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Changes in metabolism of macrophages are required to sustain macrophage activation in response to different stimuli. We showed that the cytokine TGF-β (transforming growth factor–β) regulates glycolysis in macrophages independently of inflammatory cytokine production and affects survival in mouse models of sepsis. During macrophage activation, TGF-β increased the expression and activity of the glycolytic enzyme PFKL (phosphofructokinase-1 liver type) and promoted glycolysis but suppressed the production of proinflammatory cytokines. The increase in glycolysis was mediated by an mTOR–c-MYC–dependent pathway, whereas the inhibition of cytokine production was due to activation of the transcriptional coactivator SMAD3 and suppression of the activity of the proinflammatory transcription factors AP-1, NF-κB, and STAT1. In mice with LPS-induced endotoxemia and experimentally induced sepsis, the TGF-β–induced enhancement in macrophage glycolysis led to decreased survival, which was associated with increased blood coagulation. Analysis of septic patient cohorts revealed that the expression of PFKL, TGFBRI (which encodes a TGF-β receptor), and F13A1 (which encodes a coagulation factor) in myeloid cells positively correlated with COVID-19 disease. Thus, these results suggest that TGF-β is a critical regulator of macrophage metabolism and could be a therapeutic target in patients with sepsis. Editor's summary: Macrophages can initiate or resolve inflammatory responses. Usually, glycolytic metabolism results in an inflammatory phenotype in immune cells, but Gauthier et al. found that macrophages activated by the cytokine TGF-β had a glycolytic metabolism but were less inflammatory. TGF-β decreased the survival of mice with sepsis, which was associated with increased coagulation, a complication of sepsis that can lead to organ dysfunction. COVID-19 disease correlated with enhanced expression of genes encoding a TGF-β receptor, a glycolytic enzyme, and a coagulation factor in septic patients. Thus, targeting TGF-β may improve survival and reduce coagulation-associated complications in septic patients. —Wei Wong [ABSTRACT FROM AUTHOR]

Plasmacytoid dendritic cells (pDCs) have been shown to play an important role during immune responses, ranging from initial viral control through the production of type I interferons to antigen presentation. However, recent studies uncovered unexpected heterogeneity among pDCs. We identified a previously uncharacterized immune subset, referred to as pDC-like cells, that not only resembles pDCs but also shares conventional DC (cDC) features. We show that this subset is a circulating precursor distinct from common DC progenitors, with prominent cDC2 potential. Our findings from human CD2-iCre and CD300c-iCre lineage tracing mouse models suggest that a substantial fraction of cDC2s originates from pDC-like cells, which can therefore be referred to as pre-DC2. This precursor subset responds to homeostatic cytokines, such as macrophage colony stimulating factor, by expanding and differentiating into cDC2 that efficiently prime T helper 17 (TH17) cells. Development of pre-DC2 into CX3CR1+ ESAM− cDC2b but not CX3CR1− ESAM+ cDC2a requires the transcription factor KLF4. Last, we show that, under homeostatic conditions, this developmental pathway regulates the immune threshold at barrier sites by controlling the pool of TH17 cells within skin-draining lymph nodes. Building out the DC family tree: Analysis of dendritic cell (DC) subsets has demonstrated extensive phenotypic and functional heterogeneity. Identifying the progenitor cell populations that serve as precursors for the three main subsets of conventional DCs (cDCs) has been challenging. Rodrigues et al. used lineage tracing in mouse models and in vivo transfer studies to analyze the differentiation of a DC subset called "pDC-like cells" with morphological features of plasmacytoid DCs (pDCs) but other features suggestive of cDCs. These pDC-like cells were identified as precursors of a substantial fraction of the cDC2 subset through a differentiation process dependent on the KLF4 transcription factor. These studies revealed a key developmental pathway used by an unusual set of DC precursor cells with features conserved between mice and humans. —IRW [ABSTRACT FROM AUTHOR]

