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The developmental programs that generate a broad repertoire of regulatory T cells (T reg cells) able to respond to both self antigens and non-self antigens remain unclear. Here we found that mature T reg cells were generated through two distinct developmental programs involving CD25 + T reg cell progenitors (CD25 + T reg P cells) and Foxp3 lo T reg cell progenitors (Foxp3 lo T reg P cells). CD25 + T reg P cells showed higher rates of apoptosis and interacted with thymic self antigens with higher affinity than did Foxp3 lo T reg P cells, and had a T cell antigen receptor repertoire and transcriptome distinct from that of Foxp3 lo T reg P cells. The development of both CD25 + T reg P cells and Foxp3 lo T reg P cells was controlled by distinct signaling pathways and enhancers. Transcriptomics and histocytometric data suggested that CD25 + T reg P cells and Foxp3 lo T reg P cells arose by coopting negative-selection programs and positive-selection programs, respectively. T reg cells derived from CD25 + T reg P cells, but not those derived from Foxp3 lo T reg P cells, prevented experimental autoimmune encephalitis. Our findings indicate that T reg cells arise through two distinct developmental programs that are both required for a comprehensive T reg cell repertoire capable of establishing immunotolerance. Farrar and colleagues show that thymic T reg cells are generated through two distinct developmental programs that are both required for a comprehensive T reg cell repertoire.

Background Whole transcriptome sequencing (RNA-seq) represents a powerful approach for whole transcriptome gene expression analysis. However, RNA-seq carries a few limitations, e.g., the requirement of a significant amount of input RNA and complications led by non-specific mapping of short reads. The Ion AmpliSeq™ Transcriptome Human Gene Expression Kit (AmpliSeq) was recently introduced by Life Technologies as a whole-transcriptome, targeted gene quantification kit to overcome these limitations of RNA-seq. To assess the performance of this new methodology, we performed a comprehensive comparison of AmpliSeq with RNA-seq using two well-established next-generation sequencing platforms (Illumina HiSeq and Ion Torrent Proton). We analyzed standard reference RNA samples and RNA samples obtained from human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs). Results Using published data from two standard RNA reference samples, we observed a strong concordance of log2 fold change for all genes when comparing AmpliSeq to Illumina HiSeq (Pearson’s r  = 0.92) and Ion Torrent Proton (Pearson’s r  = 0.92). We used ROC, Matthew’s correlation coefficient and RMSD to determine the overall performance characteristics. All three statistical methods demonstrate AmpliSeq as a highly accurate method for differential gene expression analysis. Additionally, for genes with high abundance, AmpliSeq outperforms the two RNA-seq methods. When analyzing four closely related hiPSC-CM lines, we show that both AmpliSeq and RNA-seq capture similar global gene expression patterns consistent with known sources of variations. Conclusions Our study indicates that AmpliSeq excels in the limiting areas of RNA-seq for gene expression quantification analysis. Thus, AmpliSeq stands as a very sensitive and cost-effective approach for very large scale gene expression analysis and mRNA marker screening with high accuracy.

The precise molecular mechanism underlying the regulation of early B cell lymphopoiesis is unclear. The PLCγ signaling pathway is critical for antigen receptor-mediated lymphocyte activation, but its function in cytokine signaling is unknown. Here we show that PLCγ1/PLCγ2 double deficiency in mice blocks early B cell development at the pre-pro-B cell stage and renders B cell progenitors unresponsive to IL-7. PLCγ pathway inhibition blocks IL-7-induced activation of mTOR, but not Stat5. The PLCγ pathway activates mTOR through the DAG/PKC signaling branch, independent of the conventional Akt/TSC/Rheb signaling axis. Inhibition of PLCγ/PKC-induced mTOR activation impairs IL-7-mediated B cell development. PLCγ1/PLCγ2 double-deficient B cell progenitors have reduced expression of genes related to B cell lineage, IL-7 signaling, and cell cycle. Thus, IL-7 receptor controls early B lymphopoiesis through activation of mTOR via PLCγ/DAG/PKC signaling, not via Akt/Rheb signaling. IL-7R activation drives early B cell development, but the signalling is unclear. Here the authors show PLCγ is involved in IL-7R-induced mTOR activation via a DAG/PKC-dependent pathway, and that double deficiency of the two PLCγ isoforms arrests B cell development at the pre-pro-B stage in mice.

