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Absence of Interferon-γ Receptor (IFNGR) or Signal Transducer and Activator of Transcription 1 (STAT1) signaling in donor cells has been shown to result in reduced acute GVHD induction. In this study, we unexpectedly observed increased activation and expansion of donor lymphocytes in both lymphohematopoietic organs and GVHD target tissues of IFNGR/STAT1-deficient recipient mice, leading to rapid mortality following the induction of GVHD. Lipopolysaccharide (LPS)-matured bone marrow-derived Ifngr1-/-/Stat1-/- dendritic cells (BMDCs) were more potent allogeneic stimulators and expressed increased levels of MHC II and costimulatory molecules. Similar effects were observed in human APCs with knockdown of Stat1 by CRISPR/Cas9 and treatment with a JAK1/2 inhibitor. Furthermore, we demonstrated that the absence of IFNGR/STAT1 signaling in hematopoietic APCs impaired the presentation of exogenous antigens while promoting the presentation of endogenous antigens. In contrast, the indirect presentation of host antigens to donor lymphocytes was defective in IFNGR/STAT1-deficient donor-derived APCs in fully donor chimeric mice. The differential effects of IFNGR/STAT1 signaling on endogenous and exogenous antigen presentation could provide further insight into the roles of the IFN-γ/STAT1 signal pathway in the pathogenesis of GVHD, organ rejection, and autoimmune diseases.

The nuclear long non-coding RNA LUCAT1 has previously been identified as a negative feedback regulator of type I interferon and inflammatory cytokine expression in human myeloid cells. Here, we define the mechanistic basis for the suppression of inflammatory gene expression by LUCAT1. Using comprehensive identification of RNA-binding proteins by mass spectrometry as well as RNA immunoprecipitation, we identified proteins important in processing and alternative splicing of mRNAs as LUCAT1-binding proteins. These included heterogeneous nuclear ribonucleoprotein C, M, and A2B1. Consistent with this finding, cells lacking LUCAT1 have altered splicing of selected immune genes. In particular, upon lipopolysaccharide stimulation, the splicing of the nuclear receptor 4A2 (NR4A2) gene was particularly affected. As a consequence, expression of NR4A2 was reduced and delayed in cells lacking LUCAT1. NR4A2-deficient cells had elevated expression of immune genes. These observations suggest that LUCAT1 is induced to control the splicing and stability of NR4A2, which is in part responsible for the anti-inflammatory effect of LUCAT1. Furthermore, we analyzed a large cohort of patients with inflammatory bowel disease as well as asthma and chronic obstructive pulmonary disease. In these patients, LUCAT1 levels were elevated and in both diseases, positively correlated with disease severity. Collectively, these studies define a key molecular mechanism of LUCAT1-dependent immune regulation through post-transcriptional regulation of mRNAs highlighting its role in the regulation of inflammatory disease.

Background & Aims CD4+ T cells are regulated by activating and inhibitory cues, and dysregulation of these proper regulatory inputs predisposes these cells to aberrant inflammation and exacerbation of disease. We investigated the role of the inhibitory receptor paired immunoglobulin-like receptor B (PIR-B) in the regulation of the CD4+ T-cell inflammatory response and exacerbation of the colitic phenotype. Methods We used Il10-/- spontaneous and CD4+CD45RBhi T-cell transfer models of colitis with PIR-B-deficient (Pirb-/-) mice. Flow cytometry, Western blot, and RNA sequencing analysis was performed on wild-type and Pirb-/- CD4+ T cells. In silico analyses were performed on RNA sequencing data set of ileal biopsy samples from pediatric CD and non–inflammatory bowel disease patients and sorted human memory CD4+ T cells. Results We identified PIR-B expression on memory CD4+ interleukin (IL)17a+ cells. We show that PIR-B regulates CD4+ T-helper 17 cell (Th17)-dependent chronic intestinal inflammatory responses and the development of colitis. Mechanistically, we show that the PIR-B– Src-homology region 2 domain-containing phosphatase-1/2 axis tempers mammalian target of rapamycin complex 1 signaling and mammalian target of rapamycin complex 1–dependent caspase-3/7 apoptosis, resulting in CD4+ IL17a+ cell survival. In silico analyses showed enrichment of transcriptional signatures for Th17 cells (RORC, RORA, and IL17A) and tissue resident memory (HOBIT, IL7R, and BLIMP1) networks in PIR-B+ murine CD4+ T cells and human CD4+ T cells that express the human homologue leukocyte immunoglobulin-like receptor subfamily B member 3 (LILRB3). High levels of LILRB3 expression were associated strongly with mucosal injury and a proinflammatory Th17 signature, and this signature was restricted to a treatment-naïve, severe pediatric CD population. Conclusions Our findings show an intrinsic role for PIR-B/LILRB3 in the regulation of CD4+ IL17a+ T-cell pathogenic memory responses., Graphical abstract

Genomic analysis of families with unexplained portal hypertension reveals GIMAP5 as a critical regulator of liver sinusoidal endothelial cells and hepatic vascular homeostasis. This study identifies a novel genetic cause of portal hypertension and provides new insight into its pathogenesis., 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.

BackgroundDeveloping novel strategies to overcome the immunosuppressive tumor microenvironment is a critically important area of cancer therapy research. Here, we assess the therapeutic potential of CD244 (2B4/signaling lymphocyte activation molecule family 4), an immunoregulatory receptor found on a variety of immune cells, including exhausted CD8+T cells, dendritic cells (DCs), and myeloid-derived suppressor cells (MDSCs).MethodsUsing de-identified human tumor and blood samples from patients with head and neck squamous cell carcinoma (HNSCC) and HNSCC models in WT and CD244-/-mice, we assessed the therapeutic potential of CD244 using flow cytometry, RT-PCR, Luminex immunoassays and histopathological analyses.ResultsCompared with healthy tissues, tumor infiltrating CD8+T cells from HNSCC patients and a HNSCC mouse model showed significant increased expression of CD244 expression that correlated with PD1 expression. Moreover, CD244 was increased on intratumoral DC and MDSC and high CD244 expression correlated with PD-L1 expression and increased spontaneous expression of immune-suppressive mediators. In addition, CD244 activation inhibited production of proinflammatory cytokines in human DC in vitro. Importantly, CD244-/-mice showed significantly impaired tumor growth of HNSCC and interventional treatment of WT mice with anti-CD244 monoclonal antibody significantly impaired the growth of established HNSCC tumors and increased tumor-infiltrating CD8+T cells.ConclusionsTogether these data suggest that CD244 contributes to the overall immune-suppressive environment and therefore has potential as a new immunotherapy target in the treatment of malignancies.

