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Esophagogastric adenocarcinomas (EAC) are obesity-associated malignancies underpinned by severe immune dysregulation and inflammation. Our previous work indicates that NK cells migrate to EAC omentum, where they undergo phenotypic and functional alterations and apoptosis. In this study, we investigate whether such erroneous chemotaxis to omentum is paralleled by compromised NK cell infiltration of EAC patient tumor and examine the role of the inflammatory chemokine fractalkine in shaping the NK cell–mediated response. Our data show diminished NK cell frequencies in EAC tumor compared with those in the circulation and reveal that intratumoral NK cell frequencies decline as visceral obesity increases in EAC patients. Our in vitro findings demonstrate that antagonism of fractalkine receptor CX3CR1 significantly reduces NK cell migration to EAC patient–derived, omental adipose tissue–conditioned media, but not toward tumor-conditioned media. These data suggest fractalkine is a key driver of NK cell chemotaxis to omentum but has a lesser role in NK cell homing to tumor in EAC. We propose that this may offer a novel therapeutic strategy to limit NK cell depletion in the omentum of obese EAC patients, and our data suggest the optimal timing for CX3CR1 antagonism is after neoadjuvant chemoradiotherapy. Our functional studies demonstrate that fractalkine induces the conversion from CX3CR1+CD27− to CX3CR1−CD27+ NK cells and increases their IFN-γ and TNF-α production, indicative of its role in shaping the dominant NK cell phenotype in EAC omentum. This study uncovers crucial and potentially druggable pathways underpinning NK cell dysfunction in obesity-associated cancer and provides compelling insights into fractalkine’s diverse biological functions.

Summary Neonatal encephalopathy (NE) is characterized by altered neurological function in term infants and inflammation plays an important pathophysiological role. Inflammatory cytokines interleukin (IL)‐1β, IL‐1ra and IL‐18 are activated by the nucleotide‐binding and oligomerization domain (NOD)‐, leucine‐rich repeat domain (LRR)‐ and NOD‐like receptor protein 3 (NLRP3) inflammasome; furthermore, we aimed to examine the role of the inflammasome multiprotein complex involved in proinflammatory responses from the newborn period to childhood in NE. Cytokine concentrations were measured by multiplex enzyme‐linked immunosorbent assay (ELISA) in neonates and children with NE in the absence or presence of lipopolysaccharide (LPS) endotoxin. We then investigated expression of the NLRP3 inflammasome genes, NLRP3, IL‐1β and ASC by polymerase chain reaction (PCR). Serum samples from 40 NE patients at days 1 and 3 of the first week of life and in 37 patients at age 4–7 years were analysed. An increase in serum IL‐1ra and IL‐18 in neonates with NE on days 1 and 3 was observed compared to neonatal controls. IL‐1ra in NE was decreased to normal levels at school age, whereas serum IL‐18 in NE was even higher at school age compared to school age controls and NE in the first week of life. Percentage of LPS response was higher in newborns compared to school‐age NE. NLRP3 and IL‐1β gene expression were up‐regulated in the presence of LPS in NE neonates and NLRP3 gene expression remained up‐regulated at school age in NE patients compared to controls. Increased inflammasome activation in the first day of life in NE persists in childhood, and may increase the window for therapeutic intervention., Neonatal encephalopathy (NE) is characterized by altered neurological function in term infants and inflammation plays an important pathophysiological role. Cytokine concentrations were measured by multiplex ELISA in neonates and children with NE in the absence or presence of LPS endotoxin. Increased inflammasome activation in the first day of life in NE persists in childhood

