Discovery Immunology

BackgroundType 2 cytokines such as IL-13 and IL-5 are important drivers of pathophysiology and exacerbation in asthma. Defining how these type 2 cytokine responses are regulated is a research priority. Epithelial cells promote type 2 responses by releasing alarmins including IL-25, IL-33 and TSLP, but much less is known about inhibitory factors. MethodsIL-13 release was measured from peripheral blood mononuclear cells (PBMC) cultured with Interleukin (IL)-2 for five days. Epithelial cell lines or human bronchial epithelial cells (HBEC) isolated from healthy or asthma donors were added to these PBMC cultured with IL-2 and release of IL-13 or IL-5 measured. To characterise the mechanisms, we assessed the effect of mechanical disruption of epithelial cells, addition of the COX inhibitor indomethacin and the G-protein inhibitor pertussis toxin. ResultsPBMC cultured with IL-2 secreted type 2 cytokines in a cell number and time dependent manner. Epithelial cell lines inhibited IL-13 and IL-5 release after co-culture with PBMC in the presence of IL-2, directly, across a transwell and using epithelial cell supernatant. Cells or supernatant from HBEC from healthy or asthma donors also inhibited the cytokine release. Trypsin treatment of conditioned media indicated that inhibitory factor(s) are trypsin insensitive. Mechanical disruption of epithelial cells or indomethacin treatment had no effect, but pertussis toxin reduced epithelial cell inhibition of IL-2 driven type 2 cytokine release. ConclusionEpithelial cells regulate cytokine release by soluble factor(s) and this could be an important immunoregulatory function of the airway epithelium.

Leukocyte trafficking is a critical step in development of chronic intestinal diseases such as Crohns disease. While strategies that block gut-homing have yielded partial success, this disease remains uncurable leaving an unmet clinical need. This is the first paper to describe a role for cannabinoid receptor two (CB2R) signalling in promoting retinoic acid-mediated induction of the gut-homing associated integrin heterodimer 4{beta}7. Using in vitro and in vivo models, we characterised the effects of pharmacological CB2R agonists and inverse agonists on T cell homing receptor expression and transmigration across gut-associated endothelial barriers. This ERK-dependent process coincides with increased T cell adherence in response to CB2R agonism with JWH133. These effects were reversed with an inverse agonist GP-1a in a CB2R dependent manner. Selective deletion of CB2R using CRISPR in vitro or CD4Cre/+ floxed mice in vivo resulted in impaired endothelial cell adherence and decreased diapedesis into the ileal lamina propria. T cell-specific deletion of cnr2, the gene encoding CB2R, attenuated chronic murine ileitis characterised by decreased naive T cell infiltration and loss of tissue architecture in 20wk TNF{Delta}ARE/+mice. This study supports further therapeutic development of CB2R-blocking drugs for the treatment of inflammatory bowel disease.

Abstract / Short SummaryEpithelial cells have been shown previously to inhibit release of type 2 cytokines from T cells and this process could be important in a type 2-mediated diseases such as asthma. Factors secreted by epithelial cells could regulate inflammatory responses and release an all is well signal such that the immune system is controlled. Here we show that lung epithelial cells are able to inhibit IL-2 driven type 2 cytokine release by PBMC cells across a transwell indicating the involvement of soluble mediators. We then fractionate the mediators within the conditioned media using methanol chloroform separation and size fractionation. Inhibitory activity was demonstrated within the polar fraction of supernatant separated by methanol chloroform extraction. We then found that lactate, a component within the polar fraction was able to mediate inhibition of the type 2 cytokines. The inhibitory activity of lactate deserves further study and could play a role in the inhibition of T cell derived cytokines in vivo.

