This model was challenged in a landmark

study by Cua et al., who used a series of cytokine subunit knockout mice to prove that Th1 immune cells were not the primary drivers of EAE pathology.[41] The differentiation of Th1 cells is dependent upon the cytokine interleukin-12 (IL-12), which is composed of two subunits, p35 and p40. The p40 subunit can also bind to p19 to form IL-23.[42] Induction of EAE by immunization with myelin oligodendrocyte glycoprotein(35–55) peptide in p35 knockout mice produced a strong paralytic disease, characteristic of disease in wild-type control animals, whereas knockouts of either p19 or p40 had no EAE symptoms.[41] Replacement of IL-23 expression within the central nervous system of p19−/− or p40−/− mice restored the development Akt inhibitor of disease pathology, providing strong evidence for IL-23 as a key mediator of EAE. Interleukin-23 was found to expand a population of T cells that were distinct in their production of IL-17A, IL-17F and IL-6, and had elevated

production of tumour necrosis factor-α.[43] These cells were strongly encephalitic in the adoptive transfer model of EAE, AZD8055 providing evidence that this T-cell subtype was a principal driver of EAE development. Curiously, addition of IL-23 to in vitro cultures of naive T cells could not polarize them towards an IL-17 producing phenotype (Th17);[44] however, it was found that the addition of transforming growth factor-β (TGF-β) and IL-6 to naive T-cell cultures did elicit Th17 differentiation, and this was confirmed in additional studies.[45, 46] It is also notable that key Th1 and Th2 polarizing factors, interferon-γ and IL-4, respectively, could inhibit Th17 polarization.[44,

46] A feature common to T-cell subset differentiation is that they require a master transcription factor that drives the cellular programme for a specific phenotype, i.e. T-bet is required for Th1 development and GATA3 is required for Th2. The nuclear receptor retinoic acid receptor-related orphan nuclear receptor γt (RORγt) Cytidine deaminase was found to be essential for induction and maintenance of the Th17 differentiation programme.[47] Knockout of RORγt abolished Th17 differentiation, and IL-6/TGF-β treatment of T-cell receptor-stimulated naive T cells increased expression of RORγt before observed increases in IL-17A and IL-17F, implying that RORγt activation is upstream of effector cytokine production. Induction of RORγt required IL-6, a cytokine that activates phosphorylation of STAT3 in a Jak-dependent manner. This was negatively regulated by the suppressor of cytokine signalling 3 protein, as T cell-specific deletion of suppressor of cytokine signalling 3 resulted in hyperactivation of STAT3 and induction of the Th17 programme, which occurred even in the absence of additional IL-6 and TGF-β.[48] STAT3 also bound to the promoters for IL-17A and IL-17F, indicating that STAT3 is a direct regulator of Th17 effector functions.