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Immunotherapies, including immune checkpoint inhibitors, have limitations in their effective treatment of malignancies. The immunosuppressive environment associated with the tumor microenvironment may prevent the achievement of optimal outcomes for immune checkpoint inhibitors alone, and nanotechnology-based platforms for delivery of immunotherapeutic agents are increasingly being investigated for their potential to improve the efficacy of immune checkpoint blockade therapy. In this manuscript, nanoparticles were designed with appropriate size and surface characteristics to enhance their retention of payload so that they can transmit their loaded drugs to the tumor. We aimed to enhance immune cell stimulation by a small molecule inhibitor of PD-1/PD-L1 (BMS202) using nanodiamonds (ND). Melanoma cells with different disease stages were exposed to bare NDs, BMS202-NDs or BMS202 alone for 6 h. Following this, melanoma cells were co-cultured with freshly isolated human peripheral blood mononuclear cells (hPBMCs). The effects of this treatment combination on melanoma cells were examined on several biological parameters including cell viability, cell membrane damage, lysosomal mass/pH changes and expression of γHA2X, and caspase 3. Exposing melanoma cells to BMS202-NDs led to a stronger than normal interaction between the hPBMCs and the melanoma cells, with significant anti-proliferative effects. We therefore conclude that melanoma therapy has the potential to be enhanced by non-classical T-cell Immune responses via immune checkpoint inhibitors delivered by nanodiamonds-based nanoparticles., (© 2023. The Author(s).)

Clinical outcomes from infection with SARS-CoV-2, the cause of the COVID-19 pandemic, are remarkably variable ranging from asymptomatic infection to severe pneumonia and death. One of the key drivers of this variability is differing trajectories in the immune response to SARS-CoV-2 infection. Many studies have noted markedly elevated cytokine levels in severe COVID-19, although results vary by cohort, cytokine studied and sensitivity of assay used. We assessed the immune response in acute COVID-19 by measuring 20 inflammatory markers in 118 unvaccinated patients with acute COVID-19 (median age: 70, IQR: 58-79 years; 48.3% female) recruited during the first year of the pandemic and 44 SARS-CoV-2 naïve healthy controls. Acute COVID-19 was associated with marked elevations in nearly all pro-inflammatory markers, whilst eleven markers (namely IL-1β, IL-2, IL-6, IL-10, IL-18, IL-23, IL-33, TNF-α, IP-10, G-CSF and YKL-40) were associated with disease severity. We observed significant correlations between nearly all markers elevated in those infected with SARS-CoV-2 consistent with widespread immune dysregulation. Principal component analysis highlighted a pro-inflammatory cytokine signature (with strongest contributions from IL-1β, IL-2, IL-6, IL-10, IL-33, G-CSF, TNF-α and IP-10) which was independently associated with severe COVID-19 (aOR: 1.40, 1.11-1.76, p=0.005), invasive mechanical ventilation (aOR: 1.61, 1.19-2.20, p=0.001) and mortality (aOR 1.57, 1.06-2.32, p = 0.02). Our findings demonstrate elevated cytokines and widespread immune dysregulation in severe COVID-19, adding further evidence for the role of a pro-inflammatory cytokine signature in severe and critical COVID-19., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Hawerkamp, Dyer, Patil, McElheron, O’Dowd, O’Doherty, Mhaonaigh, George, O’Halloran, Reddy, Kenny, Little, Martin-Loeches, Bergin, Kennelly, Donnelly, Bourke, Long, Sui, Doherty, Conlon, Cheallaigh and Fallon.)

γδ T cells are thought to contribute to immunity against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), but the mechanisms by which they are activated by the virus are unknown. Using flow cytometry, we investigated if the two most abundant viral structural proteins, spike and nucleocapsid, can activate human γδ T cell subsets, directly or in the presence of dendritic cells (DC). Both proteins failed to induce interferon-γ production by Vδ1 or Vδ2 T cells within fresh mononuclear cells or lines of expanded γδ T cells generated from healthy donors, but the same proteins stimulated CD3+ cells from COVID-19 patients. The nucleocapsid protein stimulated interleukin-12 production by DC and downstream interferon-γ production by co-cultured Vδ1 and Vδ2 T cells, but protease digestion and use of an alternative nucleocapsid preparation indicated that this activity was due to contaminating non-protein material. Thus, SARS-CoV-2 spike and nucleocapsid proteins do not have stimulatory activity for DC or γδ T cells. We propose that γδ T cell activation in COVID-19 patients is mediated by immune recognition of viral RNA or other structural proteins by γδ T cells, or by other immune cells, such as DC, that produce γδ T cell-stimulatory ligands or cytokines., Competing Interests: The authors have declared that no competing interests exist.

Background: Down syndrome (DS) is a disorder characterised by marked immune dysfunction, increased mortality from sepsis, chronic inflammation, increased oxidative stress, sleep disturbance and possibly abnormal endogenous melatonin levels. Melatonin has a myriad of immune functions, and we hypothesised that this therapeutic agent could modulate the innate immune system in this cohort., Methods: We investigated neutrophil and monocyte function (CD11b, TLR4 expression by flow cytometry), genes involved in TLR signalling (MyD88, IRAK4, TRIF), the inflammasome (NLRP3, IL-1β), and circadian rhythm (BMAL, CLOCK, CRY) by qPCR, and inflammatory cytokines (IL-2, IL-6, IL-8, IL-18, IL-1β, TNF-α, IFN-γ, IL-10, IL-1ra, VEGF, Epo, GM-CSF) by enzyme-linked immunosorbent assay (ELISA) following immunomodulation with LPS endotoxin and melatonin. 47 children with DS and 23 age- and sex-matched controls were recruited., Results: We demonstrated that melatonin has several significant effects by reducing CD11b and TLR4 expression, attenuating TLR signalling, genes involved in the inflammasome and has the potential to reduce LPS-induced inflammatory responses., Conclusions: Immunomodulatory effects of melatonin were found in both paediatric cohorts with more marked effects in the children with DS. Melatonin mediates immune response through a wide array of mechanisms and this immunomodulator may buffer the inflammatory response by regulating pro and anti-inflammatory signalling., Impact: We highlight that melatonin mediates its immune response through a wide array of mechanisms, its effects appear to be dose dependant and children with Down syndrome may be more receptive to treatment with it. Immunomodulatory effects of melatonin were demonstrated with marked effects in the children with Down syndrome with a reduction of MyD88, IL-1ß and NLRP3 expression in whole-blood samples. Melatonin is a proposed anti-inflammatory agent with a well-established safety profile, that has the potential for mitigation of pro- and anti-inflammatory cytokines in paediatric Down syndrome cohorts, though further clinical trials are warranted., (© 2021. The Author(s).)

