Constitutive aryl hydrocarbon receptor signaling constrains type I interferon-mediated antiviral innate defense.

Aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor that mediates the toxic activity of many environmental xenobiotics. However, its role in innate immune responses during viral infection is not fully understood. Here we demonstrate that constitutive AHR signaling negatively regulates the type I interferon (IFN-I) response during infection with various types of virus. Virus-induced IFN-ß production was enhanced in AHR-deficient cells and mice and resulted in restricted viral replication. We found that AHR upregulates expression of the ADP-ribosylase TIPARP, which in turn causes downregulation of the IFN-I response. Mechanistically, TIPARP interacted with the kinase TBK1 and suppressed its activity by ADP-ribosylation. Thus, this study reveals the physiological importance of endogenous activation of AHR signaling in shaping the IFN-I-mediated innate response and, further, suggests that the AHR-TIPARP axis is a potential therapeutic target for enhancing antiviral responses.

Dichotomous Expression of TNF Superfamily Ligands on Antigen-Presenting Cells Controls Post-priming Anti-viral CD4+ T Cell Immunity.

T cell antigen-presenting cell (APC) interactions early during chronic viral infection are crucial for determining viral set point and disease outcome, but how and when different APC subtypes contribute to these outcomes is unclear. The TNF receptor superfamily (TNFRSF) member GITR is important for CD4+ T cell accumulation and control of chronic lymphocytic choriomeningitis virus (LCMV). We found that type I interferon (IFN-I) induced TNFSF ligands GITRL, 4-1BBL, OX40L, and CD70 predominantly on monocyte-derived APCs and CD80 and CD86 predominantly on classical dendritic cells (cDCs). Mice with hypofunctional GITRL in Lyz2+ cells had decreased LCMV-specific CD4+ T cell accumulation and increased viral load. GITR signals in CD4+ T cells occurred after priming to upregulate OX40, CD25, and chemokine receptor CX3CR1. Thus IFN-I (signal 3) induced a post-priming checkpoint (signal 4) for CD4+ T cell accumulation, revealing a division of labor between cDCs and monocyte-derived APCs in regulating T cell expansion.

A therapeutic convection-enhanced macroencapsulation device for enhancing ß cell viability and insulin secretion.

Islet transplantation for type 1 diabetes treatment has been limited by the need for lifelong immunosuppression regimens. This challenge has prompted the development of macroencapsulation devices (MEDs) to immunoprotect the transplanted islets. While promising, conventional MEDs are faced with insufficient transport of oxygen, glucose, and insulin because of the reliance on passive diffusion. Hence, these devices are constrained to two-dimensional, wafer-like geometries with limited loading capacity to maintain cells within a distance of passive diffusion. We hypothesized that convective nutrient transport could extend the loading capacity while also promoting cell viability, rapid glucose equilibration, and the physiological levels of insulin secretion. Here, we showed that convective transport improves nutrient delivery throughout the device and affords a three-dimensional capsule geometry that encapsulates 9.7-fold-more cells than conventional MEDs. Transplantation of a convection-enhanced MED (ceMED) containing insulin-secreting ß cells into immunocompetent, hyperglycemic rats demonstrated a rapid, vascular-independent, and glucose-stimulated insulin response, resulting in early amelioration of hyperglycemia, improved glucose tolerance, and reduced fibrosis. Finally, to address potential translational barriers, we outlined future steps necessary to optimize the ceMED design for long-term efficacy and clinical utility.

4-1BB Regulates Effector CD8 T Cell Accumulation in the Lung Tissue through a TRAF1-, mTOR-, and Antigen-Dependent Mechanism to Enhance Tissue-Resident Memory T Cell Formation during Respiratory Influenza Infection.