Chemotherapy upregulates immune checkpoint (IC) expression on the surface of tumour cells and IC-intrinsic signalling confers a survival advantage against chemotherapy in several cancer-types including oesophageal adenocarcinoma (OAC). However, the signalling pathways mediating chemotherapy-induced IC upregulation and the mechanisms employed by ICs to protect OAC cells against chemotherapy remain unknown. Longitudinal profiling revealed that FLOT-induced IC upregulation on OE33 OAC cells was sustained for up to 3 weeks post-treatment, returning to baseline upon complete tumour cell recovery. Pro-survival MEK signalling mediated FLOT-induced upregulation of PD-L1, TIM-3, LAG-3 and A2aR on OAC cells promoting a more immune-resistant phenotype. Single agent PD-1, PD-L1 and A2aR blockade decreased OAC cell viability, proliferation and mediated apoptosis. Mechanistic insights demonstrated that blockade of the PD-1 axis decreased stem-like marker ALDH and expression of DNA repair genes. Importantly, combining single agent PD-1, PD-L1 and A2aR blockade with FLOT enhanced cytotoxicity in OAC cells. These findings reveal novel mechanistic insights into the immune-independent functions of IC-intrinsic signalling in OAC cells with important clinical implications for boosting the efficacy of the first-line FLOT chemotherapy regimen in OAC in combination with ICB, to not only boost anti-tumour immunity but also to suppress IC-mediated promotion of key hallmarks of cancer that drive tumour progression.

Cognitive impairment occurs in 30%-50% of patients with non-cirrhotic chronic hepatitis C virus (HCV) infection. Exercise is beneficial in preventing and treating cognitive impairment and cardiometabolic abnormalities in many chronic inflammatory diseases, but there are few studies investigating the impact of exercise in HCV infection. The study aimed to assess the effect of a 12-week aerobic exercise intervention on cognition and extrahepatic manifestations in individuals with HCV. In this nonrandomized controlled pilot study, individuals with HCV participated in a 12-week aerobic exercise intervention. Outcome measures included cognition (Montreal Cognitive Assessment [MOCA], Trail Making Test A & B [TMT-A; TMT-B], Digit Symbol Test [DST]), cardiorespiratory fitness (estimated V ˙ O 2 max ), physical activity (accelerometry), anthropometry, quality of life (depression; fatigue; sleep quality) and biochemical markers. Outcomes were assessed at baseline (T0), intervention completion (T1) and 12 weeks after intervention completion (T2). Thirty-one patients completed the study (exercise group n = 13, control group n = 18). In the exercise group, cognition improved at T1 in the TMT-A (31% mean improvement, p = 0.019), TMT-B (15% mean improvement, p = 0.012) time and MOCA (14% mean improvement, p ≤ 0.001). These improvements were not maintained at T2. Depression (p = 0.038), sleep quality (p = 0.002), fatigue (p = 0.037) and estimated V ˙ O 2 max (7.8 mL kg-1 min-1 [22%] mean increase, p = 0.004) also improved at T1. In conclusion, this study demonstrates the benefits of a 12-week aerobic exercise intervention in improving cognition, quality of life and cardiorespiratory fitness in individuals with HCV. Larger studies are needed to confirm these findings and strategies for continued exercise engagement in individuals with HCV are warranted for sustained benefits.

Esophagogastric adenocarcinomas (EAC) are obesity-associated malignancies underpinned by severe immune dysregulation and inflammation. Our previous work indicates that NK cells migrate to EAC omentum, where they undergo phenotypic and functional alterations and apoptosis. In this study, we investigate whether such erroneous chemotaxis to omentum is paralleled by compromised NK cell infiltration of EAC patient tumor and examine the role of the inflammatory chemokine fractalkine in shaping the NK cell-mediated response. Our data show diminished NK cell frequencies in EAC tumor compared with those in the circulation and reveal that intratumoral NK cell frequencies decline as visceral obesity increases in EAC patients. Our in vitro findings demonstrate that antagonism of fractalkine receptor CX3CR1 significantly reduces NK cell migration to EAC patient-derived, omental adipose tissue-conditioned media, but not toward tumor-conditioned media. These data suggest fractalkine is a key driver of NK cell chemotaxis to omentum but has a lesser role in NK cell homing to tumor in EAC. We propose that this may offer a novel therapeutic strategy to limit NK cell depletion in the omentum of obese EAC patients, and our data suggest the optimal timing for CX3CR1 antagonism is after neoadjuvant chemoradiotherapy. Our functional studies demonstrate that fractalkine induces the conversion from CX3CR1