IntroductionT cell activation is central to the adaptive immune response. In vitro studies on T cell activation often utilize two distinct approaches: first, engaging T cell receptors (TCR) using plate-bound CD3 together with soluble CD28 (TCR-dependent). Second, triggering intracellular signalling cascades using phorbol 12-myristate 13-acetate (PMA) and Ionomycin or P+I (TCR-independent). Both methods are widely used; however, a systematic comparison of the activation methods across a range of stimulation strengths to evaluate their effects on T cell function and metabolism has not been investigated in great detail. In this study, we compared the consequences of engaging T cells using TCR-dependent and TCR-independent activation pathways across varying signal strengths. MethodsT cells from BALB/c mice were isolated and activated under four conditions: CD3, CD3+CD28, PMA with low Ionomycin (P+IL) and PMA with high Ionomycin (P+IH). We studied differences with respect to several parameters: morphology, flow analysis, metabolic activities, cytokines. The roles of Protein kinase C (PKC) and Ca{superscript 2} pathways were addressed by supplementing CD3+CD28 cultures with different doses of exogenous PMA or Ionomycin. ResultsP+I activation outperformed the CD3+CD28 activation system across most readouts by displaying enhanced blasts, higher cycling, greater glucose uptake, increased lactate and ROS production, together with higher upregulation of CD25 and CD44 activation markers. P+IH activation dampened several responses including CD69 expression. CD4 co-receptor was downregulated greatly with P+I activation but not CD3+CD28. Most cytokines followed signal strength comparably between both systems; however, differences were observed with others: P+I stimulation favoured IL-6 and IL-12 induction whereas CD3+CD28 activation preferentially induced CCL2 and IL-1{beta}. Importantly, PKC activity was substantially lower upon CD3+CD28 stimulation and the addition of PMA, but not Ionomycin, to CD3+CD28 cultures enhanced proliferation, metabolism and expression of activation markers. DiscussionTCR-dependent and TCR-independent T cell activation models have clear functional and metabolic differences. The observation that PKC signalling can boost T cell activation with CD3+CD28 is likely to be significant and may have translational implications such as CAR-T cell anti-tumor therapy where CD3+CD28 stimulation is widely used. The implications of our findings with regard to augmenting T cell mediated immunotherapies are discussed.

IL-23 is an IL-12 family cytokine that is important in promoting Th17 responses and has been strongly linked to autoimmunity and psoriasis. It is a heterodimeric cytokine made up of a p19 subunit unique to IL-23 and a p40 subunit that is shared with IL-12. We show here that in response to LPS, the induction of IL-23p19 mRNA is regulated by a MSK1/2 - CREB dependent pathway downstream of ERK1/2 and p38 MAPK. Knockout of MSK1/2 resulted in a decrease in both IL-23p19 mRNA transcription and IL-23 secretion in GM-CSF differentiated bone marrow cells. Similar effects were seen when the MSK1/2 phosphorylation site in CREB was mutated to alanine. Stimulation with PGE2 promotes the nuclear localisation of CRTC3, a co-activator for CREB. In combination with LPS, PGE2 promoted IL-23p19 mRNA transcription and this was blocked by knockdown of CRTC3. Imiquimod induced skin inflammation in mice has been used as a model for psoriasis and is dependent on IL-23. While MSK1/2 knockout reduced the induction of IL-23 in vivo following i.p. injection of LPS, the knockout mice were not protected from Imiquimod induced skin inflammation. MSK1/2 knockout did not reduce the induction of IL-17 producing {gamma}{delta}T cells following Imiquimod treatment, although MSK1/2 knockout did reduce the levels of these cells in mice receiving a control cream. The lack of protection in the Imiquimod model may be due to the known anti-inflammatory roles or MSKs, such as its contribution to the induction of IL-10.

The use of bacteria as an alternative cancer therapy has been re-investigated in recent years. A number of bacterial strains for this purpose have been generated, one of which is SL7207: an auxotrophic Salmonella enterica serovar Typhimurium aroA mutant with immune-stimulatory potential. Here we show that systemic administration of SL7207 induces melanoma tumour growth arrest in vivo, with greater survival of the SL7207-treated group compared to control PBS-treated mice. Administration of SL7207 is accompanied by a change in the immune phenotype of the tumour-infiltrating cells towards pro-inflammatory, with expression of the TH1 cytokines IFN-{gamma}, TNF-, and IL-12 significantly increased. Interestingly, Ly6C+MHCII+ monocytes were recruited to the tumours following SL7207 treatment and were pro-inflammatory. Accordingly, the abrogation of these infiltrating monocytes using clodronate liposomes prevented SL7207-induced tumour growth inhibition. These data demonstrate a previously unappreciated role for infiltrating inflammatory monocytes underlying bacterial-mediated tumour growth inhibition. This information highlights a novel role for monocytes in controlling tumour growth, contributing to our understanding of the immune responses required for successful immunotherapy of cancer.

