Pre-clinical assessment of autologous DC-based therapy in ovarian cancer patients with progressive disease

Abstract

Dendritic cell-based vaccines offer promise for therapy of ovarian cancer. Previous studies have demonstrated that oxidation of several antigens, including ovarian cancer cells, using hypochlorous acid strongly enhances their immunogenicity and their uptake and presentation by dendritic cells. The response of T cells and dendritic cells to autologous tumour from patients with active disease has not previously been investigated. Monocyte-derived dendritic cells were generated from patients with active disease and activated by co-culture with oxidised tumour cells and the TLR agonist poly I:C. The dendritic cells showed an activated phenotype, but secreted high levels of TGFβ. Co-culture of the antigen-loaded dendritic cells with autologous T cells generated a population of effector T cells that showed a low level of specific lytic activity against autologous tumour, as compared to autologous mesothelium. The addition of neutralising antibody to TGFβ in DC/T cell co-cultures increased the levels of subsequent tumour killing in three samples tested. Co-culture of monocytes from healthy volunteers with the ovarian cell line SKOV-3 prior to differentiation into dendritic cells reduced the ability of dendritic cells to stimulate cytotoxic effector cells. The study suggests that co-culture of dendritic cells with oxidised tumour cells can generate effector cells able to kill autologous tumour, but that the high tumour burden in patients with active disease may compromise dendritic cell and/or T cell function.

Fig. 1

Primary ovarian cancer cells isolated from patients with progressive disease express high levels of MHC class I, low levels of HLA-G and high levels of immunosuppressive cytokines. Primary ovarian cancer cells were obtained from the ascitic fluid of 14 ovarian cancer patients and analysed by FACS for the presence of MHC class I, CD44, E-cadherin and HLA-G. a shows mean fluorescence for each sample, together with a representative FACS profile of tumour cells from AD patient 2 co-stained with MHC I and HLA-G. Primary tumour cells were seeded at 1 × 10⁶/ml, cultured in vitro for 48 h and supernatants collected for cytokine analysis. b shows the concentration of IL-1β, IL-10, TGF-β, TNF-α and IL-6 in pg/ml in cell culture supernatants as determined by ELISA. Four samples secreted more than 2,000 pg/ml IL-6 and are shown as 1,500 pg/ml

Fig. 2

PIC enhances activity of DC loaded with oxidised tumour cells. Dendritic cells were generated from peripheral blood monocytes obtained from HLA*0201⁺ healthy donors and ovarian cancer patients in remission. The ovarian cancer cell line SKOV-3 was oxidised with HOCL and washed 3 times prior to use as an antigen source. Immature DC were incubated with oxidised tumour cells at a ratio of 1:1 and combinations of IFN-γ (10 ng/ml), MPL (200 ng/ml) and P:iC (50 μg/ml) for 24 h. Pulsed, matured DC were then washed and co-cultured with autologous CD3⁺ T cells for 7 days. Stimulated T cells were tested in chromium release assays for their ability to lyse SKOV-A2 target cells. Graph shows percentage of lysis of SKOV-A2 achieved with different DC maturation conditions using cells from three patients (Rem) and three healthy donors (HD), at an effector to target ratio of 100:1. The difference between unstimulated DC and PIC stimulated DC was significant, p < 0.01, paired T test, n = 6

Fig. 3

Differences between DC from patients with active disease and DC generated from healthy donors or patients in remission. Graphs show cytokine secretion (pg/ml in culture supernatants) and surface molecule expression (mean fluorescence) of DC generated from six healthy donors (HD), six patients in remission (Rem) and twelve patients with active disease (AD). Statistically significant differences between groups as calculated by unpaired, two-tailed T test are denoted by *p < 0.05 or **p < 0.01

Fig. 4

DC generated from patients with active disease stimulate effector cells with activity against primary ovarian cancer cells. DC were generated from patients with active disease and incubated with or without oxidised SKOV-3 cells and PIC. These DC were then used to stimulate autologous effector cells in vitro. a CD4 and CD8 expression on effector cells pre- and post-culture with DC. b After 1 week of in vitro stimulation, effector cells were tested in chromium release assays for activity against autologous tumour or mesothelium. The data are shown as individual bar charts for each patient representing the mean of triplicate values at an effector to target ratio of 100:1, with error bars showing standard deviation. The killing of tumour cells was significantly greater than of mesothelium (p < 0.01, two-way analysis of variance, n = 14). c Autologous tumour was compared to SKOV-3 as a source of oxidised antigen using cells from AD patients 2,3 and 4 and tested against autologous tumour (left panel c) or autologous mesothelium (right panel c). There were no significant differences between killing of autologous tumour generated by the two antigen sources

Fig. 5

Addition of anti-TGFβ to DC/T cell co-cultures enhances killing of primary autologous tumour. DC generated from active disease patients 11, 12 and 14 were incubated with oxidised SKOV-3 and PIC for 24 h prior to co-culture with autologous effector cells. During the culture period, anti-TGFβ or control antibody (mouse anti-influenza A nuclear antigen) were added to the co-cultures. Stimulated effector cells were then tested in chromium release assays for activity against autologous tumour or mesothelium as previously. Graphs show individual points for each patient (mean of triplicate values). Lysis of primary tumour was significantly higher (p < 0.05) by effector cells stimulated in the presence of anti-TGFβ antibody compared to control antibody as determined by one-tailed, paired T test. Levels of TGF-β (pg/ml) secreted by the different patient’s DC are shown alongside each data set

Fig. 6

Reduced cytotoxic killing after stimulation with oxidised tumour-loaded DC generated from monocytes pre-exposed to SKOV-3 tumour cells. Monocytes were isolated from three healthy donor PBMC samples and cultured below transwell inserts containing tumour cells (SKOV-3) or media alone for 24 h. Transwells were then removed, and ‘tumour-educated’ and control monocytes were differentiated into DC by culture in GM-CSF and IL-4 for 5 days. The resulting ‘tumour-educated’ (TE DC) and control DC were compared phenotypically and functionally. a Mean fluorescence for MHC II, CD86, DC-sign and CD1a. Differences between groups were not significant (p > 0.05). b ‘Tumour-educated’ and ‘control’ DC were co-cultured with autologous CD3⁺ T cells for 1 week. At the end of the culture period, stimulated T cells were tested in chromium release assays for their ability to lyse SKOV-A2 target cells. The mean values from three donors are shown, with error bars showing standard deviation. The killing by control DC plus oxidised SKOV-3 is significantly higher than by TE-DC plus oxidised SKOV-3 (p < 0.01, paired T test, n = 3)