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C-type lectin domain family 4, member a4 (Clec4a4) is a C-type lectin inhibitory receptor specific for glycans thought to be exclusively expressed on murine CD8α− conventional dendritic cells. Using newly generated Clec4a4-mCherry knock-in mice, we identify a subset of Clec4a4-expressing eosinophils uniquely localized in the small intestine lamina propria. Clec4a4+ eosinophils evinced an immunomodulatory signature, whereas Clec4a4− eosinophils manifested a proinflammatory profile. Clec4a4+ eosinophils expressed high levels of aryl hydrocarbon receptor (Ahr), which drove the expression of Clec4a4 as well as other immunomodulatory features, such as PD-L1. The abundance of Clec4a4+ eosinophils was dependent on dietary AHR ligands, increased with aging, and declined in inflammatory conditions. Mice lacking AHR in eosinophils expanded innate lymphoid cells of type 2 and cleared Nippostrongylus brasiliensis infection more effectively than did wild-type mice. These results highlight the heterogeneity of eosinophils in response to tissue cues and identify a unique AHR-dependent subset of eosinophils in the small intestine with an immunomodulatory profile.

Getting around the blood–brain barrier The meninges comprise three membranes that surround and protect the central nervous system (CNS). Recent studies have noted the existence of myeloid cells resident there, but little is known about their ontogeny and function, and whether other meningeal immune cell populations have important roles remains unclear (see the Perspective by Nguyen and Kubes). Cugurra et al. found in mice that a large proportion of continuously replenished myeloid cells in the dura mater are not blood derived, but rather transit from cranial bone marrow through specialized channels. In models of CNS injury and neuroinflammation, the authors demonstrated that these myeloid cells have an immunoregulatory phenotype compared with their more inflammatory blood-derived counterparts. Similarly, Brioschi et al. show that the meninges host B cells that are also derived from skull bone marrow, mature locally, and likely acquire a tolerogenic phenotype. They further found that the brains of aging mice are infiltrated by a second population of age-associated B cells, which come from the periphery and may differentiate into autoantibody-secreting plasma cells after encountering CNS antigens. Together, these two studies may inform future treatment of neurological diseases. Science , abf7844, abf9277, this issue p. eabf7844 , p. eabf9277 ; see also abj8183, p. 396

Control of gene expression by microRNA (miRNA) has recently emerged as a critical mechanism that regulates B cell activation and function. However, the role of specific miRNAs in this process is unclear. Using germline knockout (KO) mice, we have previously shown that miR-142 ablation impairs humoral immunity despite significant expansion of the B cell compartment, suggesting that miR-142 is critical for B cell effector function. We found that mice lacking miR-142 cannot generate specific antibody responses upon antigen challenge and display impaired germinal center (GC) formation and plasma cell differentiation. To more precisely dissect the role of miR-142 in B cell effector responses, we developed an activated B cell-specific miR-142 KO mouse by breeding conditional miR-142 KO (miR-142fl/fl) mice with mice expressing Cre recombinase driven by activation-induced cytosine deaminase (AIDCre). AID is required for class switch recombination and somatic hypermutation, and is induced in B cells upon antigen encounter. To label activated B cells in miR-142fl/flAIDCre mice, we bred them to Ai6 mice that carry a loxP-flanked STOP cassette, allowing for the expression of fluorescent ZsGreen1 reporter protein in the presence of Cre recombinase. FACS analysis of Peyer’s patches from naive miR-142fl/flAIDCreZsgreen+/− mice revealed a significant decrease in GC B cells, while the number of ZsGreen1+ activated B cells increased, suggesting an aberrant B cell activation and GC response. We will discuss at the meeting the role of miR-142 in promoting humoral responses to antigen by presenting the findings from our ongoing studies of B cell differentiation and effector function in immunized miR-142fl/flAIDCreZsgreen+/− mice.

