Mutations in the inositol 5-phosphatase OCRL cause Lowe syndrome and Dent's disease. Although OCRL, a direct clathrin interactor, is recruited to late-stage clathrin-coated pits, clinical manifestations have been primarily attributed to intracellular sorting defects. Here we show that OCRL loss in Lowe syndrome patient fibroblasts impacts clathrin-mediated endocytosis and results in an endocytic defect. These cells exhibit an accumulation of clathrin-coated vesicles and an increase in U-shaped clathrin-coated pits, which may result from sequestration of coat components on uncoated vesicles. Endocytic vesicles that fail to lose their coat nucleate the majority of the numerous actin comets present in patient cells. SNX9, an adaptor that couples late-stage endocytic coated pits to actin polymerization and which we found to bind OCRL directly, remains associated with such vesicles. These results indicate that OCRL acts as an uncoating factor and that defects in clathrin-mediated endocytosis likely contribute to pathology in patients with OCRL mutations.

Bone surfaces attract hematopoietic and nonhematopoietic cells, such as osteoclasts (OCs) and osteoblasts (OBs), and are targeted by bone metastatic cancers. However, the mechanisms guiding cells toward bone surfaces are essentially unknown. Here, we show that the Gαi protein-coupled receptor (GPCR) EBI2 is expressed in mouse monocyte/OC precursors (OCPs) and its oxysterol ligand 7α,25-dihydroxycholesterol (7α,25-OHC) is secreted abundantly by OBs. Using in vitro time-lapse microscopy and intravital two-photon microscopy, we show that EBI2 enhances the development of large OCs by promoting OCP motility, thus facilitating cell-cell interactions and fusion in vitro and in vivo. EBI2 is also necessary and sufficient for guiding OCPs toward bone surfaces. Interestingly, OCPs also secrete 7α,25-OHC, which promotes autocrine EBI2 signaling and reduces OCP migration toward bone surfaces in vivo. Defective EBI2 signaling led to increased bone mass in male mice and protected female mice from age- and estrogen deficiency-induced osteoporosis. This study identifies a novel pathway involved in OCP homing to the bone surface that may have significant therapeutic potential.

The study of human macrophages and their ontogeny is an important unresolved issue. Here, we use a humanized mouse model expressing human cytokines to dissect the development of lung macrophages from human hematopoiesis in vivo. Human CD34+ hematopoietic stem and progenitor cells (HSPCs) generated three macrophage populations, occupying separate anatomical niches in the lung. Intravascular cell labeling, cell transplantation, and fate-mapping studies established that classical CD14+ blood monocytes derived from HSPCs migrated into lung tissue and gave rise to human interstitial and alveolar macrophages. In contrast, non-classical CD16+ blood monocytes preferentially generated macrophages resident in the lung vasculature (pulmonary intravascular macrophages). Finally, single-cell RNA sequencing defined intermediate differentiation stages in human lung macrophage development from blood monocytes. This study identifies distinct developmental pathways from circulating monocytes to lung macrophages and reveals how cellular origin contributes to human macrophage identity, diversity, and localization in vivo.

Systemic inflammation halts lymphopoiesis and prioritizes myeloid cell production. How blood cell production switches from homeostasis to emergency myelopoiesis is incompletely understood. Here, we show that lymphotoxin-β receptor (LTβR) signaling in combination with TNF and IL-1 receptor signaling in bone marrow mesenchymal stem cells (MSCs) down-regulates Il7 expression to shut down lymphopoiesis during systemic inflammation. LTβR signaling in MSCs also promoted CCL2 production during systemic inflammation. Pharmacological or genetic blocking of LTβR signaling in MSCs partially enabled lymphopoiesis and reduced monocyte numbers in the spleen during systemic inflammation, which correlated with reduced survival during systemic bacterial and viral infections. Interestingly, lymphotoxin-α1β2 delivered by B-lineage cells, and specifically by mature B cells, contributed to promote Il7 down-regulation and reduce MSC lymphopoietic activity. Our studies revealed an unexpected role of LTβR signaling in MSCs and identified recirculating mature B cells as an important regulator of emergency myelopoiesis.

