Surface protein marker of the induced regulatory T cells in NC/Nga mice

Abstract

CD25+ CD4+ regulatory T cells (Tregs) are known as a key modulator of immune self-tolerance and also have been found in atopic dermatitis (AD) patients. Since it has been rarely known that the different functional role between naturally occurring Tregs (nTregs) in thymus and induced Tregs (iTregs), and their surface marker proteins, we designed the experiments to separate two different subpopulations of Tregs, nTregs from thymus of healthy control mice and iTregs from spleen from the AD model mice. We initially observed AD-like skin lesions in NC/Nga mice sensitizing D. farinae ointments for six weeks and we measured their significant increases of serum IgE level and enhanced IL-4 production from Th2 subsets. CD4+ CD25+ Treg cells were separated from those AD-like mice and healthy control mice using MACS®, with high separation purity confirmed by flow cytometry analysis. Membrane proteins were extracted from CD4+ CD25+ Tregs and labeled with TMT reagents for 1DLC-MS/MS analysis. As a result of TMT-labeling method, we obtained 533 protein, 63 membrane proteins and 16 plasma membrane proteins identification list based on database research. Remarkably, H-2 class II histocompatibility antigen has the highest ratio of iTregs to nTregs among those identified proteins, and other membrane proteins including receptor-type tyrosine-protein phosphatase C and leukocyte surface antigen CD47, also showed high expression levels in iTregs, compared with nTregs. Considering the increased ratios of selected proteins in iTreg cell population and their functional roles that have been elucidated from many research groups, we believed that they might be key marker proteins or functional regulators of iTregs.