Increasing evidence points to a key role for NK cells in controlling adaptive immune responses. In studies examining the role of CD1d on CD4+ T cell responses, we found that a line of CD1d-deficient mice on the C57BL/6J background had a homozygous 129 locus on chromosome 6 containing the entire NK cell gene cluster. Mice possessing this locus (C57BL/6.NKC129) displayed a >10-fold reduction in antigen-specific CD4+ T cell responses after intracranial infection with lymphocytic choriomeningitis virus (LCMV). Neither parental strain displayed defects in viral-specific CD4+ T cell responses. Interestingly, following infection, increased numbers of NK cells accumulated in the lymph nodes of C57BL/6.NKC129 mice and displayed enhanced in vivo functionality. Moreover, depletion of NK cells with anti-asialo-GM-1 antibody in C57BL/6.NKC129 mice resulted in a >20-fold increase in viral-specific CD4+ T cell responses. Mechanistically, we found that dendritic cell antigen presentation and early type I IFN production were significantly decreased in C57BL/6.NKC129 mice, but were restored in perforin-deficient C57BL/6.NKC129 mice or following NK depletion. Together, these data reveal that the variable genomic regions containing the activating/inhibitory NK cell receptors are key determinants of antigen-specific CD4+ T cell responses, controlling type I IFN production and the antigen-presenting capacity of dendritic cells.

Myriad studies have linked type I IFN to the pathogenesis of autoimmune diseases, including systemic lupus erythematosus (SLE). Although increased levels of type I IFN are found in patients with SLE, and IFN blockade ameliorates disease in many mouse models of lupus, its precise roles in driving SLE pathogenesis remain largely unknown. In this study, we dissected the effect of type I IFN sensing by CD4 T cells and B cells on the development of T follicular helper cells (TFH), germinal center (GC) B cells, plasmablasts, and antinuclear dsDNA IgG levels using the bm12 chronic graft-versus-host disease model of SLE-like disease. Type I IFN sensing by B cells decreased their threshold for BCR signaling and increased their expression of MHC class II, CD40, and Bcl-6, requirements for optimal GC B cell functions. In line with these data, ablation of type I IFN sensing in B cells significantly reduced the accumulation of GC B cells, plasmablasts, and autoantibodies. Ablation of type I IFN sensing in T cells significantly inhibited TFH expansion and subsequent B cell responses. In contrast to the effect in B cells, type I IFN did not promote proliferation in the T cells but protected them from NK cell–mediated killing. Consequently, ablation of either perforin or NK cells completely restored TFH expansion of IFNAR−/− TFH and, subsequently, restored the B cell responses. Together, our data provide evidence for novel roles of type I IFN and immunoregulatory NK cells in the context of sterile inflammation and SLE-like disease.

The scaffolding protein Grb2-associated binding protein 3 (Gab3) is a member of the Gab family, whose functions have remained elusive. Here, we identify Gab3 as a key determinant of peripheral NK cell expansion. Loss of Gab3 resulted in impaired IL-2 and IL-15-induced NK cell priming and expansion due to a selective impairment in MAPK signaling but not STAT5 signaling. In vivo, we found that Gab3 is required for recognition and elimination of "missing-self" and tumor targets. Unexpectedly, our studies also revealed that Gab3 plays an important role during pregnancy. Gab3-deficient mice exhibited impaired uterine NK cell expansion associated with abnormal spiral artery remodeling and increased trophoblast invasion in the decidua basalis. This coincided with stillbirth, retained placenta, maternal hemorrhage, and undelivered fetoplacental units at term. Thus, Gab3 is a key component required for cytokine-mediated NK cell priming and expansion that is essential for antitumor responses and limits trophoblast cell invasion during pregnancy.

Sepsis is a life-threatening event predominantly caused by gram-negative bacteria. Bacterial infection causes a pronounced macrophage (MΦ) and dendritic cell (DC) activation that leads to excessive pro-inflammatory cytokine interleukin (IL)-1β, IL-6, and Tumor necrosis factor (TNF)-α production (cytokine storm), resulting in endotoxic shock. Previous experimental studies have revealed that inhibiting Nuclear Factor kappa Beta (NF-κB) signaling ameliorates disease symptoms; however, the contribution of myeloid p65 in endotoxic shock remains elusive. In this study, we demonstrate increased mortality in mice lacking p65 in the myeloid lineage (p65Δmye) compared to wild type (WT) mice upon ultra-pure LPS (U-LPS) challenge. We show that increased susceptibility to Lipopolysaccharide (LPS)-induced shock was associated with elevated serum level of IL-1β and IL-6. Mechanistic analyses revealed that LPS-induced pro-inflammatory cytokine production was ameliorated in p65-deficient bone marrow–derived macrophages (BMDMs); however, p65-deficient “activated” peritoneal macrophages (MΦs) exhibited elevated IL-1β and IL-6. We show that the elevated pro-inflammatory cytokine secretion was due in part to increased accumulation of IL-1β mRNA and protein in activated inflammatory MΦs. The increased IL-1β was linked with heightened binding of PU.1 and CCAAT/Enhancer Binding Protein Beta (cEBPβ to Il1b and Il6 promoters in activated inflammatory MΦs. Our data provides insight into a role for NF-κB in the negative regulation of pro-inflammatory cytokines in myeloid cells.

TCR ligation is critical for the selection, activation, and integrin expression of T lymphocytes. Here, we explored the role of the TCR adaptor protein slp-76 on iNKT-cell biology. Compared to B6 controls, slp-76(ace/ace) mice carrying a missense mutation (Thr428Ile) within the SH2-domain of slp-76 showed an increase in iNKT cells in the thymus and lymph nodes, but a decrease in iNKT cells in spleens and livers, along with reduced ADAP expression and cytokine response. A comparable reduction in iNKT cells was observed in the livers and spleens of ADAP-deficient mice. Like ADAP(-/-) iNKT cells, slp-76(ace/ace) iNKT cells were characterized by enhanced CD11b expression, correlating with an impaired induction of the TCR immediate-early gene Nur77 and a decreased adhesion to ICAM-1. Furthermore, CD11b-intrinsic effects inhibited cytokine release, concanavalin A-mediated inflammation, and iNKT-cell accumulation in the liver. Unlike B6 and ADAP(-/-) mice, the expression of the transcription factors Id3 and PLZF was reduced, whereas NP-1-expression was enhanced in slp-76(ace/ace) mice. Blockade of NP-1 decreased the recovery of iNKT cells from peripheral lymph nodes, identifying NP-1 as an iNKT-cell-specific adhesion factor. Thus, slp-76 contributes to the regulation of the tissue distribution, PLZF, and cytokine expression of iNKT cells via ADAP-dependent and -independent mechanisms.