Long-lived plasma cells (LLPC) sustain humoral immunity but also contribute to the persistence of pathogenic autoantibodies in autoimmune diseases. New therapies targeting LLPC are therefore desirable. Recent studies have shown increased expression of Slc12a2, encoding the Na+ -K+ -Cl- cotransporter (NKCC1), in LLPC. This study investigated whether NKCC1 activity was required for plasma cell survival, persistence or secretion of antibodies. Across in vitro and in vivo settings, mouse plasma cell survival was undiminished by treatment with the NKCC1 inhibitor bumetanide. Acute in vivo bumetanide treatment did not diminish plasma cell numbers, nor show any demonstrable impact on the survival of phenotypically mature I-A/I-EloSLAMF6lo plasma cells. With genetic plasma cell timestamping, even the survival of persistent LLPC was unaffected by bumetanide. Plasma cell secretory capacity, assessed by measuring IgM and IgG2b secretion in culture over three days, was also unaltered by bumetanide. Overall, these results show that pharmacological inhibition of NKCC1 is not sufficient to impair plasma cell survival, persistence or antibody secretion. Despite elevated Slc12a2 mRNA expression in LLPC, NKCC1 alone does not represent a critical plasma cell survival pathway, highlighting the resilience of plasma cells and the challenges associated with therapeutically targeting LLPC.

Human {beta}-defensin 3 (HBD3), is an anti-microbial host-defence peptide, that can rapidly enter macrophages to modulate TLR4 responses to lipopolysaccharide. However, the molecular mechanisms by which HBD3 exerts this anti-inflammatory influence remain unclear. Here, we show mice deleted for the orthologue of HBD3 have an increased acute lipopolysaccharide response in vivo. Furthermore, we found that HBD3 limited the response of macrophages to classical activation, and contemporaneously drove expression of IL-4. An increase in markers of alternative activation, and a change in metabolic flux was also observed. Consistent with these results, HBD3 enhanced the IL-4 mediated polarisation of naive macrophages. Finally, we demonstrate that the ability of HBD3 to limit macrophage classical activation requires IL-4R. These data reveal a previously unrecognised role for HBD3 in influencing the polarisation state of macrophages to enable a state conducive for repair and resolution. SYNOPSIS O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=131 SRC="FIGDIR/small/442606v2_ufig1.gif" ALT="Figure 1"> View larger version (25K): org.highwire.dtl.DTLVardef@f81c9aorg.highwire.dtl.DTLVardef@11dca23org.highwire.dtl.DTLVardef@c1e241org.highwire.dtl.DTLVardef@ed46ed_HPS_FORMAT_FIGEXP M_FIG C_FIG The anti-microbial host-defence peptide, Human {beta}-defensin 3 (HBD3), is shown here to modulate the inflammatory response to classical activation by promoting alternative activation through IL-4R, to enable a state conducive for repair and resolution. O_LIKnockout mice for the orthologous gene for HBD3, demonstrate increased acute lipopolysaccharide inflammatory response. C_LIO_LIHBD3 limited the classical activation of macrophages polarised with LPS/IFN{gamma} and drove expression of IL-4. Cells also displayed increase in alternative activation markers and promotion of oxidative phosphorylation. C_LIO_LIHBD3 enhanced the IL-4-mediated activation of naive macrophages. C_LIO_LIThe ability of HBD3 to limit macrophage classical activation and contemporaneously promote alternative activation required IL-4R. C_LI

Following antigenic stimulation, T cells switch from a catabolic metabolic state maintained by low levels of nutrient uptake to an anabolic metabolism that sustains the biosynthetic and energetic demands of clonal expansion, differentiation and effector function. Much progress has been made in understanding the transcriptional and enzymatic regulation of activated T cell metabolism. However less is understood of the role for regulators of anaplerosis and cataplerosis such as phospho-enol pyruvate carboxykinases (PEPCK) in T cells. In the current work, we show that mitochondrial isoform PEPCK-M is upregulated following T cell activation whilst cytosolic PEPCK-C is not expressed. PEPCK inhibitors limited CD8+ T cell cytotoxic capacity and both CD4+ and CD8+ T cell inflammatory cytokine production. Suppression of T cell effector functions by PEPCK inhibitors was associated with decreased maximal mitochondrial respiration. These data suggest that PEPCK-M acts as a metabolic rheostat to enable optimal T cell activation.

