2A. However, the expanded Th17 clones did not exhibit significant alterations of FOXP3 expression and IFN-γ production when the expansion system only included PBMCs but not OKT3. We extended these experiments to the other human Th17 clones and obtained similar results (data not shown). These results indicate a critical role for TCR engagement in IFN-γ-production and FOXP3 expression,

but not IL-17 reduction, in expanded Th17 cells. To further confirm the contribution of TCR stimulation to an unstable lineage phenotype and differentiation plasticity of Th17 cells, E1-Th17 cells were directly stimulated with or without plate-bound OKT3 in the presence or absence of cytokines (IL-1β, IL-6 and IL-23) critical for human Th17 cell development and function, for 7 days, followed Ixazomib price by repeated stimulation for another 7 days. We subsequently determined the proportion of IL-17 and IFN-γ-producing cell populations and FOXP3 expression in different cultures of these E1-Th17 cells following two rounds of stimulation. As shown in Fig. 4B and Supporting Information Fig. 3, the percentages of IL-17-producing cell populations decreased dramatically in E1-Th17 cells after in vitro culture with different stimulations, but there was no difference of percentages between the groups stimulated GSI-IX with or without OKT3. The addition of cytokines IL-1β, IL-6 and IL-23 to cultures could not

maintain the stability of Th17 cells and did not prevent the reduction of IL-17-producing cells in vitro. We also observed significantly decreased numbers of IL-17-producing cell populations in Th17 clones when cultured with medium only (low IL-2). The combined addition of cytokines did not alter percentages of IFN-γ-producing cells

during the first 7 days of culture, whereas these were increased in the day 14 cultures. However, the addition of these cytokines did not promote FOXP3 expression in Th17 cells in either day 7 or day 14 cultures. When E1-Th17 cells were stimulated with plate-bound OKT3, the percentages of IFN-γ-producing cell populations were not significantly induced during the first 7-day culture but were dramatically increased in day 14 cultures following the eltoprazine second round of stimulation. Furthermore, the percentages of FOXP3+ cell populations in E1-Th17 cells stimulated with plate-bound OKT3 were significantly induced in both 7-day (first stimulation) and 14-day (second stimulation) cultures (Fig. 4B and Supporting Information Fig. 3). However, further combination of the cytokines with OKT3 did not significantly alter IFN-γ-producing cell populations and FOXP3 expression in either day 7 or day 14 cultures, compared with cultures stimulated with OKT3 alone. Interestingly, the combination of these cytokines with OKT3 promoted a reduction in the proportion of IL-17-producing cell populations in Th17 clones compared with those in Th17 cell cultures stimulated with OKT3 only (Fig. 4B and Supporting Information Fig. 3).

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