Freshly isolated Peyer's patch, but not spleen, dendritic cells produce interleukin 10 and induce the differentiation of T helper type 2 cells

Abstract

Orally administered antigens often generate immune responses that are distinct from those injected systemically. The role of antigen-presenting cells in determining the type of T helper cell response induced at mucosal versus systemic sites is unclear. Here we examine the phenotypic and functional differences between dendritic cells (DCs) freshly isolated from Peyer's patches (PP) and spleen (SP). Surface phenotypic analysis of CD11c(+) DC populations revealed that PP DCs expressed higher levels of major histocompatibility complex class II molecules, but similar levels of costimulatory molecules and adhesion molecules compared with SP DCs. Freshly isolated, flow cytometrically sorted 98-100% pure CD11c(+) DC populations from PP and SP were compared for their ability to stimulate naive T cells. First, PP DCs were found to be much more potent in stimulating allogeneic T cell proliferation compared with SP DCs. Second, by using naive T cells from ovalbumin peptide-specific T cell receptor transgenic mice, these ex vivo DCs derived from PP, but not from SP, were found to prime for the production of interleukin (IL)-4 and IL-10 (Th2 cytokines). In addition, PP DCs were found to prime T cells for the production of much lower levels of interferon (IFN)-gamma (Th1) compared with SP DCs. The presence of neutralizing antibody against IL-10 in the priming culture dramatically enhanced IFN-gamma production by T cells stimulated with PP DCs. Furthermore, stimulation of freshly isolated PP DCs via the CD40 molecule resulted in secretion of high levels of IL-10, whereas the same stimulus induced no IL-10 secretion from SP DCs. These results suggest that DCs residing in different tissues are capable of inducing distinct immune responses and that this may be related to the distinct cytokines produced by the DCs from these tissues.

Figure 1

Phenotypic analysis of sorted DC populations from SP and PP. (A) Cells from PP or SP were isolated using magnetic beads as described in Materials and Methods, and were dual stained with the FITC-conjugated anti-B220 and PE-conjugated anti-CD11c antibodies. Cells that subsequently were sorted by FACS^® and used for T cell stimulation are indicated by the enclosed square (CD11c⁺/B220⁻). (B) RT-PCR analysis of B and T cell contamination in sorted DC populations. Magnetically selected DCs from SP and PP were further purified by flow cytometric sorting into either CD11c⁺/B220⁻ or CD11c⁺/B220⁺ cells. Total RNA was extracted from sorted DCs and analyzed by RT-PCR for expression of B (CD19) and T (CD3) cell markers. RT-PCR of β2m was included as a positive control for mRNA in each sample. An RT-PCR profile of total PP tissue was included to indicate that B and T cells were present in the starting population (right). (C) Exclusion of epithelial cells from sorted DC populations. CD11c⁺/B220⁻ cells from SP and PP were stained for the pan-epithelial cell marker, cytokeratin. In parallel, freshly isolated intestinal epithelial cells were stained with the same antibody (dotted line) as a positive control.

Figure 1

Phenotypic analysis of sorted DC populations from SP and PP. (A) Cells from PP or SP were isolated using magnetic beads as described in Materials and Methods, and were dual stained with the FITC-conjugated anti-B220 and PE-conjugated anti-CD11c antibodies. Cells that subsequently were sorted by FACS^® and used for T cell stimulation are indicated by the enclosed square (CD11c⁺/B220⁻). (B) RT-PCR analysis of B and T cell contamination in sorted DC populations. Magnetically selected DCs from SP and PP were further purified by flow cytometric sorting into either CD11c⁺/B220⁻ or CD11c⁺/B220⁺ cells. Total RNA was extracted from sorted DCs and analyzed by RT-PCR for expression of B (CD19) and T (CD3) cell markers. RT-PCR of β2m was included as a positive control for mRNA in each sample. An RT-PCR profile of total PP tissue was included to indicate that B and T cells were present in the starting population (right). (C) Exclusion of epithelial cells from sorted DC populations. CD11c⁺/B220⁻ cells from SP and PP were stained for the pan-epithelial cell marker, cytokeratin. In parallel, freshly isolated intestinal epithelial cells were stained with the same antibody (dotted line) as a positive control.