Because of species selectivity, HIV research is largely restricted to in vitro or clinical studies, both limited in their ability to rapidly assess new strategies to fight the virus. To prospectively study some aspects of HIV in vivo, immunodeficient mice, transplanted with either human peripheral blood leukocytes or human fetal tissues, have been developed. Although these are susceptible to HIV infection, xenoreactivity, and short infection spans, resource and ethical constraints, as well as biased HIV coreceptor tropic strain infection, pose substantial problems in their use. [Rag2.sup.-/-][[gamma].sub.c.sup.-/-] mice, transplanted as newborns with human CD[34.sup.+] cells, were recently shown to develop human B, T, and dendritic cells, constituting lymphoid organs in situ. Here we tested these mice as a model system for HIV-1 infection. HIV RNA levels peaked to up to 2 x [10.sup.6] copies per milliliter of plasma early after infection, and viremia was observed for up to 190 days, the longest time followed. A marked relative CD[4.sup.+] T cell depletion in peripheral blood occurred in CXCR4-tropic strain-infected mice, whereas this was less pronounced in CCR5-tropic strain-infected animals. Thymus infection was almost exclusively observed in CXCR4-tropic strain-infected mice, whereas spleen and lymph node HIV infection occurred irrespective of coreceptor selectivity, consistent with respective coreceptor expression on human CD[4.sup.+] T cells. Thus, this straightforward to generate and cost-effective in vivo model closely resembles HIV infection in man and therefore should be valuable to study virus-induced pathology and to rapidly evaluate new approaches aiming to prevent or treat HIV infection.

The opportunistic fungal pathogen Candida albicans frequently causes diseases such as oropharyngeal candidiasis (OPC) in immunocompromised individuals. Although it is well appreciated that the cytokine IL-17 is crucial for protective immunity against OPC, the cellular source and the regulation of this cytokine during infection are still a matter of debate. Here, we directly visualized IL-17 production in the infected tongue of experimentally infected mice, thereby demonstrating that this key cytokine is expressed by three complementary subsets of CD90+ leukocytes: RAG-dependent αβ and γδ T cells, as well as RAG-independent ILCs. To determine the regulation of IL-17 production at the onset of OPC, we investigated in detail the myeloid compartment of the tongue and found a heterogeneous and dynamic mononuclear phagocyte (MNP) network in the infected tongue that consists of Zbtb46-Langerin- macrophages, Zbtb46+Langerin+ dendritic cells (DCs) and Ly6C+ inflammatory monocytes. Of those, the Langerin+ DC population stands out by its unique capacity to co-produce the cytokines IL-1β, IL-6 and IL-23, all of which promote IL-17 induction in response to C. albicans in the oral mucosa. The critical role of Langerin+ DCs for the innate IL-17 response was confirmed by depletion of this cellular subset in vivo, which compromised IL-17 induction during OPC. In conclusion, our work revealed key regulatory factors and their cellular sources of innate IL-17-dependent antifungal immunity in the oral mucosa. [ABSTRACT FROM AUTHOR]

Dendritic cells (DCs) and monocytes develop from a series of bone-marrow-resident progenitors in which lineage potential is regulated by distinct transcription factors. Zeb2 is an E-box-binding protein associated with epithelial-mesenchymal transition and is widely expressed among hematopoietic lineages. Previously, we observed that Zeb2 expression is differentially regulated in progenitors committed to classical DC (cDC) subsets in vivo. Using systems for inducible gene deletion, we uncover a requirement for Zeb2 in the development of Ly-6Chi monocytes but not neutrophils, and we show a corresponding requirement for Zeb2 in expression of the M-CSF receptor in the bone marrow. In addition, we confirm a requirement for Zeb2 in development of plasmacytoid DCs but find that Zeb2 is not required for cDC2 development. Instead, Zeb2 may act to repress cDC1 progenitor specification in the context of inflammatory signals. [ABSTRACT FROM AUTHOR]

The dendritic cells (DCs) of the immune system function in innate and adaptive responses by directing activity of various effector cells rather than serving as effectors themselves. DCs and closely related myeloid lineages share expression of many surface receptors, presenting a challenge in distinguishing their unique in vivo functions. Recent work has taken advantage of unique transcriptional programs to identify and manipulate murine DCs in vivo. This work has assigned several nonredundant in vivo functions to distinct DC lineages, consisting of plasmacytoid DCs and several subsets of classical DCs that promote different immune effector modules in response to pathogens. In parallel, a correspondence between human and murine DC subsets has emerged, underlying structural similarities for the DC lineages between these species. Recent work has begun to unravel the transcriptional circuitry that controls the development and diversification of DCs from common progenitors in the bone marrow. [ABSTRACT FROM AUTHOR]

Mononuclear phagocytes (MP) are a quite unique subset of hematopoietic cells, which comprise dendritic cells (DC), monocytes as well as monocyte-derived and tissue-res- ident macrophages. These cells are extremely diverse with regard to their origin, their phenotype as well as their function. Developmentally, DC and monocytes are constantly replenished from a bone marrow hematopoietic progenitor. The ontogeny of macrophages is more complex and is temporally linked and specified by the organ where they reside, occurring early during embryonic or perinatal life. The functional heterogeneity of MPs is certainly a consequence of the tissue of residence and also reflects the diverse ontogeny of the subsets. In this review, we will highlight the developmental pathways of murine MP, with a particular emphasis on the transcriptional factors that regulate their development and function. Finally, we will discuss and point out open questions in the field. [ABSTRACT FROM AUTHOR]