The TNFR superfamily member 4-1BB is important in the establishment of tissue-resident memory T cells (Trm) in the lung tissue following influenza infection. Moreover, supraphysiological boosting of 4-1BB in the airways during the boost phase of a prime-boost immunization regimen increases the long-lived Trm population, correlating with increased protection against heterotypic challenge. However, little is known about how 4-1BB contributes to the establishment of the lung Trm population. In this study, we show that effects of 4-1BB on lung Trm accumulation are already apparent at the effector stage, suggesting that the major role of 4-1BB in Trm formation is to allow persistence of CD8 T effector cells in the lung as they transition to Trm. Using supraphysiological stimulation of 4-1BB in the boost phase of a prime-boost immunization, we show that the effect of 4-1BB on Trm generation requires local delivery of both Ag and costimulation, is inhibited by rapamycin treatment during secondary CD8 effector T cell expansion, and is dependent on the signaling adaptor TRAF1. The decrease in lung Trm following early rapamycin treatment is accompanied by increased circulating memory T cells, as well as fewer effectors, suggesting a role for mammalian target of rapamycin (mTOR) in the formation of Trm through effects on the accumulation of effector precursors. Taken together, these data point to an important role for 4-1BB, TRAF1, and mTOR in the persistence of CD8 effector T cells in the lung parenchyma, thereby allowing the transition to Trm.

3-Methylcholanthrene Induces Chylous Ascites in TCDD-Inducible Poly-ADP-Ribose Polymerase (Tiparp) Knockout Mice.

TCDD-inducible poly-ADP-ribose polymerase (TIPARP) is an aryl hydrocarbon receptor (AHR) target gene that functions as part of a negative feedback loop to repress AHR activity. Tiparp-/- mice exhibit increased sensitivity to the toxicological effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), including lethal wasting syndrome. However, it is not known whether Tiparp-/- mice also exhibit increased sensitivity to other AHR ligands. In this study, we treated male Tiparp-/- or wild type (WT) mice with a single injection of 100 mg/kg 3-methylcholanthrene (3MC). Consistent with TIPARP's role as a repressor of AHR signaling, 3MC-treated Tiparp-/- mice exhibited increased hepatic Cyp1a1 and Cyp1b1 levels compared with WT mice. No 3MC-treated Tiparp-/- mice survived beyond day 16 and the mice exhibited chylous ascites characterized by an accumulation of fluid in the peritoneal cavity. All WT mice survived the 30-day treatment and showed no signs of fluid accumulation. Treated Tiparp-/- mice also exhibited a transient and mild hepatotoxicity with inflammation. 3MC-treated WT, but not Tiparp-/- mice, developed mild hepatic steatosis. Lipid deposits accumulated on the surface of the liver and other abdominal organs in the 3MC-Tiparp-/- mice. Our study reveals that Tiparp-/- mice have increased sensitivity to 3MC-induced liver toxicity, but unlike with TCDD, lethality is due to chylous ascites rather than wasting syndrome.

GITR intrinsically sustains early type 1 and late follicular helper CD4 T cell accumulation to control a chronic viral infection.

CD4 T cells are critical for control of persistent infections; however, the key signals that regulate CD4 T help during chronic infection remain incompletely defined. While several studies have addressed the role of inhibitory receptors and soluble factors such as PD-1 and IL-10, significantly less work has addressed the role of T cell co-stimulatory molecules during chronic viral infection. Here we show that during a persistent infection with lymphocytic choriomeningitis virus (LCMV) clone 13, mice lacking the glucocorticoid-induced tumor necrosis factor receptor related protein (GITR) exhibit defective CD8 T cell accumulation, increased T cell exhaustion and impaired viral control. Differences in CD8 T cells and viral control between GITR+/+ and GITR-/- mice were lost when CD4 T cells were depleted. Moreover, mixed bone marrow chimeric mice, as well as transfer of LCMV epitope-specific CD4 or CD8 T cells, demonstrated that these effects of GITR are largely CD4 T cell-intrinsic. GITR is dispensable for initial CD4 T cell proliferation and differentiation, but supports the post-priming accumulation of IFNγ+IL-2+ Th1 cells, facilitating CD8 T cell expansion and early viral control. GITR-dependent phosphorylation of the p65 subunit of NF-κB as well as phosphorylation of the downstream mTORC1 target, S6 ribosomal protein, were detected at day three post-infection (p.i.), and defects in CD4 T cell accumulation in GITR-deficient T cells were apparent starting at day five p.i. Consistently, we pinpoint IL-2-dependent CD4 T cell help for CD8 T cells to between days four and eight p.i. GITR also increases the ratio of T follicular helper to T follicular regulatory cells and thereby enhances LCMV-specific IgG production. Together, these findings identify a CD4 T cell-intrinsic role for GITR in sustaining early CD8 and late humoral responses to collectively promote control of chronic LCMV clone 13 infection.