Figure 1

Phenotypic analysis of sorted DC populations from SP and PP. (A) Cells from PP or SP were isolated using magnetic beads as described in Materials and Methods, and were dual stained with the FITC-conjugated anti-B220 and PE-conjugated anti-CD11c antibodies. Cells that subsequently were sorted by FACS^® and used for T cell stimulation are indicated by the enclosed square (CD11c⁺/B220⁻). (B) RT-PCR analysis of B and T cell contamination in sorted DC populations. Magnetically selected DCs from SP and PP were further purified by flow cytometric sorting into either CD11c⁺/B220⁻ or CD11c⁺/B220⁺ cells. Total RNA was extracted from sorted DCs and analyzed by RT-PCR for expression of B (CD19) and T (CD3) cell markers. RT-PCR of β2m was included as a positive control for mRNA in each sample. An RT-PCR profile of total PP tissue was included to indicate that B and T cells were present in the starting population (right). (C) Exclusion of epithelial cells from sorted DC populations. CD11c⁺/B220⁻ cells from SP and PP were stained for the pan-epithelial cell marker, cytokeratin. In parallel, freshly isolated intestinal epithelial cells were stained with the same antibody (dotted line) as a positive control.

Figure 2

Surface phenotype analysis of sorted DC populations from SP and PP. Sorted CD11c⁺/B220⁻ DCs from SP or PP were analyzed for expression of various surface molecules. The results are shown as histograms with fluorescence intensity on the x-axis and cell number on the y-axis. The thin lines represent staining of SP DCs, and the thick lines staining of PP DCs. Isotype-matched control is indicated for each antibody set with a dotted line. The data depicted here represents four independent experiments producing similar results.

Figure 3

DCs from PP are more potent stimulators of allogeneic T cell proliferation than are those from SP. Mixed lymphocyte reaction was carried out with varying numbers of purified BALB/c DCs (H-2^d) and 10⁵ T cells from B10.A (H-2^k) mice per well in 96-well microtiter plates. Proliferation was measured by [³H]thymidine uptake during the last 8 h of a 48-h culture. Results are represented as the mean cpm of triplicate cultures on the y-axis, with the number of DCs per well on the x-axis. Similar results were obtained in three separate experiments conducted in the same manner.

Figure 4

PP DCs induced higher expansion of OVA TCR transgenic T cells during primary and secondary culture than did SP DCs. (A) Naive CD4⁺/MEL14⁺ FACS^®-sorted OVA TCR transgenic T cells (5 × 10⁴ per well) were coincubated with CD11c⁺ DCs sorted from PP or SP (5 × 10³ per well) for 5–6 d in the presence of blocking antibodies against TGF-β, IL-10, or isotype control antibody. Live cells were counted using hemacytometer by exclusion of dead cells with Trypan blue staining. The figure depicts the factor by which the number of T cells multiplied during priming by either PP or SP DCs on the y-axis. (B) OVA TCR transgenic T cells primed with DCs as described in A were restimulated with plate-bound anti-CD3∈ and soluble anti-CD28 antibodies for 48 h. Proliferation of T cells was determined by incorporation of [³H]thymidine, which was added to the culture wells during the last 8 h of incubation. Data is representative of four separate experiments producing similar results.

