Numerical and functional defects of blood dendritic cells in early- and late-stage breast cancer - PubMed (original) (raw)

Comparative Study

. 2007 Nov 5;97(9):1251-9.

doi: 10.1038/sj.bjc.6604018. Epub 2007 Oct 9.

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Comparative Study

Numerical and functional defects of blood dendritic cells in early- and late-stage breast cancer

A Pinzon-Charry et al. Br J Cancer. 2007.

Abstract

The generation of antitumour immunity depends on the nature of dendritic cell (DC)-tumour interactions. These have been studied mostly by using in vitro-derived DC which may not reflect the natural biology of DC in vivo. In breast cancer, only one report has compared blood DC at different stages and no longitudinal evaluation has been performed. Here we conducted three cross-sectional and one one-year longitudinal assessments of blood DC in patients with early (stage I/II, n=137) and advanced (stage IV, n=36) disease compared to healthy controls (n=66). Patients with advanced disease exhibit markedly reduced blood DC counts at diagnosis. Patients with early disease show minimally reduced counts at diagnosis but a prolonged period (1 year) of marked DC suppression after tumour resection. While differing in frequency, DC from both patients with early and advanced disease exhibit reduced expression of CD86 and HLA-DR and decreased immunostimulatory capacities. Finally, by comparing a range of clinically available maturation stimuli, we demonstrate that conditioning with soluble CD40L induces the highest level of maturation and improved T-cell priming. We conclude that although circulating DC are compromised by loco-regional and systemic breast cancer, they respond vigorously to ex vivo conditioning, thus enhancing their immunostimulatory capacity and potential for immunotherapy.

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Figures

Figure 1

Figure 1

Blood DC counts at diagnosis. (A) A representative dot plot of PBMC from a patient with breast cancer (stage II) where blood DC are identified in the elliptical gate as lineage-negative, HLA-DR positive cells. (B–D) Mononuclear cell population counts including (B) DC, (C) lymphocytes and (D) monocytes were assessed in a cohort of 49 patients with breast cancer (stage I, _n_=23; stage II, _n_=11 and stage IV, _n_=15) and compared with age-matched female controls (_n_=12). Patients with advanced disease were divided into patients previously (pIV, _n_=9) or newly diagnosed (nIV, _n_=6). (E) Blood DC (gated as in A) in patients with breast cancer can be further analysed for DC subsets within a CD11c/CD123 bivariate plot. A representative dot plot of a patient with stage II disease is shown. (F–H) Blood DC subsets were analysed in 43 patients to determine absolute counts of (F) CD11c+DC, (G) CD123+DC and (H) CD11c−CD123− immature cells. Absolute DC and DC subset counts are expressed as 106 l−1 and all other counts are expressed as 109 l−1. Shaded areas indicate normal reference ranges. Box plots with means, standard deviations and ranges are shown. Significant differences compared to controls are indicated as *P<0.05 and **P<0.01. All samples were analysed prior to therapy.

Figure 2

Figure 2

Blood DC counts follow-up. In a cohort of 40 patients with early-stage disease (stage I, _n_=26 and stage II, _n_=14), absolute counts of (A) DC, (B) lymphocytes, (C) monocytes, (D) neutrophils and (E) platelets were estimated at 6, 24 and 48 weeks post-surgery and compared to the pre-operative assessment (−2). (F–J) To assess the effect of the type of adjuvant therapy, the 40 patients were divided into those receiving hormone therapy (Tamoxifen, Tamox) and either radiotherapy (_n_=9; Radio), chemotherapy (_n_=11, Chemo) or combined therapy (_n_=20; Comb.). Three patients (_n_=3) not receiving hormone therapy (yet administered radiotherapy) were also included for comparison. Subsequently, (F) blood DC, (G) lymphocyte, (H) monocyte, (I) neutrophil and (J) platelet counts were estimated for the 24-week point (immediately after completion of radio/chemotherapy) and compared to the pre-surgery (Pre-SX, −2 weeks) and pre-adjuvant therapy (Pre-AT, 6 weeks) assessments. Note that Pre-AT corresponds to post-surgical evaluation. Absolute DC counts are expressed as 106 l−1 and all other counts expressed as 109 l−1. Shaded areas indicate normal reference ranges. Box plots with means, standard deviations and ranges are shown. Significant differences compared to pre-operative are shown as *P<0.05, **P<0.01 and ***P<0.001.