The transcription factors Batf3 and IRF8 are required for the development of CD8α+ conventional dendritic cells (cDCs), but the basis for their actions has remained unclear. Here we identified two progenitor cells positive for the transcription factor Zbtb46 that separately generated CD8α+ cDCs and CD4+ cDCs and arose directly from the common DC progenitor (CDP). Irf8 expression in CDPs required prior autoactivation of Irf8 that was dependent on the transcription factor PU.1. Specification of the clonogenic progenitor of CD8α+ cDCs (the pre-CD8 DC) required IRF8 but not Batf3. However, after specification of pre-CD8 DCs, autoactivation of Irf8 became Batf3 dependent at a CD8α+ cDC-specific enhancer with multiple transcription factor AP1-IRF composite elements (AICEs) within the Irf8 superenhancer. CDPs from Batf3−/− mice that were specified toward development into pre-CD8 DCs failed to complete their development into CD8α+ cDCs due to decay of Irf8 autoactivation and diverted to the CD4+ cDC lineage. [ABSTRACT FROM AUTHOR]

T-cell lymphopenia following BM transplantation or diseases such as AIDS result in immunodeficiency. Novel approaches to ameliorate this situation are urgently required. Herein, we describe a novel stromal cell free culture system in which Lineage−Sca1+c-kit+ BM hematopoietic progenitors very efficiently differentiate into pro-T cells. This culture system consists of plate-bound Delta-like 4 Notch ligand and the cytokines SCF and IL-7. The pro-T cells developing in these cultures express CD25, CD117, and partially CD44; express cytoplasmic CD3ε; and have their TCRβ locus partially D-J rearranged. They could be expanded for over 3 months and used to reconstitute the T-cell compartments of sublethally irradiated T-cell-deficient CD3ε−/− mice or lethally irradiated WT mice. Pro-T cells generated in this system could partially correct the T-cell lymphopenia of pre-Tα−/− mice. However, reconstituted CD3ε−/− mice suffered from a wasting disease that was prevented by co-injection of purified CD4+ CD25high WT Treg cells. In a T-cell-sufficient or T-lymphopenic setting, the development of disease was not observed. Thus, this in vitro culture system represents a powerful tool to generate large numbers of pro-T cells for transplantation and possibly with clinical applications. [ABSTRACT FROM AUTHOR]

The mononuclear phagocyte system (MPS) has historically been categorized into monocytes, dendritic cells and macrophages on the basis of functional and phenotypical characteristics. However, considering that these characteristics are often overlapping, the distinction between and classification of these cell types has been challenging. In this Opinion article, we propose a unified nomenclature for the MPS. We suggest that these cells can be classified primarily by their ontogeny and secondarily by their location, function and phenotype. We believe that this system permits a more robust classification during both steady-state and inflammatory conditions, with the benefit of spanning different tissues and across species. [ABSTRACT FROM AUTHOR]

Basic leucine zipper transcription factor ATF-like (BATF), BATF2 and BATF3 belong to the activator protein 1 (AP-1) family of transcription factors, which regulate numerous cellular processes. Initially, BATF family members were thought to function only as inhibitors of AP-1-driven transcription, but recent studies have uncovered that these factors have unique, non-redundant and positive transcriptional activities in dendritic cells, B cells and T cells. The question of how BATF and BATF3 - which lack a transcriptional activation domain, unlike the AP-1 factors FOS and JUN - can exert unique positive transcriptional specificity has now been answered by the discovery that BATF molecules interact with members of the interferon-regulatory factor (IRF) family. The capacity of the BATF leucine zipper regions to mediate dimerization with AP-1 factors and also to define cooperative interactions with heterologous factors explains both the positive transcriptional activity of BATF proteins and how they activate distinct sets of target genes compared with FOS. [ABSTRACT FROM AUTHOR]