Rationale CD4+T-cell differentiation into effector or memory populations is intrinsically regulated by the delicate balance between activating and inhibitory signals. Previously we identified a role of the inhibitory receptor, Paired Immunoglobulin-like Receptor (PIR) B in the negative regulation of innate immune responses in colitis. The aim of this study is to define the involvement of PIRB in CD4+ Th17 development and regulation of CD4+ Th17-dependent colitis. Results We demonstrate an intrinsic deficiency in PirB−/− Th17 in vitro polarization and cell survival. Specifically, polyclonal activation of PirB−/− Th17 cells lead to increased cell death and enhanced caspase activation (# of Dead cells, WT vs PirB−/−: 17.6 ± 2.3 × 103 vs 38.5 ± 5.8 × 103; # of Caspase 3/7+ cells: 9.8 ± 1.8 × 102 vs 19.7 ± 1.2 × 102; mean ± SEM, p < 0.05). In vivo, PirB−/− Il10−/− mice were protected from development of Il10−/− spontaneous colitis phenotype (Clinical Score, Il10−/− vs PirB−/− Il10−/−: 3.3 ± 0.6 vs 0.14 ± 0.08, p < 0.01; Histological Score: 2.3 ± 0.4 vs 1.2 ± 0.2; mean ± SEM, p < 0.01) and this was associated with reduced CD4+ Th17 cells in the mesenteric lymph nodes (% IL-17a+ cells, Il10−/− vs PirB−/− Il10−/−: 2.5 ± 0.3 vs 1.2 ± 0.1; mean ± SEM, p < 0.001). To test the intrinsic effects to the CD4+ T-cell compartment, we performed the CD4+ CD45RBhi T-cell transfer model of colitis. Rag−/− mice which received PirB−/− naïve CD4+ T-cells were protected from T-cell mediated colitis (% Change in Body Weight: −3.5 ± 4.9 % WT; 20.7 ± 4.6 % PirB−/−; p < 0.001; Histological Score: 3.8 ± 0.2 WT; 1.0 ± 0.4 PirB−/−; mean ± SEM, p < 0.001). Conclusions These data support the concept that PIRB regulates CD4+ Th17 differentiation and development of T-cell-dependent colitis phenotype.

In cancer, immune exhaustion contributes to the immunosuppressive tumor microenvironment. Exhausted immune cells demonstrate poor effector function and sustained expression of certain immunomodulatory receptors, which can be therapeutically targeted. CD244 is a Signaling Lymphocyte Activation Molecule (SLAM) family immunoregulatory receptor found on many immune cell types—including NK cells, a subset of T cells, DCs, and MDSCs—that represents a potential therapeutic target. Here, we discuss the role of CD244 in tumor-mediated immune cell regulation.

Nonalcoholic fatty liver disease (NAFLD), a metabolic predisposition for development of hepatocellular carcinoma (HCC), represents a disease spectrum ranging from steatosis to steatohepatitis to cirrhosis. Acox1, a rate-limiting enzyme in peroxisomal fatty acid β-oxidation, regulates metabolism, spontaneous hepatic steatosis, and hepatocellular damage over time. However, it is unknown whether Acox1 modulates inflammation relevant to NAFLD pathogenesis or if Acox1-associated metabolic and inflammatory derangements uncover and accelerate potential for NAFLD progression. Here, we show that mice with a point mutation in Acox1 (Acox1Lampe1) exhibited altered cellular metabolism, modified T cell polarization, and exacerbated immune cell inflammatory potential. Further, in context of a brief obesogenic diet stress, NAFLD progression associated with Acox1 mutation resulted in significantly accelerated and exacerbated hepatocellular damage via induction of profound histological changes in hepatocytes, hepatic inflammation, and robust upregulation of gene expression associated with HCC development. Collectively, these data demonstrate that β-oxidation links metabolism and immune responsiveness and that a better understanding of peroxisomal β-oxidation may allow for discovery of mechanisms central for NAFLD progression.

BACKGROUND: The GTPase of immunity-associated protein 5 is essential for lymphocyte homeostasis and survival. Recently, human GIMAP5 SNPs have been linked with an increased risk for asthma, while the loss of Gimap5 in mice has been associated with severe CD4(+) T celldriven immune pathology. OBJECTIVE: To identify the molecular and cellular mechanisms by which Gimap5-deficiency predisposes to allergic airway disease. METHODS: CD4(+) T cell polarization and the development of pathogenic CD4(+) T cells were assessed in Gimap5-deficient mice and a human patient with a GIMAP5 loss-of-function (LOF) mutation. House dust mite (HDM)-induced airway inflammation was assessed using a complete Gimap5 LOF (Gimap5(sph/sph)) and conditional Gimap5(fl/fl) Cd4(Cre/ert2) mice. RESULTS: GIMAP5 LOF mutations in both mice and humans are associated with spontaneous polarization towards pathogenic T(H)17 and T(H)2 cells in vivo. Mechanistic studies in vitro reveal that impairment of Gimap5-deficient T(H) cell differentiation is associated with increased DNA damage, particularly during T(H)1 polarizing conditions. The DNA damage in Gimap5-deficient CD4(+) T cells could be controlled by TGFβ, thereby promoting T(H)17 polarization. When challenged with HDM in vivo, Gimap5-deficient mice displayed an exacerbated asthma phenotype (inflammation and airway hyperresponsiveness) with increased development of T(H)2, T(H)17, and pathogenic T(H)17/ T(H)2 cells. CONCLUSION: Activation of Gimap5-deficient CD4(+) T cells is associated with increased DNA damage and reduced survival that can be overcome by TGFβ. This leads to a selective survival of pathogenic T(H)17 cells, but also T(H)2 in humans and mice, ultimately promoting allergic airway disease.

Trafficking from bedside to bench Typically in translational research, a discovery in cell or molecular biology is later exploited to improve patient care. Occasionally, information flows in the opposite direction. Lo et al. found that patients with an autoimmune disorder caused by deficiency of a protein called LRBA responded dramatically to the drug abatacept (see the Perspective by Sansom). Abatacept contains a segment of a potent inhibitory immune receptor, CTLA4. Experiments prompted by this observation revealed the relationship between the two proteins: LRBA controls the intracellular trafficking and degradation of CTLA4. This information may further improve patient care, because other clinically approved drugs have the desired mechanism of action with potentially fewer side effects. Science , this issue p. 436 ; see also p. 377

Absence of MHC class I expression is an important mechanism by which NK cells recognize a variety of target cells, yet the pathways underlying "missing-self" recognition, including the involvement of activating receptors, remain poorly understood. Using ethyl-N-nitrosourea mutagenesis in mice, we identified a germline mutant, designated Ace, with a marked defect in NK cell mediated recognition and elimination of "missing-self" targets. The causative mutation was linked to chromosome 11 and identified as a missense mutation (Thr428Ile) in the SH2 domain of Slp-76-a critical adapter molecule downstream of ITAM-containing surface receptors. The Slp-76 Ace mutation behaved as a hypomorphic allele-while no major defects were observed in conventional T-cell development/function, a marked defect in NK cell mediated elimination of β2-microglobulin (β2M) deficient target cells was observed. Further studies revealed Slp-76 to control NK-cell receptor expression and maturation; however, activation of Slp-76(ace/ace) NK cells through ITAM-containing NK-cell receptors or allogeneic/tumor target cells appeared largely unaffected. Imagestream analysis of the NK-β2M(-/-) target cell synapse revealed a specific defect in actin recruitment to the conjugate synapse in Slp-76(ace/ace) NK cells. Overall these studies establish Slp-76 as a critical determinant of NK-cell development and NK cell mediated elimination of missing-self target cells in mice.