Cellular competition for limiting hematopoietic factors is a physiologically regulated but poorly understood process. Here, we studied this phenomenon by hampering hematopoietic progenitor access to Leptin receptor+ mesenchymal stem/progenitor cells (MSPCs) and endothelial cells (ECs). We show that HSC numbers increase by 2-fold when multipotent and lineage-restricted progenitors fail to respond to CXCL12 produced by MSPCs and ECs. HSCs are qualitatively normal, and HSC expansion only occurs when early hematopoietic progenitors but not differentiated hematopoietic cells lack CXCR4. Furthermore, the MSPC and EC transcriptomic heterogeneity is stable, suggesting that it is impervious to major changes in hematopoietic progenitor interactions. Instead, HSC expansion correlates with increased availability of membrane-bound stem cell factor (mSCF) on MSPCs and ECs presumably due to reduced consumption by cKit-expressing hematopoietic progenitors. These studies suggest that an intricate homeostatic balance between HSCs and proximal hematopoietic progenitors is regulated by cell competition for limited amounts of mSCF.

Sphingosine-1-phosphate (S1P) receptor 1 (S1PR1) is critical for lymphocyte egress from lymphoid organs. Lymphocytes encounter low S1P concentrations near exit sites before transmigration, yet S1PR1 signaling is rapidly terminated after exposure to S1P. How lymphocytes maintain S1PR1 signaling in a low S1P environment near egress sites is unknown. Here we identify dynamin 2, an essential component of endocytosis, as a novel regulator of T cell egress. Mice with T cell-specific dynamin 2 deficiency had profound lymphopenia and impaired egress from lymphoid organs. Dynamin 2 deficiency caused impaired egress through regulation of S1PR1 signaling, and transgenic S1PR1 overexpression rescued egress in dynamin 2 knockout mice. In low S1P concentrations, dynamin 2 was essential for S1PR1 internalization, which enabled continuous S1PR1 signaling and promoted egress from both thymus and lymph nodes. In contrast, dynamin 2-deficient cells were only capable of a pulse of S1PR1 signaling, which was insufficient for egress. Our results suggest a possible mechanism by which T lymphocytes positioned at exit portals sense low S1P concentrations, promoting their egress into circulatory fluids.

Prolonged T-cell receptor (TCR) signaling is required for the proliferation of T lymphocytes. Ligation of the TCR activates signaling, but also causes internalization of the TCR from the cell surface. How TCR signaling is sustained for many hours despite lower surface expression is unknown. Using genetic inhibition of endocytosis, we show here that TCR internalization promotes continued TCR signaling and T-lymphocyte proliferation. T-cell-specific deletion of dynamin 2, an essential component of endocytosis, resulted in reduced TCR signaling strength, impaired homeostatic proliferation, and the inability to undergo clonal expansion in vivo. Blocking endocytosis resulted in a failure to maintain mammalian target of rapamycin (mTOR) activity and to stably induce the transcription factor myelocytomatosis oncogene (c-Myc), which led to metabolic stress and a defect in cell growth. Our results support the concept that the TCR can continue to signal after it is internalized from the cell surface, thereby enabling sustained signaling and cell proliferation.

B cell progenitors require paracrine signals such as interleukin-7 (IL-7) provided by bone marrow stromal cells for proliferation and survival. Yet, how B cells regulate access to these signals in vivo remains unclear. Here we show that proB and IL-7+ cells form a cell circuit wired by IL-7R signaling, which controls CXCR4 and focal adhesion kinase (FAK) expression and restricts proB cell movement due to increased adhesion to IL-7+CXCL12Hi cells. PreBCR signaling breaks this circuit by switching the preB cell behavior into a fast-moving and lower-adhesion state via increased CXCR4 and reduced FAK/α4β1 expression. This behavioral change reduces preB cell exposure to IL-7, thereby attenuating IL-7R signaling in vivo. Remarkably, IL-7 production is downregulated by signals provided by preB cells with unrepaired double-stranded DNA breaks and by preB acute lymphoblastic leukemic cells. Combined, these studies revealed that distinct cell circuits control the quality and homeostasis of B cell progenitors.

