Maturation of human dendritic cells with Saccharomyces cerevisiae (yeast) reduces the number and function of regulatory T cells and enhances the ratio of antigen-specific effectors to regulatory T cells

Abstract

We compared the effects of yeast-treated human dendritic cells (DCs) with CD40L-matured human DCs for the induction of effector cells and the number and functionality of CD4(+)CD25(+)CD127(-)FoxP3(+) regulatory T cells (Tregs). DCs were treated with yeast or CD40L and cocultured with isolated autologous CD4(+) T cells. CD4(+)CD25(+)CD127(-) T cells isolated from the coculture of CD4(+) T cells plus yeast-treated DCs (yeast coculture) had a lower expression of FoxP3 and decreased suppressive function compared to CD4(+)CD25(+)CD127(-) T cells isolated from the coculture of CD4(+) T cells plus CD40L-treated DCs (CD40L coculture). Also, compared to the CD40L coculture, the yeast coculture showed increases in the ratio of CD4(+)CD25(+) activated T cells to Tregs and in the production of Th1-related cytokines (IL-2, TNF-α, IFN-γ) and IL-6. In addition, yeast-treated DCs used as antigen-presenting cells (APCs) incubated with the tumor antigen CEA enhanced the proliferation of CEA-specific CD4(+) T cells compared to the use of CD40L-matured DCs used as APCs. This is the first study to report on the role of yeast-treated/matured human DCs in reducing Treg frequency and functionality and in enhancing effector to Treg ratios. These results provide an additional rationale for the use of yeast as a vector in cancer vaccines.

Fig. 1

CD4⁺CD25⁺CD127⁻ Tregs isolated from the coculture of CD4⁺ T cells and yeast-treated DCs vs. CD40L-treated DCs showed decreased FoxP3 expression. CD4⁺ T cells plus yeast-treated DCs (A), and CD4⁺ T cells plus CD40L-treated DCs (B), were cocultured for 5 days. On day 5, CD4⁺CD25⁺CD127⁻ Tregs isolated from the cocultures were analyzed by FACS. Cells were stained with CD4 FITC, CD25 PE, and FoxP3 APC antibodies and acquired on an LSRII. The numbers in the upper right of the graphs represent the % of FoxP3⁺ cells in CD4⁺CD25^high Tregs. The figure is representative of 3 experiments on 3 healthy donors.

Fig. 2

Tregs generated from yeast coculture exhibit decreased suppressive function on CD4⁺CD25⁻ effector cells compared to Tregs generated from CD40L coculture. A representative histogram shows the suppression of CD4⁺CD25⁻ T-cell proliferation by CD4⁺CD25⁺CD127⁻ Tregs in a healthy donor. CD4⁺CD25⁺CD127⁻ Tregs generated from yeast and CD40L cocultures were isolated by Miltenyi columns and rested for 24 h. Tregs were then cultured alone or cocultured at a 1:1 ratio with autologous CD4⁺CD25⁻ T cells, upon stimulation with anti-CD3 and irradiated autologous T-depleted PBMCs. Experiments were performed in triplicate and results are expressed as the mean ± SD (cpm). Percentages indicate the level of proliferation.

Fig. 3

CEA-specific CD4⁺ T-cell proliferation increased in a colorectal cancer patient pre- and post-vaccination when CD4⁺ T cells were stimulated with DCs treated with yeast and incubated with CEA protein. DCs from a colorectal cancer patient were isolated pre- and post-vaccination and cultured for 5 days with GM-CSF and IL-4. DCs were then treated with yeast (yeast:DCs = 10:1) for 48 h or with CD40L (1 μg/ml) for 24 h. CD4⁺ T cells were isolated using Miltenyi columns by negative selection and incubated with DCs (CD4⁺ T cells:DCs = 10:1) for 7 days, using 2 μg/ml PHA as a positive control and 20 μg/ml myoglobulin as a negative control. CEA protein was used at 20 μg/ml. On day 7, ³H was added to the culture, incubated for 6 h, and read with a Wallac Trilux B-scintillation counter. The numbers above the CEA columns represent the fold increase of CD4 proliferation with CEA protein and myoglobulin protein. The experiment was performed in triplicate and the results are expressed as the mean ± SD (cpm).