GTPase of immunity-associated protein 5 (Gimap5) is linked with lymphocyte survival, autoimmunity, and colitis, but its mechanisms of action are unclear. Here, we show that Gimap5 is essential for the inactivation of glycogen synthase kinase-3β (GSK3β) following T cell activation. In the absence of Gimap5, constitutive GSK3β activity constrains c-Myc induction and NFATc1 nuclear import, thereby limiting productive CD4+ T cell proliferation. Additionally, Gimap5 facilitates Ser389 phosphorylation and nuclear translocation of GSK3β, thereby limiting DNA damage in CD4+ T cells. Importantly, pharmacological inhibition and genetic targeting of GSK3β can override Gimap5 deficiency in CD4+ T cells and ameliorates immunopathology in mice. Finally, we show that a human patient with a GIMAP5 loss-of-function mutation has lymphopenia and impaired T cell proliferation in vitro that can be rescued with GSK3 inhibitors. Given that the expression of Gimap5 is lymphocyte-restricted, we propose that its control of GSK3β is an important checkpoint in lymphocyte proliferation., Loss of function GIMAP5 mutation is associated with lymphopenia, but how it mediates T cell homeostasis is unclear. Here the authors study Gimap5−/− mice and a patient with GIMAP5 deficiency to show how this GTPAse negatively regulates GSK3β activity to prevent DNA damage and cell death.

SummaryBackground Clinical and experimental analyses have identified a central role for IgE/FceRI/mast cells in promoting IgE-mediated anaphylaxis. Recent data from human studies suggest that bacterial infections can alter susceptibility to anaphylaxis. Objective We examined the effect of LPS exposure on the induction of IgE-mast cell (MC) mediated reactions in mice. Methods C57BL/6 WT, tlr4−/− and IL10−/− mice were exposed to LPS, and serum cytokines (TNF and IL-10) were measured. Mice were subsequently treated with anti-IgE, and the symptoms of passive IgE-mediated anaphylaxis, MC activation, Ca2+-mobilization and the expression of FceRI on peritoneal MCs were quantitated. Results We show that LPS exposure of C57BL/6 WT mice constraints IgE-MC–mediated reactions. LPS-induced suppression of IgE-MC–mediated responses was TLR-4-dependent and associated with increased systemic IL-10 levels, decreased surface expression of FceRI on MCs and loss of sensitivity to IgE activation. Notably, LPS-induced desensitization of MCs was short term with MC sensitivity to IgE reconstituted within 48 hours, which was associated with recapitulation of FceRI expression on the MCs. Mechanistic analyses revealed a requirement for IL-10 in LPS-mediated decrease in MC FceRI surface expression. Conclusions & Clinical Relevance Collectively, these studies suggest that LPS-induced IL-10 promotes the down-regulation of MC surface FceRI expression and leads to desensitization of mice to IgE-mediated reactions. These studies indicate that targeting of the LPS-TLR-4-IL-10 pathway may be used as a therapeutic approach to prevent adverse IgE-mediated reactions.

Preterm birth (PTB) is a leading worldwide cause of morbidity and mortality in infants. Maternal inflammation induced by microbial infection is a critical predisposing factor for PTB. However, biological processes associated with competency of pathogens, including viruses, to induce PTB or sensitize for secondary bacterial infection-driven PTB are unknown. We show that pathogen/pathogen-associated molecular pattern-driven activation of type I IFN/IFN receptor (IFNAR) was sufficient to prime for systemic and uterine proinflammatory chemokine and cytokine production and induction of PTB. Similarly, treatment with recombinant type I IFNs recapitulated such effects by exacerbating proinflammatory cytokine production and reducing the dose of secondary inflammatory challenge required for induction of PTB. Inflammatory challenge-driven induction of PTB was eliminated by defects in type I IFN, TLR, or IL-6 responsiveness, whereas the sequence of type I IFN sensing by IFNAR on hematopoietic cells was essential for regulation of proinflammatory cytokine production. Importantly, we also show that type I IFN priming effects are conserved from mice to nonhuman primates and humans, and expression of both type I IFNs and proinflammatory cytokines is upregulated in human PTB. Thus, activation of the type I IFN/IFNAR axis in pregnancy primes for inflammation-driven PTB and provides an actionable biomarker and therapeutic target for mitigating PTB risk.

Expression of the activating transcription factor 3 (ATF3) gene is induced by Toll-like receptor (TLR) signaling. In turn, ATF3 protein inhibits the expression of various TLR-driven proinflammatory genes. Given its counter-regulatory role in diverse innate immune responses, we defined the effects of ATF3 on neutrophilic airway inflammation in mice. ATF3 deletion was associated with increased lipopolysaccharide (LPS)-driven airway epithelia production of CXCL1, but not CXCL2, findings concordant with a consensus ATF3-binding site identified solely in the Cxcl1 promoter. Unexpectedly, ATF3-deficient mice did not exhibit increased airway neutrophilia after LPS challenge. Bone marrow chimeras revealed a specific reduction in ATF3−/− neutrophil recruitment to wild-type lungs. In vitro, ATF3−/− neutrophils exhibited a profound chemotaxis defect. Global gene expression analysis identified ablated Tiam2 expression in ATF3−/− neutrophils. TIAM2 regulates cellular motility by activating Rac1-mediated focal adhesion disassembly. Notably, ATF3−/− and ATF3-sufficient TIAM2 knockdown neutrophils, both lacking TIAM2, exhibited increased focal complex area, along with excessive CD11b-mediated F-actin polymerization. Together, our data describe a dichotomous role for ATF3-mediated regulation of neutrophilic responses: inhibition of neutrophil chemokine production but promotion of neutrophil chemotaxis.

Type I diabetes (T1D) is an autoimmune disorder characterized by inflammation and cellular infiltration of the pancreatic islets, resulting in insulitis and damage to islet beta-cells. T1D pathogenesis involves soluble and cellular mediators of both the innate and adaptive arms of the immune system; thus, finding therapeutic targets for reversal of acute disease has proved challenging. We have shown that an activating monoclonal antibody (UT18) targeting mouse TLR4/MD2 reverses acute T1D in non-obese diabetic (NOD) mice. UT18 preserves insulin-producing islet beta-cells and decreases insulitis; however, the molecular mechanisms underlying disease reversal remain unclear. Using AMNIS imaging flow cytometry, we observed an atypical internalization event in which UT18-bound TLR4 persists in EEA1-enriched endosomes rather than recycling back to the cell surface as seen with the TLR4 ligand LPS. Because the subcellular localization of TLR4 alters its proximity to adaptor proteins, we performed immunoblots of downstream signaling mediators. UT18 activates both MyD88 and TRIF pathways; however, the magnitude of MAPK activation is dampened and the kinetics of signaling differ from the rapid, robust activation characteristic of pro-inflammatory LPS. Our data suggest that UT18 functions as a mild TLR4 agonist, eliciting an intermediate signaling profile hypothesized to contribute to tolerance of antigen presenting cells. Knowledge of how UT18 manipulates the TLR4 signaling axis may be applicable for the development of novel immunotherapies for T1D and other autoimmune or inflammatory diseases in which innate immune receptors contribute to pathogenesis.

