Spontaneous apoptosis of blood dendritic cells in patients with breast cancer

Abstract

Introduction: Dendritic cells (DCs) are key antigen-presenting cells that play an essential role in initiating and directing cellular and humoral immunity, including anti-tumor responses. Due to their critical role in cancer, induction of DC apoptosis may be one of the central mechanisms used by tumors to evade immune recognition.

Methods: Spontaneous apoptosis of blood DCs (lineage negative HLA-DR positive cells) was assessed in peripheral blood mononuclear cells (PBMCs) using Annexin-V and TUNEL assays immediately after blood collection. The role of tumor products was assessed by culturing cells with supernatants derived from breast cancer cell lines (TDSN) or PBMCs (PBMC-SN, as a control). The capacity of DC stimulation to prevent apoptosis was assessed by incubating DC with inflammatory cytokines, poly I:C, IL-12 or CD40 ligand (CD40L) prior to culture with TDSN. Apoptosis was determined by flow cytometry and microscopy, and Bcl-2 expression determined by intracellular staining.

Results: In this study we document the presence of a significantly higher proportion of apoptotic (Annexin-V+ and TUNEL+) blood DCs in patients with early stage breast cancer (stage I to II; n = 13) compared to healthy volunteers (n = 15). We examined the role of tumor products in this phenomenon and show that supernatants derived from breast cancer lines induce apoptosis of blood DCs in PBMC cultures. Aiming to identify factors that protect blood DC from apoptosis, we compared a range of clinically available maturation stimuli, including inflammatory cytokines (tumor necrosis factor-alpha, IL-1beta, IL-6 and prostaglandin (PG)E2 as a cytokine cocktail), synthetic double-stranded RNA (poly I:C) and soluble CD40 ligand. Although inflammatory cytokines and poly I:C induced robust phenotypic maturation, they failed to protect blood DCs from apoptosis. In contrast, CD40 stimulation induced strong antigen uptake, secretion of IL-12 and protected blood DCs from apoptosis through sustained expression of Bcl-2. Exogenous IL-12 provided similar Bcl-2 mediated protection, suggesting that CD40L effect is mediated, at least in part, through IL-12 secretion.

Conclusion: Cumulatively, our results demonstrate spontaneous apoptosis of blood DCs in patients with breast cancer and confirm that ex vivo conditioning of blood DCs can protect them from tumor-induced apoptosis.

Figure 1

Spontaneous apoptosis of blood dendritic cells (DCs) in patients with breast cancer. Peripheral blood mononuclear cells isolated from patients with breast cancer (stage I to II; n = 13) and age-matched healthy volunteers (n = 15) were analyzed for apoptosis by flow cytometry. (a)In order to include all apoptotic cells and avoid debris, gating was set to include only 7-AAD negative events (R1) which were then visualized and re-gated on forward (FSC) and side scatter (SSC) characteristics (R2). Within this mononuclear cell population, blood DCs were identified as lineage negative HLA-DR positive cells (R3). Representative dot plots are shown. (b)Apoptosis in blood DCs from patients (black) and healthy volunteers (grey) was determined by two different methods, Annexin-V binding and TUNEL assays. In all experiments, each patient was tested in parallel with at least one healthy volunteer. Representative histograms are shown. (c)Summary of apoptosis data. The proportions of Annexin-V⁺ and TUNEL⁺ blood DCs estimated for all patients (black) and healthy volunteers (grey) are shown accordingly. Error bars indicate the standard error of the mean. Statistically significant differences between controls and patients are indicated as: *, p < 0.05; **, p < 0.01.

