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Regulation of food intake is a recently identified endocrine function of bone that is mediated by Lipocalin-2 (LCN2). Osteoblast-secreted LCN2 suppresses appetite and decreases fat mass while improving glucose metabolism. We now show that serum LCN2 levels correlate with insulin levels and ß-cell function, indices of healthy glucose metabolism, in obese mice and obese, prediabetic women. However, LCN2 serum levels also correlate with body mass index and insulin resistance in the same individuals and are increased in obese mice. To dissect this apparent discrepancy, we modulated LCN2 levels in mice. Silencing Lcn2 expression worsens metabolic dysfunction in genetic and diet-induced obese mice. Conversely, increasing circulating LCN2 levels improves metabolic parameters and promotes ß-cell function in mouse models of ß-cell failure acting as a growth factor necessary for ß-cell adaptation to higher metabolic load. These results indicate that LCN2 up-regulation is a protective mechanism to counteract obesity-induced glucose intolerance by decreasing food intake and promoting adaptive ß-cell proliferation.

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Activin A functions in BMP signaling in two ways: it either engages ACVR1B to activate Smad2/3 signaling or binds ACVR1 to form a non-signaling complex (NSC). Although the former property has been studied extensively, the roles of the NSC remain unexplored. The genetic disorder fibrodysplasia ossificans progressiva (FOP) provides a unique window into ACVR1/Activin A signaling because in that disease Activin can either signal through FOP-mutant ACVR1 or form NSCs with wild-type ACVR1. To explore the role of the NSC, we generated 'agonist-only' Activin A muteins that activate ACVR1B but cannot form the NSC with ACVR1. Using one of these muteins, we demonstrate that failure to form the NSC in FOP results in more severe disease pathology. These results provide the first evidence for a biological role for the NSC in vivo and pave the way for further exploration of the NSC's physiological role in corresponding knock-in mice.

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Regulatory T cells (Tregs) are crucial for suppressing autoimmunity and inflammation mediated by conventional T cells. To be useful, some Tregs should have overlapping specificity with relevant self-reactive or pathogen-specific clones. Whether matching recognition between Tregs and non-Tregs might arise through stochastic or deterministic mechanisms has not been addressed. We tested the hypothesis that some Tregs that arise in the thymus or that are induced during Ag-driven expansion of conventional CD4+ T cells might be clonally related to non-Tregs by virtue of asymmetric Foxp3 induction during cell division. We isolated mouse CD4+ thymocytes dividing in vivo, wherein sibling cells exhibited discordant expression of Foxp3 and CD25. Under in vitro conditions that stimulate induced Tregs from conventional mouse CD4+ T cells, we found a requirement for cell cycle progression to achieve Foxp3 induction. Moreover, a substantial fraction of sibling cell pairs arising from induced Treg stimulation also contained discordant expression of Foxp3. Division-linked yet asymmetric induction of Treg fate offers potential mechanisms to anticipate peripheral self-reactivity during thymic selection as well as produce precise, de novo counterregulation during CD4+ T cell-mediated immune responses.

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Growth signals drive hematopoietic progenitor cells to proliferate and branch into divergent cell fates, but how unequal outcomes arise from a common progenitor is not fully understood. We used steady-state analysis of in vivo hematopoiesis and Fms-related tyrosine kinase 3 ligand (Flt3L)-induced in vitro differentiation of dendritic cells (DCs) to determine how growth signals regulate lineage bias. We found that Flt3L signaling induced anabolic activation and proliferation of DC progenitors, which was associated with DC differentiation. Perturbation of processes associated with quiescence and catabolism, including AMP-activated protein kinase signaling, fatty acid oxidation, or mitochondrial clearance increased development of cDC2 cells at the expense of cDC1 cells. Conversely, scavenging anabolism-associated reactive oxygen species skewed differentiation toward cDC1 cells. Sibling daughter cells of dividing DC progenitors exhibited unequal expression of the transcription factor interferon regulatory factor 8, which correlated with clonal divergence in FoxO3a signaling and population-level bifurcation of cell fate. We propose that unequal transmission of growth signals during cell division might support fate branches during proliferative expansion of progenitors.

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Unequal transmission of nutritive signaling during cell division establishes fate disparity between sibling lymphocytes, but how asymmetric signaling becomes organized is not understood. We show that receptor-associated class I phosphatidylinositol 3-kinase (PI3K) signaling activity, indexed by phosphatidylinositol (3,4,5)-trisphosphate (PIP3) staining, is spatially restricted to the microtubule-organizing center and subsequently to one pole of the mitotic spindle in activated T and B lymphocytes. Asymmetric PI3K activity co-localizes with polarization of antigen receptor components implicated in class I PI3K signaling and with facultative glucose transporters whose trafficking is PI3K dependent and whose abundance marks cells destined for differentiation. Perturbation of class I PI3K activity disrupts asymmetry of upstream antigen receptors and downstream glucose transporter traffic. The roles of PI3K signaling in nutrient utilization, proliferation, and gene expression may have converged with the conserved role of PI3K signaling in cellular symmetry breaking to form a logic for regenerative lymphocyte divisions.