Anti-GITR agonist therapy intrinsically enhances CD8 T cell responses to chronic lymphocytic choriomeningitis virus (LCMV), thereby circumventing LCMV-induced downregulation of costimulatory GITR ligand on APC.

The costimulatory TNFR family member GITR can provide important survival signals for CD8 T cells. However, little is known about the regulation of this pathway during a chronic infection. In this study, we show that GITR ligand (GITRL) is maximally induced on APCs at day 2 post-lymphocytic choriomeningitis virus (LCMV) clone 13 infection, but is downregulated to below baseline levels by day 8 postinfection (p.i.), and remains so at the chronic stage of infection. At its peak, GITRL expression is highest on macrophages, with lower expression on conventional and plasmacytoid dendritic cells. GITR expression was highest on T regulatory cells but was also detected on Th1 and LCMV-specific CD8 T cells at day 8 p.i. and was maintained at low, but above baseline levels at the chronic stage of LCMV infection. As GITRL was limiting at the chronic stage of infection, we investigated the potential of therapeutic stimulation of GITR at this stage using agonistic anti-GITR Ab. Anti-GITR treatment at day 21 p.i. increased the frequency and number of LCMV-specific CD8 T cells, resulting in increased in vivo CTL activity and a concomitant decrease in viral load, despite the persistence of PD-1 expression. These effects of anti-GITR were CD8 T cell intrinsic, with no detectable effects on Th1 or T regulatory cells. In contrast to other TNFR agonists, such as anti-4-1BB, which can cause immune pathology, a single therapeutic dose of anti-GITR did not induce splenomegaly or increase serum alanine transaminase. These studies identify GITR as a promising therapeutic target for chronic infection.

GARP-TGF-ß complexes negatively regulate regulatory T cell development and maintenance of peripheral CD4+ T cells in vivo.

The role of surface-bound TGF-ß on regulatory T cells (Tregs) and the mechanisms that mediate its functions are not well defined. We recently identified a cell-surface molecule called Glycoprotein A Repetitions Predominant (GARP), which is expressed specifically on activated Tregs and was found to bind latent TGF-ß and mediate a portion of Treg suppressive activity in vitro. In this article, we address the role of GARP in regulating Treg and conventional T cell development and immune suppression in vivo using a transgenic mouse expressing GARP on all T cells. We found that, despite forced expression of GARP on all T cells, stimulation through the TCR was required for efficient localization of GARP to the cell surface. In addition, IL-2 signals enhanced GARP cell surface expression specifically on Tregs. GARP-transgenic CD4(+) T cells and Tregs, especially those expressing higher levels of GARP, were significantly reduced in the periphery. Mature Tregs, but not conventional CD4(+) T cells, were also reduced in the thymus. CD4(+) T cell reduction was more pronounced within the effector/memory subset, especially as the mouse aged. In addition, GARP-overexpressing CD4(+) T cells stimulated through the TCR displayed reduced proliferative capacity, which was restored by inhibiting TGF-ß signaling. Furthermore, inhibiting TGF-ß signals greatly enhanced surface expression of GARP on Tregs and blocked the induction of Foxp3 in activated CD4(+) T cells overexpressing GARP. These findings suggest a role for GARP in natural and induced Treg development through activation of bound latent TGF-ß and signaling, which negatively regulates GARP expression on Tregs.

Regulation of the expression of GARP/latent TGF-ß1 complexes on mouse T cells and their role in regulatory T cell and Th17 differentiation.

