Dendritic cells are defective in breast cancer patients: a potential role for polyamine in this immunodeficiency

Abstract

Introduction: Dendritic cells (DCs) are antigen-presenting cells that are currently employed in cancer clinical trials. However, it is not clear whether their ability to induce tumour-specific immune responses when they are isolated from cancer patients is reduced relative to their ability in vivo. We determined the phenotype and functional activity of DCs from cancer patients and investigated the effect of putrescine, a polyamine molecule that is released in large amounts by cancer cells and has been implicated in metastatic invasion, on DCs.

Methods: The IL-4/GM-CSF (granulocyte-macrophage colony-stimulating factor) procedure for culturing blood monocyte-derived DCs was applied to cells from healthy donors and patients (17 with breast, 7 with colorectal and 10 with renal cell carcinoma). The same peroxide-treated tumour cells (M74 cell line) were used for DC pulsing. We investigated the effects of stimulation of autologous lymphocytes by DCs pulsed with treated tumour cells (DC-Tu), and cytolytic activity of T cells was determined in the same target cells.

Results: Certain differences were observed between donors and breast cancer patients. The yield of DCs was dramatically weaker, and expression of MHC class II was lower and the percentage of HLA-DR-Lin- cells higher in patients. Whatever combination of maturating agents was used, expression of markers of mature DCs was significantly lower in patients. Also, DCs from patients exhibited reduced ability to stimulate cytotoxic T lymphocytes. After DC-Tu stimulation, specific cytolytic activity was enhanced by up to 40% when DCs were from donors but only up to 10% when they were from patients. IFN-gamma production was repeatedly found to be enhanced in donors but not in patients. By adding putrescine to DCs from donors, it was possible to enhance the HLA-DR-Lin- cell percentage and to reduce the final cytolytic activity of lymphocytes after DC-Tu stimulation, mimicking defective DC function. These putrescine-induced deficiencies were reversed by treating DCs with all-trans retinoic acid.

Conclusion: These data are consistent with blockade of antigen-presenting cells at an early stage of differentiation in patients with breast cancer. Putrescine released in the microenvironmement of DCs could be involved in this blockade. Use of all-trans retinoic acid treatment to reverse this blockade and favour ex vivo expansion of antigen-specific T lymphocytes is of real interest.

Figure 1

Phenotype of cells collected after immature dendritic cell preparation procedure in cancer patients. Peripheral blood mononuclear cells from healthy donors (n = 11) or patients with colorectal cancer (n = 7), renal cell carcinoma (n = 10), or breast cancer (n = 15) were depleted of lymphocytes (2-hour adherence step) and cultured for 7 days in the presence of granulocyte–macrophage colony-stimulating factor and IL-4. Data are expressed as the percentage of cells (with standard error) expressing the HLA-DR⁺Lin^- and HLA-DR^-Lin^- phenotype. *P < 0.01 versus healthy donors.

Figure 2

Maturation of dendritic cells (DCs) from healthy donors or from breast cancer patients. Data are expressed as the percentage of the cells (with standard error) expressing the CD80, CD83 and CD86 surface markers after treatment of immature DCs with a combination of maturating agents: (a) tumour necrosis factor (TNF)-α/lipopolysaccharide (LPS)/CD40L (n = 3); (b) IL-1β/IL-6/TNF-α/prostaglandin (PG)E₂ (n = 4–5); and (c) Ribomunyl^®/Imukin^® (n = 3). ^aDifferent from corresponding donors in each individual assay.

Figure 3

Cytolytic activity of lymphocytes from healthy donors or breast cancer patients against the M74 cell line. Lymphocytes are from donors (n = 5) or from cancer patients (n = 6), and were stimulated with autologous immature dendritic cells (DCs) pulsed with peroxide-treated M74 cells (DC-Tu_per). Controls are nonstimulated lymphocytes (NSL). Values are expressed as cytolytic activity (with standard error) against M74 target cell line. *P < 0.03 versus NSL; ^†P < 0.05 versus corresponding donors.

Figure 4

Effect of putrescine and all-trans retinoic acid (ATRA) on immature dendritic cell (DC) phenotype. Cells were collected after immature DC preparation procedure (imm DC; n = 11) and treated with 10 mmol/l putrescine (Put; n = 10). To putrescine-treated DCs was added 1 μmol/l ATRA (Put + ATRA; n = 5). Data are expressed as percentage of cells (with standard error) expressing the HLA-DR⁺Lin^- and HLA-DR^-Lin^- phenotypes. *P < 0.01 versus imm DCs; **P < 0.02 versus putrescine-treated imm DCs.

Figure 5

Cytolytic activity of lymphocytes stimulated with putrescine and all-trans retinoic acid (ATRA) treated dendritic cells (DCs). Immature DCs were from healthy donors and were treated with putrescine (Put) with or without ATRA before DCs pulsed with treated tumour cells (DC-Tu_per) preparation. Autologous lymphocytes were stimulated with DC-Tu_per, and data are expressed as cytolytic activity (with standard error) against M74 target cell line. Presented data are from seven different donors. Controls are nonstimulated lymphocytes (NSL). Decrease in M74 lysis was repeatedly observed for each of the donors in DC + Put compared with DC, and increased in DC + Put + ATRA as compared with DC + Put. *P < 0.05 versus NSL.