Ars2 links the nuclear cap-binding complex to RNA interference and cell proliferation.

Here we identify a component of the nuclear RNA cap-binding complex (CBC), Ars2, that is important for miRNA biogenesis and critical for cell proliferation. Unlike other components of the CBC, Ars2 expression is linked to the proliferative state of the cell. Deletion of Ars2 is developmentally lethal, and deletion in adult mice led to bone marrow failure whereas parenchymal organs composed of nonproliferating cells were unaffected. Depletion of Ars2 or CBP80 from proliferating cells impaired miRNA-mediated repression and led to alterations in primary miRNA processing in the nucleus. Ars2 depletion also reduced the levels of several miRNAs, including miR-21, let-7, and miR-155, that are implicated in cellular transformation. These findings provide evidence for a role for Ars2 in RNA interference regulation during cell proliferation.

PKC signaling contributes to chromatin decondensation and is required for competence to respond to IL-2 during T cell activation.

The clonal proliferation of antigen-specific T cells during an immune response critically depends on the differential response to growth factors, such as IL-2. While activated T cells proliferate robustly in response to IL-2 stimulation, naïve (quiescent) T cells are able to ignore the potent effects of growth factors because they possess chromatin that is tightly condensed such that transcription factors, such as STAT5, cannot access DNA. Activation via the T cell receptor (TCR) induces a rapid decondensation of chromatin, permitting STAT5-DNA engagement and ultimately promoting proliferation of only antigen-specific T cells. Previous work demonstrated that the mobilization of intracellular calcium following TCR stimulation is a key event in the decondensation of chromatin. Here we examine PKC-dependent signaling mechanisms to determine their role in activation-induced chromatin decondensation and the subsequent acquisition of competence to respond to IL-2 stimulation. We found that a calcium-dependent PKC contributes to activation-induced chromatin decondensation and that the p38 MAPK and NFκB pathways downstream of PKC each contribute to regulating the proper decondensation of chromatin. Importantly, we found that p44/42 MAPK activity is required for peripheral T cells to gain competence to properly respond to IL-2 stimulation. Our findings shed light on the mechanisms that control the clonal proliferation of antigen-specific peripheral T cells during an immune response.

Inhibition of phosphatidylinositol-3-kinase by the furanosesquiterpenoid hibiscone C.

The phosphatidylinositol-3-kinase (PI3K) pathway regulates cellular metabolism and is upregulated in many cancers, making it an attractive chemotherapeutic target. Wortmannin is a potent inhibitor of PI3K; however, its potential as a chemotherapeutic is limited due to its instability, lack of selectivity, and lengthy chemical synthesis. In contrast, hibiscone C, a structurally simpler and less studied member of the furanosteroid family, has been expediently prepared by total synthesis. We demonstrate that hibiscone C competitively inhibits PI3K activity in intact cells, slows proliferation, and induces cell death. Hibiscone C may therefore serve as a productive scaffold for the development of therapeutically relevant PI3K inhibitors.

Chromatin condensation via the condensin II complex is required for peripheral T-cell quiescence.

Naive T cells encountering their cognate antigen become activated and acquire the ability to proliferate in response to cytokines. Stat5 is an essential component in this response. We demonstrate that Stat5 cannot access DNA in naive T cells and acquires this ability only after T-cell receptor (TCR) engagement. The transition is not associated with changes in DNA methylation or global histone modification but rather chromatin decondensation. Condensation occurs during thymocyte development and proper condensation is dependent on kleisin-ß of the condensin II complex. Our findings suggest that this unique chromatin condensation, which can affect interpretations of chromatin accessibility assays, is required for proper T-cell development and maintenance of the quiescent state. This mechanism ensures that cytokine driven proliferation can only occur in the context of TCR stimulation.

Primary Literature in the Undergraduate Immunology Curriculum: Strategies, Challenges, and Opportunities.

