The PAX5-JAK2 translocation acts as dual-hit mutation that promotes aggressive B-cell leukemia via nuclear STAT5 activation.

While PAX5 is an important tumor suppressor gene in B-cell acute lymphoblastic leukemia (B-ALL), it is also involved in oncogenic translocations coding for diverse PAX5 fusion proteins. PAX5-JAK2 encodes a protein consisting of the PAX5 DNA-binding region fused to the constitutively active JAK2 kinase domain. Here, we studied the oncogenic function of the PAX5-JAK2 fusion protein in a mouse model expressing it from the endogenous Pax5 locus, resulting in inactivation of one of the two Pax5 alleles. Pax5Jak2/+ mice rapidly developed an aggressive B-ALL in the absence of another cooperating exogenous gene mutation. The DNA-binding function and kinase activity of Pax5-Jak2 as well as IL-7 signaling contributed to leukemia development. Interestingly, all Pax5Jak2/+ tumors lost the remaining wild-type Pax5 allele, allowing efficient DNA-binding of Pax5-Jak2. While we could not find evidence for a nuclear role of Pax5-Jak2 as an epigenetic regulator, high levels of active phosphorylated STAT5 and increased expression of STAT5 target genes were seen in Pax5Jak2/+ B-ALL tumors, implying that nuclear Pax5-Jak2 phosphorylates STAT5. Together, these data reveal Pax5-Jak2 as an important nuclear driver of leukemogenesis by maintaining phosphorylated STAT5 levels in the nucleus.

Bhlhe40 function in activated B and TFH cells restrains the GC reaction and prevents lymphomagenesis.

The generation of high-affinity antibodies against pathogens and vaccines requires the germinal center (GC) reaction, which relies on a complex interplay between specialized effector B and CD4 T lymphocytes, the GC B cells and T follicular helper (TFH) cells. Intriguingly, several positive key regulators of the GC reaction are common for both cell types. Here, we report that the transcription factor Bhlhe40 is a crucial cell-intrinsic negative regulator affecting both the B and T cell sides of the GC reaction. In activated CD4 T cells, Bhlhe40 was required to restrain proliferation, thus limiting the number of TFH cells. In B cells, Bhlhe40 executed its function in the first days after immunization by selectively restricting the generation of the earliest GC B cells but not of early memory B cells or plasmablasts. Bhlhe40-deficient mice with progressing age succumbed to a B cell lymphoma characterized by the accumulation of monoclonal GC B-like cells and polyclonal TFH cells in various tissues.

Pax5 regulates B cell immunity by promoting PI3K signaling via PTEN down-regulation.

The transcription factor Pax5 controls B cell development, but its role in mature B cells is largely enigmatic. Here, we demonstrated that the loss of Pax5 by conditional mutagenesis in peripheral B lymphocytes led to the strong reduction of B-1a, marginal zone (MZ), and germinal center (GC) B cells as well as plasma cells. Follicular (FO) B cells tolerated the loss of Pax5 but had a shortened half-life. The Pax5-deficient FO B cells failed to proliferate upon B cell receptor or Toll-like receptor stimulation due to impaired PI3K-AKT signaling, which was caused by increased expression of PTEN, a negative regulator of the PI3K pathway. Pax5 restrained PTEN protein expression at the posttranscriptional level, likely involving Pten-targeting microRNAs. Additional PTEN loss in Pten,Pax5 double-mutant mice rescued FO B cell numbers and the development of MZ B cells but did not restore GC B cell formation. Hence, the posttranscriptional down-regulation of PTEN expression is an important function of Pax5 that facilitates the differentiation and survival of mature B cells, thereby promoting humoral immunity.

Cryptic activation of an Irf8 enhancer governs cDC1 fate specification.

Induction of the transcription factor Irf8 in the common dendritic cell progenitor (CDP) is required for classical type 1 dendritic cell (cDC1) fate specification, but the mechanisms controlling this induction are unclear. In the present study Irf8 enhancers were identified via chromatin profiling of dendritic cells and CRISPR/Cas9 genome editing was used to assess their roles in Irf8 regulation. An enhancer 32 kilobases (kb) downstream of the Irf8 transcriptional start site (+32-kb Irf8) that was active in mature cDC1s was required for the development of this lineage, but not for its specification. Instead, a +41-kb Irf8 enhancer, previously thought to be active only in plasmacytoid dendritic cells, was found to also be transiently accessible in cDC1 progenitors, and deleting this enhancer prevented the induction of Irf8 in CDPs and abolished cDC1 specification. Thus, cryptic activation of the +41-kb Irf8 enhancer in dendritic cell progenitors is responsible for cDC1 fate specification.