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., Competing Interests: None., (© 2021 The Author(s).)

Background and Aims: Genetic predisposition to autoimmune hepatitis (AIH) in adults is associated with possession of human leukocyte antigen (HLA) class I (A*01, B*08) and class II (DRB1*03, -04, -07, or -13) alleles, depending on geographic region. Juvenile autoimmune liver disease (AILD) comprises AIH-1, AIH-2, and autoimmune sclerosing cholangitis (ASC), which are phenotypically different from their adult counterparts. We aimed to define the relationship between HLA profile and disease course, severity, and outcome in juvenile AILD., Approach and Results: We studied 236 children of European ancestry (152 female [64%], median age 11.15 years, range 0.8-17), including 100 with AIH-1, 59 with AIH-2, and 77 with ASC. The follow-up period was from 1977 to June 2019 (median 14.5 years). Class I and II HLA genotyping was performed using PCR/sequence-specific primers. HLA B*08, -DRB1*03, and the A1-B8-DR3 haplotype impart predisposition to all three forms of AILD. Homozygosity for DRB1*03 represented the strongest risk factor (8.8). HLA DRB1*04, which independently confers susceptibility to AIH in adults, was infrequent in AIH-1 and ASC, suggesting protection; and DRB1*15 (DR15) was protective against all forms of AILD. Distinct HLA class II alleles predispose to the different subgroups of juvenile AILD: DRB1*03 to AIH-1, DRB1*13 to ASC, and DRB1*07 to AIH-2. Possession of homozygous DRB1*03 or of DRB1*13 is associated with fibrosis at disease onset, and possession of these two genes in addition to DRB1*07 is associated with a more severe disease in all three subgroups., Conclusions: Unique HLA profiles are seen in each subgroup of juvenile AILD. HLA genotype might be useful in predicting responsiveness to immunosuppressive treatment and course., (© 2021 The Authors. Hepatology published by Wiley Periodicals LLC on behalf of American Association for the Study of Liver Diseases.)

Background: Type 2 diabetes mellitus (T2DM) is a major risk factor for the acquisition of latent tuberculosis (TB) infection (LTBI) and development of active tuberculosis (ATB), although the immunological basis for this susceptibility remains poorly characterised. Innate lymphoid cells (ILCs) immune responses to TB infection in T2DM comorbidity is anticipated to be reduced. We compared ILC responses (frequency and cytokine production) among adult patients with LTBI and T2DM to patients (13) with LTBI only (14), T2DM only (10) and healthy controls (11)., Methods: Using flow cytometry, ILC phenotypes were categorised based on (Lin - CD127 + CD161 + ) markers into three types: ILC1 (Lin - CD127 + CD161 + CRTH2 - CD117 - ); ILC2 (Lin - CD127 + CD161 + CRTH2 + ) and ILC3 (Lin - CD127 + CD161 + CRTH2 - NKp44 +/- CD117 + ). ILC responses were determined using cytokine production by measuring percentage expression of interferon-gamma (IFN-γ) for ILC1, interleukin (IL)-13 for ILC2, and IL-22 for ILC3. Glycaemic control among T2DM patients was measured using glycated haemoglobin (HbA1c) levels. Data were analysed using FlowJo version 10.7.1, and GraphPad Prism version 8.3., Results: Compared to healthy controls, patients with LTBI and T2DM had reduced frequencies of ILC2 and ILC3 respectively (median (IQR): 0.01 (0.005-0.04) and 0.002 (IQR; 0.002-0.007) and not ILC1 (0.04 (0.02-0.09) as expected. They also had increased production of IFN-γ [median (IQR): 17.1 (5.6-24.9)], but decreased production of IL-13 [19.6 (12.3-35.1)]. We however found that patients with T2DM had lower ILC cytokine responses in general but more marked for IL-22 production (median (IQR): IFN-γ 9.3 (4.8-22.6); IL-13 22.2 (14.7-39.7); IL-22 0.7 (IQR; 0.1-2.1) p-value 0.02), which highlights the immune suppression status of T2DM. We also found that poor glycaemic control altered ILC immune responses., Conclusion: This study demonstrates that LTBI and T2DM, and T2DM were associated with slight alterations of ILC immune responses. Poor T2DM control also slightly altered these ILC immune responses. Further studies are required to assess if these responses recover after treatment of either TB or T2DM., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Ssekamatte, Nakibuule, Nabatanzi, Egesa, Musubika, Bbuye, Hepworth, Doherty, Cose and Biraro.)

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., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

We enumerated conventional and innate lymphocyte populations in neonates with neonatal encephalopathy (NE), school-age children post-NE, children with cerebral palsy and age-matched controls. Using flow cytometry, we demonstrate alterations in circulating T, B and natural killer cell numbers. Invariant natural killer T cell and Vδ2 + γδ T cell numbers and frequencies were strikingly higher in neonates with NE, children post-NE and children with cerebral palsy compared to age-matched controls, whereas mucosal-associated invariant T cells and Vδ1 T cells were depleted from children with cerebral palsy. Upon stimulation ex vivo, T cells, natural killer cells and Vδ2 T cells from neonates with NE more readily produced inflammatory cytokines than their counterparts from healthy neonates, suggesting that they were previously primed or activated. Thus, innate and conventional lymphocytes are numerically and functionally altered in neonates with NE and these changes may persist into school-age., (Copyright © 2021 The Author(s). Published by Elsevier B.V. All rights reserved.)

Life-threatening invasive fungal diseases (IFD) are increasing in incidence, especially in immunocompromised patients and successful resolution of IFD requires a variety of different immune cells. With the limited repertoire of available antifungal drugs there is a need for more effective therapeutic strategies. This review interrogates the evidence on the human immune response to the main pathogens driving IFD, with a focus on the role of unconventional lymphocytes e.g. natural killer (NK) cells, gamma/delta (γδ) T cells, mucosal associated invariant T (MAIT) cells, invariant natural killer T (iNKT) cells and innate lymphoid cells (ILC). Recent discoveries and new insights into the roles of these novel lymphocyte groups in antifungal immunity will be discussed, and we will explore how an improved understanding of antifungal action by lymphocytes can inform efforts to improve antifungal treatment options., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

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., (© 2020 John Wiley & Sons Ltd.)