Hexanucleotide expansion in an intron of the C9orf72 gene causes amyotrophic lateral sclerosis and frontotemporal dementia. However, beyond bioinformatics predictions that suggested structural similarity to folliculin, the Birt-Hogg-Dubé syndrome tumor suppressor, little is known about the normal functions of the C9orf72 protein. To address this problem, we used genome-editing strategies to investigate C9orf72 interactions, subcellular localization, and knockout (KO) phenotypes. We found that C9orf72 robustly interacts with SMCR8 (a protein of previously unknown function). We also observed that C9orf72 localizes to lysosomes and that such localization is negatively regulated by amino acid availability. Analysis of C9orf72 KO, SMCR8 KO, and double-KO cell lines revealed phenotypes that are consistent with a function for C9orf72 at lysosomes. These include abnormally swollen lysosomes in the absence of C9orf72 and impaired responses of mTORC1 signaling to changes in amino acid availability (a lysosome-dependent process) after depletion of either C9orf72 or SMCR8. Collectively these results identify strong physical and functional interactions between C9orf72 and SMCR8 and support a lysosomal site of action for this protein complex.

Leukocyte residence in lymphoid organs is controlled by a balance between retention and egress-promoting chemoattractants sensed by pertussis toxin (PTX)-sensitive Gαi protein-coupled receptors (GPCRs). Here, we use two-photon intravital microscopy to show that immature B cell retention within bone marrow (BM) was strictly dependent on amoeboid motility mediated by CXCR4 and CXCL12 and by α4β1 integrin-mediated adhesion to VCAM-1. However, B lineage cell egress from BM is independent of PTX-sensitive GPCR signaling. B lineage cells expressing PTX rapidly exited BM even though their motility within BM parenchyma was significantly reduced. Our experiments reveal that when immature B cells are near BM sinusoids their motility is reduced, their morphology is predominantly rounded, and cells reverse transmigrate across sinusoidal endothelium in a largely nonamoeboid manner. Immature B cell egress from BM was dependent on a twofold CXCR4 down-regulation that was antagonized by antigen-induced BCR signaling. This passive mode of cell egress from BM also contributes significantly to the export of other hematopoietic cells, including granulocytes, monocytes, and NK cells, and is reminiscent of erythrocyte egress.

Intestinal immune homeostasis is dependent upon tightly regulated and dynamic host interactions with the commensal microbiota. Immunoglobulin A (IgA) produced by mucosal B cells dictates the composition of commensal bacteria residing within the intestine. While emerging evidence suggests the majority of IgA is produced innately and may be polyreactive, mucosal-dwelling species can also elicit IgA via T cell-dependent mechanisms. However, the mechanisms that modulate the magnitude and quality of T cell-dependent IgA responses remain incompletely understood. Here we demonstrate that group 3 innate lymphoid cells (ILC3) regulate steady state interactions between T follicular helper cells (TfH) and B cells to limit mucosal IgA responses. ILC3 used conserved migratory cues to establish residence within the interfollicular regions of the intestinal draining lymph nodes, where they act to limit TfH responses and B cell class switching through antigen presentation. The absence of ILC3-intrinsic antigen presentation resulted in increased and selective IgA coating of bacteria residing within the colonic mucosa. Together these findings implicate lymph node resident, antigen-presenting ILC3 as a critical regulatory checkpoint in the generation of T cell-dependent colonic IgA and suggest ILC3 act to maintain tissue homeostasis and mutualism with the mucosal-dwelling commensal microbiota.

Mice repopulated with human hematopoietic cells are a powerful tool for the study of human hematopoiesis and immune function in vivo. However, existing humanized mouse models cannot support development of human innate immune cells, including myeloid cells and natural killer (NK) cells. Here we describe two mouse strains called MITRG and MISTRG, in which human versions of four genes encoding cytokines important for innate immune cell development are knocked into their respective mouse loci. The human cytokines support the development and function of monocytes, macrophages and NK cells derived from human fetal liver or adult CD34(+) progenitor cells injected into the mice. Human macrophages infiltrated a human tumor xenograft in MITRG and MISTRG mice in a manner resembling that observed in tumors obtained from human patients. This humanized mouse model may be used to model the human immune system in scenarios of health and pathology, and may enable evaluation of therapeutic candidates in an in vivo setting relevant to human physiology.

