Interleukin-3 and granulocyte-macrophage colony-stimulating factor enhance the generation and function of dendritic cells

Abstract

Dendritic cells, well-known for their potent antigen-presenting activity, are generally present at very low frequency in the spleens of naive mice. We examined the ability of mice to generate functional dendritic cells (DC) following exposure to the cytokines interleukin-3 (IL-3) and granulocyte-macrophage colony-stimulating factor (GM-CSF). Tumours secreting these cytokines provided a continuous stimulus resulting in a greatly increased number and frequency of DC in the spleen. These cells were purified by conventional DC isolation techniques and were found to exhibit many of the characteristics of DC from unmanipulated mice, including high allo-stimulatory activity in mixed lymphocyte reactions and expression of many similar cell surface markers. Using ovalbumin-peptide specific class I- and class II-restricted hybridomas containing the lacZ reporter gene, we found that these cytokine-generated DC had a greatly increased efficacy in the uptake and processing of particulate antigen. These cells appear to have retained the ability to ingest antigen that is generally associated with immature DC, but also exhibit the peptide/major histocompatibility complex (MHC)-presenting capabilities of mature DC. Development of an assay to measure the activity of a single DC revealed that these dual activities were the properties of the majority of the cytokine-generated DC. These findings indicate that exposure in vivo to the cytokines IL-3 and GM-CSF can result in the generation of large numbers of DC with increased capability of stimulating T cells. Thus, these cells may be important in vivo in the process of cross-priming and the subsequent generation of tumour-reactive cytotoxic T lymphocytes (CTL).

Figure 1

Yield and frequency of DC generated in mouse spleens by in vivo delivery of IL-3 or GM-CSF increase significantly over untreated mice. Spleens of untreated CByB6F1/J or CByB6F1/J mice injected with 5×10⁵ parental B16.F0, GM-CSF transfected B16 (B16/GM-CSF), or IL-3 transfected B16 (B16/IL-3) tumour cells i.m. were harvested 10 days post tumour implantation and evaluated for increase in total cell number (a), total DC number (b), or frequency of DC (c). The frequency of DC was calculated as the total number of DC/total number of splenocytes for each condition. Data are expressed as the mean cell number±SD of four individual experiments.

Figure 2

Cytokine-enhanced and conventional DC show a similar capacity to stimulate naive T cells in a primary allogeneic MLR. DC isolated from B16/GM-CSF (▴), B16/IL-3 (♦), B16 (•) or non-tumour-bearing (▪) CByB6F1/J mice; or bulk splenocytes from allogeneic (○) or syngeneic (□) animals were irradiated with 20 Gy and graded doses of purified DC were incubated with 3×10⁵ allogeneic (C57BL/6) T cells and cultured for 3 days. [³H]TdR uptake was measured at 72 hr. Values are mean c.p.m. of triplicate wells ± SD of one experiment. Two separate experiments gave similar results.

Figure 3

Morphology and immunocytochemical analysis of conventional and cytokine-enhanced DC. Cytospin preparations of DC isolated from untreated (a, e, i), B16 (b, f, j), B16/IL-3 (c, g, k) or B16/GM-CSF (d, h, l), tumour-bearing CByB6F1/J mice were stained with Wright–Giesma (a–d), for class II (e–h) or the 2A1 antigen (i–l).

Figure 4

Flow cytometric analysis of cytokine-enhanced and conventional DC. DC from non-tumour-bearing, B16, B16/IL-3, or B16/GM-CSF tumour-bearing CByB6F1/J mice were isolated and stained for expression of B, T, macrophage, granulocyte, and DC-restricted antigens. EMA staining was also used to exclude any dead cells from analysis. The open histogram shows the isotype control staining. Note that the control used with the anti-CD11c was anti-CD3 as both are hamster antibodies. Each result is representative of at least three such analyses.

Figure 5

GM-CSF and IL-3 enhance class I and class II presentation of particulate and soluble antigen by mature splenic DC. DC (5×10⁴) were incubated with 5×10⁴ B3Z or BDZ in the presence of serial titrations of particulate OVA (closed symbols) or soluble OVA (open symbols) for 16–18 hr in 96-well plates. DC were isolated from non-tumour-bearing (□, ▪), or from B16 (○, •), B16/IL-3 (◊, ♦), or B16/GM-CSF (▵, ▴) tumour-bearing animals. One representative experiment of three is shown and data is presented as absorbance read at 570 nm.

Figure 6

Presentation of exogenous particulate antigen by cytokine enhanced DC is TAP-1/-2 dependent and occurs via the cytosolic pathway. DC were harvested from B16/IL-3 or B16/GM-CSF tumour-bearing C57BL/6 mice or TAP-1-deficient mice 10 days post tumour implantation. 5×10⁵ DC were incubated with 5×10⁵ B3Z in the presence of 5;μg/ml (0·11 μm) of particulate OVA for 16–18 hr in 24-well plates. Fixation and X-gal processing were performed as described in Materials and Methods. Data are presented as mean frequency (stimulated cells/5×10⁵ DC)±SD and are representative of three experiments.

Figure 7

Single-cell assay and presentation of antigen to multiple CD4 and CD8 T cells by single DC. (a) A single cytokine enhanced DC is shown (top) prior to the addition of T cells and following development of the assay (bottom). (b) DC isolated from B16 (open bars), B16/IL-3 (grey bars), or B16/GM-CSF (black bars) tumour-bearing CByB6F1/J mice were incubated with 10 μg/ml (.22 μm) of particulate OVA during overnight transient adherence incubation and counted by haemocytometer. 96-well U-bottom plates were seeded at 1 OVA-bead containing cell/well in the presence of excess B3Z (class I) or BDZ (class II) reporter cells. The total number of activated hybridoma cells in wells containing single antigen-containing DC was counted. Fixation and X-gal processing were performed as described in Materials and Methods. Each value represents the mean (± SD) of 20 wells in a representative experiment of three.