Cannabinoid receptor 2 (CB2R) is highly expressed on immune cells, but its role in T cell metabolism remains unclear. Here, we show that CB2R activation rapidly increases glucose uptake in human Jurkat T cells and drives a broader metabolic reprogramming away from glycolysis toward oxidative metabolism and the pentose phosphate pathway. Pharmacological CB2R activation increased mitochondrial mass, spare respiratory capacity, proton leak, and NADPH production, while CB2R inverse agonism produced the opposite effects. These metabolic changes were accompanied by upregulation of key pentose phosphate pathway enzymes, including GALT and TALDO1, and were abolished in CNR2-deficient cells, confirming receptor dependence. In primary human lamina propria mononuclear cells, CB2R signalling also influenced memory and gut-homing-associated T cell phenotypes, including integrin 4{beta}7 expression. Together, these findings identify CB2R as a regulator of T cell bioenergetics and suggest that cannabinoid signalling may promote metabolic states linked to memory and tissue-homing functions in chronic intestinal inflammation.

Fibroblasts are traditionally considered structural cells that maintain tissue homeostasis and facilitate repair. However, accumulating evidence suggests they also participate in innate immunity, although their pattern recognition capabilities remain incompletely characterized. Here, we systematically assessed the innate immune responses of commercially available primary human dermal fibroblasts from a male and a female donor. Fibroblasts were stimulated with a panel of microbe-associated molecular patterns (MAMPs) targeting various pattern recognition receptors (PRRs), including Toll-like receptors (TLRs), NOD-like receptors (NODs), Alpha kinase 1 (ALPK1) and STING. Innate immune activation was quantified by measuring the nuclear translocation of NF-{kappa}B via high content microscopy and cytokines and chemokines secretion by ELISA; baseline PRRs expression was determined by quantitative PCR. Only a restricted subset of agonists, specifically E. coli LPS (TLR4), Poly I:C (TLR3 / RIG-I) and unexpectedly ADP heptose (ALPK1) induced robust NF-{kappa}B activation and secretion of the chemokines IL-8 and MCP-1. Apart from IL-6 and RANTES, which were produced exclusively following Poly I:C stimulation, pro-inflammatory cytokines (IL-1{beta}, TNF, IFN-{beta}) and the anti-inflammatory cytokine IL-10 remained undetectable. Consistent with this limited reactivity, qPCR of PRRs revealed basal expression of TLR4 and ALPK1, whereas most other receptors were expressed at very low or undetectable levels. Notably, NOD1 was highly expressed although no cell activation was observed with several NOD1 agonists. Dose-response analysis revealed surprisingly high sensitivity to LPS. In conclusion, primary human dermal fibroblasts exhibit a highly selective but sensitive innate immune response, largely restricted to chemokine production upon PRR activation. This unexpected dissociation between chemokine and cytokine responses suggests that fibroblasts function as sentinel cells in early skin defense, capable of detecting key microbial patterns at low concentrations, to orchestrate local immune surveillance. Further investigation into interindividual variability and context-dependent activation is needed.