The Grb2-associated binding protein 3 (Gab3) is a scaffolding protein that belongs to the Grb2-assocated binding family of proteins along with Gab1 and Gab2. Gab3 is expressed exclusively in immune cells including NK cells, CD8+T cells, mast cells and macrophages, however the function of Gab3 has remained elusive. Based on two independent mouse models; a hypomorph germline mouse model carrying a missense mutation in the PH domain of Gab3 and complete Gab3-KO mice, we uncovered a key role for Gab3 in peripheral NK cell function. Specifically, loss of Gab3 results in impaired IL-2/IL-15-induced NK cell priming and expansion, due to a selective impairment in MAPK- but not STAT5-signaling. In vivo, we show that Gab3 is required for recognition and elimination of “missing-self” and tumor targets. Gab3-deficient mice exhibit impaired uterine NK cell expansion that is associated with abnormal spiral artery remodeling and increased trophoblast invasion in the decidua basalis. This coincides with stillbirth, retained placenta, maternal hemorrhage and undelivered fetoplacental units at term. Thus, our studies identify Gab3 as a novel component required for IL-2/IL-15-mediated NK cell priming and expansion that is essential for anti-tumor responses and for controlling fetal trophoblast invasion during pregnancy.

Post-translational protein modifications can play a significant role in immune cell signaling. Recently, we showed that inhibition of transmethylation curtails experimental autoimmune encephalomyelitis, notably by reducing T cell receptor (TCR)-induced activation of CD4(+) T cells. Here, we demonstrate that transmethylation inhibition by a reversible S-adenosyl-l-homocysteine hydrolase inhibitor (DZ2002) led to immunosuppression by reducing TLR-, B cell receptor (BCR)- and TCR-induced activation of immune cells, most likely by blocking NF-κB activity. Moreover, prophylactic treatment with DZ2002 prevented lupus-like disease from developing in both BXSB and MRL-Fas(lpr) mouse models. DZ2002 treatment initiated during active disease significantly improved outcomes in both in vivo models, suggesting methylation inhibition as a novel approach for the treatment of autoimmune/inflammatory diseases.

The immune system exerts a critical function as it recognizes and eliminates transformed or neoplastic cells, a process also referred to as immunosurveillance. NK cells play a particularly important role in that they are able to recognize tumor cells via “missing-self”—i.e., the absence of major histocompatibility complex Class I on target cells. Moreover, recent studies suggest that NK cells also participate in the onset and regulation of adaptive immune responses. The exact molecular pathways by which this occurs, however, remain poorly understood. To obtain further insight into the genes that are required for self-induced immune responses via NK cell-mediated cell death, our laboratory initiated a forward genetic approach using N-ethyl-N-nitrosourea (ENU) as a mutagen. Specifically, we tested the ability of NK cells from G3 ENU germline mice to recognize missing-self target cells and induce CD8+ T-cell responses following immunization with irradiated tumor cells. Here we present two ENU germline mutants, designated Ace and Chip, that are defective in the recognition of β-2 microglobulin-deficient target cells, yet exhibit improved clearance of B16 melanoma cells in vivo. Coarse mapping and whole genome sequencing of the Chip mutation revealed a missense mutation causing a T’A amino acid substitution in the highly conserved third immuno-receptor tyrosine-based switch motif of CD244 (2B4). The forward genetic approach described here promises to reveal important insight into critical genes that are required for host responses involved in anticancer immunity.

Previously, we reported the abrogation of quiescence and reduced survival in lymphocytes from Gimap5sph/sph mice, an ENU germline mutant with a missense mutation in the GTPase of immunity-associated protein 5 (Gimap5). These mice showed a progressive loss of peripheral lymphocyte populations and developed spontaneous colitis, resulting in early mortality. In this study, we identify the molecular pathways that contribute to the onset of colitis in Gimap5sph/sph mice. We show that CD4+ T cells become Th1/Th17 polarized and are critically important for the development of colitis. Concomitantly, regulatory T cells become reduced in frequency in the peripheral tissues, and their immunosuppressive capacity becomes impaired. Most importantly, these progressive changes in CD4+ T cells are associated with the loss of Forkheadbox group O (Foxo)1, Foxo3, and Foxo4 expression. Our data establish a novel link between Gimap5 and Foxo expression and provide evidence for a regulatory mechanism that controls Foxo protein expression and may help to maintain immunological tolerance.

Complement, NKT, and NK cells play critical roles in the first line defense against pathogens. Functional roles for both C5a receptors, that is, complement receptor C5a (C5aR) and C5a receptor-like 2 (C5L2), in sepsis have been demonstrated. However, the role of C5a in innate lymphocyte activation during sepsis remains elusive. In this article, we show that naive NKT and NK cells already express high levels of C5aR and minor levels of C5L2 mRNA, but no protein. Upon Escherichia coli-induced sepsis, we found C5aR surface expression on subpopulations of NKT and NK cells, suggesting rapid translation into C5aR protein on bacterial encounter. Importantly, significantly increased survival in the absence of C5aR, NKT, and NK cells, but not of C5L2, was associated with reduced IFN-γ and TNF-α serum levels. Sepsis induction in C5aR+/C5aR− mixed bone marrow chimeras identified cognate engagement of C5aR on NKT cells as an important factor for the recruitment of NKT cells. Furthermore, we found synergistic interaction between C5aR and TLRs enhancing the production of TNF-α and IFN-γ from NKT and NK cells in cocultures with dendritic cells. Our results identify C5aR activation as a novel pathway driving detrimental effects of NKT and NK cells during early experimental sepsis.

Background GTPase of immunity-associated protein 5 (GIMAP5) is essential for lymphocyte homeostasis and survival. Recently, human GIMAP5 single nucleotide polymorphisms have been linked to an increased risk for asthma, whereas loss of Gimap5 in mice has been associated with severe CD4+ T cell–driven immune pathology. Objective We sought to identify the molecular and cellular mechanisms by which Gimap5 deficiency predisposes to allergic airway disease. Methods CD4+ T-cell polarization and development of pathogenic CD4+ T cells were assessed in Gimap5-deficient mice and a human patient with a GIMAP5 loss-of-function (LOF) mutation. House dust mite–induced airway inflammation was assessed by using a complete Gimap5 LOF (Gimap5sph/sph) and conditional Gimap5fl/flCd4Cre/ert2 mice. Results GIMAP5 LOF mutations in both mice and human subjects are associated with spontaneous polarization toward pathogenic TH17 and TH2 cells in vivo. Mechanistic studies in vitro reveal that impairment of Gimap5-deficient TH cell differentiation is associated with increased DNA damage, particularly during TH1-polarizing conditions. DNA damage in Gimap5-deficient CD4+ T cells could be controlled by TGF-β, thereby promoting TH17 polarization. When challenged with house dust mite in vivo, Gimap5-deficient mice displayed an exacerbated asthma phenotype (inflammation and airway hyperresponsiveness), with increased development of TH2, TH17, and pathogenic TH17/TH2 cells. Conclusion Activation of Gimap5-deficient CD4+ T cells is associated with increased DNA damage and reduced survival that can be overcome by TGF-β. This leads to selective survival of pathogenic TH17 cells but also TH2 cells in human subjects and mice, ultimately promoting allergic airway disease.

