Identifying a Novel Role for Fractalkine (CX3CL1) in Memory CD8(+) T Cell Accumulation in the Omentum of Obesity-Associated Cancer Patients

The omentum is enriched with pro-inflammatory effector memory CD8+ T cells in patients with the obesity-associated malignancy, esophagogastric adenocarcinoma (EAC) and we have identified the chemokine macrophage inflammatory protein-1alpha as a key player in their active migration to this inflamed tissue. More recently, others have established that subsets of memory CD8+ T cells can be classified based on their surface expression of CX3CR1; the specific receptor for the inflammatory chemokine fractalkine. CD8+ T cells expressing intermediate levels (CX3CR1INT) are defined as peripheral memory, those expressing the highest levels (CX3CR1HI) are effector memory/terminally differentiated and those lacking CX3CR1 (CX3CR1NEG) are classified as central memory. To date, the fractalkine:CX3CR1 axis has not been examined in the context of CD8+ T cell enrichment in the omentum and here we examine this chemokines involvement in the accumulation of memory CD8+ T cells in the omentum of EAC patients. Our data show that fractalkine is significantly enriched in the omentum of EAC patients and drives migration of T cells derived from EAC patient blood. Furthermore, CX3CR1 is endocytosed specifically by CD8+ T cells upon encountering fractalkine, which is consistent with the significantly diminished frequencies of CX3CR1INT and CX3CR1HI CD8+ T cells in the fractalkine-rich environment of omentum in EAC, relative to matched blood. Fractalkine-mediated endocytosis of CX3CR1 by CD8+ T cells is sustained and is followed by enhanced surface expression of L-selectin (CD62L). These novel data align with our findings that circulating CX3CR1NEG CD8+ T cells express higher levels of L-selectin than CX3CR1INT CD8+ T cells. This is consistent with previous reports and implicates fractalkine in the conversion of CX3CR1INT CD8+ T cells to a CX3CR1NEG phenotype characterized by alterations in the migratory capacity of these T cells. For the first time, these findings identify fractalkine as a driver of T cell migration to the omentum in EAC and indicate that CD8+ T cells undergo sequenced fractalkine-mediated alterations in CX3CR1 and L-selectin expression. These data implicate fractalkine as more than a chemotactic cytokine in obesity-associated meta-inflammation and reveal a role for this chemokine in the maintenance of the CX3CR1NEG CD8+ T cell populations

The role and treatment potential of natural killer T (NKT) cells in patients with upper gastrointestinal cancers

Oesophageal adenocarcinoma (OAC), squamous cell carcinoma (SSC) and gastric cancers (GAC), collectively cause over 1.3 million deaths worldwide reported in 2018. Current therapeutic regimens focus on chemo-radiotherapy prior to surgery, however, only 20-30% of patients respond to treatment. Therefore, new treatments are urgently required. Invariant natural killer T (iNKT) cells are innate T cells with semi-invariant T cell receptors that recognise glycolipids presented by CD1d. iNKT cells have antitumour activities that are currently being tested as cellular therapies iNKT cell frequencies were quantified in pre- and post-treatment blood and omentum from 152 patients with GAC, SCC or OAC by flow cytometry. They were found to be depleted in peripheral blood from GAC, SCC and OAC patients compared to controls. Omentum had higher frequencies of iNKT cells in the cancer patients compared to blood. These findings suggest that iNKT cells may mediate immunity against GAC, SSC and OAC and that the omentum could be a source of cells for use in adoptive cell therapy. Since clinical trials involving iNKT cells in other cancers have to date shown limited clinical efficacy, we investigated if the antitumour activities of these cells could be improved by coordinating treatment with current chemotherapy and radiotherapy regimens or by the use of alternative glycolipid ligands. iNKT cells were isolated from healthy donor blood samples and expanded in vitro. They were treated with various concentrations of cisplatin, carboplatin, paclitaxel, 5-fluorouracil for 24 or 48h or irradiated with a single dose of 2 Gy or 10 Gy or five fractionated doses of 2 Gy. Cells were then assayed for viability, apoptotic markers, or co-cultured with CD1d transfected HeLa cells pulsed with the iNKT cell agonist ligand, ?-galactosylceramide (?-GalCer), for assays of cytolytic degranulation and intracellular cytokine, granzyme B and perforin production. Cisplatin, 5-FU, carboplatin, paclitaxel and radiation exhibited a dose-dependent inhibition of iNKT cell viability. Cisplatin also inhibited degranulation and IFN-?, but not IL-4, production by viable iNKT cells. While 5-FU, carboplatin, paclitaxel and radiation increased apoptosis of iNKT cells, it did not affect activation. A number of synthetic glycolipids analogues of ?-GalCer were also tested for their ability to bind to CD1d-transfected HeLa cells and activate the antitumour activities of iNKT cells. One novel glycolipid, XZ7, induced cytolytic degranulation and IFN-? production by CD8+ and double negative iNKT cells, suggesting that it may be superior to ?-GalCer as a lead compound for activating the antitumour activities of iNKT cells. We also tested for the presence of non-invariant (type II) NKT cells reactive against a number of glycolipids that were previously shown to bind to CD1d and stimulate T cells, in OAC patients and control subjects. OAC patients had higher frequencies of sulfatide and tetramyristoyl-cardiolipin (TO CL)-specific T cells compared to controls. Most of these cells expressed the V?1 T cell receptor. TO CL induced the production of transforming growth factor-? by expanded V?1 T cells in vitro. Low iNKT cell and high type II NKT cell numbers may predispose individuals to upper gastrointestinal cancers and boosting iNKT cell numbers may have therapeutic value. However, exposure to systemic chemotherapy can negatively affect their functions and should be considered when developing iNKT cell-based immunotherapies

Activation and Regulation of B Cell Responses by Invariant Natural Killer T Cells

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

Dysregulated T helper type 1 (Th1) and Th17 responses in elderly hospitalised patients with infection and sepsis.