The interaction between BAFF and BAFF-R is crucial for the development of mature B cells. Here, we report that the expression of BAFF-R is first detectable on a fraction of mouse CD19+ CD93+ IgM+ CD23- and human CD19+ CD10+ IgM+ BM B cells. This BAFF-R+ BM B-cell population shows higher levels of surface IgM expression and decreased RAG-2 transcripts than BAFF-R- immature B cells. When cultured, mouse BAFF-R-, but not BAFF-R+ immature B cells spontaneously undergo B-cell receptor editing. However, BAFF-R+ immature B cells cultured in the presence of an anti-κ light chain antibody are induced to undergo receptor editing. This receptor editing correlates with down-modulation of surface BAFF-R expression and the up-regulation of RAG-2 at the RNA level. B-cell receptor (BCR) cross-linking on splenic T1 B cells results in down-modulation of the BAFF-R, and receptor editing and RAG-2 up-regulation in a minor fraction of B cells. BCR cross-linking on splenic T2/3 B cells results in partly down and partly up-modulation of BAFF-R expression and no evidence for receptor editing. Overall, our data indicate that BAFF-R expression is tightly regulated during B-cell development in mouse and human and its expression is correlated with positive selection. [ABSTRACT FROM AUTHOR]

The requirement for Notch signaling during T-cell development has been extensively studied. Nevertheless, the developmental stage at which it is required and whether additional signaling pathways are needed are still poorly understood. By using a stromal-cell-free culture system, we show that sorted double-negative 3 (DN3) thymocytes only require a Delta-like-4-induced Notch signal to differentiate into double-positive (DP) cells. This differentiation process is preTCR-α dependent. DN3 cells undergo 4-5 proliferation cycles, and the addition of the chemokine CXCL12 improves proliferation. IL-7 blocks the differentiation of DN3 cells to DP cells but not the Notch-induced proliferation of cultured DN3 cells. The impaired differentiation correlates with an inhibition of Rag-2 up-regulation. Overall, the in vitro stromal-cell-free culture system presented here also provides a powerful and unique tool for studying the mechanisms involved in the positive and negative selection of T cells. [ABSTRACT FROM AUTHOR]

Defects in the expression of either BAFF (B cell activating factor) or BAFF-R impairs B cell development beyond the immature, transitional type-1 stage and thus, prevents the formation of follicular and marginal zone B cells, whereas B-1 B cells remain unaffected. The expression of BAFF-R on all mature B cells might suggest a role for BAFF-R signaling also for their in vivo maintenance. Here, we show that, 14 days following a single injection of an anti-BAFF-R mAb that prevents BAFF binding, both follicular and marginal zone B cell numbers are drastically reduced, whereas B-1 cells are not affected. Injection of control, isotype-matched but non-blocking anti-BAFF-R mAbs does not result in B cell depletion. We also show that this depletion is neither due to antibody-dependent cellular cytotoxicity nor to complement-mediated lysis. Moreover, prevention of BAFF binding leads to a decrease in the size of the B cell follicles, an impairment of a T cell dependent humoral immune response and a reduction in the formation of memory B cells. Collectively, these results establish a central role for BAFF-BAFF-R signaling in the in vivo survival and maintenance of both follicular and marginal zone B cell pools. [ABSTRACT FROM AUTHOR]

Because of species selectivity, HIV research is largely restricted to in vitro or clinical studies, both limited in their ability to rapidly assess new strategies to fight the virus. To prospectively study some aspects of HIV in vivo, immunodeficient mice, transplanted with either human peripheral blood leukocytes or human fetal tissues, have been developed. Although these are susceptible to HIV infection, xenoreactivity, and short infection spans, resource and ethical constraints, as well as biased I-IIV coreceptor tropic strain infection, pose substantial problems in their use. Rag2-/-γc-/- mice, transplanted as newborns with human CD34+ cells, were recently shown to develop human B, T, and dendritic cells, constituting lymphoid organs in situ. Here we tested these mice as a model system for HIV-1 infection. HIV RNA levels peaked to up to 2 × 106 copies per milliliter of plasma early after infection, and viremia was observed for up to 190 days, the longest time followed. A marked relative CD4+ T cell depletion in peripheral blood occurred in CXCR4-tropic strain-infected mice, whereas this was less pronounced in CCR5-tropic strain-infected animals. Thymus infection was almost exclusively observed in CXCR4-tropic strain-infected mice, whereas spleen and lymph node HIV infection occurred irrespective of coreceptor selectivity, consistent with respective coreceptor expression on human CD4+ T cells. Thus, this straightforward to generate and cost-effective in vivo model closely resembles HIV infection in man and therefore should be valuable to study virus-induced pathology and to rapidly evaluate new approaches aiming to prevent or treat HIV infection. [ABSTRACT FROM AUTHOR]

