HSC-independent definitive hematopoiesis persists into adult life.

It is widely believed that hematopoiesis after birth is established by hematopoietic stem cells (HSCs) in the bone marrow and that HSC-independent hematopoiesis is limited only to primitive erythro-myeloid cells and tissue-resident innate immune cells arising in the embryo. Here, surprisingly, we find that significant percentages of lymphocytes are not derived from HSCs, even in 1-year-old mice. Instead, multiple waves of hematopoiesis occur from embryonic day 7.5 (E7.5) to E11.5 endothelial cells, which simultaneously produce HSCs and lymphoid progenitors that constitute many layers of adaptive T and B lymphocytes in adult mice. Additionally, HSC lineage tracing reveals that the contribution of fetal liver HSCs to peritoneal B-1a cells is minimal and that the majority of B-1a cells are HSC independent. Our discovery of extensive HSC-independent lymphocytes in adult mice attests to the complex blood developmental dynamics spanning the embryo-to-adult transition and challenges the paradigm of HSCs exclusively underpinning the postnatal immune system.

Embryonic type 3 innate lymphoid cells sense maternal dietary cholesterol to control local Peyer's patch development.

Lymphoid tissue inducer (LTi) cells develop during intrauterine life and rely on developmental programs to initiate the organogenesis of secondary lymphoid organs (SLOs). This evolutionary conserved process endows the fetus with the ability to orchestrate the immune response after birth and to react to the triggers present in the environment. While it is established that LTi function can be shaped by maternal-derived cues and is critical to prepare the neonate with a functional scaffold to mount immune response, the cellular mechanisms that control anatomically distinct SLO organogenesis remain unclear. We discovered that LTi cells forming Peyer's patches, gut-specific SLOs, require the coordinated action of two migratory G protein coupled receptors (GPCR) GPR183 and CCR6. These two GPCRs are uniformly expressed on LTi cells across SLOs, but their deficiency specifically impacts Peyer's patch formation, even when restricted to fetal window. The unique CCR6 ligand is CCL20, while the ligand for GPR183 is the cholesterol metabolite 7α,25-Dihydroxycholesterol (7α,25-HC), whose production is controlled by the enzyme cholesterol 25-hydroxylase (CH25H). We identified a fetal stromal cell subset that expresses CH25H and attracts LTi cells in the nascent Peyer's patch anlagen. GPR183 ligand concentration can be modulated by the cholesterol content in the maternal diet and impacts LTi cell maturation in vitro and in vivo, highlighting a link between maternal nutrients and intestinal SLO organogenesis. Our findings revealed that in the fetal intestine, cholesterol metabolite sensing by GPR183 in LTi cells for Peyer's patch formation is dominant in the duodenum, the site of cholesterol absorption in the adult. This anatomic requirement suggests that embryonic, long-lived non-hematopoietic cells might exploit adult metabolic functions to ensure highly specialized SLO development in utero.

Skin γδ T cell inflammatory responses are hardwired in the thymus by oxysterol sensing via GPR183 and calibrated by dietary cholesterol.

Dietary components and metabolites have a profound impact on immunity and inflammation. Here, we investigated how sensing of cholesterol metabolite oxysterols by γδ T cells impacts their tissue residency and function. We show that dermal IL-17-producing γδ T (Tγδ17) cells essential for skin-barrier homeostasis require oxysterols sensing through G protein receptor 183 (GPR183) for their development and inflammatory responses. Single-cell transcriptomics and murine reporter strains revealed that GPR183 on developing γδ thymocytes is needed for their maturation by sensing medullary thymic epithelial-cell-derived oxysterols. In the skin, basal keratinocytes expressing the oxysterol enzyme cholesterol 25-hydroxylase (CH25H) maintain dermal Tγδ17 cells. Diet-driven increases in oxysterols exacerbate Tγδ17-cell-mediated psoriatic inflammation, dependent on GPR183 on γδ T cells. Hence, cholesterol-derived oxysterols control spatially distinct but biologically linked processes of thymic education and peripheral function of dermal T cells, implicating diet as a focal parameter of dermal Tγδ17 cells.

