Time-course proteomics analysis of human regulatory T cell (Treg) differentiation

Human naïve CD4+ T cells (CD4+ CD45RA+ CD25- CD45RO- CD8- CD14- CD15- CD16- CD19- CD34- CD36- CD56- CD123- TCRγ/δ- HLA-DR- and CD235a-) were magnetically negatively isolated from peripheral blood. Cells were stimulated with anti-CD3/anti-CD28 antibodies plus IL-2, and samples were taken at 6h, 24h, 48h and 6d of stimulation. Mock stimulation control cells (sample group G02) received no further compounds, whereas induced regulatory T cells (iTregs) were either differentiated under addition of TGF-b (sample group G03) or TGF-b + retinoic acid + rapamycin (sample group G05). As control, naïve CD4+ T cells were left unstimulated (0h; sample group G01). Ex vivo isolated CD25-high cells were included as positive control for the Treg signature (“nTreg”; sample group G07). Tregs were defined by expression of FOXP3, the “master” transcription factor of Tregs. Samples from 3 male healthy donors (age 34 to 38 years) were prepared with the Qiagen Allprep kit and protein precipitate was solubilized (5 min, 95°C) in freshly prepared buffer containing 4% (w/v) SDS, 25 mM HEPES pH 7.6, 1mM DTT. Samples were prepared using the FASP assay and peptides were labeled with TMT 10-plex reagents and MS data acquired on a Q Exactive Hybrid Quadrupole-Orbitrap Mass Spectrometer.
Phenotyping, stability and functional analyses for iTregs induced under these conditions are available in Schmidt A et al., PLoSONE 2016, PMID: 26886923). In the publication associated to this dataset, the time-course proteomic profiling during human Treg differentiation is presented and integrated with RNA-Seq data from the same cells (including additional iTreg culture conditions and 2h time points for RNA-Seq). The data underwent clustering, network analysis and disease enrichment, which revealed many known regulators of Tregs along with novel candidate genes putatively involved in FOXP3 induction, the biological importance of which was validated with a targeted shRNA screen.