In line with this, several recent publications demonstrated a surprisingly high plasticity of differentiated CD4+ T-cell subpopulations generated either in vitro or in JQ1 order vivo. First, a number of studies showed that Foxp3+ Treg

in both mouse and human can be redirected to express IL-17 16–20. Similarly, a recent report showed that transferred natural Treg develop to follicular B-helper T cells in the Peyer’s patches of T-cell-deficient hosts 21. Second, several groups demonstrated that Th17 cells generated in vitro are plastic upon exposure to Th1 cytokines and start to express IFN-γ (22–24). Finally, studies with purified in vitro generated Th17 cells transferred to NOD mice showed infiltrating cells changing their phenotype to become Th1 cells 22, 23. Very importantly, human Th17 T-cell clones were shown to be highly flexible and to co-express IFN-γ and IL-17A when stimulated in the presence of IL-12 24. Similarly a specific CD161+ subpopulation derived from human umbilical cord blood,

which is prone to contain and differentiate to Th17 cells, develops strongly toward Th1 cells under the influence of IL-12 in vitro25. Since these groups demonstrated IFN-γ production by Th17 cells following adoptive transfer, we aimed to define whether indeed trans-differentiation of IL-17 expressing cells is the cause of this finding. To address this question, we used our recently generated IL-17F fate mapping mouse line 26. When these IL-17F-Cre BAC-transgenic mice are crossed to ROSA26-EYFP find more reporter mice 27, IL-17F-expressing cells are irreversibly genetically tagged by Cre-mediated excision of a loxP flanked stop cassette, resulting in ubiquitous expression of EYFP in all recombined cells. We analyzed the behavior of transferred, sorted Th17 reporter

cells generated either in vitro or in vivo and found that a considerable amount of these DNA Synthesis inhibitor cells ceased IL-17A expression entirely, and expressed purely IFN-γ. Additionally, we found a number of previously highly pure Th1 cells co-expressing IL-17A together with IFN-γ in the mesenteric LN (mLN). In a first attempt to define whether in vitro generated Th17 cells maintain their cytokine phenotype upon EAE induction, we performed transfer EAE using in vitro polarized Th17 cells generated from MOG35–55-specific CD4+ cells isolated from 2D2 TCR-transgenic mice 28. After 5 days of stimulation in Th17-polarizing conditions, about 50% of cells expressed IL-17A, whereas only negligible numbers produced IFN-γ (Supporting Information Fig. S1A). We adoptively transferred 5×106 of these cells per mouse to RAG1-deficient mice (of the C57BL/6 background), resulting in severe EAE symptoms (Supporting Information Fig. S1B). In line with the findings by O’Connor et al.