Dendritic cells derived from metastatic cancer patients vaccinated with allogeneic dendritic cell-autologous tumor cell hybrids express more CD86 and induce higher levels of interferon-gamma in mixed lymphocyte reactions

Abstract

Dendritic cells (DCs) are highly effective antigen-presenting cells that, when derived from cancer patients, seem to be functionally deficient. Herein, we show that vaccination with allogeneic DC-autologous tumor cell hybrids affects the phenotype and improves the function of monocyte-derived DCs (Mo-DCs) from cancer patients. Mononuclear cells were isolated from patients' peripheral blood by density gradient centrifugation, and adherent cells were cultured in medium containing GM-CSF plus IL-4 and, after 5 days, TNF-alpha. After 2 more days, Mo-DCs were harvested and their CD80, CD86, and CD83 expression was assessed by flow cytometry. They were also used as stimulators in mixed lymphocyte reactions (MLR), where IFN-gamma production was measured by ELISA. Mo-DCs from unvaccinated patients expressed significantly lower levels of CD86, and tended to express lower levels of CD83 than Mo-DCs from healthy donors. However, Mo-DCs generated after hybrid cell vaccination presented increased expression of the same markers and induced significantly higher levels of IFN-gamma in MLR. These results indicate that the use of allogeneic DC-based cancer vaccines induces recovery of DC function in metastatic cancer patients and, therefore, could precede the use of autologous DCs for vaccine preparation. Such an approach could be relevant and should be investigated in clinical trials.

Fig. 1

Immunophenotype of monocyte-derived DCs generated in vitro from cells of patients or from healthy individuals. Peripheral blood monocytes from eight cancer patients and eight healthy individuals were cultured in the presence of GM-CSF plus IL-4 and activated with TNF-α on the 5th day of culture. On the 7th day, cells were harvested, and CD80, CD86, and CD83 surface expression was assessed by flow cytometry. Bars represent the mean % of positive cells for each marker; p values (healthy vs patients) were CD86, p=0.04; CD83, p=0.14; CD80, p=0.92.

Fig. 2

Costimulatory molecules expression by monocyte-derived DCs before and after vaccination. Two representative experiments of CD80 and CD86 expression by DCs generated from blood monocytes obtained from metastatic cancer patients before or after vaccination with allogeneic DC–autologous tumor cell hybrids.

Fig. 3

Immunophenotype of monocyte-derived DCs generated in vitro from cells of cancer patients before or after hybrid cell vaccination. Blood monocytes obtained before or after vaccination of six patients were cultured in the presence of GM-CSF plus IL-4 and activated with TNF-α on the 5th day of culture. On the 7th day, cells were harvested, and CD80, CD86, and CD83 surface expression was assessed by flow cytometry. Bars represent the mean % of positive cells for each marker; p values (before vs after): CD80, p=0.34; CD86, p=0.03; CD83, p=0.06.

Fig. 4

IFN-γ production in mixed lymphocyte reactions stimulated with monocyte-derived DCs. DCs generated in vitro from cells of five healthy individuals and from three cancer patients before or after vaccination were used as stimulators for allogeneic lymphocytes in MLR. Cells were cultured for 5 days, after which their supernatants were collected and assayed for the presence of IFN-γ by ELISA. Means ± SE are represented (p<0.05).