The soil-transmitted helminth Ascaris lumbricoides infects ~800 million people worldwide. Some people are heavily infected, harbouring many worms, whereas others are only lightly infected. The mechanisms behind this difference are unknown. We used a mouse model of hepatic resistance to Ascaris, with C57BL/6J mice as a model for heavy infection and CBA/Ca mice as a model for light infection. The mice were infected with the porcine ascarid, Ascaris suum or the human ascarid, A. lumbricoides and immune cells in their livers and spleens were enumerated using flow cytometry. Compared to uninfected C57BL/6J mice, uninfected CBA/Ca mice had higher splenic CD4+ and γδ T cell counts and lower hepatic eosinophil, Kupffer cell and B cell counts. Infection with A. suum led to expansions of eosinophils, Kupffer cells, monocytes and dendritic cells in the livers of both mouse strains and depletions of hepatic natural killer (NK) cells in CBA/Ca mice only. Infection with A. lumbricoides led to expansions of hepatic eosinophils, monocytes and dendritic cells and depletions of CD8+, αβ, NK and NK T cells in CBA/Ca mice, but not in C57BL/6J mice where only monocytes expanded. Thus, susceptibility and resistance to Ascaris infection are governed, in part, by the hepatic immune system.

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 V̇O 2max ), 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 V̇O 2max 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 V̇O 2max 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., (© 2020 John Wiley & Sons Ltd.)

Background: the role of innate immunity in human sepsis must be fully clarified to identify potential avenues for novel immune adjuvant sepsis therapies. Methods: A prospective observational study was performed including patients with sepsis (septic group), infection without sepsis (infection group), and healthy controls (control group) in the setting of acute medical wards and intensive care units in a 1000-bed university hospital. A total of 42 patients with sepsis, 30 patients with infection, and 30 healthy controls were studied. The differentiation states of circulating mucosal associated invariant T (MAIT) cells and Natural Killer T (NKT) cells were characterised as naive (CD45RA + , CD197 + ), central memory (CD45RA - , CD197 + ), effector memory (CD45RA - , CD197 - ), or terminally differentiated (CD45RA + , CD197 - ). The differentiation states of circulating gamma-delta T lymphocytes were characterised as naive (CD45RA + , CD27 + ), central memory (CD45RA - , CD27 + ), effector memory (CD45RA - , CD27 - ), or terminally differentiated (CD45RA + , CD27 - ). The expression of IL-12 and IL-23 receptors, the transcription factors T-Bet and RORγt, and interferon-γ and IL-17a were analysed. Results: MAIT cell counts were lower in the septic group ( p = 0.002) and the infection group ( p < 0.001) than in the control group. The MAIT cell T-Bet expression in the infection group was greater than in the septic group ( p = 0.012). The MAIT RORγt expression in the septic group was lower than in the control group ( p = 0.003). The NK cell counts differed in the three groups ( p < 0.001), with lower Natural Killer (NK) cell counts in the septic group ( p < 0.001) and in the infection group ( p = 0.001) than in the control group. The NK cell counts increased in the septic group in the 3 weeks following the onset of sepsis ( p = 0.028). In lymphocyte stimulation experiments, fewer NK cells expressed T-Bet in the septic group than in the infection group ( p = 0.002), and fewer NK cells expressed IFN-γ in the septic group than in the control group ( p = 0.002). The NKT cell counts were lower in the septic group than both the control group ( p = 0.05) and the infection group ( p = 0.04). Fewer NKT cells expressed T-Bet in the septic group than in the infection group ( p = 0.004). Fewer NKT cells expressed RORγt in the septic group than in the control group ( p = 0.003). Fewer NKT cells expressed IFN-γ in the septic group than in both the control group ( p = 0.002) and the infection group ( p = 0.036). Conclusion: The clinical presentation of infection and or sepsis in patients is linked with a mosaic of changes in the innate lymphocyte Th1 and Th17 phenotypes. The manipulation of the innate lymphocyte phenotype offers a potential avenue for immune modulation in patients with sepsis.

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.

Background and Purpose: Immunotherapies involving the adoptive transfer of ex vivo expanded autologous invariant natural killer (iNKT) cells are a potential option for cancer patients and are under investigation in clinical trials. Most cancer patients receive radiotherapy at some point during their treatment. We investigated the effects of therapeutic doses of radiation on the viability and function of human primary cultures of iNKT cells in vitro., Materials and Methods: iNKT cell lines generated from 6 healthy donors were subjected to therapeutically-relevant doses of radiation. Cell cycle arrest and cell death were assessed by flow cytometry. Double strand DNA breaks were analysed by measuring phosphorylated histone H2AX expression by fluorescence microscopy. Cytolytic degranulation, cytokine production and cytotoxicity by antigen-stimulated iNKT cells were assessed by flow cytometry., Results: Radiation inhibited viability of iNKT cells in a dose-dependent manner. Radiation caused double strand DNA breaks, which were rapidly repaired, and affected the cell cycle at high doses. Moderate doses of radiation did not inhibit degranulation or cytotoxicity by iNKT cells, but induced perforin expression and inhibited proliferation and interferon-γ production by surviving iNKT cells., Discussion: Exposure of iNKT cell to radiation can negatively affect their viability and function., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Children with Down syndrome (DS) develop more infections, have an increased mortality from sepsis and an increased incidence of chronic inflammatory conditions. Cytokine dysregulation may underpin these clinical sequelae and raised pro-inflammatory biomarkers are a feature in adults with DS. The importance of the anti-inflammatory mediators IL-1ra and IL-10, as well as cytokines Epo and VEGF, which could impact on the pathogenesis and outcomes in congenital heart disease (CHD) which is more prevalent in DS, are less well known. We examined a comprehensive array of pro-(IL-2, IL-6, IL-8, IL-18, IL-1β, TNF-α, IFN-γ), and anti-inflammatory (IL-10 and IL-1ra) mediators, cytokines involved in inflammation in response to hypoxia (EPO), propagating angiogenesis (VEGF), and myelopoiesis (GM-CSF), by enzyme linked immunosorbent assay (ELISA), as well as discussing the potential impact of significant CHD and Lipopolysaccharide endotoxin on these mediators. 114 children with DS and 60 age and sex matched controls were recruited. Children with Down syndrome exhibit significantly greater levels of pro and anti-inflammatory cytokines; IL-2, IL-6, IL-10, IL-1ra, as well as increased Epo, VEGF and GM-CSF at baseline. CHD does not seem to have an impact on circulating cytokines beyond the acute surgical phase. Both cohorts had similar responses to LPS stimulation. These differences may contribute to varied clinical outcomes, acutely like in sepsis, and over time in autoimmunity., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Down syndrome (DS) is the most common genetic syndrome associated with immune defects. The extent of immune dysregulation in DS is substantial, spanning the innate and adaptive systems and including anomalies in: T and B cells, monocytes, neutrophil chemotaxis, circulating cytokines, and suboptimal antibody responses which all contribute to an increased risk of infections, poorer clinical outcomes and chronic inflammation in this vulnerable cohort. Other aspects of innate immunity may also be abnormal and contribute to the increased morbidity and warrant further interrogation such as: gamma delta T cell function, the inflammasome, Toll-like receptors and their pathways. Pharmacotherapies such as pavilizumab, pneumococcal and influenza immunizations, as well as potential immunoprophylactic agents such as pidotimod, azithromycin and Broncho-Vaxom may help alleviate the infectious consequences. Children with DS need to be managed with a heightened sense of awareness and urgency in the setting of sepsis and signs of chronic inflammation need regular screening and appropriate follow up., (Copyright © 2020 Huggard, Doherty and Molloy.)