Cardiac hypertrophy is an independent risk factor for cardiovascular disease and heart failure. There is increasing evidence that microRNAs (miRNAs) play an important role in the regulation of messenger RNA (mRNA) and the pathogenesis of various cardiovascular diseases. However, the ability to comprehensively study cardiac hypertrophy on a gene regulatory level is impacted by the limited availability of human cardiomyocytes. Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) offer the opportunity for disease modeling. Here we utilize a previously established in vitro model of cardiac hypertrophy to interrogate the regulatory mechanism associated with the cardiac disease process. We perform miRNA sequencing and mRNA expression analysis on endothelin 1 (ET-1) stimulated hiPSC-CMs to describe associated RNA expression profiles. MicroRNA sequencing revealed over 250 known and 34 predicted novel miRNAs to be differentially expressed between ET-1 stimulated and unstimulated control hiPSC-CMs. Messenger RNA expression analysis identified 731 probe sets with significant differential expression. Computational target prediction on significant differentially expressed miRNAs and mRNAs identified nearly 2000 target pairs. A principal component analysis approach comparing the in vitro data with human myocardial biopsies detected overlapping expression changes between the in vitro samples and myocardial biopsies with Left Ventricular Hypertrophy. These results provide further insights into the complex RNA regulatory mechanism associated with cardiac hypertrophy.

The gene interleukin-12 receptor β1 ( IL12RB1 ) regulates susceptibility to several human diseases, including mycobacterial disease (e.g., tuberculosis). Here, we demonstrate that many of the mRNAs transcribed from IL12RB1 in primary immune cells contain RNA-DNA differences (RDDs). RDDs are nucleotide differences between RNA and its encoding DNA and are introduced posttranscriptionally; in the case of IL12RB1 , RDDs are concentrated in cytokine-binding regions that are important for IL12RB1 function. This observation is significant, as it is the first demonstration to our knowledge that a mechanism of sequence diversification exists for a human cytokine receptor. Given IL12RB1 ’s importance to mycobacterial disease resistance, our data raise the intriguing possibility that individual differences in IL12RB1 RDD introduction contribute to differences in mycobacterial disease susceptibility. Human interleukin 12 and interleukin 23 (IL12/23) influence susceptibility or resistance to multiple diseases. However, the reasons underlying individual differences in IL12/23 sensitivity remain poorly understood. Here we report that in human peripheral blood mononuclear cells (PBMCs) and inflamed lungs, the majority of interleukin-12 receptor β1 ( IL12RB1 ) mRNAs contain a number of RNA-DNA differences (RDDs) that concentrate in sequences essential to IL12Rβ1’s binding of IL12p40, the protein subunit common to both IL-12 and IL-23. IL12RB1 RDDs comprise multiple RDD types and are detectable by next-generation sequencing and classic Sanger sequencing. As a consequence of these RDDs, the resulting IL12Rβ1 proteins have an altered amino acid sequence that could not be predicted on the basis of genomic DNA sequencing alone. Importantly, the introduction of RDDs into IL12RB1 mRNAs negatively regulates IL12Rβ1’s binding of IL12p40 and is sensitive to activation. Collectively, these results suggest that the introduction of RDDs into an individual’s IL12RB1 mRNA repertoire is a novel determinant of IL12/23 sensitivity.

In their native environment, tissues are organized into intricate fractal structures, rarely recapitulated in their culture in vitro. The extent to which fractal (self-similar) patterns that resemble complex topography in vivo influence cell maturation remain inadequately elucidated. Yet, the application of fractal topographical stimulation may address the challenge of improving the differentiated cell phenotype in vitro. Here, we show fractality in the kidney glomerulus and podocytes, branching highly differentiated cells within the glomerulus. Biomimetic fractal patterns derived from glomerular histology are used to generate topographical (2.5-D) substrates for cell culture. Podocytes grown on fractal topography exhibit higher expression of functional markers and enhanced cell polarity. RNA sequencing suggests podocytes’ enhanced ECM deposition and remodeling on fractal versus flat topography, and enhanced maturation accompanied by stress on fractal versus non-fractal topography. The incorporation of fractal topography into standard well plates may serve as a user-friendly bioengineered platform for high-fidelity cell culture. Liu and colleagues demonstrate that biomimetic fractal patterns derived from glomerular histology can enhance the maturation of podocytes (highly differentiated glomerular cells) that are grown in culture. This work presents a bioengineered platform for improved cell culture fidelity.