Background Lifestyle interventions are the primary treatment for metabolic (dysfunction) associated fatty liver disease (MAFLD). However, the histological and cardiometabolic effects of aerobic exercise in MAFLD remain unclear. Aims To assess the effects of a 12‐week aerobic exercise intervention on histological and cardiometabolic endpoints in MAFLD. Methods Patients with biopsy‐confirmed MAFLD participated in a 12‐week aerobic exercise intervention. Liver histology, cardiorespiratory fitness (estimated VO2max), physical activity, anthropometry and biochemical markers were assessed at baseline, intervention completion, and 12 and 52 weeks after intervention completion. Results Twenty‐four patients completed the exercise intervention (exercise group n = 16, control group n = 8). In the exercise group, 12 weeks of aerobic exercise reduced fibrosis and hepatocyte ballooning by one stage in 58% (P = 0.034) and 67% (P = 0.020) of patients, with no changes in steatosis (P = 1.000), lobular inflammation (P = 0.739) or NAFLD activity score (P = 0.172). Estimated VO2max increased by 17% compared to the control group (P = 0.027) but this level of improvement was not maintained at 12 or 52 weeks after the intervention. Patients with fibrosis and ballooning improvement increased estimated VO2max by 25% (P = 0.020) and 26% (P = 0.010), respectively. Anthropometric reductions including body mass (P = 0.038), waist circumference (P = 0.015) and fat mass (P = 0.007) were also observed, but no patient achieved 7%‐10% weight loss. Conclusion This study highlights the potential benefits of a 12‐week aerobic exercise intervention in improving histological endpoints of MAFLD. The development of strategies to ensure continued engagement in aerobic exercise in MAFLD are needed.

Infection and persistent inflammation have a prominent role in the pathogenesis of brain injury and cerebral palsy, as well as other conditions associated with prematurity such as bronchopulmonary dysplasia. The NLRP3 inflammasome-interleukin (IL)-1β pathway has been extensively studied in adults and pre-clinical models, improving our understanding of innate immunity and offering an attractive therapeutic target that is already contributing to clinical management in many auto-inflammatory disorders. IL-1 blockade has transformed the course and outcome of conditions such as chronic infantile neurological, cutaneous, articular (CINCA/NOMID) syndrome. Inflammasome activation and upregulation has recently been implicated in neonatal brain and lung inflammatory disease and may be a novel therapeutic target.

This study tested the hypothesis that the Vδ3 subset of human γδ T cells, like their Vδ2 counterparts, can influence differentiation, antibody secretion and cytokine production by B cells. Vδ3 T cells constitute a minor subset of peripheral blood lymphocytes but are enriched in the liver and gut and are expanded in patients with cytomegalovirus activation and B cell chronic lymphocytic leukemia. They have been reported to include MHC class I and CD1d restricted cells. Like Vδ2 T cells, they are capable of maturing dendritic cells into cytokine-producing antigen presenting cells, making them potential targets for dendritic cell-based immunotherapies. Since it is unknown if Vδ3 T cells can also provide B cell help, we investigated if Vδ3 T cells can promote B cell differentiation, antibody secretion and cytokine production in vitro. Vδ3 T cells were sorted from healthy human blood and expanded using phytohemagglutinin and cultured with freshly isolated human B cells. We found that Vδ3 T cells and B cells reciprocally induced expression of maturation markers CD40, CD86 and HLA-DR but not TH1, TH2 or TH17 cytokines. Furthermore, Vδ3 T cells promoted the release of IgM, but not IgG, IgA or IgE by B cells. These data demonstrate, for the first time, a reciprocal activating relationship between Vδ3 T cells and B cells, which could prove a useful target for cellular immunotherapy.