Immunology is a rapidly advancing and expanding field that is regularly highlighted in the lay media, whether it be checkpoint blockade immunotherapy winning the Nobel Prize, CAR-T cells in the treatment of cancer, or the latest anti-inflammatory/immunomodulatory medication advertised directly to consumers. Advances such as these not only transform the way we think about immunology, they also illuminate how knowledge of the immune system can be harnessed to impact public health. Immunology is also a vast subject, with thousands of articles published each year that contribute to our understanding of complex processes such as inflammation, pathogen recognition, and self-tolerance, Taken together, these observations pose significant challenges to teaching immunology in the undergraduate classroom. To meet this challenge, instructors can use primary literature as a means to introduce cutting-edge discoveries that have not yet found their way into textbooks, link what students are learning to what they are exposed to in lay media, and ultimately provide added depth to the students' understanding of the immune system all while illustrating how clinical advances are fundamentally dependent on basic research studies. Furthermore, the addition of primary literature to the curriculum can enhance student enthusiasm for learning immunology and can provide an excellent platform for students to gain critical thinking and analytical skills. Presented here are strategies, challenges, and opportunities in the use of primary literature to effectively augment the immunology curriculum in the undergraduate classroom. Topics include selecting papers to read, teaching students how to read scientific literature, and assessing student learning.

The Use of Flow Cytometry to Assess the State of Chromatin in T Cells.

During a proper immune response, quiescent T cells become activated upon antigen presentation to their antigen-specific T cell receptor. This leads to clonal proliferation of only those T cells that bear a receptor that recognizes the antigen. Chromatin decondensation is a hallmark of T cell activation and is required for T cells to acquire the ability to proliferate after antigen engagement. This change in chromatin condensation can be detected using antibodies raised against histone proteins. These antibodies cannot bind to their epitopes in naïve T cells as well as they can in activated T cells. We describe how to simultaneously stain T cell-specific surface markers, track viability with a fixable dead cell stain, and measure chromatin status via intracellular staining of Histone H3 proteins. Stained cells are analyzed by flow cytometry and chromatin condensation status is measured as the mean fluorescence intensity (MFI) of the Histone H3 stain. Chromatin decondensation during T cell activation is demonstrated as an increase in the MFI.

Calcium mobilization is both required and sufficient for initiating chromatin decondensation during activation of peripheral T-cells.

Antigen engagement of the T-cell receptor (TCR) induces a rapid and dramatic decondensation of chromatin that is necessary for T-cell activation. This decondensation makes T-cells competent to respond to interleukin-2 providing a mechanism to ensure clonotypic proliferation during an immune response. Using murine T-cells, we investigated the mechanism by which TCR signaling can initiate chromatin decondensation, focusing on the role of calcium mobilization. During T-cell activation, calcium is first released from intracellular stores, followed by influx of extracellular calcium via store operated calcium entry. We show that mobilization of intracellular calcium is required for TCR-induced chromatin decondensation. However, the decondensation is not dependent on the activity of the downstream transcription factor NFAT. Furthermore, we show that the influx of extracellular calcium is dispensable for initiating chromatin decondensation. Finally, we show that mobilization of calcium from intracellular stores is sufficient to induce decondensation, independent of TCR engagement. Collectively, our data suggest that chromatin decondensation in peripheral T-cells is controlled by modulating intracellular calcium levels.

Two Drosophila suppressors of cytokine signaling (SOCS) differentially regulate JAK and EGFR pathway activities.

