Investigation of pyrimethamine treated monocyte-derived dendritic cells pulsed with breast cancer cell lysate effects on the responses of autologous peripheral blood mononuclear cells

Abstract

Dendritic cell (DC) immunotherapy is a successful treatment approach for several kinds of tumors. One of the DCs' inhibitory signaling pathways, signal transducer and activator of transcription (STAT3), reduces the cells' function and results in their tolerogenic state. Pyrimethamine is an anti-malaria medication that inhibits STAT3. In this experiment, we aim to pulse DCs with lysates of breast cancer cells and pyrimethamine to suppress the STAT3 protein and then assay their effect on the T-cell-mediated responses following DC/T cell co-culture. Materials and methods Monocytes were separated using the myeloid-derived suppressor cells (MACS) approach after peripheral blood mononuclear cells (PBMCs) from a donor's peripheral blood were fractionated along Ficoll gradients. Next, granulocyte-macrophage colony stimulating factor (GM-CSF), interleukin-4 (IL-4), and lipopolysaccharide (LPS) were used to transform the monocytes into mature DCs. In order to suppress the STAT3 protein, these cells were subsequently pulsed with breast cancer lysate (mDCs) and treated with pyrimethamine (Pyri-mDC). Using flow cytometry and real-time polymerase chain reaction (RT-PCR), the impact of pyrimethamine-induced STAT3 inhibition on DC maturation and activation was assessed. After co-culturing T cells with Pyri-mDC, the transcription factor and pattern of cytokine expression were evaluated using flow cytometry and RT-PCR. Results The expression of TNF- and IL-12 was increased in Pyri-mDCs compared to mDCs while the expression of IDO and TGF-β was reduced. As expected, the expression of GATA3, IL-4, FOXP3, and TGF-β was decreased, and the IFN-γ expression was increased in T cells isolated from the co-culture of T/Pyri-mDC in comparison with T cells separated from the co-culture of T/mDC.