Investigation of the combined effect of immunotherapy with DCs pulsed with breast tumor cell lysates and CTLA-4 inhibitor on T cell response

Abstract

The use of conventional methods to treat breast cancer is associated with limitations such as serious side effects, drug resistance, drug non-specificity, and cancer recurrence in distant areas. Dendritic cells isolated from breast cancer patients can be activated in laboratory conditions with target tumor antigens and after activation, they can be re-injected into patients to activate or strengthen immune responses. This method effectively initiates cytotoxic responses against tumor antigens. CTLA-4 is one of the inhibitory receptors on the dendritic cells derived from monocytes (mDC), which causes dysfunction of these cells in the tumor microenvironment. Therefore, by using monoclonal antibody or siRNA, its expression level can be reduced and the performance of these dendritic cells can be improved to some extent. siRNAs are double-stranded RNA molecules that can inhibit the expression of target mRNA by binding to it. In this study, the aim is to pulse dendritic cells with breast tumor lysate and inhibit the expression of this receptor on dendritic cells derived from monocytes by using CTLA-4 inhibitors and then check the effect of this inhibition on T-lymphocytes. Methods After isolating peripheral blood mononuclear cells (PBMCs) using Faicol's solution from peripheral blood of donors, monocytes were separated by their adhesion to polystyrene surfaces. Then monocytes were transformed into mature DCs using granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-4 (IL-4), and lipopolysaccharide (LPS). Then these cells were inhibited with breast tumor lysate (mDC) and expression of CTLA-4 in them using siRNA (CTLA-4 silenced mDCs). Flow cytometry method was used to investigate the effect of co-culture of CTLA-4 silenced mDCs with T cells on the proliferation rate of these lymphocytes. In order to investigate the effect of CTLA-4 silenced mDCs on the amount of cytokine production from T lymphocytes, the ELISA method was used. Result Inhibition of CTLA-4 expression in mDCs significantly improved their maturation and stimulatory activity. CTLA-4 silenced mDCs had a higher capacity to stimulate the proliferation of CD3+ T cells than mDCs. co-culture of autologous T cells and CTLA-4 silenced mDCs resulted in higher levels of IFN-γ and IL-4 production from T cells than T cells/mDCs co-culture.