Neonatal encephalopathy is a syndrome characterised by abnormal neurological function often caused by a hypoxic insult during childbirth. Triggers such as hypoxia-ischaemia result in the release of cytokines and chemokines inducing the infiltration of neutrophils, natural killer cells, B cells, T cells and innate T cells into the brain. However, the role of these cells in the development of the brain injury is poorly understood. We review the mechanisms by which lymphocytes contribute to brain damage in NE. NK, T and innate T cells release proinflammatory cytokines contributing to the neurodegeneration in the secondary and tertiary phase of injury, whereas B cells and regulatory T cells produce IL-10 protecting the brain in NE. Targeting lymphocytes may have therapeutic potential in the treatment of NE in terms of management of inflammation and brain damage, particularly in the tertiary or persistent phases.

The global obesity epidemic is contributing to increased prevalence of diseases fuelled by chronic inflammation, including cancer. Oesophageal adenocarcinoma (OAC) is an obesity-associated malignancy with increasing prevalence, dismal prognosis, and severely dysregulated immune processes. We previously reported that αβ T cells migrate to omentum and liver in OAC and contribute to inflammation in these tissues. Here, we assessed the tissue distribution and phenotype of gamma/delta (γδ) T cells in the blood, omentum, liver and tumour of OAC patients. Our data show that the Vδ1 and Vδ3 subsets of γδ T cells are most prevalent in omentum and liver of OAC patients. Furthermore, γδ T cells are predominantly pro-inflammatory in these tissues, and co-express IFN-γ and IL-17. Moreover, γδ T cells exhibit cytotoxic capabilities in OAC omentum and liver. This study provides the first indication that γδ T cells contribute to obesity-associated inflammation in OAC and might be exploited therapeutically.

Summary Innate lymphocyte populations, such as innate lymphoid cells (ILCs), γδ T cells, invariant natural killer T (iNK T) cells and mucosal-associated invariant T (MAIT) cells are emerging as important effectors of innate immunity and are involved in various inflammatory and autoimmune diseases. The aim of this study was to assess the frequencies and absolute numbers of innate lymphocytes as well as conventional lymphocytes and monocytes in peripheral blood from a cohort of anti-neutrophil cytoplasm autoantibody (ANCA)-associated vasculitis (AAV) patients. Thirty-eight AAV patients and 24 healthy and disease controls were included in the study. Patients with AAV were sampled both with and without immunosuppressive treatment, and in the setting of both active disease and remission. The frequencies of MAIT and ILC2 cells were significantly lower in patients with AAV and in the disease control group compared to healthy controls. These reductions in the AAV patients remained during remission. B cell count and frequencies were significantly lower in AAV in remission compared to patients with active disease and disease controls. Despite the strong T helper type 2 (Th) preponderance of eosinophilic granulomatosis with polyangiitis, we did not observe increased ILC2 frequency in this cohort of patients. The frequencies of other cell types were similar in all groups studied. Reductions in circulating ILC2 and MAIT cells reported previously in patients with AAV are not specific for AAV, but are more likely to be due to non-specific manifestations of renal impairment and chronic illness. Reduction in B cell numbers in AAV patients experiencing remission is probably therapy-related.

Aims To evaluate systemic inflammation in TBI by exploration of the inflammasome pathway, a component of the innate immune system that regulates and induces inflammation. We examine the pathway at baseline in TBI compared with healthy control children, and in vitro in response to both LPS stimulation and melatonin therapy. Melatonin has protective effects against NLRP3 inflammasome activation and has therapeutic implications. Methods Whole blood was sampled from children with TBI (n=10) within 24 hours of injury and compared to healthy age-matched controls (n=8) at baseline, following stimulation with bacterial endotoxin (LPS) (10ng/ml) and melatonin treatment (10–3M). Granulocytes were delineated as CD66b+ and FSC, SSC-A. Measurements of mean channel fluorescence (MCF) of CD11b and TLR4 expression on FACS Canto II were recorded and analysed with FloJo software v10. Gene Expression of NLRP3 via rtPCR was recorded in 10 patients and 10 controls at baseline and following LPS and melatonin treatment. Results Granulocyte CD11b expression was lower in children with TBI compared to controls (p=0.04) Both upregulated CD11b with LPS stimulation. Melatonin significantly decreased this LPS upregulation. There was no significant difference in baseline TLR4 expression between TBI and controls, but LPS upregulation of TLR4 was decreased by melatonin in the TBI cohort. Inflammasome was upregulated via NLRP3 expression in children with TBI compared to controls (p= 0.02). Melatonin significantly decreased LPS-induced upregulation of NLRP3 only in controls. Conclusion Inflammation is altered in TBI compared to controls with altered responsiveness to melatonin treatment following LPS stimulation. The inflammasome is downregulated in children immediately following TBI. Selective inhibition of systemic inflammation targeting the inflammasome may have a future immunomodulatory role as a target in treating TBI.