Many parasites migrate through different tissues during their life-cycle, possibly with the aim to enhance their fitness. This is true for species of three parasite genera of global importance, Ascaris, Schistosoma and Plasmodium, which cause significant global morbidity and mortality. Interestingly, these parasites all incorporate the liver in their life-cycle. The liver has a special immune status being able to preferentially induce tolerance over immunity. This function may be exploited by parasites to evade host immunity, with Plasmodium spp. in particular using this organ for its multiplication. However, hepatic larval attrition occurs in both ascariasis and schistosomiasis. A better understanding of the molecular mechanisms involved in hepatic infection could be useful in developing novel vaccines and therapies for these parasites.

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<0.0001). CD4+ T cells expressing IL-23 receptor were lower in patients with sepsis compared to patients with infection alone (p = 0.007). RORγt expression by CD4+ T cells was less frequent in patients with sepsis (p<0.001), whereas T-bet expressing CD8+ T cells that do not express RORγt was lower in the sepsis patients. HLA-DR expression by monocytes was lower in patients with sepsis. In septic patients fewer monocytes expressed IL-23., Conclusion: Persistent failure of T cell activation was observed in patients with sepsis. Sepsis was associated with attenuated CD8+Th1 and CD4+Th17 based lymphocyte response., Competing Interests: The authors have declared that no competing interests exist.

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 5.3 years, female n = 11) compared to controls ( n = 15, mean age 6.2 ± 4.2 years, female n = 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., Competing Interests: The authors have no conflict of interest to declare., (Copyright © 2019 Dean Huggard et al.)

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.

CD1d-restricted invariant natural killer T (iNKT) cells play central roles in the activation and regulation of innate and adaptive immunity. Cytokine-mediated and CD1d-dependent interactions between iNKT cells and myeloid and lymphoid cells enable iNKT cells to contribute to the activation of multiple cell types, with important impacts on host immunity to infection and tumors and on the prevention of autoimmunity. Here, we review the mechanisms by which iNKT cells contribute to B cell maturation, antibody and cytokine production, and antigen presentation. Cognate interactions with B cells contribute to the rapid production of antibodies directed against conserved non-protein antigens resulting in rapid but short-lived innate humoral immunity. iNKT cells can also provide non-cognate help for the generation of antibodies directed against protein antigens, by promoting the activation of follicular helper T cells, resulting in long-lasting adaptive humoral immunity and B cell memory. iNKT cells can also regulate humoral immunity by promoting the development of autoreactive B cells into regulatory B cells. Depletions and functional impairments of iNKT cells are found in patients with infectious, autoimmune and malignant diseases associated with altered B cell function and in murine models of these conditions. The adjuvant and regulatory activities that iNKT cells have for B cells makes them attractive therapeutic targets for these diseases.

Human γδ T cells expressing the Vδ1 T cell receptor (TCR) recognize self and microbial antigens and stress-inducible molecules in a major histocompatibility complex-unrestricted manner and are an important source of innate interleukin (IL)-17. Vδ1 T cells are expanded in the circulation and intestines of patients with human immunodeficiency virus (HIV) infection. In this study, we show that patients with HIV have elevated frequencies, but not absolute numbers, of circulating Vδ1 T cells compared to control subjects. This increase was most striking in the patients with Candida albicans co-infection. Using flow cytometry and confocal microscopy, we identify two populations of Vδ1 T cells, based on low and high expression of the ε chain of the CD3 protein complex responsible for transducing TCR-mediated signals (denoted CD3ε lo and CD3ε hi Vδ1 T cells). Both Vδ1 T cell populations expressed the CD3 ζ-chain, also used for TCR signaling. Using lines of Vδ1 T cells generated from healthy donors, we show that CD3ε can be transiently downregulated by activation but that its expression is restored over time in culture in the presence of exogenous IL-2. Compared to CD3ε hi Vδ1 T cells, CD3ε lo Vδ1 T cells more frequently expressed terminally differentiated phenotypes and the negative regulator of T cell activation, programmed death-1 (PD-1), but not lymphocyte-activation gene 3, and upon stimulation in vitro , only the CD3ε hi subset of Vδ1 T cells, produced IL-17. Thus, while HIV can infect and kill IL-17-producing CD4 + T cells, Vδ1 T cells are another source of IL-17, but many of them exist in a state of exhaustion, mediated either by the induction of PD-1 or by downregulation of CD3ε expression.

Checkpoint inhibitors, such as anti-PD-1 (Programmed death-1), are transforming cancer treatment for inoperable or advanced disease. As the incidence of obesity-associated malignancies, including esophageal adenocarcinoma (EAC) continues to increase and treatment with checkpoint inhibitors are being FDA approved for a broader range of cancers, it is important to assess how anti-PD-1 treatment might exacerbate pre-existing inflammatory processes at other sites. Outside the EAC tumor, the omentum and liver were found to be enriched with substantial populations of PD-1 expressing T cells. Treatment of omental and hepatic T cells with anti-PD-1 (clone EH12.2H7) did not enhance inflammatory cytokine expression or proliferation, but transiently increased CD107a expression by CD8 + T cells. Importantly, PD-1-expressing T cells are significantly lower in EAC tumor post neoadjuvant chemoradiotherapy, suggesting that combination with specific conventional treatments may severely impair the efficacy of anti-PD-1 immunotherapy. This study provides evidence that systemically administered anti-PD-1 treatment is unlikely to exacerbate pre-existing T cell-mediated inflammation outside the tumor in obesity-associated cancers, such as EAC. Furthermore, our data suggests that studies are required to identify the negative impact of concomitant therapies on PD-1 expression in order to boost overall response rates., (Copyright © 2018 Elsevier B.V. All rights reserved.)