Signaling by formyl peptide receptor 1 (FPR1), the prototype G protein-coupled receptor (GPCR) expressed in neutrophil leukocytes, is initiated by an activation of a G protein containing a Gi subunit. FPR1 activation results in an increase in the cytosolic concentration of free calcium ions ([Ca2+]i), and an activation of the superoxide anion producing NADPH oxidase. Receptor downstream signals generated by the danger molecule ATP recognized by the purinergic receptor P2Y2 are transduced by a G protein containing a Gq subunit. The neutrophil response induced by ATP also includes a transient rise in [Ca2+]i, but the downstream signals do not activate the NADPH oxidase. ATP can, however, activate this enzyme system through a receptor transactivation mechanism dependent not only on the ATP receptor but also on the free fatty acid receptor FFA2R, provided that this receptor is allosterically modulated. This occurs through a novel mechanism whereby FFA2R is activated from the cytosolic side of the plasma membrane by Gq transduced signals generated by the ATP receptor. Furthermore, in neutrophils with a disrupted actin cytoskeleton, ATP (as well as platelet activating factor; recognized by the Gq-coupled PAFR) becomes a potent NADPH oxidase activating agonist. At high concentrations of the actin cytoskeleton disrupting drug latrunculin A the activation was only partly reduced by Gq inhibition. More importantly, this response was also partly inhibited by pertussis toxin. The effects on the ATP-induced NADPH oxidase activity, of the Gq inhibitor and pertussis toxin were more and less pronounced, respectively, when the concentration of latrunculin A was reduced. Taken together, we show that in primary human neutrophils the actin cytoskeleton is part of the regulatory machinery that determines the activation of NADPH oxidase activation and the G protein recruitment profile downstream of activated of Gq-coupled GPCRs. HighlightsO_LIATP is a biased signaling agonist unable to activate the neutrophil NADPH oxidase C_LIO_LIATP activates the NADPH oxidase through P2Y2R mediated transactivation of FFA2R C_LIO_LIActin cytoskeleton disruption enables ATP to activate the NADPH oxidase C_LIO_LICytoskeleton regulated NADPH oxidase activation depends on Gi and Gq signaling C_LIO_LIThe actin cytoskeleton regulates the G protein recruitment profile of P2Y2R C_LI

Human eosinophils activated in suspension with IL5 or IL33 undergo morphological change prior to adhesion. Refractive granules, which contain major basic protein-1 and other toxic proteins, move to one side of the cell, the granulomere, and the two nuclear lobes move to the other. How these features persist when eosinophils become adherent and migrate is not known. We now compare behavior of activated eosinophils on surfaces coated with ITGAM/ITGB2-integrin ligands fibrinogen or periostin using live cell imaging of reporters of tubulin/actin organization and cell viability. We find that unlike eosinophils activated with IL5, IL33-activated eosinophils undergo two stages of activation; a preliminary pear-like activation in which the cell develops polarity, followed by a flattening of the eosinophil into a thin pancake-like morphology with less discrete polarization. IL5-treated eosinophils migrated persistently for more than an hour with nucleopod in the back. In contrast, IL33-treated eosinophils moved more slowly and within 30 min transitioned to a flattened morphology with nuclear lobes in the center and dispersed motile granules. Loss of cell viability after an hour, although variable, in all comparisons was greater among IL33-treated eosinophils on periostin. We sought to understand how cytoskeletal elements may drive these differences in morphology. Cytoskeletal elements had similar responses when activated with IL5/IL33; vimentin collapsed from a web-like network at the periphery of the cell and condensed adjacent to the nucleopod/nuclear interface, f-actin was found in the granulomere as well as the tip of the nucleopod and forward periphery, and microtubules radiated from the microtubule organizing center (MTOC) spanning both the nucleopod and the granulomere. The dynamic formation of microtubules correlated with cellular locomotion, suggesting mesenchymal migration within these cells. These in vitro findings suggest that adhesion plays an important role in determining functional morphology and demonstrates new insights into IL33-activated eosinophils. This work suggests roles for activators and adhesive substrates in regulating the behavior of activated eosinophils in tissues.

Mast cells are emerging players in malignant conditions, but the underlying molecular mechanisms remain poorly defined. Based on previous studies showing that steroids can impact on tumour progression in various settings, we here investigated whether mast cell-derived steroid synthesis can have an impact on tumour metastasis in a melanoma model. To this end, we used mice with mast cell-specific ablation of Cyp11a1, a key enzyme in steroid synthesis. We show that lung colonization of melanoma nodules was markedly diminished in mice with mast cell-specific ablation of Cyp11a1, accompanied by reduced infiltration of mast cells into the lungs. Cyp11a1 gene expression was significantly decreased in lungs of mice with mast cell-specific ablation of Cyp11a1, indicating that mast cells account for a substantial fraction of the total Cyp11a1 expression. Our results also revealed that the mast cell-specific deletion of Cyp11a1 led to an overall increase in CD107a/LAMP1 staining intensity of the lung tissue, suggesting that mast cell-derived steroids can suppress immune cell activation/degranulation. A further dissection of this finding by flow cytometry analysis of individual immune cell populations revealed that CD8+ T cells, NK cells and basophils were activated to a higher extent in lungs from mice with mast cell-specific Cyp11a1 ablation. We also demonstrate that both CD8+ and CD4+ T cells in lungs of mice with mast cell-specific deletion of Cyp11a1 expressed elevated levels of IFN-{gamma} in comparison with controls. Altogether, these findings introduce a hitherto unrecognized role of a mast cell-derived steroid axis in regulating tumour metastasis.