During an immune response, most effector T cells die, whereas some are maintained and become memory T cells. Factors controlling the survival of effector CD4+ and CD8+ T cells remain unclear. In this study, we assessed the role of IL-7, IL-15, and their common signal transducer, STAT5, in maintaining effector CD4+ and CD8+ T cell responses. Following viral infection, IL-15 was required to maintain a subpopulation of effector CD8+ T cells expressing high levels of killer cell lectin-like receptor subfamily G, member 1 (KLRG1), and lower levels of CD127, whereas IL-7 and IL-15 acted together to maintain KLRG1lowCD127high CD8+ effector T cells. In contrast, effector CD4+ T cell numbers were not affected by the individual or combined loss of IL-15 and IL-7. Both IL-7 and IL-15 drove phosphorylation of STAT5 within effector CD4+ and CD8+ T cells. When STAT5 was deleted during the course of infection, both KLRG1highCD127low and KLRG1lowCD127high CD8+ T cells were lost, although effector CD4+ T cell populations were maintained. Furthermore, STAT5 was required to maintain expression of Bcl-2 in effector CD8+, but not CD4+, T cells. Finally, IL-7 and IL-15 required STAT5 to induce Bcl-2 expression and to maintain effector CD8+ T cells. Together, these data demonstrate that IL-7 and IL-15 signaling converge on STAT5 to maintain effector CD8+ T cell responses.

GTPase of immunity-associated protein 5 (Gimap5) has been linked with lymphocyte survival, autoimmunity and colitis, but its mechanisms of action are unclear. Here we show that Gimap5 is essential for inactivation of glycogen synthase kinase-3β (GSK3β) following T cell activation. In the absence of Gimap5, constitutive GSK3β activity constrains c-Myc induction as well as NFATc1 nuclear import, thereby reducing the frequency of CD4+ T cells that complete cell cycle. Additionally, Gimap5 facilitates phosphorylation of GSK3β at Ser389 and its nuclear translocation. Impairment in the latter steps of GSK3β regulation by Gimap5 is associated with increased DNA damage and reduced survival of activated CD4+ T cells. Importantly, pharmacological targeting and genetic deletion of GSK3β in mice can override Gimap5-deficiency in CD4+ T cells, limiting DNA damage and enhancing T cell proliferation and survival. Additionally, this ameliorates immune-mediated pathology in vivo caused by loss of Gimap5. Finally, we show that a human patient with a GIMAP5 loss-of-function mutation has lymphopenia and impaired T cell proliferation. T cells from this patient exhibit impaired proliferation, but can be rescued in vitro with GSK3 inhibitors. Given the restricted expression of Gimap5 in lymphocytes, we propose that its control of GSK3b activity is an important checkpoint in lymphocyte proliferation and function.

Homeostatic control of the immune system involves mechanisms that ensure the self-tolerance, survival and quiescence of hematopoietic-derived cells. In this study, we demonstrate that the GTPase of immunity associated protein (Gimap)5 regulates these processes in lymphocytes and hematopoietic progenitor cells. As a consequence of a recessive N-ethyl-N-nitrosourea–induced germline mutation in the P-loop of Gimap5, lymphopenia, hepatic extramedullary hematopoiesis, weight loss, and intestinal inflammation occur in homozygous mutant mice. Irradiated fetal liver chimeric mice reconstituted with Gimap5-deficient cells lose weight and become lymphopenic, demonstrating a hematopoietic cell-intrinsic function for Gimap5. Although Gimap5-deficient CD4+ T cells and B cells appear to undergo normal development, they fail to proliferate upon Ag-receptor stimulation although NF-κB, MAP kinase and Akt activation occur normally. In addition, in Gimap5-deficient mice, CD4+ T cells adopt a CD44highCD62LlowCD69low phenotype and show reduced IL-7rα expression, and T-dependent and T-independent B cell responses are abrogated. Thus, Gimap5-deficiency affects a noncanonical signaling pathway required for Ag-receptor–induced proliferation and lymphocyte quiescence. Antibiotic-treatment or the adoptive transfer of Rag-sufficient splenocytes ameliorates intestinal inflammation and weight loss, suggesting that immune responses triggered by microbial flora causes the morbidity in Gimap5-deficient mice. These data establish Gimap5 as a key regulator of hematopoietic integrity and lymphocyte homeostasis.

Recent evidence suggests that NK cells require priming to display full effector activity. In this study, we demonstrate that IL-18 contributed to this phenomenon. IL-18 signaling-deficient NK cells were found to be unable to secrete IFN-γ in response to ex vivo stimulation with IL-12. This was not due to a costimulatory role of IL-18, because blocking IL-18 signaling during the ex vivo stimulation with IL-12 did not alter IFN-γ production by wild-type NK cells. Rather, we demonstrate that IL-18 primes NK cells in vivo to produce IFN-γ upon subsequent stimulation with IL-12. Importantly, IL-12-induced IFN-γ transcription by NK cells was comparable in IL-18 signaling-deficient and -sufficient NK cells. This suggests that priming by IL-18 leads to an improved translation of IFN-γ mRNA. These results reveal a novel type of cooperation between IL-12 and IL-18 that requires the sequential action of these cytokines.

Forward genetic approaches have contributed to our understanding of how the host senses infectious agents such as bacteria, viruses and fungi. Beginning with the initial discovery of Toll-like receptors (TLRs) as primary sensors involved in the recognition of microbial components, our laboratory has taken a forward genetic approach, using N-ethyl-N-nitrosourea (ENU) mutagenesis in mice, to decipher TLR-signaling pathways. This long term effort has helped to elucidate the circuitry of these pathways, identified new molecules, and disclosed new functions for known molecules. Here we review some of the more important insights developed from this approach and discuss its prospects.

The innate arm of our immune system is the first line of defence against infections. In addition, it is believed to drive adaptive immune responses, which help fight pathogens and provide long-term memory. As such, the innate immune system is instrumental for protection against pathogens that would otherwise destroy their host. Although our understanding of the innate immune components involved in pathogen sensing and fighting is improving, it is still limited. This is particularly exemplified by increased documentation of innate immune deficiencies in humans that often result in high and recurrent susceptibility to infections or even death, without the genetic cause being evident. To provide further insight into the mechanisms by which pathogen sensing and eradication occur, several strategies can be used. The current review focuses on the forward genetic approaches that have been used to dissect innate immunity in the fruit fly and the mouse. For both animal models, forward genetics has been instrumental in the deciphering of innate immunity and has greatly improved our understanding of how we respond to invading pathogens.