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 T H 1, T H 2 or T H 17 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., (Copyright © 2018 European Federation of Immunological Societies. Published by Elsevier B.V. All rights reserved.)

Immunotherapies that target CD1d-restricted invariant NKT (iNKT) cells can prevent tumor growth in murine models but trials in humans have shown limited clinical efficacy. Here, we show that iNKT cells are depleted from blood and bronchial lavage samples from patients with non-small cell lung cancer (NSCLC) suggesting a role for these cells in immunity against NSCLC. We interrogated the Lung Cancer Explorer and Kaplan-Meier Plotter databases of NSCLC patients and found that pulmonary CD1d expression is reduced in patients with NSCLC and that low expression of CD1d mRNA is significantly associated with poor patient survival. We hypothesized that CD1d expression in NSCLC is epigenetically regulated and can be modulated using epigenetic targeting therapies. Treatment of the CD1d-negative NSCLC cell lines, A549 and SK-MES-1, with DNA methyltransferase inhibitors and histone deacetylase inhibitors resulted in a dose-dependent induction of CD1d mRNA and protein expression. Chromatin immunoprecipitation analysis indicated that this induction of CD1d expression directly involved chromatin remodelling. Induction of CD1d expression by A549 and SK-MES-1 cells using therapeutic low doses of DNA methyltransferase inhibitors and histone deacetylase inhibitors made them targets for iNKT cell-mediated cytolytic degranulation. Thus, epigenetic manipulation of CD1d expression may augment the efficacy of iNKT cell-based immunotherapies for NSCLC.

Invariant natural killer T (iNKT) cells are cytotoxic T cells that respond to glycolipid antigens presented by CD1d. Therapeutic activation of iNKT cells with α-galactosylceramide (α-GalCer) can prevent and reverse tumor growth in mice and clinical trials involving α-GalCer-stimulated iNKT cells are ongoing in humans. B cells express CD1d, however, we show that CD1d expression is reduced on B cells from patients with chronic lymphocytic leukemia (CLL). B cells from CLL patients pulsed with α-GalCer failed to stimulate cytolytic degranulation by iNKT cell lines, but could present the more potent glycolipid analogue, 7DW8-5. Retinoic acid receptor-α (RAR-α) agonists induced CD1d expression by CLL B cells, restoring their ability to present α-GalCer to CD8α + iNKT cells, resulting in cytolytic degranulation. Thus, RAR-α agonists can augment the anti-tumor activities of iNKT cells against CLL cells in vitro. Their inclusion in iNKT cell-based therapies may benefit patients with CLL., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Common variable immunodeficiency (CVID) is a primary immunoglobulin deficiency characterized by recurrent infections and complications, including autoimmunity, enteropathy, polyclonal lymphocytic infiltration or lymphoid malignancy. Innate T cells can support B cell maturation and antibody production. We investigated the numbers, phenotypes and functions of circulating B cell, γδ T cell, invariant natural killer T (iNKT) cell and mucosal-associated invariant T (MAIT) cell subsets in 23 CVID patients and 27 healthy controls. Switched-memory B cells and plasmablasts were depleted in CVID patients (p<0.0001). γδ T cells were found at normal numbers, but iNKT and MAIT cells were depleted (p<0.0001 and p<0.002). MAIT cells were especially low in patients with complicated CVID (p<0.05). MAIT cells from patients appeared more activated and more frequently produced interleukin-17A, interleukin-22 and tumor necrosis factor-α than MAIT cells from healthy subjects in vitro. Thus, MAIT cell depletion and activation may contribute to immunodeficiency and complications associated with CVID., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Background: Disease severity in viral bronchiolitis is often difficult to predict at onset, and may be related to the host immune response. Recognizing the particular immunologic features of infants who develop severe disease might offer an opportunity for developing diagnostic tools to facilitate early intervention and improve outcomes., Methods: We compared cytokine gene expression (by real-time reverse-transcriptase polymerase chain reaction), cytokine concentrations (by enzyme-linked immunosorbent assay) and the activation status of lymphocytes (by flow cytometry) in the peripheral blood of children hospitalized with moderate and severe viral bronchiolitis and a group of age-matched controls., Results: Analysis was undertaken on 57 children with viral bronchiolitis and 33 controls. Interleukin-7 mRNA expression at enrollment in peripheral blood mononuclear cells differed significantly between those with moderate and severe bronchiolitis, and correlated with both the subsequent length of hospital stay and need for supplemental oxygen therapy. Serum interleukin-10 concentration also distinguished moderate from severe disease. Participants with viral bronchiolitis demonstrated a more activated γδ-T cell phenotype (Vδ1+), but a more naive TCR αβ-T cell compartment compared with controls., Conclusions: Viral bronchiolitis is characterized by a distinct pattern of cytokine expression and lymphocyte activation. These changes suggest an inadequate innate response in severe disease, and may offer potential as markers of disease severity.

NKG2D is an important activating receptor expressed on NK cells. Ligands (termed NKG2DL) for this receptor include ULBP1-6, MICA and MICB in humans; they are upregulated in stressed, cancerous or infected cells where they engage NKG2D to induce NK cell cytotoxicity and cytokine production. Expression of NKG2DL on effector cells has been described in mice and more recently in human cells. We confirm that NK cell lines and IL-2 stimulated primary human NK cells also express the NKG2DL, ULBP2. However, expression of ULBP2 was not a result of transfer from a non-NK cell to an NK cell and in contrast to recent reports we saw no evidence that ULBP2 expression targeted these NK cells for fratricide or for cytotoxicity by NKG2D-expressing, non-NK effector cells. ULBP2 expression was however linked to expression of mature CD57(+) NK cells. In particular, expression of ULBP2 was strongest on those NK cells that had evidence of recent activation and proliferation. We suggest that ULBP2 could be used to identify recently activated "mature" NK cells. Defining this phenotype would be useful for understanding the ontogeny on human NK cells., (Copyright © 2016 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.)

