Identification of possible Treg biomarkers. (A) Work flow of the strategy applied to identify and validate potential Treg biomarkers. CD4⁺CD25⁺ and CD4⁺CD25⁻ T cells were isolated by antibody coupled magnetic beads. Isolated cells were subfractionated into their respective cytosolic, membrane and nucleic/insoluble cytoskeleton fractions. Each protein fraction was precipitated by ice-cold acetone followed by SDS-PAGE. SimplyBlue SafeStain stained protein bands were excised, in-gel trypsinated and the resulting peptides enriched on a C18 gel-loader tip column before nano-LC-MS/MS analysis. Resulting peptide fragmentation spectra were searched against the Uniprot database using Mascot as a search engine. A selected set of proteins were taken further for validation by complementary methods and finally functional analysis. (B) Proteins observed exclusively in CD4⁺CD25⁺ cells, number of observations and observed peptides, total Mascot score and the proteins respective cellular compartments. To fulfill our criteria, proteins had to be observed in CD4⁺CD25 enriched cells in at least 2 of 3 experiments and not in the CD4⁺CD25 depleted cells. A Mascot cut-off ion score of 30 was applied on the peptide level to ensure high confidence. (C) Among the identified membrane proteins, 4 were already designated with a number in the CD nomenclature and were known to contain an extracellular domain, CD95, CD147, CD71, and CD148. (C) CD4⁺ cells were either depleted or enriched for CD25 and cell surface stained with an antibody cocktail containing conjugated anti-CD4, anti-CD3, anti-CD25, and either anti-CD95, anti-CD147, anti-CD71, or anti-CD148 before flow cytometry.