Time-course RNA-sequencing of human induced regulatory T cell (iTreg) differentiation. Homo sapiens

Regulatory T cells (Tregs) expressing the transcription factor FOXP3 are crucial mediators of self-tolerance, preventing autoimmune diseases but possibly hampering tumor rejection. Clinical manipulation of Tregs is of great interest, and first- in-man trials of Treg transfer achieved promising outcomes. Yet, the mechanisms governing induced Treg (iTreg) differentiation and the regulation of FOXP3 are incompletely understood. To gain a comprehensive and unbiased molecular understanding of FOXP3 induction, we performed time-series RNA sequencing (RNA-Seq) and proteomics profiling on the same samples during human iTreg differentiation. To enable the broad analysis of universal FOXP3-inducing pathways, we used five differentiation protocols in parallel. Integrative analysis of the transcriptome and proteome confirmed involvement of specific molecular processes, as well as overlap of a novel iTreg subnetwork with known Treg regulators and autoimmunity-associated genes. Importantly, we propose 37 novel molecules putatively involved in iTreg differentiation. Their relevance was validated by: a targeted shRNA screen confirming a functional role in FOXP3 induction; discriminant analyses classifying iTregs accordingly; and comparable expression in an independent novel iTreg RNA-Seq data set. The data generated by this novel approach facilitate understanding the molecular mechanisms underlying iTreg generation as well as the concomitant changes in the transcriptome and proteome. Our results provide a reference map exploitable for future discovery of markers and drug candidates governing control of Tregs, which has important implications for the treatment of cancer, autoimmune and inflammatory diseases. Overall design: We performed a time-course experiment including six time points and four iTreg differentiation conditions. We included also a control time series and unstimulated nTregs. Three biological replicates were included. The total sample count is 81. In detail, human naïve CD4+ T cells were magnetically negatively isolated from peripheral blood. Cells were stimulated in serum-free medium with anti-CD3/anti-CD28 antibodies plus IL-2, and samples were taken at 2h, 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), TGF-b + retinoic acid (sample group G04), TGF-b + retinoic acid + rapamycin (sample group G05) or TGF-b + butyrate (sample group G06). 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.