Flt3 ligand (Flt3L) is a nonredundant cytokine in type I interferon-producing cell (IPC) and dendritic cell (DC) development, and IPC and DC differentiation potential is confined to Flt3+ hematopoietic progenitor cells. Here, we show that overexpression of human Flt3 in Flt3- (Flt3-Lin-IL-7Rα-Thy1.1-c-Kit+) and Flt3+ (Flt3+Lin-IL-7Rα-Thy1.1-c-Kit+) hematopoietic progenitors rescues and enhances their IPC and DC differentiation potential, respectively. In defined hematopoietic cell populations, such as Flt3- megakaryocyte/ erythrocyte-restricted progenitors (MEPs), enforced Flt3 signaling induces transcription of IPC, DC, and granulocyte/macrophage (GM) development-affiliated genes, including STAT3, PU.1. and G-/M-/GM-CSFR, and activates differentiation capacities to these lineages. Moreover, ectopic expression of Flt3 downstream transcription factors STAT3 or PU. 1 in Flt3- MEPs evokes Flt3 receptor expression and instructs differentiation into IPCs, DCs, and myelomonocytic cells, whereas GATA-1 expression and consecutive megakaryocyte/ erythrocyte development is suppressed. Based on these data, we propose a demand-regulated, cytokine-driven DC and IPC regeneration model, in which high Flt3L levels initiate a self-sustaining, Flt3-STAT3- and Flt3-PU.1-mediated IPC and DC differentiation program in Flt3+ hematopoietic progenitor cells. [ABSTRACT FROM AUTHOR]

Although many biologic principles are conserved in mice and humans, species-specific differences exist, for example, in susceptibility and response to pathogens, that often do not allow direct implementation of findings in experimental mice to humans. Research in humans, however, for ethical and practical reasons, is largely restricted to in vitro assays that lack components and the complexity of a living organism. To nevertheless study the human hematopoietic and immune system in vivo, xenotransplantation assays have been developed that substitute human components to small animals. Here, we summarize our recent findings that transplantation of human cord blood CD34+ cells to newborn Rag2-/-γc-/- mice leads to de novo development of major functional components of the human adaptive immune system. These human adaptive immune system Rag2-/-γc-/- (huAIS-RG) mice can now be used as a technically straightforward preclinical model to evaluate in vivo human adaptive immune system development as well as immune responses, for example, to vaccines or live infectious pathogens. [ABSTRACT FROM AUTHOR]

Immune surveillance is critical to prevent tumorigenesis. Gliomas evade immune attack, but the underlying mechanisms remain poorly understood. We show that glioma cells can sustain growth independent of immune system constraint by reducing Notch signaling. Loss of Notch activity in a mouse model of glioma impairs MHC-I and cytokine expression and curtails the recruitment of anti-tumor immune cell populations in favor of immunosuppressive tumor-associated microglia/macrophages (TAMs). Depletion of T cells simulates Notch inhibition and facilitates tumor initiation. Furthermore, Notch-depleted glioma cells acquire resistance to interferon-γ and TAMs re-educating therapy. Decreased interferon response and cytokine expression by human and mouse glioma cells correlate with low Notch activity. These effects are paralleled by upregulation of oncogenes and downregulation of quiescence genes. Hence, suppression of Notch signaling enables gliomas to evade immune surveillance and increases aggressiveness. Our findings provide insights into how brain tumor cells shape their microenvironment to evade immune niche control. [Display omitted] • Tumor cells and their immune niche co-evolve during tumor initiation • Proneural glioma cells evade immune niche control by lowering Notch activity • IFNγ response and MHC-I expression by GSCs are co-regulated by Notch • A single pathway governs glioma immunophenotype and activated cancer stem cell traits Parmigiani et al. demonstrate that Notch signaling regulates the interferon-γ response of glioma cells and recruitment of immune cells to the tumor. Uncoupling of immune and tumor cells by Notch inhibition favors an immunosuppressive microenvironment, exacerbates immune evasion, and promotes activated stem-cell-driven, niche-independent glioma growth. [ABSTRACT FROM AUTHOR]

Males and females exhibit profound differences in immune responses and disease susceptibility. However, the factors responsible for sex differences in tissue immunity remain poorly understood. Here, we uncovered a dominant role for type 2 innate lymphoid cells (ILC2s) in shaping sexual immune dimorphism within the skin. Mechanistically, negative regulation of ILC2s by androgens leads to a reduction in dendritic cell accumulation and activation in males, along with reduced tissue immunity. Collectively, our results reveal a role for the androgen-ILC2-dendritic cell axis in controlling sexual immune dimorphism. Moreover, this work proposes that tissue immune set points are defined by the dual action of sex hormones and the microbiota, with sex hormones controlling the strength of local immunity and microbiota calibrating its tone.