T cell activation is associated with, and dependent upon, the upregulation of amino acid uptake from the extracellular environment. Uptake of the non-essential amino acid asparagine (Asn) is mediated via amino transporters such as Slc1a5 whilst Asn can be synthesized within cells that express asparagine synthetase (ASNS). Previous work demonstrated that initial activation of CD8+ T cells is perturbed in the absence of Asn, whereas effector cytotoxic T cells cells upregulate ASNS and lose their dependence on Asn uptake. By contrast, less is known of the role of Asn uptake and ASNS in CD4+ T cell responses. Here we demonstrate that CD4+ T cells are more reliant than CD8+ T cells on Asn uptake for initial activation, differentiation, metabolic reprogramming and regulation of autophagy. These phenotypes are associated with enhanced expression of ASNS in CD8+ as compared to CD4+ effector T cells.

Mucosal-associated invariant T (MAIT) cells are innate-like T lymphocytes that are abundant in mucosal tissues and the liver where they can respond rapidly to a broad range of riboflavin producing bacterial and fungal pathogens. Neutrophils, which are recruited early to sites of infection, play a non-redundant role in pathogen clearance and are crucial for controlling infection. The interaction of these two cell types is poorly studied. Here, we investigated both the effect of neutrophils on MAIT cell activation and the effect of activated MAIT cells on neutrophils. We show that neutrophils suppress the activation of MAIT cells by a cell-contact and H2O2 dependent mechanism. Moreover, highly activated MAIT cells were able to produce high levels of TNF that induced neutrophil death. We therefore provide evidence for a negative regulatory feedback mechanism in which neutrophils prevent over-activation of MAIT cells and, in turn, MAIT cells limit neutrophil survival.

Macrophage recruitment during sterile inflammation and infection is essential to clear pathogens, apoptotic cells and debris. However, persistent macrophage accumulation leads to chronic inflammation. Platelets are emerging as key modulators of the inflammatory response. Here, we identify that platelet C-type-lectin-like receptor-2 (CLEC-2) is a crucial immunomodulatory receptor through the interaction with podoplanin, upregulated on inflammatory macrophages. Mechanistically, platelet CLEC-2 upregulates the expression of podoplanin and its co-ligands CD44 and ERM proteins, leading to actin rearrangement and promotion of cell migration; this is mimicked by recombinant CLEC-2-Fc (rCLEC-2-Fc). Treatment of LPS-challenged mice with rCLEC-2-Fc induces a rapid emigration of peritoneal macrophages to mesenteric lymph nodes, through a gradient generated by the podoplanin ligand, CCL21, to prime T cells. We propose that crosslinking podoplanin using rCLEC-2-Fc is a novel, cell-specific strategy to accelerate macrophage removal from the site of inflammation, and hence promote the resolution of the inflammatory response. Visual Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=188 SRC="FIGDIR/small/423770v1_ufig1.gif" ALT="Figure 1"> View larger version (54K): org.highwire.dtl.DTLVardef@e37acborg.highwire.dtl.DTLVardef@92a5e1org.highwire.dtl.DTLVardef@1c8832eorg.highwire.dtl.DTLVardef@11c5c97_HPS_FORMAT_FIGEXP M_FIG C_FIG SummaryPersistent macrophage accumulation in inflamed tissue leads to chronic inflammation and organ damage. Bourne et al. identify recombinant CLEC-2-Fc crosslinking podoplanin on inflammatory macrophages, as a cell-specific strategy to accelerate their emigration to draining lymph nodes, and reduce local inflammation.

