Evaluation of T Cell Responses In The Co-Cultures Establıshed Wıth Neutrophıls, Monocytes, and Lung Adenocarcınoma Cells

Abstract

CD4+ and CD8+ T cells are critical mediators in anti-tumor immunity. Together with neutrophils, they have been shown to dominate the immune landscape of the non-small cell lung cancer (NSCLC). A number of studies have estimated the prognostic significance of neutrophil-to-lymphocyte ratio in NSCLC. However, immune regulatory role of neutrophils has not yet been fully elucidated. Hence, this study aims to evaluate T cell responses in the presence of neutrophils, monocytes and lung adenocarcinoma cell lines, in vitro. Peripheral blood neutrophils, CD14+ monocytes, and CD8+ or CD4+ T cells were purified from the healthy volunteers. In order to optimize the co-cultures, different combinations of these cell types were employed under various stimuli. Neutrophils were stimulated with different combinations of IFN-γ, G-CSF, N-acetylcysteine (NAC), and N-Formylmethionine-leucyl-phenylalanine (fMLP). At different ratios, pre-stimulated or freshly isolated neutrophils were co-cultured with NSCLC cell lines media (A549, NCI-H1299, or NCI-H441) or their conditioned with or without monocytes. Soluble anti-human CD3 mAb was added in order to test the antigen-independent influences on T cells. Proliferation, viability, activation, ROS production capacity, cytokine secretion, and expression of co-stimulatory molecules were tested on specific cell types. Based on TIM-3 expression, CD8+ T cells were recovered from the co-cultures and re-stimulated to test whether the TIM-3mod/high subpopulation is exhausted. G-CSF and NAC stimulation promoted the PMN longevity, diminished the ROS production, and enhanced the stimulatory capacity of PMNs on T cell proliferation. The presence of lung cancer cells and monocytes prolonged the survival of neutrophils. In co-cultures, neutrophils swiftly acquired an activated state with decreased CD62L. Besides, ROS production by neutrophils was decreased in the co-cultures. The presence of monocytes, neutrophils, and lung cancer cells enhanced CD8+ T proliferation and the expression of inhibitory receptors. Under certain conditions lacking monocytes as major supporters of T-cell proliferation, the presence of neutrophils together with lung adenocarcinoma cells, barely supported CD8+ T cell responses. TIM-3mod/high CD8+ T cells recovered from NSCLC co-cultures were not exhausted and displayed high proliferation and IFN-γ secretion. NAC stimulation did not modulate the CD8+ T cell proliferation or TIM-3/LAG3 expression in co-cultures. Expression of 4-1BBL, OX40L and B7-2 costimulatory genes were increased in the PMNs co-cultured with lung cancer cells, monocytes and CD8+ T cells. Consequently, in a co-culture setup employing neutrophils, monocytes, CD8+ T cells and lung cancer cells, which was established to partially model the tumor microenvironment, our findings indicate the importance of neutrophils as a critical modulator for CD8+ T cell responses.