Fig 2: Proposed regulatory mechanism for IL-17A and IL-17F differential regulation. Guided by our modeling study, we further analyzed recent publications to identify mechanisms susceptible to explain the three main cytokine input-output scenarios observed: a Expression of IL-17F, but not IL-17A under an IL-12 + IL-1ß condition. Note the autocrine loop involving IL-2. The red blunt arc denotes an enhancer-mediated inhibition of IL-17A transcription. b Expression of IL-17F and IL-17A in a proTh17 condition. The green arrow denotes an enhancer-mediated activation of IL-17A transcription. c Expression of IL-17A, but not IL-17F in a proTh17 condition. The proposed regulatory scheme relies on the following assumptions: i) SMAD2 is required for the expression of IL-17A, ii) SMAD2 and NFAT2A over-expressions have a cooperative effect on IL-17A expression, iii) SMAD2 over-expression and STAT5A down-regulation have a cooperative effect on IL-17A expression, iv) IL-17F transcription presumably requires STAT5A, but not SMAD2 nor NFAT2A, v) STAT5A OFF inhibits IL-17F expression and a low level of STAT5A is necessary, since STAT5A is involved in BLIMP-1 activation circuit and BLIMP-1 is required for IL-17F expression but not for IL-17A expression

Fig 3: Quantification of transcription of model internal components by RT-PCR. Human naive T cells were differentiated for five days in the presence of polyclonal activation (anti-CD3/anti-CD28 beads). Cells were cultured in the presence of different cytokine inputs: proTh1 (IL-12), IL-1ß, IL-23, IL-12 + IL-1ß, IL-1ß + IL-23, IL-12 + IL-1ß + IL-23, proTh17 (IL-1ß + IL-6 + IL-23 + TGF-ß). Cells exposed only to polyclonal stimulation were considered as Th0. RNA extraction of differentiated cells was performed and transcripts were then quantified by RT-PCR. Gene expression was normalized to the housekeeping genes HPRT1, B2M and RPL34. Relative expression of IL-17A, IL-17F, IFN-?, RORC, RORA, T-bet, STAT1, SATB1, MINA are depicted. Graphs represent mean ± SD, N = 5 and * denotes p < 0.05 (Wilcoxon test)

Fig 4: Influence of Tregs on the cytokine secretion of T cells. (A) Representative flow cytometry demonstrating the association between Treg quantity and cytokine secretion of T cells in tumors. Comparison of CD4+T cell secretion of (B) IFN-?, (C) IL-4 and (D) IL-17 and (E) CD8+T cell cytokine secretion between Treg low and Treg high BC patients. All experiments were repeated three times. Treg, regulatory T cell; BC, breast cancer; CD, cluster of differentiation; IFN, interferon; IL, interleukin.

Fig 5: Computational perturbations point to model internal components controlling IL-17A activation. The schema summarizes the impact of selected perturbations (knock-in or knock-out) on IL-12 + IL-1ß and Th17 differentiation. Rows denote single or multiple perturbations of SMAD2, STAT5A and NFAT2A. Green and red cells represent activated and inactivated phenotypes, respectively