Immunity to Toxoplasma gondii at early stages of infection in C57BL/6 mice depends on gamma interferon (IFN-γ) production by NK cells, while at later stages it is primarily mediated by CD8 T cells. We decided to explore the requirement for CD4 T cells during T. gondii infection in Batf3 -/- mice, which lack CD8α + dendritic cells (DCs) that are necessary for cross-presentation of cell-associated antigens to CD8 T cells. We show that in this immunodeficient background on a BALB/c background, CD4 T cells become important effector cells and are able to protect Batf3 -/- mice from infection with the avirulent strain RHΔ ku80 Δ rop5 Independently of the initial NK cell activation, CD4 T cells in wild-type and Batf3 -/- mice were the major source of IFN-γ. Importantly, memory CD4 T cells were sufficient to provide protective immunity following transfer into Batf3 -/- mice and secondary challenge with the virulent RHΔ ku80 strain. Collectively, these results show that under situations where CD8 cell responses are impaired, CD4 T cells provide an important alternative immune response to T. gondii IMPORTANCE Toxoplasma gondii is a widespread parasite of animals that causes zoonotic infections in humans. Although healthy individuals generally control the infection with only moderate symptoms, it causes serious illness in newborns and those with compromised immune systems such as HIV-infected AIDS patients. Because rodents are natural hosts for T. gondii , laboratory mice provide an excellent model for studying immune responses. Here, we used a combination of an attenuated mutant strain of the parasite that effectively vaccinates mice, with a defect in a transcriptional factor that impairs a critical subset of dendritic cells, to studying the immune response to infection. The findings reveal that in BALB/c mice, CD4 memory T cells play a dominant role in producing IFN-γ needed to control chronic infection. Hence, BALB/c mice may provide a more appropriate model for declining immunity seen in HIV-AIDS patients where loss of CD4 cells is associated with emergence of opportunistic infections., (Copyright © 2020 Tussiwand et al.)

Dendritic cells (DCs) are antigen-presenting cells subdivided in specialized subsets. In this issue, Bosteels et al. challenge this concept, identifying a unique subset of inflammatory DCs characterized by hybrid myeloid features, capable of efficiently priming CD4 + as well as CD8 + T cells., (Published by Elsevier Inc.)

The opportunistic fungal pathogen Candida albicans frequently causes diseases such as oropharyngeal candidiasis (OPC) in immunocompromised individuals. Although it is well appreciated that the cytokine IL-17 is crucial for protective immunity against OPC, the cellular source and the regulation of this cytokine during infection are still a matter of debate. Here, we directly visualized IL-17 production in the tongue of experimentally infected mice, thereby demonstrating that this key cytokine is expressed by three complementary subsets of CD90+ leukocytes: RAG-dependent αβ and γδ T cells, as well as RAG-independent ILCs. To determine the regulation of IL-17 production at the onset of OPC, we investigated in detail the myeloid compartment of the tongue and found a heterogeneous and dynamic mononuclear phagocyte (MNP) network in the infected tongue that consists of Zbtb46-Langerin- macrophages, Zbtb46+Langerin+ dendritic cells (DCs) and Ly6C+ inflammatory monocytes. Of those, the Langerin+ DC population stands out by its unique capacity to co-produce the cytokines IL-1β, IL-6 and IL-23, all of which promote IL-17 induction in response to C. albicans in the oral mucosa. The critical role of Langerin+ DCs for the innate IL-17 response was confirmed by depletion of this cellular subset in vivo, which compromised IL-17 induction during OPC. In conclusion, our work revealed key regulatory factors and their cellular sources of innate IL-17-dependent antifungal immunity in the oral mucosa., Competing Interests: The authors have declared that no competing interests exist.

Variable strengths of signaling via the T cell antigen receptor (TCR) can produce divergent outcomes, but the mechanism of this remains obscure. The abundance of the transcription factor IRF4 increases with TCR signal strength, but how this would induce distinct types of responses is unclear. We compared the expression of genes in the T H 2 subset of helper T cells to enhancer occupancy by the BATF-IRF4 transcription factor complex at varying strengths of TCR stimulation. Genes dependent on BATF-IRF4 clustered into groups with distinct TCR sensitivities. Enhancers exhibited a spectrum of occupancy by the BATF-IRF4 ternary complex that correlated with the sensitivity of gene expression to TCR signal strength. DNA sequences immediately flanking the previously defined AICE motif controlled the affinity of BATF-IRF4 for direct binding to DNA. Analysis by the chromatin immunoprecipitation-exonuclease (ChIP-exo) method allowed the identification of a previously unknown high-affinity AICE2 motif at a human single-nucleotide polymorphism (SNP) of the gene encoding the immunomodulatory receptor CTLA-4 that was associated with resistance to autoimmunity. Thus, the affinity of different enhancers for the BATF-IRF4 complex might underlie divergent signaling outcomes in response to various strengths of TCR signaling.

