An autologous dendritic cell vaccine polarizes a Th-1 response which is tumoricidal to patient-derived breast cancer cells

Abstract

Breast cancer remains one of the leading causes of cancer-associated death worldwide. Conventional treatment is associated with substantial toxicity and suboptimal efficacy. We, therefore, developed and evaluated the in vitro efficacy of an autologous dendritic cell (DC) vaccine to treat breast cancer. We recruited 12 female patients with stage 1, 2, or 3 breast cancer and matured their DCs with autologous tumour-specific lysate, a toll-like receptor (TLR)-3 and 7/8 agonist, and an interferon-containing cocktail. The efficacy of the vaccine was evaluated by its ability to elicit a cytotoxic T-lymphocyte response to autologous breast cancer cells in vitro. Matured DCs (≥ 60% upregulation of CD80, CD86, CD83, and CCR7) produced high levels of the Th1 effector cytokine, IL12-p70 (1.2 ng/ml; p < 0.0001), compared to DCs pulsed with tumour lysate, or matured with an interferon-containing cocktail alone. We further showed that matured DCs enhance antigen-specific CD8 + T-cell responses to HER-2 (4.5%; p < 0.005) and MUC-1 (19%; p < 0.05) tetramers. The mature DCs could elicit a robust and dose-dependent antigen-specific cytotoxic T-lymphocyte response (65%) which was tumoricidal to autologous breast cancer cells in vitro compared to T-lymphocytes that were primed with autologous lysate loaded-DCs (p < 0.005). Lastly, we showed that the mature DCs post-cryopreservation maintained high viability, maintained their mature phenotype, and remained free of endotoxins or mycoplasma. We have developed a DC vaccine that is cytotoxic to autologous breast cancer cells in vitro. The tools and technology generated here will now be applied to a phase I/IIa clinical trial.

Keywords: Autologous primary breast cancer cells; Breast cancer; Dendritic cell vaccine; Immunotherapy; Tumour lysate.

Fig. 1

Patient recruitment plan for the DC vaccine breast cancer preclinical trial. Two hundred and twenty-four patients were asked to consent to the study. Two hundred and three patients were not included in the study because they either declined or they did not meet the inclusion criteria. Of the remaining 21 patients who consented to the study a further 9 were excluded or withdrew from the trial. In total 12 patients were included in the current preclinical trial

Fig. 2

DCs from patients with breast cancer, pulsed with tumour-specific lysate and matured with Ampligen®, an IFN-containing cocktail and R848 and or with IFN-containing cocktail only express higher levels of co-stimulatory molecules compared to immature DCs or DCs pulsed with tumour-specific lysate only. Immature DCs were differentiated from monocytes, incubated in CellGenix DC complete medium with or without 100 µg/mL tumour-specific lysate for 6 h at 37 °C. The cells were then matured with or without or in combination with 100 μg/ml Ampligen®, an IFN-containing cocktail (10 ng/mL IFN-α, 25 ng/mL IFN-γ, 1 µg/mL CD40L and 10 ng/mL IL-1β), and/or 2.5 µg/mL R848 for 42 h at 37 °C. The monocytes, immature DCs and mature DCs were subjected to a haematoxylin and eosin stain (a) or were stained with CD14 PE-CY7 (monocytes), CD40 FITC (immature and mature DCs) and or CD83 APC (mature DCs; A) for confocal microscopy. The maturation phenotype was also determined by flow cytometry (b). Arrows (→) show dendrites being expressed on the surface of mature DCs. Data were analysed for statistical significance by one-way Anova with Dunnett’s post-test, where **, *** and **** indicate p < 0.01, p < 0.005 and p < 0.0001, respectively. Each of the treatments were compared to the control group (iDCs). Error bars represent standard deviation. Light microscopy magnification: ×100 (oil immersion); scale bars = 20 µm. Confocal magnification: ×63 (oil immersion); scale bars = 10 µm. iDCs = immature DCs

Fig. 3

DCs pulsed with tumour-specific lysate and matured with Ampligen®, an IFN-containing cocktail and R848 express higher levels of the Th1 effector cytokines, IL-12p70, compared to the immature DCs (iDCs) or DCs pulsed with tumour-specific lysate only. The level of IL-12p70 from the culture supernatants of the immature DCs or matured DCs was determined using the ELIZAPRO IL-12p70 detection kit from Mabtech as indicated by the manufacturer. Data were analysed for statistical significance by one-way Anova with Dunnett’s post-test or Wilcoxon signed rank paired t test, *, ***, **** indicate p < 0.05, p < 0.005 and p < 0.0001, respectively. For Anova each of the treatments were compared to the control group (iDCs). Error bars represent standard deviation

Fig. 4

The TCRs of CD8 + T-cells primed with tumour-specific lysate/Ampligen®/IFN-cocktail and R848-matured DCs can recognise HER-2 and MUC-1-specific tetramers. Immature DCs were differentiated from monocytes as indicated previously. DCs were matured and effector cells were generated as indicated in the methods. The cells were stained with CD8 FITC, CD3 PerCP/Cy5.5, HER-2 APC, MUC-1 PE and Zombie NIR according to the manufacturer’s instructions (MBL, USA). The levels of HER-2 and MUC-1 recognised by the TCR on CD8 + T-cells were determined by flow cytometry. Data were analysed for statistical significance by one-way Anova with Dunnett’s post-test where *, *** indicate p < 0.05 and p < 0.005, respectively. Each of the treatments were compared to the control group (T-cells primed with IFN-cocktail matured DCs not stained with HER-2/MUC-1 tetramer). Error bars represent standard deviation

Fig. 5

PBMCs from breast cancer patients co-cultured with tumour-specific lysate pulsed and Ampligen®/IFN-containing cocktail/R848-matured DCs results in cytotoxic T-lymphocyte-mediated killing of primary breast cancer cells in vitro. Matured DCs were prepared as indicated in the methods. The matured DCs were then co-cultured with PBMC at a ratio of 1:10 for 7 days at 37 °C. The primary breast cancer cells were incubated with or without the primed PBMCs (effector cells) at a ratio of 1:10 (a and c) or the primary cells were incubated with the effector T-cells at various ratios indicated (b and d) for 4 h at 37 °C. Cytotoxicity (a and b) was determined using the LDH assay (Cytotoxicity Detection Kit^Plus LDH; Roche, Germany) and cell death (c and d) of the primary breast cancer cells was measured by flow cytometry. Data were analysed for statistical significance by one-way Anova with Dunnett’s post-test where *, ***indicate p < 0.05 and p < 0.005, respectively. Each of the treatments were compared to the control group (primary breast cancer cells incubated in the absence of PBMCs). Error bars represent standard deviation