MicroRNAs (miRNAs) are a class of powerful posttranscriptional regulators implicated in the control of diverse biological processes, including regulation of hematopoiesis and the immune response. To define the biological functions of miR-142, which is preferentially and abundantly expressed in immune cells, we created a mouse line with a targeted deletion of this gene. Our analysis of miR-142(-/-) mice revealed a critical role for this miRNA in the development and homeostasis of lymphocytes. Marginal zone B cells expand in the knockout spleen, whereas the number of T and B1 B cells in the periphery is reduced. Abnormal development of hematopoietic lineages in miR-142(-/-) animals is accompanied by a profound immunodeficiency, manifested by hypoimmunoglobulinemia and failure to mount a productive immune response to soluble antigens and virus. miR-142(-/-) B cells express elevated levels of B-cell-activating factor (BAFF) receptor (BAFF-R) and as a result proliferate more robustly in response to BAFF stimulation. Lowering the BAFF-R gene dose in miR-142(-/-) mice rescues the B-cell expansion defect, suggesting that BAFF-R is a bona fide miR-142 target through which it controls B-cell homeostasis. Collectively, our results uncover miR-142 as an essential regulator of lymphopoiesis, and suggest that lesions in this miRNA gene may lead to primary immunodeficiency.

microRNA-146a (miR-146a) has been previously implicated as an essential molecular brake, preventing immune overreaction and malignant transformation by attenuating NF-κB signaling, putatively via repression of the Traf6 and Irak1 genes. The exact contribution of miR-146a–mediated silencing of these genes to the control of immune activation is currently unknown. Therefore, we defined the role of the miR-146a–Traf6 signaling axis in the regulation of immune homeostasis using a genetic epistasis analysis in miR-146a−/− mice. We have uncovered a surprising separation of functions at the level of miR-146a targets. Lowering the Traf6 gene dose and consequent attenuation of NF-κB activation rescued several significant miR-146a−/− phenotypes, such as splenomegaly, aberrant myeloproliferation, and excessive inflammatory responses. In contrast, decreasing Traf6 expression had no effect on the development of the progressive bone marrow failure phenotype, as well as lymphomagenesis in miR-146a−/− mice, indicating that miR-146a controls these biological processes through different molecular mechanisms.

Natural killer (NK) cells are cytotoxic type 1 innate lymphoid cells (ILCs) that defend against viruses and mediate anti-tumor responses, yet mechanisms controlling their development and function remain incompletely understood. We hypothesized that the abundantly expressed microRNA-142 (miR-142) is a critical regulator of type 1 ILC biology. Interleukin-15 (IL-15) signaling induced miR-142 expression, whereas global and ILC-specific miR-142-deficient mice exhibited a cell-intrinsic loss of NK cells. Death of NK cells resulted from diminished IL-15 receptor signaling within miR-142-deficient mice, likely via reduced suppressor of cytokine signaling-1 (Socs1) regulation by miR-142-5p. ILCs persisting in Mir142-/- mice demonstrated increased expression of the miR-142-3p target αV integrin, which supported their survival. Global miR-142-deficient mice exhibited an expansion of ILC1-like cells concurrent with increased transforming growth factor-β (TGF-β) signaling. Further, miR-142-deficient mice had reduced NK-cell-dependent function and increased susceptibility to murine cytomegalovirus (MCMV) infection. Thus, miR-142 critically integrates environmental cues for proper type 1 ILC homeostasis and defense against viral infection.

microRNA-142 (miR-142) is expressed predominantly in cells of hematopoietic origin and plays a vital role in the regulation of innate and adaptive immunity. We have previously shown that miR-142 ablation in mice results in a marked expansion of the immature and mature B cell compartments. To better understand the role of miR-142 in B cell ontogenesis, we examined the early B cell development in miR-142 null mice using Hardy fraction analysis. Our findings indicate that miR-142 deletion significantly upregulates the frequency of pro-B cells, large pre-B cells, and immature B cells (Fractions B, C, and E), while decreasing the relative number of the earliest B cell progenitors (pre-pro-B cells; Fraction A). Our data links accumulation of pro-B and large pre-B cells in miR-142−/− bone marrow to an increase in their survival capacity. Moreover, we established miR-142 as a negative regulator of interleukein-7 receptor (IL7R), a cytokine receptor which plays a critical role in B cell maturation from pro-B to pre-B cell stage. In agreement with this notion, miR-142-deficient pro-B cells displayed elevated IL7R levels on their surface and proliferated more vigorously than WT cells in response to IL-7 stimulation in vitro. To test the effect of miR-142 dysregulation on malignant B cell transformation, we bred miR-142−/− mice with Eμ-myc transgenic mice, which spontaneously develop a mix of B cell lymphoblastic leukemia and lymphoma. Interestingly, miR-142 haploinsufficiency dramatically accelerated development of the disease in Eμ-myc transgenic mice. Collectively, our results establish miR-142 as an essential negative regulator of primary B cell development, whose dysregulation may contribute to malignant B cell transformation.

