Mucosal CD8alpha+ DC, with a plasmacytoid phenotype, induce differentiation and support function of T cells with regulatory properties

Abstract

Repetitive stimulation of naïve T cells by immature splenic dendritic cells (DC) can result in the differentiation of T-cell lines with regulatory properties. In the present study we identified a population of DC in the mucosae that exhibit the plasmacytoid phenotype, secrete interferon-alpha (IFN-alpha) following stimulation with oligodeoxynucleotides containing certain cytosine-phosphate-guanosine (CpG) motifs and can differentiate naïve T cells into cells that exhibit regulatory properties. Although these DC appear to be present in both spleen and mesenteric lymph nodes (MLN), only CpG-matured DC from the MLN (but not the spleen) were able to differentiate naïve T cells into T regulatory 1-like cells with regulatory properties. The activity of these DC failed to sustain robust T-cell proliferation and thereby enhanced the suppressive efficacy of CD4+ CD25+ T regulatory cells. These DC are the major CD8alpha+ DC population in the Peyer's patches (PP). Given their significant presence in mucosal tissue, we propose that these DC may provide a mechanistic basis for the homeostatic regulation in the gut by eliciting regulatory cell suppressor function and poorly supporting T helper cell proliferation at a site of high antigenic stimulation like the intestine.

Figure 1

Phenotypic diversity of plasmacytoid dendritic cells (DC) in tissues of Flt3-ligand (Flt3L)-treated mice. A single-cell suspension was prepared from the spleen, mesenteric lymph nodes (MLN), peripheral lymph nodes (PLN) and Peyer's patches (PP) of Flt3L-treated mice and stained with cell-surface marker antibodies. Region 1 [R1] and region 2 [R2] were selected based on the expression of CD8α and CD11c, where CD8α⁺ CD11c^hi cells are included in R1 and CD8α⁺ CD11c^lo cells are included in R2. These gates were used to determine the relative expression of B220, Class II, CD40, CD80, CD86 and CD19 for CD8α⁺ CD11c^hi (black histograms) and CD8α⁺ CD11c^lo (grey histograms) populations by mean fluorescence intensity (MFI). The isotype controls for each tissue type are represented by the dotted line. Gr-1 expression was determined in a separate experiment using the same gate settings. A total of 30 000 live, gated events were collected for each plot. Data are representative of three separate experiments.

Figure 2

Interferon-α (IFN-α) secretion by CD8α⁺ CD11c^hi and CD8α⁺ CD11c^lo dendritic cell (DC) populations. CD8α⁺ CD11c^hi and CD8α⁺ CD11c^lo DC populations were isolated from the spleen, mesenteric lymph nodes (MLN) and peripheral lymph nodes (PLN) by fluorescence-activated cell sorter (FACS) analysis and incubated for 18 hr with CpG (1 µg/ml). As CD8α⁺ CD11c^hi DC cannot be isolated from Peyer's patches (PP), CD8α⁺ CD11c^lo DC from the PP were compared with CD8α⁻ CD11c^hi DC from the PP. Supernatants were collected and IFN-α levels were measured by enzyme-linked immunosorbent assay (ELISA). Data are representative of three separate experiments. Error bars represent the standard error of the mean of triplicate wells. P-values were calculated using the unpaired Student t-test. P-values of <0·05 were considered significant.

Figure 3

CD8⁺ plasmacytoid dendritic cells (pDC) are less efficient at inducing T helper cell proliferation than CD8⁺ non-pDC. Fresh CD8⁺ pDC and CD8⁺ non-pDC populations were isolated from the spleen (Sp), peripheral lymph nodes (PLN) and mesenteric lymph nodes (MLN) by fluorescence-activated cell sorter (FACS) analysis. As Peyer's patches (PP) do not have the CD8⁺ non-pDC population, we isolated the CD8⁺ pDC and CD8⁻ DC populations for comparison. The different DC populations were cultured with freshly isolated CD4⁺ DO11.10 T-cell receptor (TCR) transgenic T helper cells at a ratio of 5 : 1 (T cells : DC), and ovalbumin (OVA) peptide was added at a final concentration of 375 nm. Cellular proliferation was assessed after 72 hr by [³H]thymidine incorporation (a). Interleukin (IL)-10 (b) and interferon-γ (IFN-γ) (c) protein levels in culture supernatants were assessed by Luminex analysis at 48- and 72 hr, respectively. Data are representative of three separate experiment and the error bars represent the standard deviation of quadruplicate wells. P-values were calculated using the unpaired Student's t-test. P-values of <0·05 were considered significant.