Figure 2

Apoptosis in blood dendritic cells (DCs) is induced by breast tumor-derived supernatants. (a)Apoptosis in blood DCs was determined by Annexin-V binding and TUNEL assays following culture (24 h) in the presence of 50% (v/v) peripheral blood mononuclear cell (PBMC)-derived supernatant (PBMC-SN) or tumor-derived supernatants (TDSN; MCF7, MB435, MA11 and SKBR3). Representative dot plots (SSC on y-axis versus apoptosis on x-axis) are shown with numbers indicating the percentage of apoptotic cells. (b)Summary of apoptosis data. The proportions of Annexin-V⁺ and TUNEL⁺ blood DCs represent the average ± standard error of the mean of five independent experiments for which statistically significant differences compared to PBMC-SN are shown: *, p < 0.05; **, p < 0.01; ***, p < 0.001. (c)For morphological analyses (original magnification times 1,000), 50 cells were assessed in three separate fields. In contrast to PBMC-SN, cultures incubated with TDSN (MA11) frequently demonstrated features of apoptotic death in blood DCs (reduction in cell volume and nuclear size, cytoplasmic blebbing, cell membrane convolutions and formation of apoptotic bodies). Three independent experiments with similar findings were performed.

Figure 3

CD40 stimulation protects blood dendritic cells (DCs) against tumor-derived supernatant (TDSN)-induced apoptosis. TDSN-induced apoptosis in blood DCs was determined by Annexin-V binding following incubation (24 h) in the presence or absence of: (a)a combination of inflammatory cytokines (tumor necrosis factor-α, IL-1β, IL-6 and prostaglanding (PG)E₂ as a cytokine cocktail (CC)); (b)synthetic double-stranded RNA (poly I:C); or (c)CD40 ligand (CD40L) prior to culture (24 h) with 50% (v/v) peripheral blood mononuclear cell-conditioned supernatant (PBMC-SN) or TDSN (MA11, MB435, MCF7 and SKBR3). Representative dot plots (SSC on y-axis versus apoptosis on x-axis) are shown with numbers indicating the percentage of apoptotic cells. Five independent experiments were performed for which p-values are indicated.

Figure 4

Protective effect of IL-12 on tumor-derived supernatant (TDSN)-induced apoptosis. (a)Expression of HLA-DR and co-stimulatory molecules (CD40, CD80, CD83 and CD86) as well as secretion of cytokines (tumor necrosis factor (TNF)-α, IL-10 and IL-12) was determined for blood DCs following stimulation (24 h) with a cytokine cocktail (CC), synthetic double-stranded RNA (poly I:C) or CD40 ligand (CD40L) as described in Materials and methods. Histograms indicate expression in the absence (shaded) or presence (non-shaded) of stimulation. Numbers indicate delta mean fluorescence intensity (ΔMFI, stimulated cells minus unstimulated cells) and are representative of five independent experiments. (b)TDSN-induced apoptosis in blood DCs was determined by Annexin-V binding following incubation (24 h) in the presence or absence of exogenous IL-12 prior to culture (24 h) with 50% (v/v) peripheral blood mononuclear cell-conditioned supernatant (PBMC-SN) or TDSN (MA11, MB435, MCF7 and SKBR3). Representative dot plots (SSC on y-axis versus apoptosis on x-axis) are shown with numbers indicating the percentage of apoptotic cells. Five independent experiments were performed for which the p-value is indicated.

Figure 5

Effect of tumor-derived supernatant (TDSN) and stimulation on expression of Bcl-2. (a)Expression of Bcl-2 in blood dendritic cells (DCs) was determined: ex vivo (Fresh); following culture (24 h) in the presence of 50% (v/v) peripheral blood mononuclear cell-conditioned supernatant (PBMC-SN) or TDSN (MA11); or following stimulation (24 h) with a cytokine cocktail (CC), synthetic double-stranded RNA (poly I:C), IL-12 or CD40 ligand (CD40L) prior (24 h) to culture with 50% (v/v) TDSN. Representative histograms (filled) are shown with numbers indicating the mean fluorescence intensity (MFI) of Bcl-2 expression. The unfilled histogram shows staining with isotype control. (b)Summary of Bcl-2 expression data. Results show the average ± standard error of the mean of five independent experiments for which statistically significant differences compared to fresh cells (*, p < 0.05; ***, p < 0.001) or after incubation with TDSN (^##, p < 0.01; ^###, p < 0.001) are indicated.