Interleukin (IL)-9-producing CD4+ T cells are a novel subset of T helper (Th) cells that develops independently of the Th1, Th2, Th17 and regulatory T-cell lineages. Similar to the murine model, transforming growth factor (TGF)-β and IL-4 directed human naive CD4+ T cells to produce IL-9. Whereas IL-4 suppressed TGF-β-induced Foxp3 expression, TGF-β failed to inhibit IL-4-mediated upregulation of the Th2 transcription factor GATA-3. Addition of IL-1β, IL-6, IL-10, interferon (IFN)-α, IFN-β or IL-21 to Th9-polarizing conditions augmented Th9 differentiation, while the Th1-associated cytokines IFN-γ and IL-27 partially suppressed IL-9 production. Given that T cells are a primary source of IL-21, IL-21 expression was analyzed under Th9-polarizing conditions in the context of inflammatory cytokines. Surprisingly, type I IFNs induced elevated levels of IL-21, and blockade of IL-21 abrogated their ability to enhance Th9 differentiation. Taken together, these data indicate a complex cytokine network in the regulation of human IL-9-producing CD4+ T cells.

A hallmark of SLE is the production of high-titer, high-affinity, isotype-switched IgG autoantibodies directed against nucleic acid-associated antigens. Several studies have established a role for both type I IFN (IFN-I) and the activation of TLRs by nucleic acid-associated autoantigens in the pathogenesis of this disease. Here, we demonstrate that 2 IFN-I signaling molecules, IFN regulatory factor 9 (IRF9) and STAT1, were required for the production of IgG autoantibodies in the pristane-induced mouse model of SLE. In addition, levels of IgM autoantibodies were increased in pristane-treated Irf9 -/-mice, suggesting that IRF9 plays a role in isotype switching in response to self antigens. Upregulation of TLR7 by IFN-α was greatly reduced in Irf9 -/-and Stat1 -/-B cells. Irf9 -/-B cells were incapable of being activated through TLR7, and Stat1 -/-B cells were impaired in activation through both TLR7 and TLR9. These data may reveal a novel role for IFN-I signaling molecules in both TLR-specific B cell responses and production of IgG autoantibodies directed against nucleic acid-associated autoantigens. Our results suggest that IFN-I is upstream of TLR signaling in the activation of autoreactive B cells in SLE.

RNA has recently been shown to play diverse roles in gene regulation, including the small molecule-dependent inhibition of translation in prokaryotes. To create an artificial genetic switch that acts at the level of transcription, we fused a small molecule binding aptamer to a previously evolved RNA that activates transcription when localized to a promoter. We designed a conformational shift in which a helical element required for transcriptional activation was stabilized upon ligand binding. Selection and screening in S. cerevisiae optimized the linker region, generating an RNA that is 10-fold more active in the presence of tetramethylrosamine (TMR). TMR increases the activity of this evolved RNA in a graded, dose-dependent manner. Our results exemplify a strategy for controlling the activity of laboratory-evolved RNAs in living cells.

From random RNA libraries expressed in yeast, we evolved RNA-based transcriptional activators that are comparable in potency to the strongest natural protein activation domains. The evolved RNAs activated transcription up to 53-fold higher than a three-hybrid positive control using the Gal4 activation domain and only 2-fold lower than the highly active VP16 activation domain. Using a combination of directed evolution and site-directed mutagenesis, we dissected the functional elements of the evolved transcriptional activators. A surprisingly large fraction of RNAs from our library are capable of activating transcription, suggesting that nucleic acids may be well suited for binding transcriptional machinery elements normally recruited by proteins. In addition, our work demonstrates an RNA evolution-based approach to perturbing natural cellular function that may serve as a general tool for studying selectable or screenable biological processes in living cells.

Toll-like receptor (TLR) ligands are known to activate antigen presenting cells (APCs), but direct T cell responsiveness to TLR ligands is controversial. Because of their clinical relevance, we performed in depth studies of the effects of the TLR9-associated ligands, oligodeoxynucleotides (ODNs), on highly-purified T lymphocytes. Both CpG and non-CpG ODNs directly costimulate mouse and human CD4+ T cells resulting in activation marker upregulation, cytokine secretion, elevated TCR phosphorylation, and proliferation. Surprisingly, ODN costimulation occurred independently of TLR9 and MyD88, as well as ICOS, CD28 and TRIF. TLR9-antagonist ODNs likewise promoted T cell activation, which has important implications for the study of these “inhibitory” ODNs in inflammatory diseases. Cytokine profiling revealed that ODNs promote polarization of distinct T-helper subsets, and that ODNs differentially affect human naïve and memory T cells. Our studies reveal a striking and unexpected ability of ODNs to directly activate and polarize T cells, presenting an opportunity to enhance the paradigm for selection of therapeutic ODNs in humans.