Publisher Correction: The metabolite BH4 controls T cell proliferation in autoimmunity and cancer.

An Amendment to this paper has been published and can be accessed via a link at the top of the paper.

SGLT2 inhibition and renal urate excretion: role of luminal glucose, GLUT9, and URAT1.

Inhibitors of the Na+-glucose cotransporter SGLT2 enhance urinary glucose and urate excretion and lower plasma urate levels. The mechanisms remain unclear, but a role for enhanced glucose in the tubular fluid, which may interact with tubular urate transporters, such as the glucose transporter GLUT9 or the urate transporter URAT1, has been proposed. Studies were performed in nondiabetic mice treated with the SGLT2 inhibitor canagliflozin and in gene-targeted mice lacking the urate transporter Glut9 in the tubule or in mice with whole body knockout of Sglt2, Sglt1, or Urat1. Renal urate handling was assessed by analysis of urate in spontaneous plasma and urine samples and normalization to creatinine concentrations or by renal clearance studies with assessment of glomerular filtration rate by FITC-sinistrin. The experiments confirmed the contribution of URAT1 and GLUT9 to renal urate reabsorption, showing a greater contribution of the latter and additive effects. Genetic and pharmacological inhibition of SGLT2 enhanced fractional renal urate excretion (FE-urate), indicating that a direct effect of the SGLT2 inhibitor on urate transporters is not absolutely necessary. Consistent with a proposed role of increased luminal glucose delivery, the absence of Sglt1, which by itself had no effect on FE-urate, enhanced the glycosuric and uricosuric effects of the SGLT2 inhibitor. The SGLT2 inhibitor enhanced renal mRNA expression of Glut9 in wild-type mice, but tubular GLUT9 seemed dispensable for the increase in FE-urate in response to canagliflozin. First evidence is presented that URAT1 is required for the acute uricosuric effect of the SGLT2 inhibitor in mice.

The metabolite BH4 controls T cell proliferation in autoimmunity and cancer.

Genetic regulators and environmental stimuli modulate T cell activation in autoimmunity and cancer. The enzyme co-factor tetrahydrobiopterin (BH4) is involved in the production of monoamine neurotransmitters, the generation of nitric oxide, and pain1,2. Here we uncover a link between these processes, identifying a fundamental role for BH4 in T cell biology. We find that genetic inactivation of GTP cyclohydrolase 1 (GCH1, the rate-limiting enzyme in the synthesis of BH4) and inhibition of sepiapterin reductase (the terminal enzyme in the synthetic pathway for BH4) severely impair the proliferation of mature mouse and human T cells. BH4 production in activated T cells is linked to alterations in iron metabolism and mitochondrial bioenergetics. In vivo blockade of BH4 synthesis abrogates T-cell-mediated autoimmunity and allergic inflammation, and enhancing BH4 levels through GCH1 overexpression augments responses by CD4- and CD8-expressing T cells, increasing their antitumour activity in vivo. Administration of BH4 to mice markedly reduces tumour growth and expands the population of intratumoral effector T cells. Kynurenine-a tryptophan metabolite that blocks antitumour immunity-inhibits T cell proliferation in a manner that can be rescued by BH4. Finally, we report the development of a potent SPR antagonist for possible clinical use. Our data uncover GCH1, SPR and their downstream metabolite BH4 as critical regulators of T cell biology that can be readily manipulated to either block autoimmunity or enhance anticancer immunity.

Control of B-1a cell development by instructive BCR signaling.

B-1a cells remain one of the most enigmatic lymphocyte subsets. In this review, we discuss recent advances in our understanding of the development of these cells and their regulation by the transcription factors Bhlhe41 and Arid3a as well as by the RNA-binding protein Lin28b. A large body of literature supports an instructive role of BCR signaling in B-1a cell development and lineage commitment, which is initiated only after signaling from an autoreactive BCR. While both fetal and adult hematopoiesis can generate B-1a cells, the contribution of adult hematopoiesis to the B-1a cell compartment is low under physiological conditions. We discuss several models that can reconcile the instructive role of BCR signaling with this fetal bias in B-1a cell development.