We have previously shown that mutations of CD14 or TLR4 impair type I interferon (IFN) production and macrophage survival during infection with vesicular stomatitis virus (VSV). We now report that VSV glycoprotein G (gpG) is essential for the induction of a previously unrecognized CD14/TLR4-dependent response pathway in which the adapter TRAM has predominant importance, absent any need for MyD88 or Mal, and with only a partial requirement for TRIF. Downstream of TRAM, IRF7 activation leads to a type I IFN response. The pathway is utilized by myeloid dendritic cells (mDCs) and macrophages rather than plasmacytoid DCs. This new mode of TLR4 signal transduction, which does not stimulate NF-κB activation, reveals the importance of viral protein recognition by mDCs and shows that TLR4 can drive qualitatively different events within the cell in response to different ligands.

We formulate a two-phase paradigm of autoimmunity associated with systemic lupus erythematosus, the archetypal autoimmune disease. The initial Toll-like receptor (TLR)-independent phase is mediated by dendritic cell uptake of apoptotic cell debris and associated nucleic acids, whereas the subsequent TLR-dependent phase serves an amplification function and is mediated by uptake of TLR ligands derived from self-antigens (principally nucleic acids) complexed with autoantibodies. Both phases depend on elaboration of type I interferons (IFNs), and therapeutic interruption of induction or activity of these cytokines in predisposed individuals might have a substantial mitigating effect in lupus and other autoimmune diseases.

Mouse cytomegalovirus (MCMV) susceptibility often results from defects of natural killer (NK) cell function. Here we describe Jinx, an N-ethyl-N-nitrosourea–induced MCMV susceptibility mutation that permits unchecked proliferation of the virus, causing death. In Jinx homozygotes, activated NK cells and cytotoxic T lymphocytes (CTLs) fail to degranulate, although they retain the ability to produce cytokines, and cytokine levels are markedly elevated in the blood of infected mutant mice. Jinx was mapped to mouse chromosome 11 on a total of 246 meioses and confined to a 4.60–million basepair critical region encompassing 122 annotated genes. The phenotype was ascribed to the creation of a novel donor splice site in Unc13d, the mouse orthologue of human MUNC13-4, in which mutations cause type 3 familial hemophagocytic lymphohistiocytosis (FHL3), a fatal disease marked by massive hepatosplenomegaly, anemia, and thrombocytopenia. Jinx mice do not spontaneously develop clinical features of hemophagocytic lymphohistiocytosis (HLH), but do so when infected with lymphocytic choriomeningitis virus, exhibiting hyperactivation of CTLs and antigen-presenting cells, and inadequate restriction of viral proliferation. In contrast, neither Listeria monocytogenes nor MCMV induces the syndrome. In mice, the HLH phenotype is conditional, which suggests the existence of a specific infectious trigger of FHL3 in humans.

Toll-like receptors (TLRs) sense the presence of microbial and viral pathogens by signal transduction mechanisms that remain to be fully elucidated. A single point mutation (H412R) in the polytopic endoplasmic reticulum (ER)–resident membrane protein UNC93B abolishes signaling via TLR3, 7, and 9. We show that UNC93B specifically interacts with TLR3, 7, 9, and 13, whereas introduction of the point mutation H412R in UNC93B abolishes their interactions. We establish the physical interaction of the intracellular TLRs with UNC93B in splenocytes and bone marrow–derived dendritic cells. Further, by expressing chimeric TLRs, we show that TLR3 and 9 bind to UNC93B via their transmembrane domains. We propose that a physical association between UNC93B and TLRs in the ER is essential for proper TLR signaling.

Mutations in the LRBA gene (encoding the lipopolysaccharide-responsive and beige-like anchor protein) cause a syndrome of autoimmunity, lymphoproliferation, and humoral immune deficiency. The biological role of LRBA in immunologic disease is unknown. We found that patients with LRBA deficiency manifested a dramatic and sustained improvement in response to abatacept, a CTLA4 (cytotoxic T lymphocyte antigen-4)-immunoglobulin fusion drug. Clinical responses and homology of LRBA to proteins controlling intracellular trafficking led us to hypothesize that it regulates CTLA4, a potent inhibitory immune receptor. We found that LRBA colocalized with CTLA4 in endosomal vesicles and that LRBA deficiency or knockdown increased CTLA4 turnover, which resulted in reduced levels of CTLA4 protein in FoxP3(+) regulatory and activated conventional T cells. In LRBA-deficient cells, inhibition of lysosome degradation with chloroquine prevented CTLA4 loss. These findings elucidate a mechanism for CTLA4 trafficking and control of immune responses and suggest therapies for diseases involving the CTLA4 pathway.

Inhaled endotoxins induce an acute inflammatory response in the airways mediated through Toll-like receptor 4 (TLR4) and myeloid differentiation factor 88 (MyD88). However, the relative roles of the TLR4 adaptor proteins TIRAP and TRIF and of the MyD88-dependent IL-1 and IL-18 receptor pathways in this response are unclear. Here, we demonstrate that endotoxin-induced acute bronchoconstriction, vascular damage resulting in protein leak, Th1 cytokine and chemokine secretion and neutrophil recruitment in the airways are abrogated in mice deficient for either TIRAP or MyD88, but not in TRIF deficient mice. The contribution of other TLR-independent, MyD88-dependent signaling pathways was investigated in IL-1R1, IL-18R and caspase-1 (ICE)-deficient mice, which displayed normal airway responses to endotoxin. In conclusion, the TLR4-mediated, bronchoconstriction and acute inflammatory lung pathology to inhaled endotoxin critically depend on the expression of both adaptor proteins, TIRAP and MyD88, suggesting cooperative roles, while TRIF, IL-1R1, IL-18R signaling pathways are dispensable.

The efficacy of tumor cell vaccination largely depends on the maturation and activation status of the dendritic cell. Here we investigated the ability of soluble and tumor cell-associated dsRNA to serve as an adjuvant in the induction of protective adaptive antitumor responses. Our data showed that cell-associated dsRNA, but not soluble dsRNA, enhanced both tumor-specific CD8+ and CD4+ T cell responses. The cell-associated dsRNA increased the clonal burst of tumor-specific CD8+ T cells and endowed them with an enhanced capacity for expansion upon a secondary encounter with tumor Ags, even when the CD8+ T cells were primed in the absence of CD4+ T cell help. The adjuvant effect of cell-associated dsRNA was fully dependent on the expression of TLR3 by the APCs and their subsequent production of type I IFNs, as the adjuvant effect of cell-associated dsRNA was completely abrogated in mice deficient in TLR3 or type I IFN signaling. Importantly, treatment with dsRNA-associated tumor cells increased the number of tumor-infiltrating lymphocytes and enhanced the survival of tumor-bearing mice. The data from our studies suggest that using cell-associated dsRNA as a tumor vaccine adjuvant may be a suitable strategy for enhancing vaccine efficacy for tumor cell therapy in cancer patients.