Esophageal and gastric cancers collectively cause over 1.1 million deaths annually and only 20-30% of patients respond favorably to current therapies. Cellular therapies using invariant natural killer T (iNKT) cells are showing promise for patients with other cancers; therefore, we investigated if these cells are altered in esophageal and gastric cancer patients. Flow cytometric analysis of peripheral blood from 139 patients revealed that iNKT cells are depleted from patients with esophageal and gastric adenocarcinoma and esophageal squamous cell carcinoma, both before and after treatment. Interrogation of the KMPlot database of transcriptomic data from 876 gastric cancer patients revealed that low CD1d expression is associated with poor prognosis. These observations suggest that therapies that boost CD1d expression and iNKT cell responses may benefit these patients. However, we found that chemotherapies used for esophageal and gastric cancers have adverse effects on iNKT cells in vitro. Cisplatin caused a significant reduction of CD1d expression by esophageal tumor cell lines. Cisplatin, 5-fluorouracil and carboplatin induced dose-dependent apoptosis in primary lines of iNKT cells and inhibited CD1d-dependent interferon-γ production and cytolytic degranulation by viable iNKT cells. Interestingly, cisplatin increased granzyme B and perforin production and decreased the production of the granzyme B inhibitor PI9, which protects cytotoxic cells from self-damage by granzyme B. Thus, cisplatin-induced apoptosis of iNKT cells may be mediated in part by altering granzyme B and PI9 expression. Our data suggest that iNKT cell-based immunotherapies may benefit patients with gastrointestinal cancers, but may be negatively affected by chemotherapies used for these cancers.

Objective The role of Th1 and Th17 lymphocyte responses in human infection and sepsis of elderly patients has yet to be clarified. Design A prospective observational study of patients with sepsis, infection only and healthy controls. Setting The acute medical wards and intensive care units in a 1000 bed university hospital. Patients 32 patients with sepsis, 20 patients with infection, and 20 healthy controls. Patients and controls were older than 65 years of age. Patients with recognised underlying immune compromise were excluded. Methods Phenotype, differentiation status and cytokine production by T lymphocytes were determined by flow cytometry. Measurements The differentiation states of circulating CD3+, CD4+, and CD8+ T cells were characterised as naive (CD45RA+, CD197+), central memory (CD45RA-, CD197+), effector memory (CD45RA-, CD197-), or terminally differentated (CD45RA+, CD197-). Expression of IL-12 and IL-23 receptors, and the transcription factors T-bet and RORγt, was analysed in circulating T lymphocytes. Expression of interferon- γ and IL-17A were analysed following stimulation in vitro. Results CD4+ T cells from patients with infection predominantly expressed effector-memory or terminally differentiated phenotypes but CD4+ T cells from patients with severe sepsis predominantly expressed naive phenotypes (p