The Philadelphia (Ph) chromosome or t(9;22) results in the generation of a fusion gene, namely BCR/ABL1, which encodes a chimeric protein with aberrant tyrosine kinase activity that drives leukemia cell growth and survival. This molecular/cytogenetic aberration occurs in ~20%-30% of ALL cases and confers poor prognosis. Ph+ ALL patients (pts) are often referred for allogeneic hematopoietic stem cell transplantation (alloHCT), although more recently BCR-ABL-specific tyrosine-kinase inhibitors (TKIs) and immunotherapeutic approaches seemingly induced long-term remission in some patients. Nevertheless, it is still a challenge to determine which Ph+ ALL of the pts could be treated more conservatively without alloHCT. Thus identification of new prognostic biomarkers and/or therapeutic targets may be helpful. Regulation of short non-coding microRNAs(miRNAs) associated with initiation and progression of acute leukemia has been reported. miR-142(both miR-142-3p and miR-142-5p) is expressed at a relatively high level in hematopoietic tissue, and plays a role in myeloid lineage differentiation. In fact, low miR-142-3p expression was associated with myeloid differentiation failure, and miR-142 mutations was reported to promote acute myeloid leukemia (AML). More recently, Kramer et al demonstrated a role of miR-142 in lymphopoiesis by showing that miR-142 deficiency impaired B cell production in a miR-142 knock-out(ko) mouse model (Blood. 2015). Here, we first investigated if miR-142 levels were altered in ALL pts. Analysis of a publically available miRNA expression dataset(GSE23024) showed lower level of miR-142-3p, but not miR-142-5p in Ph+ ALL pts(n=10) vs. healthy donors(n=7;p=0.0093); while no significant differences were observed in Ph- pre-B ALL pts(n=61) vs. healthy donors (n=7). In ALL Tg(P190-BCR/ABL) transgenic mice(Ph+ ALL; Nature. 1990), we found bone marrow (BM) miR-142-3p level to be ~2.3-fold lower than those in the wild-type (wt) controls(p=0.036). Compared to wt mice, Ph+ ALL mice showed significantly lower miR-142-3p level in all the immunophenotypically identified BM lymphoid subpopulations, including progenitor B (pro-B, B220+CD19+CD43+IgM-,~19.1-fold lower,p Disclosures Stein: Celgene: Speakers Bureau; Amgen Inc.: Speakers Bureau. Jin:The National Natural Science Foundation of China: Research Funding; College of Medicine, Zhejiang University: Employment.

Regulatory T (Treg) cells are critical in preventing aberrant immune responses. Post-transcriptional gene control by microRNA (miRNA), a large class of small regulatory RNA, has recently emerged as a key genetic element required for Treg cell function. Nevertheless, specific miRNA gene(s) that play a vital role in the regulation of Treg cell activity are still unknown. Here we report that mice with Treg cell-specific ablation of miR-142 (hereafter Foxp3CremiR-142fl/fl mice) developed a fatal systemic autoimmune disorder due to a breakdown in peripheral T cell tolerance. Foxp3CremiR-142fl/fl mice displayed a sharp decrease in abundance and suppressive capacity of Treg cells. Analysis of miR-142-deficient Treg cells revealed excessive IFNγ production and dysregulated IFNγ signaling. We have identified several of the derepressed IFNγ-related genes, including Ifngr2 receptor and Hif1a transcription factor, as direct miR-142 targets. Furthermore, lowering the Hif1a gene dose in Treg cells significantly diminished the hyperactivation of peripheral effector T cells in Foxp3CremiR-142fl/flmice. Thus, miR-142 is an indispensable regulator of Treg cell development and function that mediates its control by attenuating IFNg response.