Modeling Renal Cell Carcinoma in Mice: Bap1 and Pbrm1 Inactivation Drive Tumor Grade.

Clear cell renal cell carcinoma (ccRCC) is characterized by BAP1 and PBRM1 mutations, which are associated with tumors of different grade and prognosis. However, whether BAP1 and PBRM1 loss causes ccRCC and determines tumor grade is unclear. We conditionally targeted Bap1 and Pbrm1 (with Vhl) in the mouse using several Cre drivers. Sglt2 and Villin proximal convoluted tubule drivers failed to cause tumorigenesis, challenging the conventional notion of ccRCC origins. In contrast, targeting with PAX8, a transcription factor frequently overexpressed in ccRCC, led to ccRCC of different grades. Bap1-deficient tumors were of high grade and showed greater mTORC1 activation than Pbrm1-deficient tumors, which exhibited longer latency. Disrupting one allele of the mTORC1 negative regulator, Tsc1, in Pbrm1-deficient kidneys triggered higher grade ccRCC. This study establishes Bap1 and Pbrm1 as lineage-specific drivers of ccRCC and histologic grade, implicates mTORC1 as a tumor grade rheostat, and suggests that ccRCCs arise from Bowman capsule cells.Significance: Determinants of tumor grade and aggressiveness across cancer types are poorly understood. Using ccRCC as a model, we show that Bap1 and Pbrm1 loss drives tumor grade. Furthermore, we show that the conversion from low grade to high grade can be promoted by activation of mTORC1. Cancer Discov; 7(8); 900-17. ©2017 AACR.See related commentary by Leung and Kim, p. 802This article is highlighted in the In This Issue feature, p. 783.

Essential role for the transcription factor Bhlhe41 in regulating the development, self-renewal and BCR repertoire of B-1a cells.

Innate-like B-1a cells provide a first line of defense against pathogens, yet little is known about their transcriptional control. Here we identified an essential role for the transcription factor Bhlhe41, with a lesser contribution by Bhlhe40, in controlling B-1a cell differentiation. Bhlhe41-/-Bhlhe40-/- B-1a cells were present at much lower abundance than were their wild-type counterparts. Mutant B-1a cells exhibited an abnormal cell-surface phenotype and altered B cell receptor (BCR) repertoire exemplified by loss of the phosphatidylcholine-specific VH12Vκ4 BCR. Expression of a pre-rearranged VH12Vκ4 BCR failed to 'rescue' the mutant phenotype and revealed enhanced proliferation accompanied by increased cell death. Bhlhe41 directly repressed the expression of cell-cycle regulators and inhibitors of BCR signaling while enabling pro-survival cytokine signaling. Thus, Bhlhe41 controls the development, BCR repertoire and self-renewal of B-1a cells.

Molecular role of the PAX5-ETV6 oncoprotein in promoting B-cell acute lymphoblastic leukemia.

PAX5 is a tumor suppressor in B-ALL, while the role of PAX5 fusion proteins in B-ALL development is largely unknown. Here, we studied the function of PAX5-ETV6 and PAX5-FOXP1 in mice expressing these proteins from the Pax5 locus. Both proteins arrested B-lymphopoiesis at the pro-B to pre-B-cell transition and, contrary to their proposed dominant-negative role, did not interfere with the expression of most regulated Pax5 target genes. Pax5-Etv6, but not Pax5-Foxp1, cooperated with loss of the Cdkna2a/b tumor suppressors in promoting B-ALL development. Regulated Pax5-Etv6 target genes identified in these B-ALLs encode proteins implicated in pre-B-cell receptor (BCR) signaling and migration/adhesion, which could contribute to the proliferation, survival, and tissue infiltration of leukemic B cells. Together with similar observations made in human PAX5-ETV6+ B-ALLs, these data identified PAX5-ETV6 as a potent oncoprotein that drives B-cell leukemia development.

Anabolism-Associated Mitochondrial Stasis Driving Lymphocyte Differentiation over Self-Renewal.

