The frequency of IL-17A single-positive and IL-17A/IFN- double-positive CD4+ T cells was significantly increased in response to IL-1, which was further upregulated by adding IL-23 (Fig.?1b, c). to experimental autoimmune encephalomyelitis in an IL-1 receptor-dependent manner. In humans, IL-1R1high memory CD4+ T cells are major producers of IL-17A and IFN- in response to IL-1 and IL-23. TCF1 Collectively, our findings reveal the innate-like pathogenic function of antigen non-related memory CD4+ T cells, which contributes to the development of Dacarbazine autoimmune diseases. Introduction Multiple sclerosis (MS) is an unpredictable, chronic, demyelinating, human autoimmune disease caused by the induction of inflammation in the central nervous system (CNS)1. Studies of experimental autoimmune encephalomyelitis (EAE), a model of multiple sclerosis (MS), have demonstrated that CNS-invading myelin-specific TH1 and TH17 cells are the major mediators of autoimmune neuroinflammation2C4. TH17 cells are categorized into two functionally distinct subsets: non-pathogenic TH17 and pathogenic TH17 cells5. TH17 cells differentiate in the presence of transforming growth factor (TGF)- and interleukin (IL)-6 produce IL-17A and IL-10, which are not pathogenic6. However, further stimulation with IL-1 and IL-23 induces highly encephalitogenic TH17 cells, which have been shown Dacarbazine to express signature genes, including RORt, T-bet, IL-17A, IL-22, and granulocyte macrophage colony-stimulating factor (GM-CSF)5,7C9. Dacarbazine Recently, IL-17-producing innate-like lymphocytes, such as gamma delta () T cells, invariant natural killer T cells, and innate lymphoid cells were shown to be important for responding to the pro-inflammatory cytokines IL-1 and IL-23, by producing IL-17 in an antigen-nonspecific manner10C13. The ability of innate-like lymphocytes to produce innate IL-17 has been shown to be critical in many autoimmune disease models, including experimental autoimmune encephalomyelitis (EAE)14,15 and inflammatory bowel disease16,17. CD4+ T lymphocytes respond to their specific cognate antigen and further differentiate into distinct subsets of helper T cells, including TH1, TH2, and TH17, as defined by their pattern of effector cytokine production18. However, differentiated CD4+ T cells can respond directly to pro-inflammatory cytokines by producing innate effector cytokines. IL-1 family cytokines (IL-18, IL-33, IL-1), along with the STAT activator cytokines (IL-12, IL-2, IL-23), were shown to promote Dacarbazine effector cytokine production by TH1, TH2, and TH17 cells19. Moreover, IL-33-dependent IL-13 production by memory TH2 cells has been shown to contribute to allergic inflammation and protect against early helminth infection20. These findings demonstrate that the innate-like capacity of CD4+ T lymphocytes, which is correlated with innate-like lymphocytes, produce effector cytokines in response to pro-inflammatory cytokines. However, whether the innate immunological function of CD4+ T lymphocytes contributes to the pathogenicity of autoimmune diseases remains unclear. CD4+ T lymphocytes specific for nonmyelin proteins have been proposed to invade the CNS21,22, regardless of their specificity for CNS antigens, thus providing encephalitogenic potential23,24. Furthermore, in an EAE model, most CNS-infiltrating CD4+ T cells were found to be myelin oligodendrocyte glycoprotein (MOG)-nonspecific25C27. Although nonmyelin-specific T cells have been associated with the pathogenesis of autoimmune disorders, the precise mechanism is unknown. Here, we hypothesized that antigen non-related CD4+ T cells contribute to autoimmune disease pathogenesis in response to pro-inflammatory cytokines. We first screened for pro-inflammatory cytokines capable of initiating innate effector cytokine production by CD4+ T cells. We found that memory-like CD4+ T cells, but not naive CD4+ T cells, produced IL-17A and interferon (IFN)- in response to IL-1 and IL-23 in the absence of T-cell receptor (TCR) engagement. Bystander activation of memory-like CD4+ T cells increased the expression of pathogenic TH17 signature genes, including RORt, CCR6, and GM-CSF. Furthermore, TCR-transgenic (OT-II) memory-like TH17 cells were shown to contribute to EAE pathogenicity regardless of antigen specificity by infiltrating and producing IL-17A, IFN-, and GM-CSF in the spinal cord in an IL-1R1-dependent manner. Taken together, our findings demonstrate the importance of the TCR-independent innate-like pathogenic role of bystander-activated memory CD4+ T cells in autoimmune encephalomyelitis. Results TCR-independent CD4 T cells activation via IL-1 and IL-23 To examine the innate-like capacity of CD4+ T lymphocytes, CD4+CD25? T cells were sorted by fluorescence-activated cell sorting (FACS) and cultured in the presence of pro-inflammatory cytokines, including tumor necrosis factor (TNF), IL-6, IL-23, IL-12, and IL-1 in the absence of TCR stimulation. Additionally, IL-7 was added to the culture medium for T-cell survival and maintenance28,29. Consistent with previous results30,31, we found that IL-12 promoted IFN- production, which further synergized with TNF and IL-1 (Supplementary Fig.?1a). Interestingly, the pro-inflammatory cytokines IL-1 and IL-23 were the most potent cytokines inducing IL-17A production by CD4+ T cells and also promoted IFN- production (Supplementary Fig.?1a, b). The amount of IL-17A produced by CD4+ T cells in response to IL-1 and IL-23 was fivefold higher than that.