GARP/LRRC32 was defined as a marker of activated human regulatory T cells (Tregs) that is responsible for surface localization of latent TGF-ß1. We find that GARP and latent TGF-ß1 are also found on mouse Tregs activated via TCR stimulation; however, in contrast to human Tregs, GARP is also expressed at a low level on resting Tregs. The expression of GARP can be upregulated on mouse Tregs by IL-2 or IL-4 exposure in the absence of TCR signaling. GARP is expressed at a low level on Tregs within the thymus, and Treg precursors from the thymus concomitantly express GARP and Foxp3 upon exposure to IL-2. The expression of GARP is independent of TGF-ß1 and TGF-ß1 loading into GARP and is independent of furin-mediated processing of pro-TGF-ß1 to latent TGF-ß1. Specific deletion of GARP in CD4(+) T cells results in lack of expression of latent TGF-ß1 on activated Tregs. GARP-deficient Tregs develop normally, are present in normal numbers in peripheral tissues, and are fully competent suppressors of the activation of conventional T cells in vitro. Activated Tregs expressing GARP/latent TGF-ß1 complexes are potent inducers of Th17 differentiation in the presence of exogenous IL-6 and inducers of Treg in the presence of IL-2. Induction of both Th17-producing cells and Tregs is caused preferentially by Tregs expressing the latent TGF-ß1/GARP complex on their cell surface rather than by secreted latent TGF-ß1.

MicroRNA Expression Prior to Biting in a Vector Mosquito Anticipates Physiological Processes Related to Energy Utilization, Reproduction and Immunity.

Understanding the molecular and physiological processes underlying biting behavior in vector mosquitoes has important implications for developing novel strategies to suppress disease transmission. Here, we conduct small-RNA sequencing and qRT-PCR to identify differentially expressed microRNAs (miRNAs) in the head tissues of two subspecies of Culex pipiens that differ in biting behavior and the ability to produce eggs without blood feeding. We identified eight differentially expressed miRNAs between biting C. pipiens pipiens (Pipiens) and non-biting C. pipiens molestus (Molestus); six of these miRNAs have validated functions or predicted targets related to energy utilization (miR8-5-p, miR-283, miR-2952-3p, miR-1891), reproduction (miR-1891), and immunity (miR-2934-3p, miR-92a, miR8-5-p). Although miRNAs regulating physiological processes associated with blood feeding have previously been shown to be differentially expressed in response to a blood meal, our results are the first to demonstrate differential miRNA expression in anticipation of a blood meal before blood is actually imbibed. We compare our current miRNA results to three previous studies of differential messenger RNA expression in the head tissues of mosquitoes. Taken together, the combined results consistently show that biting mosquitoes commit to specific physiological processes in anticipation of a blood meal, while non-biting mosquitoes mitigate these anticipatory costs.

Fragment-based screening by protein-detected NMR spectroscopy.

Fragment-based drug discovery (FBDD) identifies low molecular weight compounds that can be developed into ligands with high affinity and selectivity for therapeutic targets. Screening fragment libraries (<10,000 molecules) with biophysical techniques against macromolecules provides information about novel chemical spaces that bind the macromolecule and scaffolds that can be modified to increase potency. A fragment-screening pipeline requires a standardized protocol for target selection, library assembly and maintenance, library screening, and hit validation to ensure hit integrity. Herein, the fundamental aspects of a fragment screening pipeline-focusing on protein-detected NMR data collection and analysis-are discussed in detail for researchers to use as a resource in their FBDD projects. Selected screening targets must undergo rigorous stability and buffer testing by NMR spectroscopy to ensure the protein structure is stable for the entire screen. Biophysical instrumentation that rapidly measures protein thermostability is helpful in buffer screening. Molecules in fragment libraries are analyzed computationally and physically, stored at appropriate temperatures, and multiplexed in well plates for library conservation. The screening protocol is streamlined using liquid handling robotics for sample preparation and customized Python scripts for protein-detected NMR data analysis. Molecules identified from the screen are titrated to determine their binding site(s) and Kd values and confirmed with an orthogonal biophysical assay. This detailed FBDD screening pipeline developed by the Program in Chemical Biology at the Medical College of Wisconsin has successfully screened many unrelated target proteins to identified novel molecules that selectively bind to these target proteins.

Fragment-based drug discovery of small molecule ligands for the human chemokine CCL28.

