD4+ T cells were labeled with CFSE and analyzed by flow cytometry 3 days later. Middle panel: 2 × 105 naive CD4+ T cells were stimulated with antibodies against CD3 and CD28 and labeled with CFSE 3 days prior to analysis. At the same time, 2 × 105 total CD4+ T cells that were stimulated previously by uninfected DCs were added. However, adding nonstimulated CD4+ T cells to the culture had no influence on the proliferation of the anti-CD3, anti-CD28–stimulated T cells (data not shown). The MFI of 3 independent tests was 1155 ± 21.6. Right panel: 2 × 105 naive CD4+ T cells were stimulated with antibodies against CD3 and CD28 and labeled with CFSE 3 days prior to analysis. At the same time, a total of 2 × 105 CD4+ T cells containing about 5 × 104 Foxp3+ cells that had been stimulated previously by FV-infected DCs were added. The MFI of 3 independent tests was 1842 ± 51.7. M1 shows the percentage of nonproliferating cells in each culture. Representative data for 3 independent experiments with similar results are shown. The difference in the MFI between the groups labeled “uninfected DC” and “infected DC” was statistically significant (P < .001).

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