Xist ribonucleoproteins promote female sex-biased autoimmunity.

Autoimmune diseases disproportionately affect females more than males. The XX sex chromosome complement is strongly associated with susceptibility to autoimmunity. Xist long non-coding RNA (lncRNA) is expressed only in females to randomly inactivate one of the two X chromosomes to achieve gene dosage compensation. Here, we show that the Xist ribonucleoprotein (RNP) complex comprising numerous autoantigenic components is an important driver of sex-biased autoimmunity. Inducible transgenic expression of a non-silencing form of Xist in male mice introduced Xist RNP complexes and sufficed to produce autoantibodies. Male SJL/J mice expressing transgenic Xist developed more severe multi-organ pathology in a pristane-induced lupus model than wild-type males. Xist expression in males reprogrammed T and B cell populations and chromatin states to more resemble wild-type females. Human patients with autoimmune diseases displayed significant autoantibodies to multiple components of XIST RNP. Thus, a sex-specific lncRNA scaffolds ubiquitous RNP components to drive sex-biased immunity.

Engineering circular RNA for enhanced protein production.

Circular RNAs (circRNAs) are stable and prevalent RNAs in eukaryotic cells that arise from back-splicing. Synthetic circRNAs and some endogenous circRNAs can encode proteins, raising the promise of circRNA as a platform for gene expression. In this study, we developed a systematic approach for rapid assembly and testing of features that affect protein production from synthetic circRNAs. To maximize circRNA translation, we optimized five elements: vector topology, 5' and 3' untranslated regions, internal ribosome entry sites and synthetic aptamers recruiting translation initiation machinery. Together, these design principles improve circRNA protein yields by several hundred-fold, provide increased translation over messenger RNA in vitro, provide more durable translation in vivo and are generalizable across multiple transgenes.

Circular RNA migration in agarose gel electrophoresis.

Circular RNAs are garnering increasing interest as potential regulatory RNAs and a format for gene expression. The characterization of circular RNA using analytical techniques commonly employed in the literature, such as gel electrophoresis, can, under differing conditions, yield different results when attempting to distinguish circular RNA from linear RNA of similar molecular weights. Here, we describe circular RNA migration in different conditions, analyzed by gel electrophoresis and high-performance liquid chromatography (HPLC). We characterize key parameters that affect the migration pattern of circular RNA in gel electrophoresis systems, which include gel type, electrophoresis time, sample buffer composition, and voltage. Finally, we demonstrate the utility of orthogonal analytical tests for circular RNA that take advantage of its covalently closed structure to further distinguish circular RNA from linear RNA following in vitro synthesis.

Data Streaming for Metabolomics: Accelerating Data Processing and Analysis from Days to Minutes.

The speed and throughput of analytical platforms has been a driving force in recent years in the "omics" technologies and while great strides have been accomplished in both chromatography and mass spectrometry, data analysis times have not benefited at the same pace. Even though personal computers have become more powerful, data transfer times still represent a bottleneck in data processing because of the increasingly complex data files and studies with a greater number of samples. To meet the demand of analyzing hundreds to thousands of samples within a given experiment, we have developed a data streaming platform, XCMS Stream, which capitalizes on the acquisition time to compress and stream recently acquired data files to data processing servers, mimicking just-in-time production strategies from the manufacturing industry. The utility of this XCMS Online-based technology is demonstrated here in the analysis of T cell metabolism and other large-scale metabolomic studies. A large scale example on a 1000 sample data set demonstrated a 10 000-fold time savings, reducing data analysis time from days to minutes. Further, XCMS Stream has the capability to increase the efficiency of downstream biochemical dependent data acquisition (BDDA) analysis by initiating data conversion and data processing on subsets of data acquired, expanding its application beyond data transfer to smart preliminary data decision-making prior to full acquisition.

Uncovering the mechanisms that regulate tumor-induced T-cell anergy.

Helper T cells become hyporesponsive in the tumor microenvironment (at least in part) owing to the NFAT1-dependent expression of anergy-associated genes. Anergy constitutes a crucial mechanism to prevent tumor destruction by T cells, and hence may represent a powerful target to boost antitumor immune responses and improve the efficacy of immunotherapy.

NFAT1 supports tumor-induced anergy of CD4(+) T cells.

Cancer cells express antigens that elicit T cell-mediated responses, but these responses are limited during malignant progression by the development of immunosuppressive mechanisms in the tumor microenvironment that drive immune escape. T-cell hyporesponsiveness can be caused by clonal anergy or adaptive tolerance, but the pathophysiological roles of these processes in specific tumor contexts has yet to be understood. In CD4+ T cells, clonal anergy occurs when the T-cell receptor is activated in the absence of a costimulatory signal. Here we report that the key T-cell transcription factor NFAT mediates expression of anergy-associated genes in the context of cancer. Specifically, in a murine model of melanoma, we found that cancer cells induced anergy in antigen-specific CD4+ T-cell populations, resulting in defective production of several key effector cytokines. NFAT1 deficiency blunted the induction of anergy in tumor antigen-specific CD4+ T cells, enhancing antitumor responses. These investigations identified tumor-induced T-cell hyporesponsiveness as a form of clonal anergy, and they supported an important role for CD4+ T-cell anergy in driving immune escape. By illustrating the dependence of tumor-induced CD4+ T-cell anergy on NFAT1, our findings open the possibility of targeting this transcription factor to improve the efficacy of cancer immunotherapy or immunochemotherapy.

Regulation of T-cell tolerance by calcium/NFAT signaling.

Cells that escape negative selection in the thymus must be inactivated or eliminated in the periphery through a series of mechanisms that include the induction of anergy, dominant suppression by regulatory T cells, and peripheral deletion of self-reactive T cells. Calcium signaling plays a central role in the induction of anergy in T cells, which become functionally inactivated and incapable of proliferating and expressing cytokines following antigen re-encounter. Suboptimal stimulation of T cells results in the activation of a calcium/calcineurin/nuclear factor of activated T cells-dependent cell-intrinsic program of self-inactivation. The proteins encoded by those genes are required to impose a state of functional unresponsiveness through different mechanisms that include downregulation of T-cell receptor signaling and inhibition of cytokine transcription.

Transcriptional complexes formed by NFAT dimers regulate the induction of T cell tolerance.

In T cells, anergy can be induced after T cell receptor engagement in the absence of costimulation. Under these conditions, the expression of a specific set of anergy-associated genes is activated. Several lines of evidence suggest that nuclear factor of activated T cells (NFAT) proteins may regulate the expression of many of those genes; however, the nature of the complexes responsible for the induction of this new program of gene expression is unknown. Here, we show that transcriptional complexes formed by NFAT homodimers are directly responsible for the activation of at least two anergy-inducing genes, Grail and Caspase3. Our data shows that Grail expression is activated by direct binding of NFAT dimers to the Grail promoter at two different sites. Consequently, a mutant NFAT protein with impaired ability to dimerize is not able to induce an unresponsive state in T cells. Our results not only identify a new biological function for NFAT dimers but also reveal the different nature of NFAT-containing complexes that induce anergy versus those that are activated during a productive immune response. These data also establish a basis for the design of immunomodulatory strategies that specifically target each type of complex.