The mucosal chemokine CCL28 is a promising target for immunotherapy drug development due to its elevated expression level in epithelial cells and critical role in creating and maintaining an immunosuppressive tumor microenvironment. Using sulfotyrosine as a probe, NMR chemical shift mapping identified a potential receptor-binding hotspot on the human CCL28 surface. CCL28 was screened against 2,678 commercially available chemical fragments by 2D NMR, yielding thirteen verified hits. Computational docking predicted that two fragments could occupy adjoining subsites within the sulfotyrosine recognition cleft. Dual NMR titrations confirmed their ability to bind CCL28 simultaneously, thereby validating an initial fragment pair for linking and merging strategies to design high-potency CCL28 inhibitors.

A human model of arteriovenous malformation (AVM)-on-a-chip reproduces key disease hallmarks and enables drug testing in perfused human vessel networks.

Brain arteriovenous malformations (AVMs) are a disorder wherein abnormal, enlarged blood vessels connect arteries directly to veins, without an intervening capillary bed. AVMs are one of the leading causes of hemorrhagic stroke in children and young adults. Most human sporadic brain AVMs are associated with genetic activating mutations in the KRAS gene. Our goal was to develop an in vitro model that would allow for simultaneous morphological and functional phenotypic data capture in real time during AVM disease progression. By generating human endothelial cells harboring a clinically relevant mutation found in most human patients (activating mutations within the small GTPase KRAS) and seeding them in a dynamic microfluidic cell culture system that enables vessel formation and perfusion, we demonstrate that vessels formed by KRAS4AG12V mutant endothelial cells (ECs) were significantly wider and more leaky than vascular beds formed by wild-type ECs, recapitulating key structural and functional hallmarks of human AVM pathogenesis. Immunofluorescence staining revealed a breakdown of adherens junctions in mutant KRAS vessels, leading to increased vascular permeability, a hallmark of hemorrhagic stroke. Finally, pharmacological blockade of MEK kinase activity, but not PI3K inhibition, improved endothelial barrier function (decreased permeability) without affecting vessel diameter. Collectively, our studies describe the creation of human KRAS-dependent AVM-like vessels in vitro in a self-assembling microvessel platform that is amenable to phenotypic observation and drug delivery.

A simple assay to quantify mycobacterial lipid antigen-specific T cell receptors in human tissues and blood.

T cell receptors (TCRs) encode the history of antigenic challenge within an individual and have the potential to serve as molecular markers of infection. In addition to peptide antigens bound to highly polymorphic MHC molecules, T cells have also evolved to recognize bacterial lipids when bound to non-polymorphic CD1 molecules. One such subset, germline-encoded, mycolyl lipid-reactive (GEM) T cells, recognizes mycobacterial cell wall lipids and expresses a conserved TCR-ɑ chain that is shared among genetically unrelated individuals. We developed a quantitative PCR assay to determine expression of the GEM TCR-ɑ nucleotide sequence in human tissues and blood. This assay was validated on plasmids and T cell lines. We tested blood samples from South African subjects with or without tuberculin reactivity or with active tuberculosis disease. We were able to detect GEM TCR-ɑ above the limit of detection in 92% of donors but found no difference in GEM TCR-ɑ expression among the three groups after normalizing for total TCR-ɑ expression. In a cohort of leprosy patients from Nepal, we successfully detected GEM TCR-ɑ in 100% of skin biopsies with histologically confirmed tuberculoid and lepromatous leprosy. Thus, GEM T cells constitute part of the T cell repertoire in the skin. However, GEM TCR-ɑ expression was not different between leprosy patients and control subjects after normalization. Further, these results reveal the feasibility of developing a simple, field deployable molecular diagnostic based on mycobacterial lipid antigen-specific TCR sequences that are readily detectable in human tissues and blood independent of genetic background.

Depression in type 2 diabetes: A systematic review and meta-analysis of blood inflammatory markers.

