P2.01-055 Lymphocytes' Subtypes Differentiation after Stimulation with Synthetic Antigen-Pulsed Dendritic Cells in Lung Adenocarcinoma Patients

Abstract

Immunotherapy in lung cancer is currently experiencing huge interest. The greatest efficacy demonstrated the antibodies against immune checkpoints receptors (PD-L1, CTLA-4 or LAG-3). However, an effective anti-tumor response also required tumor antigen presentation to lymphocytes in peripheral lymph nodes. To enhance this effect, vaccination with synthetic tumor antigen was conducted in many clinical trials using antigen presenting cells (APC). Unfortunately, this type of active immunotherapy has not achieved spectacular success (START or MAGRIT studies). The explanation could lie in distorted presentation of tumor antigens in peripheral lymph nodes or in activation of lymphocytes with strong immunosuppressive properties. In our study, we checked which pathways of T helper differentiation are favored by autologous dendritic cells (DCs) after synthetic tumor antigens stimulation. Peripheral blood was acquired from twenty unresectable and treatment-naïve lung adenocarcinoma patients. Using CD14 magnetic beads, two cell populations were isolated. CD14-negative cells were used for CD3-positive cells isolation, which were frozen until further stimulation. CD14-positive cells were cultured in CellDCGrow medium supplemented with IL-4 and GM-CSF. After TNF-a stimulation, mature DCs were incubated with tumor-specific antigens: MUC1, MAGE-A3, EGFR and CMV (positive stimulator) or only with medium (negative control). Flow cytometry and specific monoclonal antibodies were used to analyze immunophenotype of DCs: CD1a, CD11c, CD83, CD80, CD86, B7-H1 (PD-L1), B7-DC (PD-L2). Mature autologous DCs were cultured with fraction of CD3-positive cells and after 48h of mixed cultures, the expression of intracellular factors specific for different T helper cells subpopulation (T-bet – Th1, STAT-6 – Th2, ROR-gT – Th17, FoxP3 – Treg) and expression of extracellular interleukin receptors (IL-12R, IL-4R, IL-23R, TGF-bR, IL-2R) were analyzed. Autologous DCs were well generated and showed phenotype specific for fully-mature DCs. In culture with DCs after MUC1 stimulation, we observed in lymphocytes the highest expression of ROR-gT and IL-23R (p<0.05) compared with lymphocytes from cultures with other synthetic tumor antigens. Expression of IL-4R on lymphocytes was also significantly (0<0.05) higher in the culture stimulated with MUC1. The highest expression of intracellular FoxP3 factor, TGF-bR (p<0.05) and IL-2R (p=0.009) on Th cells were observed in mixed culture with DCs after MAGE-A3 stimulation. Dendritic cells stimulated with synthetic tumor antigens could activate different T helper cell populations. It could depend on the nature of antigens and could influence the effectiveness of stimulation different lymphocyte subtypes in peripheral lymph nodes. Therefore, our study clarifies some failure reasons of large clinical trials using active antigen-specific immunotherapy.