Toll-like receptors (TLRs) are the key in initiating innate immune responses. TLR2 is crucial in recognising lipopeptides from gram-positive bacteria and is implicated in chronic inflammation. Children with Down syndrome (DS) are prone to infections from these pathogens and have an increased risk of autoimmunity. Sparstolonin B (SsnB) is a TLR antagonist which attenuates cytokine production and improves outcomes in sepsis. We hypothesised that TLR signalling may be abnormal in children with DS and contribute to their clinical phenotype. We evaluated TLR pathways in 3 ways: determining the expression of TLR2 on the surface of neutrophils and monocytes by flow cytometry, examining the gene expression of key regulatory proteins involved in TLR signal propagation, MyD88, IRAK4, and TRIF, by quantitative PCR, and lastly determining the cytokine production by ELISA following immunomodulation with proinflammatory stimuli (lipopolysaccharide (LPS), Pam3Csk4) and the anti-inflammatory agent SsnB. We report TLR2 expression being significantly increased on neutrophils, total monocytes, and intermediate and nonclassical monocytes in children with DS (n=20,mean age 8.8±SD 5.3years, femalen=11) compared to controls (n=15,mean age 6.2±4.2years, femalen=5). At baseline, the expression of MyD88 was significantly lower, and TRIF significantly raised in children with DS. The TLR antagonist SsnB was effective in reducing TLR2 and CD11b expression and abrogating cytokine production in both cohorts. We conclude that TLR signalling and the TLR2 pathway are dysregulated in DS, and this disparate innate immunity may contribute to chronic inflammation in DS. SsnB attenuates proinflammatory mediators and may be of therapeutic benefit.

Background Down syndrome (DS) is the most common syndromic immunodeficiency with an increased risk of infection, mortality from sepsis, and autoinflammation. Innate immune function is altered in DS and therefore we examined responses in CD11b and Toll like receptor 4 (TLR-4), which are important immune cell surface markers upregulated in response to Lipopolysaccharide (LPS) endotoxin, and the immunomodulator melatonin. Neutrophil and monocyte responses to LPS and melatonin in children with Down syndrome (DS) who were clinically stable were compared to age-matched controls. Whole blood was incubated with LPS and melatonin and the relative expression of CD11b and TLR-4 evaluated by flow cytometry. Results Children with DS had an increased response to LPS in neutrophils and intermediate monocytes, while also having elevated TLR-4 expression on non-classical monocytes compared to controls at baseline. Melatonin reduced CD11b expression on neutrophils, total monocytes, both classical and intermediate sub-types, in children with DS and controls. Conclusion Melatonin could represent a useful clinical adjunct in the treatment of sepsis as an immunomodulator. Children with DS had increased LPS responses which may contribute to the more adverse outcomes seen in sepsis.

Invariant natural killer T (iNKT) cells recognize glycolipid antigens bound to CD1d molecules on antigen-presenting cells. Therapeutic activation of iNKT cells with the xenogeneic glycolipid α-galactosylceramide (α-GalCer) can prevent and reverse tumor growth in murine models, but clinical trials using α-GalCer-stimulated human iNKT cells have shown limited efficacy. We synthesized a series of thioglycoside analogs of α-GalCer with different substituents to the galactose residue and found that two of these compounds, XZ7 and XZ11, bound to CD1d-transfected HeLa cells and activated lines of expanded human iNKT cells. Both compounds stimulated cytolytic degranulation by iNKT cells and while XZ7 preferentially stimulated the production of the antitumor cytokine interferon-γ (IFN-γ), XZ11 preferentially stimulated interleukin-4 (IL-4) production. This biased T helper type 1 effector profile of XZ7 was also evident when iNKT were stimulated with dendritic cells presenting this glycolipid. Separate analysis of the responses of CD4+, CD8α+ and CD4-CD8- iNKT cells indicated that XZ7 preferentially activated CD8α+ iNKT cells, and to a lesser degree, CD4-CD8- iNKT cells. The partial agonist effect of glycolipid XZ7, inducing cytotoxicity and IFN-γ production but not IL-4 production, indicates that specific protumour activities of iNKT cells can be abolished, while preserving their antitumor activities, by introducing structural modifications to α-GalCer. Since XZ7 was much less potent than α-GalCer as an iNKT cell agonist, it is unlikely to be superior to α-GalCer as a therapeutic agent for cancer, but may serve as a parent compound for developing more potent structural analogs.