Human IL12RB1 is an autosomal gene that is essential for mycobacterial disease resistance and T cell differentiation. Using primary human tissue and PBMCs, we demonstrate that lung and T cell IL12RB1 expression is allele-biased, and the extent to which cells express one IL12RB1 allele is unaffected by activation. Furthermore following its expression the IL12RB1 pre-mRNA is processed into either IL12RB1 Isoform 1 (IL12Rβ1, a positive regulator of IL12 responsiveness) or IL12RB1 Isoform 2 (a protein of heretofore unknown function). T cells choice to process pre-mRNA into Isoform 1 or Isoform 2 is controlled by intragenic competition of IL12RB1 exon 9–10 splicing with IL12RB1 exon 9b splicing, as well as an IL12RB1 exon 9b-associated polyadenylation site. Heterogeneous nuclear ribonucleoprotein H (hnRNP H) binds near the regulated polyadenylation site, but is not required for exon 9b polyadenylation. Finally, microRNA-mediated knockdown experiments demonstrated that IL12RB1 Isoform 2 promotes T cell IL12 responses. Collectively, our data support a model wherein tissue expression of human IL12RB1 is allele-biased and produces an hnRNP H-bound pre-mRNA, the processing of which generates a novel IL12 response regulator.

Whole transcriptome sequencing (RNA-seq) represents a powerful approach for whole transcriptome gene expression analysis. However, RNA-seq carries a few limitations, e.g., the requirement of a significant amount of input RNA and complications led by non-specific mapping of short reads. The Ion AmpliSeq⢠Transcriptome Human Gene Expression Kit (AmpliSeq) was recently introduced by Life Technologies as a whole-transcriptome, targeted gene quantification kit to overcome these limitations of RNA-seq. To assess the performance of this new methodology, we performed a comprehensive comparison of AmpliSeq with RNA-seq using two well-established next-generation sequencing platforms (Illumina HiSeq and Ion Torrent Proton). We analyzed standard reference RNA samples and RNA samples obtained from human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs). Using published data from two standard RNA reference samples, we observed a strong concordance of log2 fold change for all genes when comparing AmpliSeq to Illumina HiSeq (Pearson's r = 0.92) and Ion Torrent Proton (Pearson's r = 0.92). We used ROC, Matthew's correlation coefficient and RMSD to determine the overall performance characteristics. All three statistical methods demonstrate AmpliSeq as a highly accurate method for differential gene expression analysis. Additionally, for genes with high abundance, AmpliSeq outperforms the two RNA-seq methods. When analyzing four closely related hiPSC-CM lines, we show that both AmpliSeq and RNA-seq capture similar global gene expression patterns consistent with known sources of variations. Our study indicates that AmpliSeq excels in the limiting areas of RNA-seq for gene expression quantification analysis. Thus, AmpliSeq stands as a very sensitive and cost-effective approach for very large scale gene expression analysis and mRNA marker screening with high accuracy.

Human interleukin 12 and interleukin 23 (IL12/23) influence susceptibility or resistance to multiple diseases. However, the reasons underlying individual differences in IL12/23 sensitivity remain poorly understood. Here we report that in human peripheral blood mononuclear cells (PBMCs) and inflamed lungs, the majority of interleukin-12 receptor β1 (IL12RB1) mRNAs contain a number of RNA-DNA differences (RDDs) that concentrate in sequences essential to IL12Rβ1’s binding of IL12p40, the protein subunit common to both IL-12 and IL-23.IL12RB1RDDs comprise multiple RDD types and are detectable by next-generation sequencing and classic Sanger sequencing. As a consequence of these RDDs, the resulting IL12Rβ1 proteins have an altered amino acid sequence that could not be predicted on the basis of genomic DNA sequencing alone. Importantly, the introduction of RDDs intoIL12RB1mRNAs negatively regulates IL12Rβ1’s binding of IL12p40 and is sensitive to activation. Collectively, these results suggest that the introduction of RDDs into an individual’sIL12RB1mRNA repertoire is a novel determinant of IL12/23 sensitivity.