DC1 dendritic cells inhibit T_regs directly.

<p>DC1 dendritic cells were generated as previously described. Medium from these cells was then harvested and combined 1∶1 with culture medium to create the “pretreatment” medium. CD4+CD25+ T cells or effectors were cultured in the pretreatment medium for 24 hours at a concentration of 3×10⁶ cells/mL. These “treated” cells were then harvested, washed, and used in cocultures as previously described. (<i>4A</i>) 2.5×10⁵ “treated” or “untreated” CFSE-labeled unfractionated responder lymphocytes were co-cultured with 1×10⁵ immature dendritic cells and 1.25×10⁵ sorted, purified CD4+CD25+ T cells for 5 days. CD4-positive responder cell proliferation is shown. (<i>4B</i>) 2.5×10⁵ CFSE-labeled unfractionated responder lymphocytes were co-cultured with 1×10⁵ immature dendritic cells and 1.25×10⁵ sorted, purified “treated” or “untreated” CD4+CD25+ T cells for 5 days. CD4-positive responder cell proliferation is shown. (<i>4C</i>) The number of mitoses per 10⁴ cells is summarized. In each case, data shown are representative of three separate experiments.</p

Inhibition of T_reg function by DC1 dendritic cells results from a soluble factor.

<p>(<i>3A</i>) 1.25×10⁵ sorted, purified CD4+CD25+ T cells were co-cultured with 2.5×10⁵ CFSE-labeled unfractionated responder lymphocytes and 1×10⁵ immature dendritic cells (solid line) or DC1 dendritic cells for 5 days (dashed line). Data shown are gated on CD4-positive cells and are representative of at least 10 experiments. (<i>3B</i>) 1.25×10⁵ T_regs were co-cultured with 2.5×10⁵ CFSE-labeled responders and 1×10⁵ immature dendritic cells for 5 days (solid line). In addition, 1×10⁵ DC1 dendritic cells were added to a transwell membrane placed in the culture well (dashed line). Data shown are gated on CD4-positive cells and are representative of 4 experiments. (<i>3C</i>) 1.25×10⁵ T_regs were co-cultured with 2.5×10⁵ CFSE-labeled responders alone for 5 days. 1×10⁵ DC1 dendritic cells were added to a transwell membrane placed in the culture well. Data shown are gated on CD4-positive cells (<i>N</i> = 2). (3<i>D</i>) The number of mitoses per 10⁴ cells is summarized for CD4-positive responder cells in the presence of T_regs and iDC alone, iDC with DC1 added to the Transwell membrane, and DC1 alone. (<i>3E&F</i>) 1.25×10⁵ T_regs were co-cultured with 2.5×10⁵ CFSE-labeled responders and 1×10⁵ DC1 dendritic cells in the presence (dashed line) or absence (solid line) of neutralizing anti-IL-12 (<i>3E</i>) or anti-IL-6 (<i>3F</i>) antibodies (5 µg/mL). Data shown are gated on CD4-positive cells and are representative of at least three experiments.</p

Inhibition of T_reg function by DC1 dendritic cells is not due to apoptosis.

<p>1.25×10⁵ sorted, purified CD4+CD25+ T cells were co-cultured with 1×10⁵ immature dendritic cells (<i>2A</i>) or DC1 dendritic cells (<i>2B</i>). Expression of the apoptotic markers Annexin-V and 7-AAD 24 hours later is shown. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0074698#pone-0074698-g002" target="_blank">Figure 2C</a> summarizes the percent of cells expressing both markers (+/+), Annexin-V only (+/−), 7-AAD only (−/+), or neither marker (−/−). (iDC black, DC1 gray; <i>N = 4</i>).</p

CD4+CD25+ T cells upregulate T-bet in the presence of DC1 dendritic cells.

<p>1.25×10⁵ CD4+CD25+ T cells were co-cultured with 1×10⁵ immature (<i>6A</i>) or DC1 (<i>6B,C</i>) dendritic cells. Neutralizing anti-IL-12 antibody (5 µg/mL) was included in some samples (<i>6C</i>). At 48 hours cells were harvested, permeabilized, and intracellular expression of T-bet and FoxP3 was detected by intracellular staining. Data shown are gated on CD4-positive cells and are representative of at least three separate experiments in each instance. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0074698#pone-0074698-g006" target="_blank">Figure 6D</a> summarizes the percent of FoxP3+ cells that are T-bet positive at 48 hours for each group.</p

Suppressor CD4+CD25+ T cells secrete effector cytokines in the presence of DC1 dendritic cells.

<p>(<i>5A</i>) 2.5×10⁵ sorted CD4+CD25+ (T_reg) or CD4+CD25− (T_eff) T cells were combined with 2.0×10⁵ immature or DC1 dendritic cells. Supernatants were harvested at 5 days and ELISA was used to measure the amount of IFN-γ present in the supernatant. Data shown are the average of at least four experiments. (<i>5B</i>) At day 5, some culture samples were permeabilized and intracellular IFN-γ was detected by flow cytometry. (<i>5C&D</i>) 2.5×10⁵ sorted CD4+CD25+ T cells were cocultured with immature (<i>5C</i>) or DC1 (<i>5D</i>) dendritic cells; intracellular expression of FoxP3 and IFN-γ was detected in permeabilized cells 5 days later.</p

T_regs inhibit responder cell proliferation in the presence of immature but not DC1 dendritic cells.

<p>(<i>1A&B</i>) 2.5×10⁵ CFSE-labeled unfractionated responder lymphocytes were initially co-cultured with 1×10⁵ immature dendritic cells in the presence (dashed line) or absence (solid line) of 1.25×10⁵ sorted, purified CD4+CD25+ T cells for 5 days. Responder cell proliferation is shown for CD4-gated (<i>1A</i>) or CD8-gated (<i>1B</i>) T cells. Data shown are representative of at least 10 experiments. (<i>1C&1D</i>) 2.5×10⁵ CFSE-labeled unfractionated responders were co-cultured with 1.25×10⁵ sorted CD4+CD25+ T cells and 1×10⁵ immature (dashed line) or DC1 dendritic cells (solid line). Again responder cell proliferation is shown for CD4-gated (<i>1C</i>) or CD8-gated (<i>1D</i>) T cells. Data shown are representative of at least 10 experiments. (<i>1E</i>) 2.5×10⁵ CFSE-labeled unfractionated responders were co-cultured with 1×10⁵ dendritic cells matured using a conventional cytokine maturation cocktail (IL-1, IL-6, TNF-α, PGE-2) in the presence (dashed line) or absence (solid line) of 1.25×10⁵ sorted CD4+CD25+ T cells. Proliferation for CD4-gated T cells is shown (<i>N</i> = 4). (<i>1F</i>) Proliferation of CD4-positive responders in the presence of T_regs and immature dendritic cells treated briefly with LPS (15 minutes) prior to co-culture is shown (<i>N</i> = 3). (<i>1G</i>) A mathematical algorithm previously applied elsewhere was used to calculate the number of mitoses per 10⁴ responder CD4-gated T cells in the presence of iDC, DC1, or conventional cytokine maturation cocktail DC (CMDC).</p