Herpes simplex virus-1 (HSV-1) encephalitis (HSE) is the most common form of sporadic viral encephalitis in western countries. Its pathogenesis remains unclear, as it affects otherwise healthy patients and only a small minority of HSV-1–infected individuals. Here, we elucidate a genetic etiology for HSE in two children with autosomal recessive deficiency in the intracellular protein UNC-93B, resulting in impaired cellular interferon-α/β and -λ antiviral responses. HSE can result from a single-gene immunodeficiency that does not compromise immunity to most pathogens, unlike most known primary immunodeficiencies. Other severe infectious diseases may also reflect monogenic disorders of immunity.

Summary Here, we describe a previously unrecognized pathway for activation of antigen-specific adaptive immune responses that was independent of Toll-Interleukin 1 Receptor signaling and directed toward detection of antigens expressed by apoptotic cells. This pathway is represented within Flt-3 Ligand-derived dendritic cells (DCs) that represent immature lymphoid DCs, but not within GM-CSF-treated bone marrow-derived dendritic cells. Exposure of these DCs to apoptotic cells resulted in production of type I interferon and favored the development of cytotoxic T cell responses. The N-Ethyl-N-Nitrosourea-induced germline mutation 3d ( Unc3b1 3d/3d ) abolished both MHC class I and II responses elicited by this pathway, whereas a null allele of Cd36 selectively abolished class II responses. We propose that this mode of adaptive immune activation evolved to permit the sensitive detection of intracellular microbial infections, particularly viral infections, which frequently induce apoptotic cell death, but may also be important in transplantation, autoimmunity, and vaccine development.

The mouse cytomegalovirus (MCMV) resistome is the set of host genes with nonredundant functions in resistance to MCMV infection. By screening 3,500 G(3) germline mutant mice ( approximately 1,750 gamete equivalents), we have identified eight transmissible mutations that create MCMV susceptibility in C57BL/6 mice. Among these, a mutation called Domino was noted to cause macrophage susceptibility to vesicular stomatitis virus (VSV) in vitro. This accessory phenotype was not corrected by type I interferon (IFN), which suggested a defect of the type I IFN pathway. Domino corresponds to a point mutation that alters the DNA binding domain of STAT1, leading to a defect of STAT1 activation. Identification of the Domino mutation demonstrates that an in vivo MCMV susceptibility screen is feasible and illustrates how it can provide insight into the resistome. Moreover, some mutations are far more deleterious than Domino in MCMV-infected mice, consistent with the interpretation that certain protein(s) unrelated to IFN production or signaling are more important than IFNs with regard to their net antiviral effects.

Classical genetic methods, driven by phenotype rather than hypotheses, generally permit the identification of all proteins that serve nonredundant functions in a defined biological process. Long before this goal is achieved, and sometimes at the very outset, genetics may cut to the heart of a biological puzzle. So it was in the field of mammalian innate immunity. The positional cloning of a spontaneous mutation that caused lipopolysaccharide resistance and susceptibility to Gram-negative infection led directly to the understanding that Toll-like receptors (TLRs) are essential sensors of microbial infection. Other mutations, induced by the random germ line mutagen ENU (N-ethyl-N-nitrosourea), have disclosed key molecules in the TLR signaling pathways and helped us to construct a reasonably sophisticated portrait of the afferent innate immune response. A still broader genetic screen—one that detects all mutations that compromise survival during infection—is permitting fresh insight into the number and types of proteins that mammals use to defend themselves against microbes.

Here we have identified 'triple D' (3d), a recessive N-ethyl-N-nitrosourea-induced mutation and phenotype in which no signaling occurs via the intracellular Toll-like receptors 3, 7 and 9 (sensors for double-stranded RNA, single-stranded RNA and unmethylated DNA, respectively). The 3d mutation also prevented cross-presentation and diminished major histocompatibility complex class II presentation of exogenous antigen; it also caused hypersusceptibility to infection by mouse cytomegalovirus and other microbes. By positional identification, we found 3d to be a missense allele of Unc93b1, which encodes the 12-membrane-spanning protein UNC-93B, a highly conserved molecule found in the endoplasmic reticulum with multiple paralogs in mammals. Innate responses to nucleic acids and exogenous antigen presentation, which both initiate in endosomes, thus seem to depend on an endoplasmic reticulum–resident protein, which suggests communication between these organellar systems.

flake (flk ), an N -ethyl- N -nitrosourea-induced recessive germ line mutation of C57BL/6 mice, impairs the clearance of skin infections by Streptococcus pyogenes and Staphylococcus aureus , gram-positive pathogens that elicit innate immune responses by activating Toll-like receptor 2 (TLR2) (K. Takeda and S. Akira, Cell. Microbiol. 5: 143-153, 2003). Positional cloning and sequencing revealed that flk is a novel allele of the stearoyl coenzyme A desaturase 1 gene ( Scd1 ). flake homozygotes show reduced sebum production and are unable to synthesize the monounsaturated fatty acids (MUFA) palmitoleate (C 16:1 ) and oleate (C 18:1 ), both of which are bactericidal against gram-positive (but not gram-negative) organisms in vitro. However, intradermal MUFA administration to S. aureus -infected mice partially rescues the flake phenotype, which indicates that an additional component of the sebum may be required to improve bacterial clearance. In normal mice, transcription of Scd1 —a gene with numerous NF-κB elements in its promoter—is strongly and specifically induced by TLR2 signaling. Similarly, the SCD1 gene is induced by TLR2 signaling in a human sebocyte cell line. These observations reveal the existence of a regulated, lipid-based antimicrobial effector pathway in mammals and suggest new approaches to the treatment or prevention of infections with gram-positive bacteria.

Type I interferons (IFN) play a critical role in the Toll-like receptor (TLR)-mediated expression of B7 costimulatory family members. For example, LPS-induced up-regulation of CD80 (B7.1) and CD86 (B7.2) is abrogated in antigen-presenting cells (APC) deficient in TRIF or TRAM, two adaptors that are responsible for TLR4-mediated production of Type I IFN. In this report, we demonstrate that LPS-induced up-regulation of B7-related protein 1 (B7RP-1), a ligand for ICOS, is dependent primarily upon the MyD88-dependent signaling pathway. Signaling via the TRIF pathway sharply limits MyD88-dependent B7RP-1 up-regulation. Hence, LPS induces significantly higher B7RP-1 expression on TRIF- or TRAM-deficient mouse peritoneal macrophages and on TRIF-deficient mouse splenic B cells as compared to wild-type cells. Further studies reveal that Type I IFN are general suppressors of TLR-mediated up-regulation of B7RP-1. These data indicate that Type I IFN play a dual role in the TLR-mediated expression of B7 costimulatory family members and suggest that they may act to limit B7RP-1 expression and thus limit signals derived from B7RP-1-ICOS interaction.

This focus analyzes some of the ways the innate immune system influences adaptive immune responses. Here the main principles and themes that govern this intricate relationship are discussed.