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

Abstract

Colorectal cancer (CRC) is the third most frequent cancer globally, accounting for approximately 935,000 deaths per year, and has been ranked as the second major cause of cancer deaths in 2020. Immunotherapy using dendritic cells (DCs) is an effective therapeutic strategy for a variety of malignancies. Nevertheless, the immunosuppressive mechanisms in the tumor milieu, e.g., inhibitory immune checkpoint molecules have been implicated in diminishing the efficacy of DC-mediated anti-tumoral immune responses. Among the inhibitory immune checkpoints, cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) expression on DCs diminishes their maturation and antigen presentation capability. The aim of this study was to investigate the effect of CTLA-4 silenced colorectal tumor lysate pulsed DCs on the T-cell mediated responses following DC/T cell co-culture. Materials and methods After obtainment of peripheral blood mononuclear cells (PBMCs) by fractionation over Ficoll gradients from donors' peripheral blood, monocytes were isolated due to their adherence to polystyrene surfaces. The monocytes were then converted to mature DCs using granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukine-4 (IL-4), and lipopolysaccharide (LPS). These cells were then pulsed with colorectal tumor cell lysate (mDCs) and the expression of CTLA-4 in them was silenced via siRNA (CTLA-4 silenced mDCs). The effect of CTLA-4 silenced mDCs on the proliferation of T cells was evaluated by flow cytometry. The impact of CTLA-4 silenced mDCs on the production of cytokines by T-lymphocytes was assessed via ELISA. Results Our results demonstrated that scilencing of CTLA-4 could promote stimulatory properties of DCs in the stimulation of T cell responses. CTLA-4-silenced mDCs showed a higher capacity to stimulate CD3+ T cell proliferation than mDCs. Co-cultures of autologous T cells and CTLA-4-silenced mDCs resulted in considerably higher IFN-γ and IL-4 levels than T cell/ mDCs co-culture.