Bacteriophage P1 Cre/loxP based systems can be used to manipulate the genomes of mice in vivo and in vitro, allowing the generation of tissue‐specific conditional mutants. Wehave generated mouse lines expressing Cre recombinase in hematopoietic tissues using the vav regulatory elements, or in lymphoid cells using the hCD2 promoter and locus control region (LCR). The R26R‐EYFP Cre reporter mouse line was used to determine the pattern of Cre expression in each line and enabled the assessment of Cre activity at a single‐cell level. Analysis showed that the vav promoter elements were able to direct Cre‐mediated recombination in all cells of the hematopoietic system. The hCD2 promoter and LCR on the other hand were able to drive Cre‐mediated recombination only in T cells and B cells, but not in other hematopoietic cell types. Furthermore, in the appropriate tissues, deletion of the floxed target was complete in all cells, thereby excluding the possibility of variegated expression of the Cre transgene. Both of these Cre‐transgenic lines will be useful in generating tissue‐specific gene deletions within all the cells of hematopoietic or lymphoid tissues.

RNA is known to be an abundant and important structural component of the nuclear matrix, including long noncoding RNAs (lncRNA). Yet the molecular identities, functional roles, and localization dynamics of lncRNAs that influence nuclear architecture remain poorly understood. Here, we describe one lncRNA, Firre, that interacts with the nuclear matrix factor hnRNPU, through a 156 bp repeating sequence and Firre localizes across a ~5 Mb domain on the X-chromosome. We further observed Firre localization across at least five distinct trans-chromosomal loci, which reside in spatial proximity to the Firre genomic locus on the X-chromosome. Both genetic deletion of the Firre locus or knockdown of hnRNPU resulted in loss of co-localization of these trans-chromosomal interacting loci. Thus, our data suggest a model in which lncRNAs such as Firre can interface with and modulate nuclear architecture across chromosomes.

The spleen is the largest secondary lymphoid organ in the body and, as such, hosts a wide range of immunologic functions alongside its roles in hematopoiesis and red blood cell clearance. The physical organization of the spleen allows it to filter blood of pathogens and abnormal cells and facilitate low-probability interactions between antigen-presenting cells (APCs) and cognate lymphocytes. APCs specific to the spleen regulate the T and B cell response to these antigenic targets in the blood. This review will focus on cell types, cell organization, and immunologic functions specific to the spleen and how these affect initiation of adaptive immunity to systemic blood-borne antigens. Potential differences in structure and function between mouse and human spleen will also be discussed.

Cross-linking of high-affinity immunoglobulin E (IgE) results in the life-threatening allergic reaction anaphylaxis. Yet the cellular mechanisms that induce B cells to produce IgE in response to allergens remain poorly understood. T follicular helper (TFH) cells direct the affinity and isotype of antibodies produced by B cells. Although TFH cell–derived interleukin-4 (IL-4) is necessary for IgE production, it is not sufficient. We report a rare population of IL-13–producing TFH cells present in mice and humans with IgE to allergens, but not when allergen-specific IgE was absent or only low-affinity. These “TFH13” cells have an unusual cytokine profile (IL-13hiIL-4hiIL-5hiIL-21lo) and coexpress the transcription factors BCL6 and GATA3. TFH13 cells are required for production of high- but not low-affinity IgE and subsequent allergen-induced anaphylaxis. Blocking TFH13 cells may represent an alternative therapeutic target to ameliorate anaphylaxis.

Regulation of metabolic pathways in the immune system provides a mechanism to actively control cellular function, growth, proliferation and survival. Here, we report that miR-181 is a non-redundant determinant of cellular metabolism and is essential to support the biosynthetic demands of early NKT cell development. As a result, miR-181-deficient mice showed a complete absence of mature NKT cells in the thymus and periphery. Mechanistically, miR-181 modulated expression of the phosphatase PTEN to control PI3K signaling, which was a primary stimulus for anabolic metabolism in immune cells. Thus miR-181-deficient mice also showed severe defects in lymphoid development and T cell homeostasis associated with impaired PI3K signaling. These results uncover miR-181 as essential for NKT cell development, and establish this family of miRNAs as central regulators of PI3K signaling and global metabolic fitness during development and homeostasis.