Dietary Cholesterol Metabolite Regulation of Tissue Immune Cell Development and Function.

Obesity is considered the primary environmental factor associated with morbidity and severity of wide-ranging inflammatory disorders. The molecular mechanism linking high-fat or cholesterol diet to imbalances in immune responses, beyond the increased production of generic inflammatory factors, is just beginning to emerge. Diet cholesterol by-products are now known to regulate function and migration of diverse immune cell subsets in tissues. The hydroxylated metabolites of cholesterol oxysterols as central regulators of immune cell positioning in lymphoid and mucocutaneous tissues is the focus of this review. Dedicated immunocyte cell surface receptors sense spatially distributed oxysterol tissue depots to tune cell metabolism and function, to achieve the "right place at the right time" axiom of efficient tissue immunity.

Epithelial HNF4A shapes the intraepithelial lymphocyte compartment via direct regulation of immune signaling molecules.

Hepatocyte nuclear factor 4 α (HNF4A) is a highly conserved nuclear receptor that has been associated with ulcerative colitis. In mice, HNF4A is indispensable for the maintenance of intestinal homeostasis, yet the underlying mechanisms are poorly characterized. Here, we demonstrate that the expression of HNF4A in intestinal epithelial cells (IECs) is required for the proper development and composition of the intraepithelial lymphocyte (IEL) compartment. HNF4A directly regulates expression of immune signaling molecules including butyrophilin-like (Btnl) 1, Btnl6, H2-T3, and Clec2e that control IEC-IEL crosstalk. HNF4A selectively enhances the expansion of natural IELs that are TCRγδ+ or TCRαß+CD8αα+ to shape the composition of IEL compartment. In the small intestine, HNF4A cooperates with its paralog HNF4G, to drive expression of immune signaling molecules. Moreover, the HNF4A-BTNL regulatory axis is conserved in human IECs. Collectively, these findings underscore the importance of HNF4A as a conserved transcription factor controlling IEC-IEL crosstalk and suggest that HNF4A maintains intestinal homeostasis through regulation of the IEL compartment.

Activation of CD81+ skin ILC2s by cold-sensing TRPM8+ neuron-derived signals maintains cutaneous thermal homeostasis.

As the outermost barrier tissue of the body, the skin harbors a large number of innate lymphoid cells (ILCs) that help maintain local homeostasis in the face of changing environments. How skin-resident ILCs are regulated and function in local homeostatic maintenance is poorly understood. We here report the discovery of a cold-sensing neuron-initiated pathway that activates skin group 2 ILCs (ILC2s) to help maintain thermal homeostasis. In stearoyl-CoA desaturase 1 (SCD1) knockout mice whose skin is defective in heat maintenance, chronic cold stress induced excessive activation of CCR10-CD81+ST2+ skin ILC2s and associated inflammation. Mechanistically, stimulation of the cold-sensing receptor TRPM8 expressed in sensory neurons of the skin led to increased production of IL-18, which, in turn, activated skin ILC2s to promote thermogenesis. Our findings reveal a neuroimmune link that regulates activation of skin ILC2s to support thermal homeostasis and promotes skin inflammation after hyperactivation.

Neonatal-derived IL-17 producing dermal γδ T cells are required to prevent spontaneous atopic dermatitis.

Atopic Dermatitis (AD) is a T cell-mediated chronic skin disease and is associated with altered skin barrier integrity. Infants with mutations in genes involved in tissue barrier fitness are predisposed towards inflammatory diseases, but most do not develop or sustain the diseases, suggesting that there exist regulatory immune mechanisms to prevent aberrant inflammation. The absence of one single murine dermal cell type, the innate neonatal-derived IL-17 producing γδ T (Tγδ17) cells, from birth resulted in spontaneous, highly penetrant AD with many of the major hallmarks of human AD. In Tγδ17 cell-deficient mice, basal keratinocyte transcriptome was altered months in advance of AD induction. Tγδ17 cells respond to skin commensal bacteria and the fulminant disease in their absence was driven by skin commensal bacteria dysbiosis. AD in this model was characterized by highly expanded dermal αß T clonotypes that produce the type three cytokines, IL-17 and IL-22. These results demonstrate that neonatal Tγδ17 cells are innate skin regulatory T cells that are critical for skin homeostasis, and that IL-17 has dual homeostatic and inflammatory function in the skin.