The prevalence of depression is higher among people with type 2 diabetes (T2DM). Individually, both conditions are associated with systemic inflammation. This study aimed to summarize the clinical data comparing peripheral inflammatory markers in blood between people with T2DM, with and without comorbid depression. From 2187 records, we identified 20 original peer-reviewed articles from which blood inflammatory marker concentrations could be combined and compared between people with T2DM and comorbid depression (D) vs. no depression (ND) as standardized mean differences (SMD) in random effects meta-analysis. Concentrations of C-reactive protein (CRP; ND/NND = 1742/15244, SMD = 0.31 95% confidence interval [0.16, 0.45], Z16 = 4.03, p < 0.01; I2 = 84.0%) and interleukin-6 (IL-6; ND/NND = 677/4349, SMD = 0.17 [0.04, 0.30], Z4 = 2.58, p = 0.01; I2 = 48.1%), were higher, and concentrations of brain derived neurotrophic factor (BDNF; ND/NND = 358/1512, SMD = -0.37 95% confidence interval [-0.64,-0.10], Z2 = -2.68, p = 0.01; I2 = 61.2%) were lower, among those with depression. Depression in T2DM was associated with systemic inflammation and lower peripheral blood BDNF concentrations. Inconsistency between studies suggests the need to explore further population heterogeneity and pathophysiological elements. PROSPERO (CRD42020188509).

A hidden risk: Survival and resuscitation of Escherichia coli O157:H7 in the viable but nonculturable state after boiling or microwaving.

We report the existence and resuscitation of viable but nonculturable (VBNC) Escherichia coli O157:H7 cells in drinking water induced by the common point-of-use disinfection treatments of boiling or microwaving. Tap water and saline samples containing E. coli O157:H7 culturable cells from a bovine isolate or two clinical isolates were boiled (1, 10, or 15 min) on a hot plate or microwaved (1.5 min) to reach boiling. No culturable E. coli O157:H7 cells were observed in the treated samples using conventional plating methods. In samples boiled for 1 or 10 min, two viability assays separately detected that 2-5.5% of the cells retained an intact membrane, while 28 to 87 cells out of the initial 108 cells retained both measurable intracellular esterase activity and membrane integrity. In samples boiled for 15 min, no viable cells were detected. The microwaved samples contained 6-10% of cells with an intact membrane, while 21 to 108 cells out of the initial 108 cells retained both membrane integrity and esterase activity. The number of viable cells retaining both metabolic activity and membrane integrity were consistent in all samples, supporting the survival of a small number of E. coli O157:H7 cells in the VBNC state after boiling for 1 or 10 min or microwaving. Furthermore, the VBNC E. coli O157:H7 cells regained growth at 37 °C in culture media containing autoinducers produced by common non-pathogenic E. coli, commonly present in the human intestine, and norepinephrine. The resuscitated cells were culturable on conventional plates and expressed mRNA encoding the E. coli O157 lipopolysaccharide gene (rfbE) and the H7 flagellin gene (fliC). This study highlights potential concerns for public health risk management of VBNC E. coli O157:H7 in drinking water disinfected by heat treatment at point-of-use. The public health significance of these concerns warrants further investigation.

A 3D culture platform enables development of zinc-binding prodrugs for targeted proliferation of ß cells.

Advances in treating ß cell loss include islet replacement therapies or increasing cell proliferation rate in type 1 and type 2 diabetes, respectively. We propose developing multiple proliferation-inducing prodrugs that target high concentration of zinc ions in ß cells. Unfortunately, typical two-dimensional (2D) cell cultures do not mimic in vivo conditions, displaying a markedly lowered zinc content, while 3D culture systems are laborious and expensive. Therefore, we developed the Disque Platform (DP)-a high-fidelity culture system where stem cell-derived ß cells are reaggregated into thin, 3D discs within 2D 96-well plates. We validated the DP against standard 2D and 3D cultures and interrogated our zinc-activated prodrugs, which release their cargo upon zinc chelation-so preferentially in ß cells. Through developing a reliable screening platform that bridges the advantages of 2D and 3D culture systems, we identified an effective hit that exhibits 2.4-fold increase in ß cell proliferation compared to harmine.

GITRL on inflammatory antigen presenting cells in the lung parenchyma provides signal 4 for T-cell accumulation and tissue-resident memory T-cell formation.