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Anabolic metabolism in lymphocytes promotes plasmablast and cytotoxic T cell differentiation at the expense of self-renewal. Heightened expression and function of the transcription factor IFN regulatory factor 4 (IRF4) accompany enhanced anabolic induction and full commitment to functional differentiation in B cells and CD8+ T cells. In this study, we used a genetic approach to determine whether IRF4 plays an analogous role in Th1 cell induction. Our findings indicate that IRF4 promotes determined Th1 cell differentiation in tandem with anabolic metabolism of CD4+ T cells. IRF4-deficient CD4+ T cells stimulated in vitro exhibit impaired induction of Th1 gene expression and defective silencing of T cell factor 1 expression. IRF4-deficient CD4+ T cells also undergo a shift toward catabolic metabolism, with reduced mammalian target of rapamycin activation, cell size, and nutrient uptake, as well as increased mitochondrial clearance. These findings suggest that the ability to remodel metabolic states can be an essential gateway for altering cell fate.

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Upon infection, an activated CD4+ T cell produces terminally differentiated effector cells and renews itself for continued defense. In this study, we show that differentiation and self-renewal arise as opposing outcomes of sibling CD4+ T cells. After influenza challenge, antigen-specific cells underwent several divisions in draining lymph nodes (LN; DLNs) while maintaining expression of TCF1. After four or five divisions, some cells silenced, whereas some cells maintained TCF1 expression. TCF1-silenced cells were T helper 1-like effectors and concentrated in the lungs. Cells from earliest divisions were memory-like and concentrated in nondraining LN. TCF1-expressing cells from later divisions in the DLN could self-renew, clonally yielding a TCF1-silenced daughter cell as well as a sibling cell maintaining TCF1 expression. Some TCF1-expressing cells in DLNs acquired an alternative, follicular helper-like fate. Modeled differentiation experiments in vitro suggested that unequal PI3K/mechanistic target of rapamycin signaling drives intraclonal cell fate heterogeneity. Asymmetric division enables self-renewal to be coupled to production of differentiated CD4+ effector T cells during clonal selection.

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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.

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Selected CD8+ T cells must divide, produce differentiated effector cells, and self-renew, often repeatedly. We now show that silencing expression of the transcription factor TCF1 marks loss of self-renewal by determined effector cells and that this requires cell division. In acute infections, the first three CD8+ T cell divisions produce daughter cells with unequal proliferative signaling but uniform maintenance of TCF1 expression. The more quiescent initial daughter cells resemble canonical central memory cells. The more proliferative, effector-prone cells from initial divisions can subsequently undergo division-dependent production of a TCF1-negative effector daughter cell along with a self-renewing TCF1-positive daughter cell, the latter also contributing to the memory cell pool upon resolution of infection. Self-renewal in the face of effector cell determination may promote clonal amplification and memory cell formation in acute infections, sustain effector regeneration during persistent subclinical infections, and be rate limiting, but remediable, in chronic active infections and cancer.

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Type 1 innate lymphocytes comprise two developmentally divergent lineages, type 1 helper innate lymphoid cells (hILC1s) and conventional NK cells (cNKs). All type 1 innate lymphocytes (ILCs) express the transcription factor T-bet, but cNKs additionally express Eomesodermin (Eomes). We show that deletion of Eomes alleles at the onset of type 1 ILC maturation using NKp46-Cre imposes a substantial block in cNK development. Formation of the entire lymphoid and nonlymphoid type 1 ILC compartment appears to require the semiredundant action of both T-bet and Eomes. To determine if Eomes is sufficient to redirect hILC1 development to a cNK fate, we generated transgenic mice that express Eomes when and where T-bet is expressed using Tbx21 locus control to drive expression of Eomes codons. Ectopic Eomes induces cNK-like properties across the lymphoid and nonlymphoid type 1 ILC compartments. Subsequent to their divergent lineage specification, hILC1s and cNKs thus possess substantial developmental plasticity.

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Metazoan sibling cells often diverge in activity and identity, suggesting links between growth signals and cell fate. We show that unequal transduction of nutrient-sensitive PI3K/AKT/mTOR signaling during cell division bifurcates transcriptional networks and fates of kindred cells. A sibling B lymphocyte with stronger signaling, indexed by FoxO1 inactivation and IRF4 induction, undergoes PI3K-driven Pax5 repression and plasma cell determination, while its sibling with weaker PI3K activity renews a memory or germinal center B cell fate. PI3K-driven effector T cell determination silences TCF1 in one sibling cell, while its PI3K-attenuated sibling self-renews in tandem. Prior to bifurcations achieving irreversible plasma or effector cell fate determination, asymmetric signaling during initial divisions specifies a more proliferative, differentiation-prone lymphocyte in tandem with a more quiescent memory cell sibling. By triggering cell division but transmitting unequal intensity between sibling cells, nutrient-sensitive signaling may be a frequent arbiter of cell fate bifurcations during development and repair.

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Central memory (CM) CD8(+) T cells "remember" prior encounters because they maintain themselves through cell division in the absence of ongoing challenge (homeostatic self-renewal), as well as reproduce the CM fate while manufacturing effector cells during secondary Ag encounters (rechallenge self-renewal). We tested the consequence of conditional deletion of the bone marrow homing receptor CXCR4 on antiviral T cell responses. CXCR4-deficient CD8(+) T cells have impaired memory cell maintenance due to defective homeostatic proliferation. Upon rechallenge, however, CXCR4-deficient T cells can re-expand and renew the CM pool while producing secondary effector cells. The critical bone marrow-derived signals essential for CD8(+) T cell homeostatic self-renewal appear to be dispensable to yield self-renewing, functionally asymmetric cell fates during rechallenge.

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