γδTCR-independent origin of neonatal γδ T cells prewired for IL-17 production.

A classical view of T cell lineages consists of two major clades of T cells expressing either the αß or γδ T cell receptor (TCR). However, genome-wide assessments indicate molecular clusters segregating T cell subsets that are preprogrammed for effector function (innate) from those that mediate conventional adaptive response, regardless of the TCR types. Within this paradigm, γδ T cells remain the prototypic innate-like lymphocytes, many subsets of which are programmed during intrathymic development for committed peripheral tissue localization and effector responses. Emerging evidence for innate γδ T cell lineage choice dictated by developmental gene programs rather than the sensory TCR is discussed in this review.

Interleukin-17-Producing γδ T Cells Originate from SOX13+ Progenitors that Are Independent of γδTCR Signaling.

Lineage-committed αß and γδ T cells are thought to originate from common intrathymic multipotent progenitors following instructive T cell receptor (TCR) signals. A subset of lymph node and mucosal Vγ2+ γδ T cells is programmed intrathymically to produce IL-17 (Tγδ17 cells), however the role of the γδTCR in development of these cells remains controversial. Here we generated reporter mice for the Tγδ17 lineage-defining transcription factor SOX13 and identified fetal-origin, intrathymic Sox13+ progenitors. In organ culture developmental assays, Tγδ17 cells derived primarily from Sox13+ progenitors, and not from other known lymphoid progenitors. Single cell transcriptome assays of the progenitors found in TCR-deficient mice demonstrated that Tγδ17 lineage programming was independent of γδTCR. Instead, generation of the lineage committed progenitors and Tγδ17 cells was controlled by TCF1 and SOX13. Thus, T lymphocyte lineage fate can be prewired cell-intrinsically and is not necessarily specified by clonal antigen receptor signals.

SOX4 controls invariant NKT cell differentiation by tuning TCR signaling.

Natural killer T (NKT) cells expressing the invariant T cell receptor (iTCR) serve an essential function in clearance of certain pathogens and have been implicated in autoimmune and allergic diseases. Complex effector programs of these iNKT cells are wired in the thymus, and upon thymic egress, they can respond within hours of antigenic challenges, classifying iNKT cells as innate-like. It has been assumed that the successful rearrangement of the invariant iTCRα chain is the central event in the divergence of immature thymocytes to the NKT cell lineage, but molecular properties that render the iTCR signaling distinct to permit the T cell lineage diversification remain obscure. Here we show that the High Mobility Group (HMG) transcription factor (TF) SOX4 controls the production of iNKT cells by inducing MicroRNA-181 (Mir181) to enhance TCR signaling and Ca2+ fluxes in precursors. These results suggest the existence of tailored, permissive gene circuits in iNKT precursors for innate-like T cell development.

Hypercholesterolemia Increases Colorectal Cancer Incidence by Reducing Production of NKT and γδ T Cells from Hematopoietic Stem Cells.

Obesity will soon surpass smoking as the most preventable cause of cancer. Hypercholesterolemia, a common comorbidity of obesity, has been shown to increase cancer risk, especially colorectal cancer. However, the mechanism by which hypercholesterolemia or any metabolic disorder increases cancer risk remains unknown. In this study, we show that hypercholesterolemia increases the incidence and pathologic severity of colorectal neoplasia in two independent mouse models. Hypocholesterolemia induced an oxidant stress-dependent increase in miR101c, which downregulated Tet1 in hematopoietic stem cells (HSC), resulting in reduced expression of genes critical to natural killer T cell (NKT) and γδ T-cell differentiation. These effects reduced the number and function of terminally differentiated NKT and γδ T cells in the thymus, the colon submucosa, and during early tumorigenesis. These results suggest a novel mechanism by which a metabolic disorder induces epigenetic changes to reduce lineage priming of HSC toward immune cells, thereby compromising immunosurveillance against cancer. Cancer Res; 77(9); 2351-62. ©2017 AACR.