In this study, to examine cross-presentation by classical dendritic cells (DCs; cDCs), we evaluated the role of RAB43, a protein found to be selectively expressed by Batf3-dependent CD8α + and CD103 + compared with other DC subsets and immune lineages. Using a specific monoclonal antibody, we localized RAB43 expression to the Golgi apparatus and LAMP1 - cytoplasmic vesicles. Mice with germline or conditional deletion of Rab43 are viable and fertile and have normal development of cDCs but show a defect for in vivo and in vitro cross-presentation of cell-associated antigen. This defect is specific to cDCs, as Rab43-deficient monocyte-derived DCs showed no defect in cross-presentation of cell-associated antigen. These results suggest that RAB43 provides a specialized activity used in cross-presentation selectively by CD8α + DCs but not other antigen-presenting cells., (© 2016 Kretzer et al.)

CD8α(+) and CD103(+) dendritic cells (DCs) play a central role in the development of type 1 immune responses. However, their role in type 2 immunity remains unclear. We examined this issue using Batf3(-/-) mice, in which both of these DC subsets are missing. We found that Th2 cell responses, and related events such as eosinophilia, alternative macrophage activation, and immunoglobulin class switching to IgG1, were enhanced in Batf3(-/-) mice responding to helminth parasites. This had beneficial or detrimental consequences depending on the context. For example, Batf3 deficiency converted a normally chronic intestinal infection with Heligmosomoides polygyrus into an infection that was rapidly controlled. However, liver fibrosis, an IL-13-mediated pathological consequence of wound healing in chronic schistosomiasis, was exacerbated in Batf3(-/-) mice infected with Schistosoma mansoni. Mechanistically, steady-state production of IL-12 by migratory CD103(+) DCs, independent of signals from commensals or TLR-initiated events, was necessary and sufficient to exert the suppressive effects on Th2 response development. These findings identify a previously unrecognized role for migratory CD103(+) DCs in antagonizing type 2 immune responses., (© 2016 Everts et al.)

The transcription factors Batf3 and IRF8 are required for the development of CD8α(+) conventional dendritic cells (cDCs), but the basis for their actions has remained unclear. Here we identified two progenitor cells positive for the transcription factor Zbtb46 that separately generated CD8α(+) cDCs and CD4(+) cDCs and arose directly from the common DC progenitor (CDP). Irf8 expression in CDPs required prior autoactivation of Irf8 that was dependent on the transcription factor PU.1. Specification of the clonogenic progenitor of CD8α(+) cDCs (the pre-CD8 DC) required IRF8 but not Batf3. However, after specification of pre-CD8 DCs, autoactivation of Irf8 became Batf3 dependent at a CD8α(+) cDC-specific enhancer with multiple transcription factor AP1-IRF composite elements (AICEs) within the Irf8 superenhancer. CDPs from Batf3(-/-) mice that were specified toward development into pre-CD8 DCs failed to complete their development into CD8α(+) cDCs due to decay of Irf8 autoactivation and diverted to the CD4(+) cDC lineage.

Retention of lymphocytes in the intestinal mucosa requires specialized chemokine receptors and adhesion molecules. We find that both CD4(+)CD8(+) and CD4(+) T cells in the intestinal epithelium, as well as CD8(+) T cells in the intestinal mucosa and mesenteric lymph nodes, express the cell adhesion molecule class I-restricted T cell-associated molecule (Crtam) upon activation, whereas the ligand of Crtam, cell adhesion molecule 1 (Cadm1), is expressed on gut CD103(+)DCs. Lack of Crtam-Cadm1 interactions in Crtam(-/-) and Cadm1(-/-) mice results in loss of CD4(+)CD8(+) T cells, which arise from mucosal CD4(+) T cells that acquire a CD8 lineage expression profile. After acute oral infection with Toxoplasma gondii, both WT and Crtam(-/-) mice mounted a robust TH1 response, but markedly fewer TH17 cells were present in the intestinal mucosa of Crtam(-/-) mice. The almost exclusive TH1 response in Crtam(-/-) mice resulted in more efficient control of intestinal T. gondii infection. Thus, Crtam-Cadm1 interactions have a major impact on the residency and maintenance of CD4(+)CD8(+) T cells in the gut mucosa in the steady state. During pathogenic infection, Crtam-Cadm1 interactions regulate the dynamic equilibrium between newly formed CD4(+) T cells and their retention in the gut, thereby shaping representation of disparate CD4(+) T cell subsets and the overall quality of the CD4(+) T cell response.