The hepatic immune system is constantly exposed to a massive load of harmless dietary and commensal antigens, to which it must remain tolerant. Immune tolerance in the liver is mediated by a number of specialized antigen-presenting cells, including dendritic cells, Kupffer cells, liver sinusoidal endothelial cells and hepatic stellate cells. These cells are capable of presenting antigens to T cells leading to T cell apoptosis, anergy, or differentiation into regulatory T cells. However, the hepatic immune system must also be able to respond to pathogens and tumours and therefore must be equipped with mechanisms to override immune tolerance. The liver is a site of accumulation of a number of innate lymphocyte populations, including natural killer cells, CD56(+) T cells, natural killer T cells, γδ T cells, and mucosal-associated invariant T cells. Innate lymphocytes recognize conserved metabolites derived from microorganisms and host cells and respond by killing target cells or promoting the differentiation and/or activation of other cells of the immune system. Innate lymphocytes can promote the maturation of antigen-presenting cells from their precursors and thereby contribute to the generation of immunogenic T cell responses. These cells may be responsible for overriding hepatic immune tolerance to autoantigens, resulting in the induction and maintenance of autoreactive T cells that mediate liver injury causing autoimmune liver disease. Some innate lymphocyte populations can also directly mediate liver injury by killing hepatocytes or bile duct cells in murine models of hepatitis, whilst other populations may protect against liver disease. It is likely that innate lymphocyte populations can promote or protect against autoimmune liver disease in humans and that these cells can be targeted therapeutically. Here I review the cellular mechanisms by which hepatic antigen-presenting cells and innate lymphocytes control the balance between immunity, tolerance and autoimmunity in the liver., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Disease severity in viral bronchiolitis in infancy is difficult to predict and has been linked to host innate immunity. The study aimed to investigate the innate cytokine interleukin-15 (IL-15) as a marker of disease severity.A prospective single-centre observational study was conducted in a university-affiliated paediatric teaching hospital, comparing children (0-18 months) hospitalised for viral bronchiolitis, those admitted to the paediatric intensive care unit with severe disease and healthy age-matched controls. IL-15-related parameters were compared between groups. PCR and microRNA (miRNA) sequencing was undertaken on natural killer (NK) cells collected from study participants.Samples from 88 children with viral bronchiolitis and 43 controls enrolled between 2009 and 2012 were analysed. Peripheral blood mononuclear cell (PBMC) IL-15 mRNA expression was significantly higher in those with moderate severity bronchiolitis compared with controls and those with severe disease. Serum IL-15 levels correlated with disease severity. The relative frequency of NK cells in peripheral blood was significantly reduced in participants with bronchiolitis. The NK cell miRNA transcriptome in bronchiolitis was distinct. Targets of de-regulated miRNA were differentially expressed in bronchiolitis, including JAK3, STAT5A and NFKB1 on the IL-15 signalling pathway.IL-15 is associated with disease severity in children hospitalised with viral bronchiolitis., (Copyright ©ERS 2016.)

Unconventional T cells are gaining center stage as important effector and regulatory cells that orchestrate innate and adaptive immune responses. Human Vγ9/Vδ2 T cells are amongst the best understood unconventional T cells, as they are easily accessible in peripheral blood, can readily be expanded and manipulated in vitro, respond to microbial infections in vivo and can be exploited for novel tumor immunotherapies. We here review findings that suggest that Vγ9/Vδ2 T cells, and possibly other unconventional human T cells, play an important role in bridging innate and adaptive immunity by promoting the activation and differentiation of various types of antigen-presenting cells (APCs) and even turning into APCs themselves, and thereby pave the way for antigen-specific effector responses and long-term immunological memory. Although the direct physiological relevance for most of these mechanisms still needs to be demonstrated in vivo, these findings may have implications for novel therapies, diagnostic tests and vaccines., (Copyright © 2015 Elsevier Inc. All rights reserved.)

γδ T cells expressing the Vδ1 TCR are expanded in patients with HIV infection. We show in this article that circulating Vδ1 T cell numbers are particularly high in patients with HIV and candidiasis, and that these cells expand and produce IL-17 in response to Candida albicans in vitro. Although C. albicans could directly stimulate IL-17 production by a subset of Vδ1 T cells, fungus-treated dendritic cells (DCs) were required to expand C. albicans-responsive Vδ1 T cells to generate sufficient numbers of cells to release IL-17 at levels detectable by ELISA. C. albicans induced the release of IL-1β, IL-6, and IL-23 by DCs, but addition of these cytokines or supernatants of C. albicans-treated DCs to Vδ1 T cells was not sufficient to induce proliferation. We found that direct contact with DCs was required for Vδ1 T cell proliferation, whereas IL-23R-blocking studies showed that IL-23 was required for optimal C. albicans-induced IL-17 production. Because IL-17 affords protection against both HIV and C. albicans, and because Vδ1 T cells are not depleted by HIV, these cells are likely to be an important source of IL-17 in HIV-infected patients with candidiasis, in whom CD4(+) Th17 responses are impaired. These data show that C. albicans stimulates proliferation and IL-17 production by Vδ1 T cells by a mechanism that involves IL-23 release by DCs., (Copyright © 2015 by The American Association of Immunologists, Inc.)

Mucosal-associated invariant T (MAIT) cells are innate MHC-unrestricted cells that regulate inflammatory responses through the rapid production of cytokines. In this article, we show that circulating MAIT cells are depleted in obese adults, and depletion is associated with diabetic status. Circulating MAIT cells more frequently produced IL-17 upon stimulation ex vivo, a cytokine implicated in insulin resistance. MAIT cells were enriched in adipose tissue (AT) compared with blood. AT MAIT cells, but not circulating MAIT cells, were capable of producing IL-10. In AT from obese subjects, MAIT cells were depleted, were less likely to produce IL-10, and more frequently produced IL-17. Finally, we show that IL-17(+) MAIT cells are also increased in childhood obesity, and altered MAIT cell frequencies in obese children are positively associated with insulin resistance. These data indicate that MAIT cells are enriched in human AT and display an IL-17(+) phenotype in both obese adults and children, correlating with levels of insulin resistance. The alterations in MAIT cells may be contributing to obesity-related sterile inflammation and insulin resistance., (Copyright © 2015 by The American Association of Immunologists, Inc.)