Transcription factor networks directing the development, function, and evolution of innate lymphoid effectors.

Mammalian lymphoid immunity is mediated by fast and slow responders to pathogens. Fast innate lymphocytes are active within hours after infections in mucosal tissues. Slow adaptive lymphocytes are conventional T and B cells with clonal antigen receptors that function days after pathogen exposure. A transcription factor (TF) regulatory network guiding early T cell development is at the core of effector function diversification in all innate lymphocytes, and the kinetics of immune responses is set by developmental programming. Operational units within the innate lymphoid system are not classified by the types of pathogen-sensing machineries but rather by discrete effector functions programmed by regulatory TF networks. Based on the evolutionary history of TFs of the regulatory networks, fast effectors likely arose earlier in the evolution of animals to fortify body barriers, and in mammals they often develop in fetal ontogeny prior to the establishment of fully competent adaptive immunity.

The necroptosis adaptor RIPK3 promotes injury-induced cytokine expression and tissue repair.

Programmed necrosis or necroptosis is an inflammatory form of cell death that critically requires the receptor-interacting protein kinase 3 (RIPK3). Here we showed that RIPK3 controls a separate, necrosis-independent pathway of inflammation by regulating cytokine expression in dendritic cells (DCs). Ripk3(-/-) bone-marrow-derived dendritic cells (BMDCs) were highly defective in lipopolysaccharide (LPS)-induced expression of inflammatory cytokines. These effects were caused by impaired NF-κB subunit RelB and p50 activation and by impaired caspase 1-mediated processing of interleukin-1ß (IL-1ß). This DC-specific function of RIPK3 was critical for injury-induced inflammation and tissue repair in response to dextran sodium sulfate (DSS). Ripk3(-/-) mice exhibited an impaired axis of injury-induced IL-1ß, IL-23, and IL-22 cytokine cascade, which was partially corrected by adoptive transfer of wild-type DCs, but not Ripk3(-/-) DCs. These results reveal an unexpected function of RIPK3 in NF-κB activation, DC biology, innate inflammatory-cytokine expression, and injury-induced tissue repair.

TRIM13 is a negative regulator of MDA5-mediated type I interferon production.

UNLABELLED: Retinoic acid-inducible gene I (RIG-I) and melanoma differentiation-associated gene 5 (MDA5) are essential intracellular detectors of viral RNA. They contribute to the type I interferon (IFN) response that is crucial for host defense against viral infections. Given the potent antiviral and proinflammatory activities elicited by the type I IFNs, induction of the type I IFN response is tightly regulated. Members of the tripartite motif (TRIM) family of proteins have recently emerged as key regulators of antiviral immunity. We show that TRIM13, an E3 ubiquitin ligase, is expressed in immune cells and is upregulated in bone marrow-derived macrophages upon stimulation with inducers of type I IFN. TRIM13 interacts with MDA5 and negatively regulates MDA5-mediated type I IFN production in vitro, acting upstream of IFN regulatory factor 3. We generated Trim13(-/-) mice and show that upon lethal challenge with encephalomyocarditis virus (EMCV), which is sensed by MDA5, Trim13(-/-) mice produce increased amounts of type I IFNs and survive longer than wild-type mice. Trim13(-/-) murine embryonic fibroblasts (MEFs) challenged with EMCV or poly(I · C) also show a significant increase in beta IFN (IFN-ß) levels, but, in contrast, IFN-ß responses to the RIG-I-detected Sendai virus were diminished, suggesting that TRIM13 may play a role in positively regulating RIG-I function. Together, these results demonstrate that TRIM13 regulates the type I IFN response through inhibition of MDA5 activity and that it functions nonredundantly to modulate MDA5 during EMCV infection. IMPORTANCE: The type I interferon (IFN) response is crucial for host defense against viral infections, and proper regulation of this pathway contributes to maintaining immune homeostasis. Retinoic acid-inducible gene I (RIG-I) and melanoma differentiation-associated gene 5 (MDA5) are intracellular detectors of viral RNA that induce the type I IFN response. In this study, we show that expression of the gene tripartite motif 13 (Trim13) is upregulated in response to inducers of type I IFN and that TRIM13 interacts with both MDA5 and RIG-I in vitro. Through the use of multiple in vitro and in vivo model systems, we show that TRIM13 is a negative regulator of MDA5-mediated type I IFN production and may also impact RIG-I-mediated type I IFN production by enhancing RIG-I activity. This places TRIM13 at a key junction within the viral response pathway and identifies it as one of the few known modulators of MDA5 activity.