Modulation of T cell activity is an effective strategy for the treatment of autoimmune diseases, immune-related disorders and cancer. This highlights a critical need for continued investigation of proteins that regulate T cell function. The kinase DNA-dependent protein kinase catalytic subunit (DNA-PKcs) is emerging as a potent regulator of the immune system spurring interest in its use as a therapeutic target for immune-related diseases. In murine models of autoimmune disease including asthma and rheumatoid arthritis, treatment with small molecule DNA-PKcs inhibitors, which are in clinical trials for cancer therapy, decreased disease severity. Additionally, DNA-PKcs inhibitors reduced T cell-mediated graft rejection and extended graft survival in a murine allogenic skin graft rejection model. These in vivo studies suggest the therapeutic use of DNA-PKcs inhibitors for autoimmune and T cell-mediated disorders. In this study, we sought to further characterize the effects of DNA-PKcs inhibitors on T cells to better understand their clinical potential. We determined that pharmacological inhibition of DNA-PKcs abrogated activation of murine and human CD4+ and CD8+ T cells as evident by reduced expression of the activation markers CD69 and CD25. Furthermore, inhibition of DNA-PKcs impeded metabolic pathways and proliferation of anti-CD3/CD28 activated CD4+ and CD8+ T cells as well as peptide-stimulated OTI-CD8+ T cells. This reduced the ability of OTI-CD8+ T cells to kill cancer cells and the expression of IFN{gamma} and the cytotoxic genes eomes, perforin and granzyme B. These results suggest a novel role for DNA-PKcs in early T cell activation. Furthermore, our data support the therapeutic potential of DNA-PKcs inhibitors on diseases of immune dysregulation.

Toll-like receptors (TLRs) play a central role in innate immune responses through recognition of pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs). Activation of TLRs leads to the induction of inflammatory cytokines via MyD88- and TRIF-dependent signaling pathways. Aquamin, a multi-mineral supplement derived from the marine red algae Lithothamnion, is known for its anti-inflammatory properties. In this study, we demonstrate that Aquamin dose-dependently inhibits lipopolysaccharide (LPS)-induced production of pro-inflammatory cytokines (TNF-, IL-6, IL-1{beta}) and chemokines in both human peripheral blood mononuclear cells (hPBMCs) and murine bone marrow-derived macrophages (mBMDMs), without causing cytotoxicity. Mechanistic studies demonstrate that Aquamin specifically suppressed TRIF dependent IRF3 activation downstream of TLR3 and TLR4. These findings support Aquamin as a promising agent for use as an intervention in counteracting inflammatory diseases states where TLR signalling is implicated.

The interferon-induced transmembrane protein, IFITM3, has been shown to restrict influenza virus infection in murine and in vitro settings for ten years, but no explanation has been found to explain why this virus infection is so highly contagious and infects most individuals it comes in contact with. We confirm that the expression level of IFITM3 plays a role in determining the level of viral infection through manipulation of IFITM3 levels with interferon (IFN) stimulation and overexpression systems. Low basal expression may put some immune cells, including lymphocytes and lung-resident macrophages, at risk of influenza virus infection. Investigating the induction of IFITM3 by IFN, we find a strong preference for Type I IFN in IFITM3 induction in both cell lines and primary human cells. While myeloid cells can increase expression following stimulation by Type I IFN, lymphocytes show minimal induction of IFITM3 following IFN stimulation, suggesting that they are always at risk of viral infection. Surprisingly, we found that the time it takes for maximal induction of IFITM3 is relatively slow for an interferon-stimulated gene at around 36 hours. Low basal expression and slow induction of IFITM3 could increase the risk of influenza virus infection in selected immune cells. ImportanceInfluenza virus infection remains one of the top ten threats to global health, causing significant deaths and hospitalisations across the world each year. Understanding mechanisms for controlling influenza virus infection remain a priority. The interferon-induced transmembrane protein IFITM3 can restrict influenza infection by limiting replication of the virus. The precise mechanisms of how IFITM3 reduced replication of influenza are unknown, although it is predicted to prevent release of viral contents into the cytosol by preventing pore formation on the endosomal compartments where it is suggested to reside. Here we have shown that the expression level of IFITM3 is important in determining the control of influenza virus infection. We find an expression pattern for IFITM3 that varies based on cell type, tissue locality, differentiation state and cell naivety, all of which highlights cells that may be at the highest risk of influenza infection.