Hepatitis B virus (HBV) is a leading cause of liver cirrhosis and hepatocellular carcinoma. The outcome of HBV infection is largely determined by the host immune response, with virus-specific cytotoxic T cells being able to mediate immunity against HBV as well as causing liver pathology. γδ T cells are reported to be depleted in patients with HBV-associated liver disease. However, it is not known if these cells control HBV infection in patients with asymptomatic chronic HBV infection. In this study, the frequencies, phenotypes, and interferon-γ production were examined by circulating γδ T cell subsets in a group of asymptomatic HBV carriers with low viral loads and little evidence of liver disease. It is shown that γδ T cells expressing Vδ1 and Vδ2 T cell receptors and effector-memory phenotypes are found at higher frequencies in these patients compared to controls. Vδ2 T cells from the patients expressed interferon-γ significantly more frequently than Vδ2 T cells from healthy donors in the absence of ex vivo stimulation. These data suggest that effector-memory IFN-γ-producing Vδ2 T cells may contribute to the control of HBV in patients with asymptomatic infection, without mediating liver pathology.

Human γδ T cells expressing the Vγ9Vδ2 T cell receptor can induce maturation of dendritic cells (DC) into antigen-presenting cells (APC) and B cells into antibody-secreting plasma cells. Since B cells are capable of presenting antigens to T cells, we investigated if Vγ9Vδ2 T cells can influence antigen-presentation by these cells. We report that Vγ9Vδ2 T cells induced expression of CD86, HLA-DR, and CD40 by B cells and stimulated the release of IL-4, IL-6, TNF-α, and IgG, IgA, and IgM. Vγ9Vδ2 T cells also augmented the ability of B cells to stimulate proliferation but not IFN-γ or IL-4 release by alloreactive T cells. In contrast, Vγ9Vδ2 T cells induced expression of CD86 and HLA-DR and the release of IFN-γ, IL-6, and TNF-α by DC and these DC stimulated proliferation and IFN-γ production by conventional T cells. Furthermore, CD86, TNF-α, IFN-γ, and cell contact were found to be important in DC activation by Vγ9Vδ2 T cells but not in the activation of B cells. These data suggest that Vγ9Vδ2 T cells can induce maturation of B cells and DC into APC, but while they prime DC to stimulate T helper 1 (TH1) responses, they drive maturation of B cells into APC that can stimulate different T cell responses. Thus, Vγ9Vδ2 T cells can control different arms of the immune system through selective activation of B cells and DC in vitro, which may have important applications in immunotherapy and for vaccine adjuvants.

HIV-1 Tat exhibits clade-specific cytokine induction in monocytes. We investigated if Tat clades A-D can alter tumour necrosis factor (TNF)-α and interferon (IFN)-γ production by total and Vγ9Vδ2 T cells in vitro. Tat clade B, but not C, augmented TNF-α production by THP-1 cells. However, Tat clades A-D did not affect TNF-α or IFN-γ production or secretion by resting or activated conventional and Vγ9Vδ2 T cells. Therefore, transactivation of cytokines by Tat is immune cell-specific.

Coeliac disease is a chronic small intestinal immune-mediated enteropathy precipitated by exposure to dietary gluten in genetically predisposed individuals. The only current therapy is a lifelong gluten free diet. While much work has focused on the gliadin-specific adaptive immune response in coeliac disease, little is understood about the involvement of the innate immune system. Here we used multi-colour flow cytometry to determine the number and frequency of γδ T cells (Vδ1, Vδ2 and Vδ3 subsets), natural killer cells, CD56(+) T cells, invariant NKT cells, and mucosal associated invariant T cells, in blood and duodenum from adults and children with coeliac disease and healthy matched controls. All circulating innate lymphocyte populations were significantly decreased in adult, but not paediatric coeliac donors, when compared with healthy controls. Within the normal small intestine, we noted that Vδ3 cells were the most abundant γδ T cell type in the adult epithelium and lamina propria, and in the paediatric lamina propria. In contrast, patients with coeliac disease showed skewing toward a predominant Vδ1 profile, observed for both adult and paediatric coeliac disease cohorts, particularly within the gut epithelium. This was concurrent with decreases in all other gut lymphocyte subsets, suggesting a specific involvement of Vδ1 cells in coeliac disease pathogenesis. Further analysis showed that γδ T cells isolated from the coeliac gut display an activated, effector memory phenotype, and retain the ability to rapidly respond to in vitro stimulation. A profound loss of CD56 expression in all lymphocyte populations was noted in the coeliac gut. These findings demonstrate a sustained aberrant innate lymphocyte profile in coeliac disease patients of all ages, persisting even after elimination of gluten from the diet. This may lead to impaired immunity, and could potentially account for the increased incidence of autoimmune co-morbidity.

Invariant NK T (iNKT) cells can provide help for B cell activation and Ab production. Because B cells are also capable of cytokine production, Ag presentation, and T cell activation, we hypothesized that iNKT cells will also influence these activities. Furthermore, subsets of iNKT cells based on CD4 and CD8 expression that have distinct functional activities may differentially affect B cell functions. We investigated the effects of coculturing expanded human CD4(+), CD8α(+), and CD4(-)CD8α(-) double-negative (DN) iNKT cells with autologous peripheral B cells in vitro. All iNKT cell subsets induced IgM, IgA, and IgG release by B cells without needing the iNKT cell agonist ligand α-galactosylceramide. Additionally, CD4(+) iNKT cells induced expansions of cells with phenotypes of regulatory B cells. When cocultured with α-galactosylceramide-pulsed B cells, CD4(+) and DN iNKT cells secreted Th1 and Th2 cytokines but at 10-1000-fold lower levels than when cultured with dendritic cells. CD4(+) iNKT cells reciprocally induced IL-4 and IL-10 production by B cells. DN iNKT cells expressed the cytotoxic degranulation marker CD107a upon exposure to B cells. Remarkably, whereas iNKT cell subsets could induce CD40 and CD86 expression by B cells, iNKT cell-matured B cells were unable to drive proliferation of autologous and alloreactive conventional T cells, as seen with B cells cultured in the absence of iNKT cells. Therefore, human CD4(+), CD8α(+), and DN iNKT cells can differentially promote and regulate the induction of Ab and T cell responses by B cells.