Innate PLZF+CD4+ αß T cells develop and expand in the absence of Itk.

T cell development in the thymus produces multiple lineages of cells, including innate T cells. Studies in mice harboring alterations in TCR signaling proteins or transcriptional regulators have revealed an expanded population of CD4(+) innate T cells in the thymus that produce IL-4 and express the transcription factor promyelocytic leukemia zinc finger (PLZF). In these mice, IL-4 produced by the CD4(+)PLZF(+) T cell population leads to the conversion of conventional CD8(+) thymocytes into innate CD8(+) T cells resembling memory T cells expressing eomesodermin. The expression of PLZF, the signature invariant NKT cell transcription factor, in these innate CD4(+) T cells suggests that they might be a subset of αß or γδ TCR(+) NKT cells or mucosal-associated invariant T (MAIT) cells. To address these possibilities, we characterized the CD4(+)PLZF(+) innate T cells in itk(-/-) mice. We show that itk(-/-) innate PLZF(+)CD4(+) T cells are not CD1d-dependent NKT cells, MR1-dependent MAIT cells, or γδ T cells. Furthermore, although the itk(-/-) innate PLZF(+)CD4(+) T cells express αß TCRs, neither ß2-microglobulin-dependent MHC class I nor any MHC class II molecules are required for their development. In contrast to invariant NKT cells and MAIT cells, this population has a highly diverse TCRα-chain repertoire. Analysis of peripheral tissues indicates that itk(-/-) innate PLZF(+)CD4(+) T cells preferentially home to spleen and mesenteric lymph nodes owing to increased expression of gut-homing receptors, and that their expansion is regulated by commensal gut flora. These data support the conclusion that itk(-/-) innate PLZF(+)CD4(+) T cells are a novel subset of innate T cells.

Regulation of tissue-dependent differences in CD8+ T cell apoptosis during viral infection.

UNLABELLED: Virus-specific CD8+ T cells in the lymphoid organs contract at the resolution of virus infections by apoptosis or by dissemination into peripheral tissues, and those residing in nonlymphoid organs, including the peritoneal cavity and fat pads, are more resistant to apoptosis than those in the spleen and lymph nodes. This stability of memory T cells in the nonlymphoid tissues may enhance protection to secondary challenges. Here, we show that lymphocytic choriomeningitis virus (LCMV)-specific CD8+ T cells in nonlymphoid tissues were enriched for memory precursors (expressing high levels of interleukin-7 receptor and low levels of killer cell lectin-like receptor G1 [IL-7Rhi KLRG1lo]) and had higher expression of CD27, CXCR3, and T cell factor-1 (TCF-1), each a marker that is individually correlated with decreased apoptosis. CD8+ T cells in the peritoneal cavity of TCF-1-deficient mice had decreased survival, suggesting a role for TCF-1 in promoting survival in the nonlymphoid tissues. CXCR3+ CD8+ T cells resisted apoptosis and accumulated in the lymph nodes of mice treated with FTY720, which blocks the export of lymph node cells into peripheral tissue. The peritoneal exudate cells (PEC) expressed increased amounts of CXCR3 ligands, CXCL9 and CXCL10, which may normally recruit these nonapoptotic cells from the lymph nodes. In addition, adoptive transfer of splenic CD8+ T cells into PEC or spleen environments showed that the peritoneal environment promoted survival of CD8+ T cells. Thus, intrinsic stability of T cells which are present in the nonlymphoid tissues along with preferential migration of apoptosis-resistant CD8+ T cells into peripheral sites and the availability of tissue-specific factors that enhance memory cell survival may collectively account for the tissue-dependent apoptotic differences. IMPORTANCE: Most infections are initiated at nonlymphoid tissue sites, and the presence of memory T cells in nonlymphoid tissues is critical for protective immunity in various viral infection models. Virus-specific CD8+ T cells in the nonlymphoid tissues are more resistant to apoptosis than those in lymphoid organs during the resolution and memory phase of the immune response to acute LCMV infection. Here, we investigated the mechanisms promoting stability of T cells in the nonlymphoid tissues. This increased resistance to apoptosis of virus-specific CD8+ T cells in nonlymphoid tissues was due to several factors. Nonlymphoid tissues were enriched in memory phenotype CD8+ T cells, which were intrinsically resistant to apoptosis irrespective of the tissue environment. Furthermore, apoptosis-resistant CD8+ T cells preferentially migrated into the nonlymphoid tissues, where the availability of tissue-specific factors may enhance memory cell survival. Our findings are relevant for the generation of long-lasting vaccines providing protection at peripheral infection sites.