Regeneration requires related cells to diverge in fate. We show that activated lymphocytes yield sibling cells with unequal elimination of aged mitochondria. Disparate mitochondrial clearance impacts cell fate and reflects larger constellations of opposing metabolic states. Differentiation driven by an anabolic constellation of PI3K/mTOR activation, aerobic glycolysis, inhibited autophagy, mitochondrial stasis, and ROS production is balanced with self-renewal maintained by a catabolic constellation of AMPK activation, mitochondrial elimination, oxidative metabolism, and maintenance of FoxO1 activity. Perturbations up and down the metabolic pathways shift the balance of nutritive constellations and cell fate owing to self-reinforcement and reciprocal inhibition between anabolism and catabolism. Cell fate and metabolic state are linked by transcriptional regulators, such as IRF4 and FoxO1, with dual roles in lineage and metabolic choice. Instructing some cells to utilize nutrients for anabolism and differentiation while other cells catabolically self-digest and self-renew may enable growth and repair in metazoa.

CXCR5(+) follicular cytotoxic T cells control viral infection in B cell follicles.

During unresolved infections, some viruses escape immunological control and establish a persistant reservoir in certain cell types, such as human immunodeficiency virus (HIV), which persists in follicular helper T cells (TFH cells), and Epstein-Barr virus (EBV), which persists in B cells. Here we identified a specialized group of cytotoxic T cells (TC cells) that expressed the chemokine receptor CXCR5, selectively entered B cell follicles and eradicated infected TFH cells and B cells. The differentiation of these cells, which we have called 'follicular cytotoxic T cells' (TFC cells), required the transcription factors Bcl6, E2A and TCF-1 but was inhibited by the transcriptional regulators Blimp1, Id2 and Id3. Blimp1 and E2A directly regulated Cxcr5 expression and, together with Bcl6 and TCF-1, formed a transcriptional circuit that guided TFC cell development. The identification of TFC cells has far-reaching implications for the development of strategies to control infections that target B cells and TFH cells and to treat B cell-derived malignancies.

Retrotransposon derepression leads to activation of the unfolded protein response and apoptosis in pro-B cells.

The H3K9me3-specific histone methyltransferase Setdb1 impacts on transcriptional regulation by repressing both developmental genes and retrotransposons. How impaired retrotransposon silencing may lead to developmental phenotypes is currently unclear. Here, we show that loss of Setdb1 in pro-B cells completely abrogates B cell development. In pro-B cells, Setdb1 is dispensable for silencing of lineage-inappropriate developmental genes. Instead, we detect strong derepression of endogenous murine leukemia virus (MLV) copies. This activation coincides with an unusual change in chromatin structure, with only partial loss of H3K9me3 and unchanged DNA methylation, but strongly increased H3K4me3. Production of MLV proteins leads to activation of the unfolded protein response pathway and apoptosis. Thus, our data demonstrate that B cell development depends on the proper repression of retrotransposon sequences through Setdb1.

NK Cell-Specific Gata3 Ablation Identifies the Maturation Program Required for Bone Marrow Exit and Control of Proliferation.

NK cells are innate lymphocytes capable of eliciting an innate immune response to pathogens. NK cells develop and become mature in the bone marrow (BM) before they migrate out to peripheral organs. Although the developmental program leading to mature NK cells has been studied in the context of several transcription factors, the stage-specific role of GATA3 in NK cell development has been incompletely understood. Using NKp46-Cre-Gata3(fl/fl) mice in which Gata3 deficiency was induced as early as the immature stage of NK cell differentiation, we demonstrated that GATA3 is required for the NK cell maturation beyond the CD27 single-positive stage and is indispensable for the maintenance of liver-resident NK cells. The frequencies of NK cells from NKp46-Cre-Gata3(fl/fl) mice were found higher in the BM but lower in peripheral organs compared with control littermates, indicating that GATA3 controls the maturation program required for BM egress. Despite the defect in maturation, upon murine CMV infection, NK cells from NKp46-Cre-Gata3(fl/fl) mice expanded vigorously, achieving NK cell frequencies surpassing those in controls and therefore provided comparable protection. The heightened proliferation of NK cells from NKp46-Cre-Gata3(fl/fl) mice was cell intrinsic and associated with enhanced upregulation of CD25 expression. Taken together, our results demonstrate that GATA3 is a critical regulator for NK cell terminal maturation and egress out of the BM and that immature NK cells present in the periphery of NKp46-Cre-Gata3(fl/fl) mice can rapidly expand and provide a reservoir of NK cells capable of mounting an efficient cytotoxic response upon virus infection.