Figure 4

PP DCs induced higher expansion of OVA TCR transgenic T cells during primary and secondary culture than did SP DCs. (A) Naive CD4⁺/MEL14⁺ FACS^®-sorted OVA TCR transgenic T cells (5 × 10⁴ per well) were coincubated with CD11c⁺ DCs sorted from PP or SP (5 × 10³ per well) for 5–6 d in the presence of blocking antibodies against TGF-β, IL-10, or isotype control antibody. Live cells were counted using hemacytometer by exclusion of dead cells with Trypan blue staining. The figure depicts the factor by which the number of T cells multiplied during priming by either PP or SP DCs on the y-axis. (B) OVA TCR transgenic T cells primed with DCs as described in A were restimulated with plate-bound anti-CD3∈ and soluble anti-CD28 antibodies for 48 h. Proliferation of T cells was determined by incorporation of [³H]thymidine, which was added to the culture wells during the last 8 h of incubation. Data is representative of four separate experiments producing similar results.

Figure 6

SP DCs induce higher IFN-γ secretion by PCC TCR transgenic T cells than do PP DCs. Naive PCC TCR transgenic T cells were stimulated with purified CD11c⁺ DCs isolated from SP (A) or PP (B) in the presence of blocking antibodies against TGF-β, IL-10, or isotype control antibody for 5 d. T cells were restimulated with plate-bound anti-CD3∈ and soluble anti-CD28 antibodies for up to 48 h. Supernatants were harvested and IFN-γ level was measured by ELISA at 48 h. The P values for cytokines secreted in the presence or absence of neutralizing antibody in the priming cultures are indicated either as a number or an asterisk if P > 0.05. The same experiment was repeated three times producing similar results.

Figure 5

Cytokine production by OVA TCR transgenic T cells during secondary stimulation. Naive CD4⁺/MEL14⁺ FACS^®-sorted OVA TCR transgenic T cells were stimulated with purified CD11c⁺ DCs isolated from SP (A, B, and C) or PP (D, E, and F) in the presence of blocking antibodies against TGF-β, IL-10, or isotype control antibody for 5–6 d. T cells were restimulated with plate-bound anti-CD3∈ and soluble anti-CD28 antibodies for up to 48 h. Supernatants were harvested and IFN-γ (A and D), IL-4 (B and E), and IL-10 (C and F) levels were measured by ELISA at 24 (IL-4) or 48 h (IFN-γ and IL-10). The P values for cytokines secreted in the presence or absence of neutralizing antibody in the priming cultures are indicated with value labels or with an asterisk if P > 0.05. This experiment was repeated four times producing similar cytokine secretion patterns.

Figure 7

Expression of TGF-β by freshly isolated PP DCs. cDNA samples prepared from freshly isolated PP and SP DCs from BALB/c (A) or B10.A (B) mice were analyzed by competitive RT-PCR for the expression of TGF-β and a control marker, β2m, as described in Materials and Methods. The ratios of TGF-β mRNA level to β2m mRNA level derived from the same cDNA sample are depicted on the y-axis as the mean values of at least three separate experiments ± SEM. The number of separate experiments done for each DC population are indicated in parentheses.

Figure 8

IL-10 is produced only by PP DCs after CD40L trimer stimulation, whereas similar levels of IL-12 p40 is detected from both DC types. FACS^®-purified CD11c⁺ DCs (10⁵ per well) from BALB/c (A and B) or B10.A (C and D) mice were incubated overnight in the presence of CD40L trimer (10 μg/ml). Supernatants were collected and IL-10 (A and C) and IL-12 p40 (B and D) levels were measured by ELISA. Each column is representative of four (A and B) or two (C and D) separate experiments.

Figure 8

IL-10 is produced only by PP DCs after CD40L trimer stimulation, whereas similar levels of IL-12 p40 is detected from both DC types. FACS^®-purified CD11c⁺ DCs (10⁵ per well) from BALB/c (A and B) or B10.A (C and D) mice were incubated overnight in the presence of CD40L trimer (10 μg/ml). Supernatants were collected and IL-10 (A and C) and IL-12 p40 (B and D) levels were measured by ELISA. Each column is representative of four (A and B) or two (C and D) separate experiments.