BACKGROUND: The Janus kinase (JAK) cascade is an essential and well-conserved pathway required to transduce signals for a variety of ligands in both vertebrates and invertebrates. While activation of the pathway is essential to many processes, mutations from mammals and Drosophila demonstrate that regulation is also critical. The SOCS (Suppressor Of Cytokine Signaling) proteins in mammals are regulators of the JAK pathway that participate in a negative feedback loop, as they are transcriptionally activated by JAK signaling. Examination of one Drosophila SOCS homologue, Socs36E, demonstrated that its expression is responsive to JAK pathway activity and it is capable of downregulating JAK signaling, similar to the well characterized mammalian SOCS. RESULTS: Based on sequence analysis of the Drosophila genome, there are three identifiable SOCS homologues in flies. All three are most similar to mammalian SOCS that have not been extensively characterized: Socs36E is most similar to mammalian SOCS5, while Socs44A and Socs16D are most similar to mammalian SOCS6 and 7. Although Socs44A is capable of repressing JAK activity in some tissues, its expression is not regulated by the pathway. Furthermore, Socs44A can enhance the activity of the EGFR/MAPK signaling cascade, in contrast to Socs36E. CONCLUSIONS: Two Drosophila SOCS proteins have some overlapping and some distinct capabilities. While Socs36E behaves similarly to the canonical vertebrate SOCS, Socs44A is not part of a JAK pathway negative feedback loop. Nonetheless, both SOCS regulate JAK and EGFR signaling pathways, albeit differently. The non-canonical properties of Socs44A may be representative of the class of less characterized vertebrate SOCS with which it shares greatest similarity.

Isolation and molecular characterization of Rem2 isoforms in the rainbow trout (Oncorhynchus mykiss): Tissue and central nervous system expression.

REM2 is a member of the REM, RAD, and GEM/KIR (RGK) subfamily of RAS superfamily proteins and plays an important role in brain development and function. In this study, two Rem2 isoforms were isolated from the rainbow trout (Oncorhynchus mykiss). The two genes, designated O. mykiss rem2a and rem2b, both encode 304 amino acid proteins with 61% and 62% identities to zebrafish (Danio rerio) Rem2, respectively, and each with 43% identity to mammalian (human) REM2. To our knowledge, this is the first incidence of Rem2 isoforms in a species that are the result of gene duplication. Both isoforms possessed similar tissue expression profiles with the highest levels in the brain. The rem2a gene has significantly higher expression levels than rem2b in all tissues assayed except the brain and head kidney. In the central nervous system, both isoforms showed similar expression levels with the highest levels occurring in the olfactory bulb, cerebrum, and midbrain, though rem2a expression is significantly higher in the spinal cord. Based on known functional roles of Rem2 in synapse development and stem cell proliferation, the characterization of Rem2 in rainbow trout could shed light on its role in adult vertebrate neurogenesis and brain regeneration.

A role for STAT5A/B in protection of peripheral T-lymphocytes from postactivation apoptosis: insights from gene expression profiling.

Activation of the transcription factors STAT5A and STAT5B by JAK1 and JAK3 tyrosine kinases is a key event in downstream signaling of gammac (common gamma chain)-family cytokine receptors in lymphoid cells. STAT5A/B-deficiency in mice causes, among other consequences, a reduced size and altered composition of the peripheral T-cell pool with predominance of an activated or memory-like population (CD4(+)/CD44(high)/CD62L(low)) and a proliferative deficiency following antigenic stimulation and subsequent IL-2 treatment. To further elucidate the critical function of STAT5A/B in homeostasis and activation of murine naïve peripheral T-lymphocytes, we have analyzed global gene expression of STAT5A/B-deficient versus wild-type splenic T-cells by profiling with high-density oligonucleotide arrays (Affymetrix). Comparison of (1) basal gene expression of untreated peripheral STAT5A/B-deficient and control T-cells and (2) immediate early gene induction upon in vitro stimulation of either population with IL-2 has revealed differential expression of a broad range of genes potentially contributing to the defects of STAT5A/B deficient T-cells. In the context of enhanced apoptotic rates of STAT5A/B(-/-)-T-cells in vivo and upon TCR-stimulation in culture our data suggest a role for STAT5 in post-activation survival beyond regulation of antiapoptotic Bcl-2 proteins and hence provide new insights into the nature of the late proliferative block in the T-cell compartment caused by STAT5-deficiency.