Normal organogenesis requires co-ordinate development and interaction of multiple cell types, and is seemingly governed by tissue specific factors. Lymphoid organogenesis during embryonic life is dependent on molecules the temporal expression of which is tightly regulated. During this process, haematopoietic 'inducer' cells interact with stromal 'organizer' cells, giving rise to the lymphoid organ primordia. Here we show that the haematopoietic cells in the gut exhibit a random pattern of motility before aggregation into the primordia of Peyer's patches, a major component of the gut-associated lymphoid tissue. We further show that a CD45+CD4-CD3-Il7Ralpha-c-Kit+CD11c+ haematopoietic population expressing lymphotoxin has an important role in the formation of Peyer's patches. A subset of these cells expresses the receptor tyrosine kinase RET, which is essential for mammalian enteric nervous system formation. We demonstrate that RET signalling is also crucial for Peyer's patch formation. Functional genetic analysis revealed that Gfra3-deficiency results in impairment of Peyer's patch development, suggesting that the signalling axis RET/GFRalpha3/ARTN is involved in this process. To support this hypothesis, we show that the RET ligand ARTN is a strong attractant of gut haematopoietic cells, inducing the formation of ectopic Peyer's patch-like structures. Our work strongly suggests that the RET signalling pathway, by regulating the development of both the nervous and lymphoid system in the gut, has a key role in the molecular mechanisms that orchestrate intestine organogenesis.

SUMMARY Inflammation is a risk factor for cancer development. Individuals with preleukemic TET2 mutations manifest clonal hematopoiesis and are at a higher risk of developing leukemia. How inflammatory signals influence the survival of preleukemic hematopoietic stem and progenitor cells (preleukemic-HSPCs) is unclear. We show a rapid increase in the frequency and absolute number of Tet2-KO mature myeloid cells and HSPCs in response to inflammatory stress, which results in enhanced production of inflammatory cytokines, including IL-6, and resistance to apoptosis. IL-6 induces hyperactivation of the Shp2-Stat3 signaling axis, resulting in increased expression of a novel anti-apoptotic lncRNA, Morrbid, in Tet2-KO myeloid cells and HSPCs. Expression of activated Shp2 in HSPCs phenocopies Tet2 loss, with regard to hyperactivation of Stat3 and Morrbid. In vivo, pharmacologic inhibition of Shp2 or Stat3 or genetic loss of Morrbid in Tet2-mutant mice rescues inflammatory stress-induced abnormalities in HSPCs and mature myeloid cells including clonal hematopoiesis.

SummaryNeutrophils, eosinophils and “classical” monocytes collectively account for ~70% of human blood leukocytes and are among the shortest-lived cells in the body1,2. Precise regulation of the lifespan of these myeloid cells is critical to maintain protective immune responses while minimizing the deleterious consequences of prolonged inflammation1,2. However, how the lifespan of these cells is strictly controlled remains largely unknown. Here, we identify a novel long non-coding RNA (lncRNA) that we termed Morrbid, which tightly controls the survival of neutrophils, eosinophils and “classical” monocytes in response to pro-survival cytokines. To control the lifespan of these cells, Morrbid regulates the transcription of its neighboring pro-apoptotic gene, Bcl2l11 (Bim), by promoting the enrichment of the PRC2 complex at the Bcl2l11 promoter to maintain this gene in a poised state. Notably, Morrbid regulates this process in cis, enabling allele-specific control of Bcl2l11 transcription. Thus, in these highly inflammatory cells, changes in Morrbid levels provide a locus-specific regulatory mechanism that allows for rapid control of apoptosis in response to extracellular pro-survival signals. As MORRBID is present in humans and dysregulated in patients with hypereosinophilic syndrome, this lncRNA may represent a potential therapeutic target for inflammatory disorders characterized by aberrant short-lived myeloid cell lifespan.