Modulating regulatory T cells towards effective cancer immunotherapy

Abstract

Regulatory T cells (Treg) suppress the immune system and play an important role in the maintenance of immunologic tolerance to self-antigens, and thus in the prevention of autoimmune diseases. However, their function is hijacked by tumours and when released inside the tumour microenvironment, Treg aid cancer cells to evade the immune system promoting tumour growth. For this reason, Treg present a major obstacle to successful immunotherapeutic treatments. There is evidence that suggests that under certain acute inflammatory conditions, induced by immunomodulatory agents, Treg function is decreased or reversed, and sometimes they might also acquire the ability to attain characteristics of other Helper T cells. Treg were isolated from human PBMC and after expanding the Treg to sufficient working numbers, the Treg cells were treated with selected immunomodulators which include Interferon-γ (IFN-γ), two putative endogenous ligands for Toll-Like Receptors (TLR) 7, 8, and 9 and two synthetic ligands for TLR7 and TLR8. Flow cytometry using antibodies that identify surface and intracellular markers of different T cell types was used to assess changes in phenotype. Suppression assays of Treg and responder Th co-cultures were carried out to check for immunomodulating effects on Treg function. Cytokine analysis assessed whether the immunomodulator favoured the generation of an anti-inflammatory environment (which promotes Treg) or a proinflammatory one (which favours Th1). Transfection with endogenous nucleic acid ligands, including both human RNA and DNA, did not block Treg suppressor function, with DNA transfection actually enhancing it. The synthetic TLR7/8 ligand CL097 modified Treg phenotype, including augmentation in CD25 marker and the upregulation of Th1 transcription factor T-bet whilst retaining FOXP3 expression. Treatment with CL097 enhanced Treg suppressor function. The two agents which had the best effects on reducing Treg suppressor function were IFN-γ and TLR7/8 ligand, synthetic single stranded polyuridine, with the former also causing a reduction in Treg signature markers FOXP3 and CD25. Importantly, single stranded polyuridine at lower doses reduced Treg proliferation and suppressor function while high doses of the ligand reversed this effect. Thus appropriate concentrations of these two agents may be developed to control Treg in cancer therapies whilst other agents, such as CL097, may be more useful in the management of autoimmune disease.