MicroRNA-142 (miR-142) is a versatile posttranscriptional regulator that plays a crucial role in the regulation of both innate and adaptive immune responses. Abrogation of miR-142 expression in mice results in a profound immunodeficiency that is characterized by hypoimmunoglobulinemia and failure to mount robust humoral immune responses to soluble antigen challenges. Paradoxically, miR-142−/− mice display a significant expansion of the B cell compartment due to the accumulation of immature and mature B lymphocytes. To better understand the role of miR-142 in B cell ontogenesis, we examined the early B cell development in miR-142−/− mice using the Hardy fraction analysis. We found that miR-142 deletion results in a significant increase in the frequency of the pro-B and large pre-B cells (Fractions B and C), while the pre-pro-B cell population (Fraction A) decreased. In contrast, miR-142 ablation had little effect on the frequency of small pre-B cell and immature B cell populations. Furthermore, our data suggest that the accumulation of pro-B and large pre-B cells in miR-142−/− bone marrow is linked to an increase in their survival capacity. Because genetic lesions that disrupt control of B cell maturation frequently contribute to the malignant transformation of B cells, we decided to examine the role of miR-142 dysregulation in B cell lymphomagenesis. To address this question, we bred miR-142−/− mice with Eμ-myc transgenic mice that spontaneously develop a mix of pre-B cell and mature B cell lymphomas. We found that miR-142 haploinsufficiency dramatically accelerated lymphoma development in this mouse model. Collectively, our results establish miR-142 as an essential negative regulator of B cell maturation and lymphomagenesis.

microRNA-146a (miR-146a) has been previously implicated as an essential molecular brake that prevents immune overreaction and malignant transformation by attenuating NF-κB signaling. miR-146a−/− mice display an autoimmune disorder, and also develop frank tumors in secondary lymphoid organs and a progressive myelodysplastic syndrome (MDS) as they age. miR-146a was proposed to exert its regulatory activity by targeting Traf6 and Irak1, genes which encode two key adapter proteins that function upstream of NF-κB activation. The exact contribution of miR-146a-mediated silencing of Traf6 and Irak1 to the control of immune activation is currently unknown. Therefore, we defined the role of the miR-146a-Traf6 axis in the regulation of immune homeostasis using mouse genetic epistasis analysis. Our studies have uncovered a unique separation of functions at the level of miR-146a targets. Lowering the Traf6 gene dose and consequent attenuation of NF-κB activation rescued many significant miR-146a−/− phenotypes such as splenomegaly, aberrant myeloproliferation, and excessive inflammatory responses. In contrast, decreasing Traf6 expression had no effect on the development of the MDS-like phenotype in aged miR-146a−/− mice, indicating that miR-146a controls hematopoietic stem cell (HSC) homeostasis through a different molecular mechanism. Our genetic data implicate excessive IFNγ production as the main driver of HSC exhaustion and bone marrow failure in miR-146a−/− mice. Furthermore, our findings suggest that control of the Traf6-NF-κB regulatory axis by miR-146a is not essential for its tumor suppressor activity, because lowering the Traf6 gene dose did not affect the rate of development of miR-146a-deficient lymphomas.

MicroRNAs (miRNAs) are a class of powerful post-transcriptional regulators implicated in the control of diverse biological processes, including regulation of hematopoiesis and the immune response. To define the biological functions of miR-142, which is specifically and abundantly expressed in immune cells, we created a mouse line with targeted deletion of this gene. Our analysis of miR-142-/- mice revealed a critical role for this miRNA in the development and homeostasis of lymphocytes. Marginal zone B cells expand in the knockout spleen, while the number of T and B1 B cells in the periphery is reduced. Abnormal development of hematopoietic lineages in miR-142-/- animals is accompanied by a profound immunodeficiency, manifested by hypoimmunoglobulinemia and failure to mount a productive immune response to soluble antigens and virus. miR-142-/- B cells express elevated levels of BAFF receptor (BAFF-R) and as a result proliferate more robustly in response to BAFF stimulation. Lowering the BAFF-R gene dose in miR-142-/- mice rescued the B cell expansion defect, suggesting that BAFF-R is a bona fide miR-142 target through which it controls B cell homeostasis. Collectively, our results uncover miR-142 as an essential regulator of lymphocyte ontogenesis and suggest that lesions in this miRNA gene may lead to primary immunodeficiency.

Green fluorescent protein (GFP) and its derivatives are the most widely used molecular reporters for live cell imagining. The development of organelle-specific fusion fluorescent proteins improves the labeling resolution to a higher level. Here we generate a R26 dual fluorescent protein reporter mouse, activated by Cre-mediated DNA recombination, labeling target cells with a chromatin-specific enhanced green fluorescence protein (EGFP) and a plasma membrane-anchored monomeric cherry fluorescent protein (mCherry). This dual labeling allows the visualization of mitotic events, cell shapes and intracellular vesicle behaviors. We expect this reporter mouse to have a wide application in developmental biology studies, transplantation experiments as well as cancer/stem cell lineage tracing.