Recent studies suggest that Epstein-Barr virus (EBV) can infect naïve B cells, driving them to differentiate into resting memory B cells via the germinal center reaction. This hypothesis has been inferred from parallels with the biology of normal B cells but has never been proven experimentally. Rag2(-/-) gamma(c)(-/-) mice that were transplanted with human CD34(+) cord blood cells as newborns were recently shown to develop human B, T, and dendritic cells, constituting lymphoid organs in situ. Here we used this model to better define the strategy of EBV infection of human B cells in vivo and to compare this model system with different conditions of EBV infection in humans. Our results support the model of EBV persistence in vivo in cases that were characterized by follicular hyperplasia and a relatively normal CD4(+) and CD8(+) T-cell distribution. Intriguingly, in cases that were characterized by nodular and diffuse proliferation with a preponderance of CD8(+) T cells, similar to infectious mononucleosis, EBV still infects naïve B cells but also induces clonal expansion and ongoing somatic mutations without germinal center reactions. Our results reveal different strategies of EBV infection in B cells that possibly result from variations in the host immune response. Future experiments might allow understanding of the mechanisms responsible for persistent EBV infection and provide targets for more highly tailored therapeutic interventions.

In vivo steady-state type I natural IFN-producing and dendritic cell (DC) development is largely dependent on Flt3 signaling. Natural IFN-producing and DC progenitors and their respective downstream cell populations express the flt3 receptor, and Flt3 ligand (Flt3L)(-/-) mice have reduced while Flt3L-injected mice develop markedly increased numbers of both cell types. In the present study, we show that SU11657, a small multitargeted receptor tyrosine kinase inhibitor with Flt3 affinity, suppressed in vitro natural IFN-producing and DC development in Flt3L-supplemented mouse whole bone marrow cell cultures in a dose-dependant manner, while DC development in GM-CSF-supplemented cultures was not affected. In vivo SU11657 application led to a significant decrease of both natural IFN-producing and DCs, comparable to the reduction observed in Flt3L(-/-) mice. Conversely, Flt3L plasma levels increased massively in inhibitor-treated animals, likely via a regulatory feedback loop, without being able to compensate for pharmacological Flt3 inhibition. No obvious toxicity was observed, and hemopoietic progenitor cell and stem cell function remained intact as assessed by myeloid colony-forming unit activity and in vivo bone marrow repopulation assays. Furthermore, upon treatment discontinuation, IFN-producing and DCs recovered to normal levels, proving that treatment effects were transient. Given the importance of IFN-producing and DCs in regulation of immune responses, these findings might lead to new pharmacological strategies in prevention and treatment of autoimmune diseases and complications of organ or blood cell transplantation.

Although many biologic principles are conserved in mice and humans, species-specific differences exist, for example, in susceptibility and response to pathogens, that often do not allow direct implementation of findings in experimental mice to humans. Research in humans, however, for ethical and practical reasons, is largely restricted to in vitro assays that lack components and the complexity of a living organism. To nevertheless study the human hematopoietic and immune system in vivo, xenotransplantation assays have been developed that substitute human components to small animals. Here, we summarize our recent findings that transplantation of human cord blood CD34(+) cells to newborn Rag2(-/-)gamma(c)(-/-) mice leads to de novo development of major functional components of the human adaptive immune system. These human adaptive immune system Rag2(-/-)gamma(c)(-/-) (huAIS-RG) mice can now be used as a technically straightforward preclinical model to evaluate in vivo human adaptive immune system development as well as immune responses, for example, to vaccines or live infectious pathogens.

Previous studies involving identical twins with concordant leukemia and retrospective scrutiny of archived neonatal blood spots have shown that common chromosome translocations of pediatric leukemia frequently arise before birth. The IGH/TCR clonotypic sequences used as surrogate molecular markers suggest this is also likely to be true for hyperdiploid acute lymphoblastic leukemia (ALL). Yet evidence that hyperdiploidy itself is an early or initiating event occurring prenatally has been limited. Now, however, we can provide direct evidence of this from our identification of CD34+/CD19+ B-lineage progenitor cells with triploid chromosomes in the stored cord blood of an individual who subsequently developed hyperdiploid ALL., (Copyright 2004 Wiley-Liss, Inc.)