Reprogramming regulatory T cells to effector T cells for enhanced antitumour responses : a study on non-small cell lung carcinoma cell lines

Abstract

Introduction: Current therapeutic approaches for lung cancer including surgery, chemotherapy, radiotherapy, and immunotherapy are often limited by significant side effects and suboptimal long-term outcomes, highlighting the urgent need for innovative strategies, particularly within the field of immunotherapy. Regulatory T cells (Tregs) play an essential role in maintaining immunological homeostasis and preventing autoimmunity, however, their immunosuppressive properties can also hinder effective antitumour responses by promoting an immunosuppressive tumour microenvironment. This effect is especially pronounced in non-small cell lung cancer (NSCLC), where increased Treg infiltration is strongly associated with poorer prognosis and decreased treatment efficacy. Given their functional plasticity under inflammatory conditions, recent research has turned to the potential of reprogramming Tregs into proinflammatory effector-like cells—such as Th1-like cells—capable of producing cytokines that support antitumour immunity and counteract tumour-mediated immune suppression. Aim: This research project aims to investigate the potential reprogramming of Tregs into Th1-like effector cells by assessing both their phenotypic and functional shift. Specifically, the study evaluates whether Tregs exposed to pro-inflammatory conditions can adopt Th1-like cells characteristics, and whether these reprogrammed cells exhibit anti-tumour activity specifically on NSCLC. Method: The CellXVivo Human Treg Cell Differentiation Kit was used to polarise CD4⁺ T cells into Tregs after they were isolated from human peripheral blood mononuclear cells, via negative magnetic selection. In order to encourage Treg reprogramming toward a Th1-like phenotype, treatment with high and low doses of IL-12 was done. After treatment, H460, a NSCLC cell line, were treated with different concentrations of conditioned media in order to evaluate the supposed anti-tumour functions of the reprogrammed Tregs, using CFSE-based proliferation assays. Results: Phenotypic analysis revealed that in some cases, Tregs expressing FOXP3 were not generated in the first place, which could be attributed to donor-to-donor heterogeneity, and thus, the direct impact of IL-12 on the phenotype of Tregs was inconclusive. Treated Treg also retained high CD25 expression typical of Treg. The conditioned media from IL-12-treated Tregs at concentration 80% reduced H460 proliferation relative to cells cultured in complete RPMI media, however unexpectedly this reduction was not as much as those cultured in untreated Treg conditioned media, suggesting IL-12 alone may be insufficient to cause the treated CD4+ cells to acquire effective effector cytokine production with anti-tumourigenic effects. Conclusion: While IL-12 has some influence on Treg plasticity, the results suggest that it is insufficient on its own to drive complete reprogramming into Th1-like effector cells, as CD25 expression was still retained and the suppressive activity of the conditioned media from IL-12-treated Tregs was not as significant as the conditioned media of untreated Tregs. These results underscore the complexity of Treg plasticity and highlight the need for additional signals to effectively achieve Tregs and convert these cells into functionally potent anti-tumour effector T cells with anti-tumourigenic properties