CD28 and ITK signals regulate autoreactive T cell trafficking.

Activation of self-reactive T cells and their trafficking to target tissues leads to autoimmune organ destruction. Mice lacking the co-inhibitory receptor cytotoxic T lymphocyte antigen-4 (CTLA-4) develop fatal autoimmunity characterized by lymphocytic infiltration into nonlymphoid tissues. Here, we demonstrate that the CD28 co-stimulatory pathway regulates the trafficking of self-reactive Ctla4(-/-) T cells to tissues. Concurrent ablation of the CD28-activated Tec family kinase ITK does not block spontaneous T cell activation but instead causes self-reactive Ctla4(-/-) T cells to accumulate in secondary lymphoid organs. Despite excessive spontaneous T cell activation and proliferation in lymphoid organs, Itk(-/-); Ctla4(-/-) mice are otherwise healthy, mount antiviral immune responses and exhibit a long lifespan. We propose that ITK specifically licenses autoreactive T cells to enter tissues to mount destructive immune responses. Notably, ITK inhibitors mimic the null mutant phenotype and also prevent pancreatic islet infiltration by diabetogenic T cells in mouse models of type 1 diabetes, highlighting their potential utility for the treatment of human autoimmune disorders.

Immunological Genome Project and systems immunology.

Immunological studies of single proteins in a single cell type have been complemented in recent years by larger studies, enabled by emerging high-throughput technologies. This trend has recently been exemplified by the discovery of gene networks controlling regulatory and effector αß T cell subset development and human hematopoiesis. The Immunological Genome Project (ImmGen) aims to decipher the gene networks underpinning mouse hematopoiesis. The first phase, completed in 2012, profiled the transcriptome of 249 immune cell types. We discuss the utilities of the datasets in high-resolution mapping of the hematopoietic system. The immune transcriptome compendium has revealed unsuspected cell lineage relations and the network reconstruction has identified novel regulatory factors of hematopoiesis.

A network of high-mobility group box transcription factors programs innate interleukin-17 production.

How innate lymphoid cells (ILCs) in the thymus and gut become specialized effectors is unclear. The prototypic innate-like γδ T cells (Tγδ17) are a major source of interleukin-17 (IL-17). We demonstrate that Tγδ17 cells are programmed by a gene regulatory network consisting of a quartet of high-mobility group (HMG) box transcription factors, SOX4, SOX13, TCF1, and LEF1, and not by conventional TCR signaling. SOX4 and SOX13 directly regulated the two requisite Tγδ17 cell-specific genes, Rorc and Blk, whereas TCF1 and LEF1 countered the SOX proteins and induced genes of alternate effector subsets. The T cell lineage specification factor TCF1 was also indispensable for the generation of IL-22 producing gut NKp46(+) ILCs and restrained cytokine production by lymphoid tissue inducer-like effectors. These results indicate that similar gene network architecture programs innate sources of IL-17, independent of anatomical origins.