Novel markers for human T regulatory cells in healthy donors and cancer patients

CD4+CD25+FoxP3+ T regulatory cells (Tregs) are essential for maintaining self-tolerance and preventing autoimmune diseases. However, FoxP3+ Tregs contribute to the progression of cancer and their levels expand in cancer patients compared to healthy donors. Tregs suppress tumour-specific immune responses by accumulating in the peripheral blood and tumour microenvironment. Human Tregs secrete the latent form of transforming growth factor-beta (TGF-β), in which the mature TGF-β protein is bound to latency-associated peptide (LAP) that binds to Glycoprotein A Repetitions Predominant (GARP). Some FoxP3+ Tregs express Helios, a member of the Ikaros transcription factor family. The purpose of this study is to identify which of FoxP3+/-Helios+/- Tregs express GARP and LAP and to investigate if these receptors are vital markers for activated conventional Tregs, and also to examine the different suppressive factors and phenotypes of Tregs. This study compared the levels of Tregs in cancer patients with controls, by measuring the levels of Tregs specific and novel markers. Peripheral blood mononuclear cells (PBMCs) were isolated from the blood of healthy donors (HDs), chronic pancreatitis (CP), malignant pancreatic cancer (PC) and liver metastases from colorectal cancer (LI/CRC) patients. PBMCs were then stained with anti-CD3, anti-CD4, anti-GARP, anti-LAP, anti-Helios, anti-FoxP3, anti-IFN-γ, and anti-IL-10 antibodies. The results demonstrated for the first time that GARP and LAP are mainly expressed on activated CD4+FoxP3+Helios+ Tregs and CD4+FoxP3-Helios+ Tregs for healthy donors and all patient groups. In contrast, CD4+FoxP3+Helios- and CD4+FoxP3-Helios- Tregs do not express GARP and LAP. FoxP3+Helios+ Tregs from cancer patients showed significantly higher expression of GARP and LAP, compared to healthy donors. Furthermore, there was no increase in the level of FoxP3+Helios+ Tregs in HDs and PC patients compared to LI/CRC patients. FoxP3+Helios+GARP+LAP+ Tregs secrete the IL-10 cytokine but not IFN-γ in comparison with FoxP3-Helios- Tregs. This study demonstrated that Helios, and not FoxP3, is the main marker of activated Tregs expressing GARP and LAP