T-cell responses in the lung are critical for protection against respiratory pathogens. TNFR superfamily members play important roles in providing survival signals to T cells during respiratory infections. However, whether these signals take place mainly during priming in the secondary lymphoid organs and/or in the peripheral tissues remains unknown. Here we show that under conditions of competition, GITR provides a T-cell intrinsic advantage to both CD4 and CD8 effector T cells in the lung tissue, as well as for the formation of CD4 and CD8 tissue-resident memory T cells during respiratory influenza infection in mice. In contrast, under non-competitive conditions, GITR has a preferential effect on CD8 over CD4 T cells. The nucleoprotein-specific CD8 T-cell response partially compensated for GITR deficiency by expansion of higher affinity T cells; whereas, the polymerase-specific response was less flexible and more GITR dependent. Following influenza infection, GITR is expressed on lung T cells and GITRL is preferentially expressed on lung monocyte-derived inflammatory antigen presenting cells. Accordingly, we show that GITR+/+ T cells in the lung parenchyma express more phosphorylated-ribosomal protein S6 than their GITR-/- counterparts. Thus, GITR signaling within the lung tissue critically regulates effector and tissue-resident memory T-cell accumulation.

Insulin Receptor-Mediated Stimulation Boosts T Cell Immunity during Inflammation and Infection.

T cells represent a critical effector of cell-mediated immunity. Activated T cells engage in metabolic reprogramming during effector differentiation to accommodate dynamic changes in energy demands. Here, we show that the hormone, insulin, and downstream signaling through its insulin receptor shape adaptive immune function through modulating T cell metabolism. T cells lacking insulin receptor expression (LckCre+ Insrfl/fl) show reduced antigen-specific proliferation and compromised production of pro-inflammatory cytokines. In vivo, T cell-specific insulin receptor deficiency reduces T cell-driven colonic inflammation. In a model of severe influenza infection with A/PR8 (H1N1), lack of insulin receptor on T cells curtails antigen-specific immunity to influenza viral antigens. Mechanistically, insulin receptor signaling reinforces a metabolic program that supports T cell nutrient uptake and associated glycolytic and respiratory capacities. These data highlight insulin receptor signaling as an important node integrating immunometabolic pathways to drive optimal T cell effector function in health and disease.

CD30 Is Dispensable for T-Cell Responses to Influenza Virus and Lymphocytic Choriomeningitis Virus Clone 13 but Contributes to Age-Associated T-Cell Expansion in Mice.

CD30 is a tumor necrosis factor receptor (TNFR) family member whose expression is associated with Hodgkin's disease, anaplastic large cell lymphomas, and other T and B lymphoproliferative disorders in humans. A limited number of studies have assessed the physiological role of CD30/CD30 ligand interactions in control of infection in mice. Here, we assess the role of CD30 in T-cell immunity to acute influenza and chronic lymphocytic choriomeningitis virus (LCMV) clone 13 infection, two viral infections in which other members of the TNFR superfamily are important for T-cell responses. We show that CD30 is expressed on activated but not resting CD4 and CD8 T cells in vitro, as well as on regulatory T cells and marginally on T helper 1 cells in vivo during influenza infection. Despite this, CD4 and CD8 T-cell expansion in response to influenza virus was comparable in CD30+/+ and CD30-/- littermates, with no discernable role for the pathway in the outcome of influenza infection. Similarly, during persistent infection with LCMV clone 13, CD30 plays no obvious role in CD4 or CD8 T-cell responses, the level of T-cell exhaustion or viral control. In contrast, in the steady state, we observed increased numbers of total CD4 and CD8 T cells as well as increased numbers of regulatory T cells in unimmunized older (~8 months) CD30+/+ but not in CD30-/- age-matched littermates. Naive T-cell numbers were unchanged in the aged CD30+/+ mice compared to their CD30-/- littermate controls, rather the T-cell expansions were explained by an increase in CD4+ and CD8+ CD44mid-hiCD62L- effector memory cells, with a similar trend in the central memory T-cell compartment. In contrast, CD30 did not impact the numbers of T cells in young mice. These data suggest a role for CD30 in the homeostatic regulation of T cells during aging, contributing to memory T-cell expansions, which may have relevance for CD30 expression in human T-cell lymphoproliferative diseases.