Figure 3

Figure 3

Blood DC exhibit reduced immunostimulatory phenotype. (A) In a cohort of 40 patients with breast cancer (stage I, _n_=19; stage II, _n_=12 and stage IV, _n_=9), the level of expression of (A) CD86 and (B) HLA-DR was analysed according to the stage of disease and compared with controls (_n_=14). Shaded areas indicate normal reference ranges and box plots show means, standard deviations and ranges. (C and D) Antigen uptake of soluble (FITC-TT) and particulate (FITC-Dextran) antigens by blood DC from patients with early (stage II, _n_=7) and advanced (stage IV, _n_=4) disease compared to controls (_n_=7) is presented as the difference in mean fluorescence intensity. (E) Allo-stimulatory capacity of blood DC as tested by mixed leucocyte reaction (MLR). One representative experiment in which increasing numbers of blood DC purified from patients with early (stage II, _n_=2) or advanced (_n_=2) disease and a healthy control (_n_=1) were tested against allogeneic T cells purified from a panel (_n_=3) of healthy volunteers. The pairs giving maximal responses are shown as means of triplicate measurements. (F) Summary of proliferation data from MLRs using DC from patients with early (stage II, _n_=6) and advanced (stage IV, _n_=3) disease compared to controls (_n_=13) at a 1 : 30 DC:T ratio. Similar results were found for all DC:T ratios. (G) Summary of IFN-γ secretion in culture supernatants collected from MLRs at a 1 : 30 DC:T ratio as assessed by ELISA. Error bars correspond to s.e.m. Significant differences compared to controls are shown as *P<0.05, **P<0.01 and ***P<0.001. All samples were analysed prior to therapy.

Figure 4

Figure 4

Effect of in vitro conditioning on blood DC from early disease. The expression of (A) CD86, (B) HLA-DR and (C) IL-12 was determined by flow cytometry following incubation (24 h) in the absence or presence of a cytokine cocktail (CC), synthetic double-stranded RNA (poly I:C, PIC) or CD40L, in blood DC from patients with early-stage breast cancer (stage II, _n_=8, black bars) and controls (_n_=6, grey bars). In parallel, (D) IL-12 p70 secretion was determined by ELISA in culture supernatants. Unstimulated or stimulated cells were cocultured for 5 days with 105 allogeneic T cells from a panel of healthy donors (_n_=3). (E) Proliferation was determined by thymidine incorporation and (F) IFN-γ secretion by ELISA. The pairs giving maximal responses are plotted. Control (grey bars) and patients (black bars) values are presented as mean and s.e.m. Statistically significant differences between stimulated (CC, PIC, CD40L or PIC plus CD40L) and fresh samples are indicated as *P<0.05, **P<0.01 and ***P<0.001. Significant differences between breast cancer and control samples are indicated as #P<0.05, ##P<0.01 and ###P<0.001. All samples were analysed prior to therapy.

Figure 5

Figure 5

Effect of in vitro conditioning on blood DC from advanced disease. Response of blood DC to pro-inflammatory factors. Phenotypic maturation in blood DC from patients with advanced breast cancer (stage IV, _n_=5) was evaluated by assessing the expression of costimulatory molecules CD40, CD80, CD83 and CD86 and secretion of IL-12 following incubation (24 h) with a cytokine cocktail (CC), CD40 ligand (CD40L) or pathogen-derived factors, including ligands for TLR4 (lipopolysaccharide, LPS), TLR3 (polyI:C, PIC) and TLR9 (CpG). Histograms indicate expression in the absence (filled) or presence (empty) of stimulation. Numbers indicate delta mean fluorescence intensity (ΔMFI, stimulated minus unstimulated cells). Data are from two patients with advanced disease and are representative of five patients who were assessed. All samples were analysed prior to therapy.

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