Figure 4

CD8⁺ plasmacytoid dendritic cells (pDC) favour T regulatory (Treg)-mediated suppression over T-cell proliferation. Fresh CD4⁺ CD25⁻ T helper cells (black circles) and CD4⁺ CD25⁺ Treg cells (white circles) were isolated from DO11.10 T-cell receptor (TCR) transgenic animals. T helper and Treg cells were cultured together (grey circles) or alone and stimulated with ovalbumin (OVA) peptide-pulsed CD8⁺ non-pDC or CD8⁺ pDC derived from the spleen, mesenteric lymph nodes (MLN) or peripheral lymph nodes (PLN). For Peyer's patches (PP), OVA peptide-pulsed CD8⁺ pDC and myeloid DC were used as antigen-presenting cells (APC). Cellular proliferation was measured by [³H]thymidine incorporation. Data are representative of two separate experiments and the error bars represent the standard deviation of triplicate wells.

Figure 5

T regulatory (Treg) cell-mediated suppression is more robust at high T helper : Treg cell ratios when CD8⁺ plasmacytoid dendritic cells (pDC) are the antigen-presenting cells (APC). Fresh CD4⁺ CD25⁻ T helper and CD4⁺ CD25⁺ Treg cells were isolated from DO11.10 T-cell receptor (TCR) transgenic animals. T helper and Treg cells were cultured either alone (black circles and white circles, respectively) or together (grey circles) at various Treg to T helper ratios and stimulated with ovalbumin (OVA) peptide-pulsed CD8⁺ non-pDC or CD8⁺ pDC derived from the spleen [panels (a) and (b), respectively] or mesenteric lymph nodes (MLN) [panels (c) and (d), respectively]. Cellular proliferation was measured by [³H]thymidine incorporation. Data are representative of two separate experiments and the error bars represent the standard deviation of triplicate wells.

Figure 6

CD8⁺ plasmacytoid dendritic cells (pDC) induce de novo differentiation of T regulatory 1 (Tr1)-like cells from naïve T cells. T-cell lines were generated by culturing CD25-depleted CD4⁺ T cells for 3 weeks with either CD8⁺ pDC or CD8⁺ non-pDC from mesenteric lymph nodes (MLN). T-cell lines were then assayed for regulatory T-cell activity, as measured by inhibition of T helper cell proliferation. Fresh CD4⁺ CD25⁻ T helper cells were isolated from DO11.10 T-cell receptor (TCR) transgenic mice and stimulated with increasing concentrations of ovalbumin (OVA) peptide in the presence (grey circles) or absence (black circles) of in vitro cultured T-cell lines. Irradiated splenocytes (a) or lipopolysaccharide (LPS) blasts (b) were used as antigen-presenting cells (APC). T cells depleted of CD25⁺ cells at the onset of culture exhibited the ability to differentiate de novo into Tr1-like cells with suppressive activity following culture with CD8⁺ pDC. Data are representative of two separate experiments and the error bars represent the standard deviation of triplicate wells. *Statistical significance at P < 0·05; **statistical significance at P < 0·02 (Welch t-test). Statistical analysis was calculated at the 50 nm and 500 nm data points only.

Figure 7

Mesenteric lymph node (MLN) CD8⁺ plasmacytoid dendritic cells (pDC) activated with CpG maintain the ability to induce T regulatory 1 (Tr1)-like T cells that exhibit regulatory T-cell function. T-cell lines were cultured with CD8⁺ pDC or CD8⁺ non-pDC for 4 weeks prior to assaying for regulatory T-cell activity. MLN CD8⁺ pDC and CD8⁺ non-pDC were incubated for 18 hr with CpG before co-culture with T-cell lines. Levels of CD80, CD86 and CD40, expressed by CD8⁺ pDC following an 18-hr incubation in the presence or absence of CpG, were assessed by fluorescence-activated cell sorter (FACS) analysis (a). Regulatory activity was assessed by the ability of the Tr1-like cell lines to inhibit the proliferation of freshly isolated ovalbumin (OVA) T-cell receptor (TCR) transgenic CD4⁺ CD25⁻ T helper cells stimulated with 500 nm of OVA peptide in the presence (grey bars) or in the absence (black bars) of in vitro-differentiated regulatory Tr1-like cell lines (b). Lipopolysaccharide (LPS) blasts (left panel) or irradiated splenocytes (right panel) were used as antigen-presenting cells (APC) (b). T-cell lines generated by stimulation with CpG-matured CD8⁺ pDC secrete higher levels of interleukin (IL)-4 and IL-10 following stimulation with plate-bound anti-CD3 compared with T-cell lines generated by stimulation with CpG-matured CD8⁺ non-pDC (c). Data are representative of three separate experiments and the error bars represent the standard deviation of triplicate wells. P-values were calculated using the unpaired Student's t-test. P-values of <0·05 were considered significant.