C9orf72 mutations are a major cause of amyotrophic lateral sclerosis and frontotemporal dementia. The C9orf72 protein undergoes regulated recruitment to lysosomes and has been broadly implicated in control of lysosome homeostasis. However, although evidence strongly supports an important function for C9orf72 at lysosomes, little is known about the lysosome recruitment mechanism. In this study, we identify an essential role for WDR41, a prominent C9orf72 interacting protein, in C9orf72 lysosome recruitment. Analysis of human WDR41 knockout cells revealed that WDR41 is required for localization of the protein complex containing C9orf72 and SMCR8 to lysosomes. Such lysosome localization increases in response to amino acid starvation but is not dependent on either mTORC1 inhibition or autophagy induction. Furthermore, WDR41 itself exhibits a parallel pattern of regulated association with lysosomes. This WDR41-dependent recruitment of C9orf72 to lysosomes is critical for the ability of lysosomes to support mTORC1 signaling as constitutive targeting of C9orf72 to lysosomes relieves the requirement for WDR41 in mTORC1 activation. Collectively, this study reveals an essential role for WDR41 in supporting the regulated binding of C9orf72 to lysosomes and solidifies the requirement for a larger C9orf72 containing protein complex in coordinating lysosomal responses to changes in amino acid availability.

The C9orf72 protein is required for normal lysosome function. In support of such functions, C9orf72 forms a heterotrimeric complex with SMCR8 and WDR41 that is recruited to lysosomes when amino acids are scarce. These properties raise questions about the identity of the lysosomal binding partner of the C9orf72 complex and the amino acid-sensing mechanism that regulates C9orf72 complex abundance on lysosomes. We now demonstrate that an interaction with the lysosomal cationic amino acid transporter PQLC2 mediates C9orf72 complex recruitment to lysosomes. This is achieved through an interaction between PQLC2 and WDR41. The interaction between PQLC2 and the C9orf72 complex is negatively regulated by arginine, lysine, and histidine, the amino acids that PQLC2 transports across the membrane of lysosomes. These results define a new role for PQLC2 in the regulated recruitment of the C9orf72 complex to lysosomes and reveal a novel mechanism that allows cells to sense and respond to changes in the availability of cationic amino acids within lysosomes.

Portal hypertension is a major contributor to decompensation and death from liver disease, a global health problem. Here, we demonstrate homozygous damaging mutations in GIMAP5, a small organellar GTPase, in four families with unexplained portal hypertension. We show that GIMAP5 is expressed in hepatic endothelial cells and that its loss in both humans and mice results in capillarization of liver sinusoidal endothelial cells (LSECs); this effect is also seen when GIMAP5 is selectively deleted in endothelial cells. Single-cell RNA-sequencing analysis in a GIMAP5-deficient mouse model reveals replacement of LSECs with capillarized endothelial cells, a reduction of macrovascular hepatic endothelial cells, and places GIMAP5 upstream of GATA4, a transcription factor required for LSEC specification. Thus, GIMAP5 is a critical regulator of liver endothelial cell homeostasis and, when absent, produces portal hypertension. These findings provide new insight into the pathogenesis of portal hypertension, a major contributor to morbidity and mortality from liver disease.

Suppressor of cytokine signaling (SOCS)-3 has been suggested to regulate CXCR4 signaling in a variety of human cell lines. In mice, conditional SOCS3 inactivation in hematopoietic cells including B-lineage lymphocytes has been reported to exacerbate CXCR4-signaling and focal adhesion kinase phosphorylation, which resulted in altered immature B cell distribution in bone marrow (BM) due to sustained α4β1 integrin-mediated adhesion to the extracellular matrix. However, a recent study examining conditional SOCS3 deletion specifically in B-lineage cells failed to detect significant roles in B-lineage cell retention in BM. In this study we carefully examined the role played by SOCS3 in CXCR4 signaling in developing B cell subsets. We show that in mice conditionally deficient in SOCS3 exclusively in B cells (Socs3fl/fl Mb1cre/+) there was no detectable difference in B cell development in BM and in periphery. We show that SOCS3 deficient and sufficient immature B cell subsets are similarly distributed between BM parenchyma and sinusoids, and are equally competent at exiting BM into peripheral blood. Furthermore, we found no significant differences in CXCR4 desensitization upon ligand exposure in developing B lymphocyte subsets. Consequently, SOCS3-deficient and sufficient B-lineage cell migration towards CXCL12 in vitro was undistinguishable, and B-lineage cell amoeboid motility within BM parenchyma was also unaffected by SOCS3-deficiency. Thus we conclude that SOCS3 has no detectable influence on biological processes known to be controlled by CXCR4 signaling.