Thymic B Cells Are Licensed to Present Self Antigens for Central T Cell Tolerance Induction.

Thymic antigen-presenting cells (APCs) such as dendritic cells and medullary thymic epithelial cells (mTECs) use distinct strategies of self-antigen expression and presentation to mediate central tolerance. The thymus also harbors B cells; whether they also display unique tolerogenic features and how they genealogically relate to peripheral B cells is unclear. Here, we found that Aire is expressed in thymic but not peripheral B cells. Aire expression in thymic B cells coincided with major histocompatibility class II (MHCII) and CD80 upregulation and immunoglobulin class-switching. These features were recapitulated upon immigration of naive peripheral B cells into the thymus, whereby this intrathymic licensing required CD40 signaling in the context of cognate interactions with autoreactive CD4(+) thymocytes. Moreover, a licensing-dependent neo-antigen selectively upregulated in immigrating B cells mediated negative selection through direct presentation. Thus, autoreactivity within the nascent T cell repertoire fuels a feed forward loop that endows thymic B cells with tolerogenic features.

Caffeine-induced diuresis and natriuresis is independent of renal tubular NHE3.

Caffeine is one of the most widely consumed behavioral substances. We have previously shown that caffeine- and theophylline-induced inhibition of renal reabsorption causes diuresis and natriuresis, an effect that requires functional adenosine A1 receptors. In this study, we tested the hypothesis that blocking the Gi protein-coupled adenosine A1 receptor via the nonselective adenosine receptor antagonist caffeine changes Na(+)/H(+) exchanger isoform 3 (NHE3) localization and phosphorylation, resulting in diuresis and natriuresis. We generated tubulus-specific NHE3 knockout mice (Pax8-Cre), where NHE3 abundance in the S1, S2, and S3 segments of the proximal tubule was completely absent or severely reduced (>85%) in the thick ascending limb. Consumption of fluid and food, as well as glomerular filtration rate, were comparable in control or tubulus-specific NHE3 knockout mice under basal conditions, while urinary pH was significantly more alkaline without evidence for metabolic acidosis. Caffeine self-administration increased total fluid and food intake comparably between genotypes, without significant differences in consumption of caffeinated solution. Acute caffeine application via oral gavage elicited a diuresis and natriuresis that was comparable between control and tubulus-specific NHE3 knockout mice. The diuretic and natriuretic response was independent of changes in total NHE3 expression, phosphorylation of serine-552 and serine-605, or apical plasma membrane NHE3 localization. Although caffeine had no clear effect on localization of the basolateral Na(+)/bicarbonate cotransporter NBCe1, pretreatment with DIDS inhibited caffeine-induced diuresis and natriuresis. In summary, NHE3 is not required for caffeine-induced diuresis and natriuresis.

Differentiation of type 1 ILCs from a common progenitor to all helper-like innate lymphoid cell lineages.

Innate lymphoid cells (ILCs) are a recently recognized group of lymphocytes that have important functions in protecting epithelial barriers against infections and in maintaining organ homeostasis. ILCs have been categorized into three distinct groups, transcriptional circuitry and effector functions of which strikingly resemble the various T helper cell subsets. Here, we identify a common, Id2-expressing progenitor to all interleukin 7 receptor-expressing, "helper-like" ILC lineages, the CHILP. Interestingly, the CHILP differentiated into ILC2 and ILC3 lineages, but not into conventional natural killer (cNK) cells that have been considered an ILC1 subset. Instead, the CHILP gave rise to a peculiar NKp46(+) IL-7Rα(+) ILC lineage that required T-bet for specification and was distinct of cNK cells or other ILC lineages. Such ILC1s coproduced high levels of IFN-γ and TNF and protected against infections with the intracellular parasite Toxoplasma gondii. Our data significantly advance our understanding of ILC differentiation and presents evidence for a new ILC lineage that protects barrier surfaces against intracellular infections.