Human γδ T cells expressing the Vδ3 TCR make up a minor lymphocyte subset in blood but are enriched in liver and in patients with some chronic viral infections and leukemias. We analyzed the frequencies, phenotypes, restriction elements, and functions of fresh and expanded peripheral blood Vδ3 T cells. Vδ3 T cells accounted for ~0.2% of circulating T cells, included CD4(+), CD8(+), and CD4(-)CD8(-) subsets, and variably expressed CD56, CD161, HLA-DR, and NKG2D but neither NKG2A nor NKG2C. Vδ3 T cells were sorted and expanded by mitogen stimulation in the presence of IL-2. Expanded Vδ3 T cells recognized CD1d but not CD1a, CD1b, or CD1c. Upon activation, they killed CD1d(+) target cells, released Th1, Th2, and Th17 cytokines, and induced maturation of dendritic cells into APCs. Thus, Vδ3 T cells are glycolipid-reactive T cells with distinct Ag specificities but functional similarities to NKT cells.

The Interleukin-23 (IL-23)/IL-23R signaling axis is an important inflammatory pathway, involved in the stimulation and regulation of the T helper (Th) 17 lymphocytes, resulting in the production of IL-17. Aside from auto-immunity, this cytokine has also been linked to carcinogenesis and polymorphisms in the IL-23R gene are associated with an increased risk for the development of a number of different cancers. Activation of the IL-23 pathway results in the up-regulation of STAT3 and it is thought that the pathological consequences associated with this are in part due to the production of IL-17. We have previously identified IL-23A as pro-proliferative and epigenetically regulated in non-small cell lung cancer (NSCLC). The current study aims to evaluate IL-23R in greater detail in NSCLC. We demonstrate that IL-23R is expressed and epigenetically regulated in NSCLC through histone post-translation modifications and CpG island methylation. In addition, Gemcitabine treatment, a chemotherapy drug used in the treatment of NSCLC, resulted in the up-regulation of the IL-23R. Furthermore, Apilimod (STA 5326), a small molecule which blocks the expression of IL-23 and IL-12, reduced the proliferative capacity of NSCLC cells, particularly in the adenocarcinoma (A549) sub-type. Apilimod is currently undergoing investigation in a number of clinical trials for the treatment of auto-immune conditions such as Crohn's disease and Rheumatoid Arthritis. Our results may have implications for treating NSCLC patients with Gemcitabine or epigenetic targeted therapies. However, Apilimod may possibly provide a new treatment avenue for NSCLC patients. Work is currently ongoing to further delineate the IL-23/IL-23R axis in this disease.

Introduction: Severe sepsis in humans may be related to an underlying profound immune suppressive state. We investigated the link between gene expression of immune regulatory cytokines and the range of illness severity in patients with infection and severe sepsis., Methods: A prospective observational study included 54 ICU patients with severe sepsis, 53 patients with infection without organ failure, and 20 healthy controls. Gene expression in peripheral blood mononuclear cells (PBMC) was measured using real-time polymerase chain reaction., Results: Infection differed from health by decreased expression of the IL2, and IL23 and greater expression of IL10 and IL27. Severe sepsis differed from infection by having decreased IL7, IL23, IFN γ , and TNF α gene expression. An algorithm utilising mRNA copy number for TNF α , IFN γ , IL7, IL10, and IL23 accurately distinguished sepsis from severe sepsis with a receiver operator characteristic value of 0.88. Gene expression was similar with gram-positive and gram-negative infection and was similar following medical and surgical severe sepsis. Severity of organ failure was associated with serum IL6 protein levels but not with any index of cytokine gene expression in PBMCs., Conclusions: Immune regulatory cytokine gene expression in PBMC provides a robust method of modelling patients' response to infection.

Hepatitis C virus (HCV) is a small, enveloped RNA virus and the number of HCV-infected individuals worldwide is estimated to be approximately 170 million. Most HCV infections persist, with up to 80% of all cases leading to chronic hepatitis associated with liver fibrosis, cirrhosis, and hepatocellular carcinoma. HCV-host interactions have a crucial role in viral survival, persistence, pathogenicity of infection, and disease progression. Maintenance of a vigorous, sustained cellular immune response recognizing multiple epitopes is essential for viral clearance. To escape immune surveillance, HCV alters its epitopes so that they are no-longer recognized by T cells and neutralizing antibodies, in addition to interfering with host cell cellular components and signaling pathways. The generation of escape variants is one of the most potent immune evasion strategies utilized by HCV. A large body of evidence suggests that single or multiple mutations within HLA-restricted epitopes contribute to viral immune escape and establishment of viral persistence. Further elucidation of the molecular mechanisms underlying immune escape will aid in the design of novel vaccines and therapeutics for the disease., (Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Invariant natural killer T (iNKT) cells are a minor subset of human T cells which express the invariant T cell receptor Vα24 Jα18 and recognize glycolipids presented on CD1d. Invariant NKT cells are important immune regulators and can initiate anti-tumor responses through early potent cytokine production. Studies show that iNKT cells are defective in certain cancers. Cigarette smoke contains many carcinogens and is implicated directly and indirectly in many cancers. We investigated the effects of cigarette smoke on the circulating iNKT cell number and function. We found that the iNKT cell frequency is significantly reduced in cigarette smoking subjects. Invariant NKT cells exposed to cigarette smoke extract (CSE) showed significant defects in cytokine production and the ability to kill target cells. CSE inhibits the upregulation of CD107 but not CD69 or CD56 on iNKT cells. These findings suggest that CSE has a specific effect on iNKT cell anti-tumor responses, which may contribute to the role of smoking in the development of cancer., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Activation of CD1d-restricted invariant NKT (iNKT) cells with the glycolipid α-galactosylceramide (α-GalCer) confers protection against disease in murine models, however, clinical trials in humans have had limited impact. We synthesized a novel thioglycoside analogue of α-GalCer, denoted α-S-GalCer, and tested its efficacy for stimulating human iNKT cells in vitro. α-S-GalCer stimulated cytokine release by iNKT cells in a CD1d-dependent manner and primed CD1d(+) target cells for lysis. α-S-GalCer-stimulated iNKT cells induced maturation of monocyte-derived dendritic cells into antigen-presenting cells that released IL-12 and small amounts of IL-10. The nature and potency of α-S-GalCer and α-GalCer in human iNKT cell activation were similar. However, in contrast to α-GalCer, α-S-GalCer did not activate murine iNKT cells in vivo. Because of its enhanced stability in biological systems, α-S-GalCer may be superior to α-GalCer as a parent compound for developing adjuvant therapies for humans., (Copyright © 2011 Elsevier Inc. All rights reserved.)