Immature B cells developing in the bone marrow are found in the parenchyma and sinusoids. The mechanisms that control the positioning of B cells in the sinusoids are not understood. Here we show that the integrin alpha(4)beta(1) (VLA-4) and its ligand VCAM-1 were required, whereas the chemokine receptor CXCR4 was dispensable, for sinusoidal retention of B cells. Instead, cannabinoid receptor 2 (CB2), a Galpha(i) protein-coupled receptor upregulated in immature B cells, was required for sinusoidal retention. Using two-photon microscopy, we found immature B cells entering and crawling in sinusoids; these immature B cells were displaced by CB2 antagonism. Moreover, CB2-deficient mice had a lower frequency of immunoglobulin lambda-chain-positive B cells in the peripheral blood and spleen. Our findings identify unique requirements for the retention of B cells in the bone marrow sinusoidal niche and suggest involvement of CB2 in the generation of the B cell repertoire.

The GTPase dynamin, a key player in endocytic membrane fission, interacts with numerous proteins that regulate actin dynamics and generate/sense membrane curvature. To determine the functional relationship between these proteins and dynamin, we have analyzed endocytic intermediates that accumulate in cells that lack dynamin (derived from dynamin 1 and 2 double conditional knockout mice). In these cells, actin-nucleating proteins, actin, and BAR domain proteins accumulate at the base of arrested endocytic clathrin-coated pits, where they support the growth of dynamic long tubular necks. These results, which we show reflect the sequence of events in wild-type cells, demonstrate a concerted action of these proteins prior to, and independent of, dynamin and emphasize similarities between clathrin-mediated endocytosis in yeast and higher eukaryotes. Our data also demonstrate that the relationship between dynamin and actin is intimately connected to dynamin's endocytic role and that dynamin terminates a powerful actin- and BAR protein-dependent tubulating activity.

B cell follicles are specialized microenvironments that support events necessary for humoral immunity. After antigen encounter, activated B cells initially seek T-cell help at the follicle-T-zone boundary and then move to interfollicular and T-zone distal (outer) regions of the follicle. Subsequently, some cells move to the follicle centre, become germinal centre B cells and undergo antibody affinity maturation. Although germinal centres within follicles were described in 1885 (ref. 12), the molecular cues mediating segregation of B cells between the outer and centre follicle have remained undefined. Here we present a role for the orphan G-protein-coupled receptor, Epstein-Barr virus induced molecule-2 (EBI2, also known as GPR183), in this process. EBI2 is expressed in mature B cells and increases in expression early after activation, before being downregulated in germinal centre B cells. EBI2 deficiency in mice led to a reduction in the early antibody response to a T-dependent antigen. EBI2-deficient B cells failed to move to the outer follicle at day 2 of activation, and instead were found in the follicle centre, whereas EBI2 overexpression was sufficient to promote B cell localization to the outer follicle. In mixed bone marrow chimaeras, EBI2-deficient B cells phenocopied germinal centre B cells in preferentially localizing to the follicle centre. When downregulation of EBI2 in wild-type B cells was antagonized, participation in the germinal centre reaction was impaired. These studies identify an important role for EBI2 in promoting B cell localization in the outer follicle, and show that differential expression of this receptor helps position B cells appropriately for mounting T-dependent antibody responses.

Group 3 innate lymphoid cells (ILC3s) sense environmental signals and are critical for tissue integrity in the intestine. Yet, which signals are sensed and what receptors control ILC3 function remain poorly understood. Here, we show that ILC3s with a lymphoid-tissue-inducer (LTi) phenotype expressed G-protein-coupled receptor 183 (GPR183) and migrated to its oxysterol ligand 7α,25-hydroxycholesterol (7α,25-OHC). In mice lacking Gpr183 or 7α,25-OHC, ILC3s failed to localize to cryptopatches (CPs) and isolated lymphoid follicles (ILFs). Gpr183 deficiency in ILC3s caused a defect in CP and ILF formation in the colon, but not in the small intestine. Localized oxysterol production by fibroblastic stromal cells provided an essential signal for colonic lymphoid tissue development, and inflammation-induced increased oxysterol production caused colitis through GPR183-mediated cell recruitment. Our findings show that GPR183 promotes lymphoid organ development and indicate that oxysterol-GPR183-dependent positioning within tissues controls ILC3 activity and intestinal homeostasis.