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

Altered endotoxin responsiveness in healthy children with Down syndrome

Abstract 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

Novel thioglycoside analogs of ?-galactosylceramide stimulate cytotoxicity and preferential Th1 cytokine production by human invariant natural killer T cells

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 ?-galactosylceramide (?-GalCer) can prevent and reverse tumor growth in murine models, but clinical trials using ?-GalCer-stimulated human iNKT cells have shown limited efficacy. We synthesized a series of thioglycoside analogs of ?-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-? (IFN-?), 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+, CD8?+ and CD4?CD8? iNKT cells indicated that XZ7 preferentially activated CD8?+ iNKT cells, and to a lesser degree, CD4?CD8? iNKT cells. The partial agonist effect of glycolipid XZ7, inducing cytotoxicity and IFN-? 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 ?-GalCer. Since XZ7 was much less potent than ?-GalCer as an iNKT cell agonist, it is unlikely to be superior to ?-GalCer as a therapeutic agent for cancer, but may serve as a parent compound for developing more potent structural analogs

Innate immune dysregulation in multisystem inflammatory syndrome in children (MIS-C)

Abstract MIS-C is a systemic inflammation disorder with poorly characterised immunopathological mechanisms. We compared changes in the systemic immune response in children with MIS-C (n = 12, 5–13 years) to healthy controls (n = 14, 5–15 years). Analysis was done in whole blood treated with LPS. Expression of CD11b and Toll-like receptor-4 (TLR4) in neutrophils and monocytes were analysed by flow cytometry. Serum cytokines (IL-1β, IL-2, IL-6, IL-8, IL-10, IL-Ira, TNF-α, TNF-β, IFN-Υ, VEGF, EPO and GM-CSF) and mRNA levels of inflammasome molecules (NLRP3, ASC and IL-1β) were evaluated. Subpopulations of lymphocytes (CD3+, CD19+, CD56+, CD4+, CD8+, TCR Vδ1+, TCR Vδ2+) were assessed at basal levels. Absolute counts of neutrophils and NLR were high in children with MIS-C while absolute counts of lymphocytes were low. Children with MIS-C had increased levels of IL-6, IL-10, TNF-β and VEGF serum cytokines at the basal level, and significantly increased TNF-β post-LPS, compared to controls. IL-1RA and EPO decreased at baseline and post-LPS in MIS-C patients compared to controls. The percentage of CD3+ cells, NK cells and Vδ1 was lower while B cells were higher in children with MIS-C than in controls. Dysregulated immune response in children with MIS-C was evident and may be amenable to immunomodulation

Neutrophils in COVID-19: not innocent bystanders

Unusually for a viral infection, the immunological phenotype of severe COVID-19 is characterised by a depleted lymphocyte and elevated neutrophil count, with the neutrophil-to-lymphocyte ratio correlating with disease severity. Neutrophils are the most abundant immune cell in the bloodstream and comprise different subpopulations with pleiotropic actions that are vital for host immunity. Unique neutrophil subpopulations vary in their capacity to mount antimicrobial responses, including NETosis (the generation of neutrophil extracellular traps), degranulation and de novo production of cytokines and chemokines. These processes play a role in antiviral immunity, but may also contribute to the local and systemic tissue damage seen in acute SARS-CoV-2 infection. Neutrophils also contribute to complications of COVID-19 such as thrombosis, acute respiratory distress syndrome and multisystem inflammatory disease in children. In this Progress review, we discuss the anti-viral and pathological roles of neutrophils in SARS-CoV-2 infection, and potential therapeutic strategies for COVID-19 that target neutrophil-mediated inflammatory responses.</p

Mucosal-Associated Invariant T Cells Display Diminished Effector Capacity in Oesophageal Adenocarcinoma

Oesophageal adenocarcinoma (OAC) is an aggressive malignancy with poor prognosis, and incidence is increasing rapidly in the Western world. Mucosal-associated invariant T (MAIT) cells recognize bacterial metabolites and kill infected cells, yet their role in OAC is unknown. We aimed to elucidate the role of MAIT cells during cancer development by characterizing the frequency, phenotype, and function of MAIT cells in human blood and tissues, from OAC and its pre-malignant inflammatory condition Barrett’s oesophagus (BO). Blood and tissues were phenotyped by flow cytometry and conditioned media from explanted tissue was used to model the effects of the tumor microenvironment on MAIT cell function. Associations were assessed between MAIT cell frequency, circulating inflammatory markers, and clinical parameters to elucidate the role of MAIT cells in inflammation driven cancer. MAIT cells were decreased in BO and OAC blood compared to healthy controls, but were increased in oesophageal tissues, compared to BO-adjacent tissue, and remained detectable after neo-adjuvant treatment. MAIT cells in tumors expressed CD8, PD-1, and NKG2A but lower NKG2D than BO cohorts. MAIT cells produced less IFN-γ and TNF-α in the presence of tumor-conditioned media. OAC cell line viability was reduced upon exposure to expanded MAIT cells. Serum levels of chemokine IP-10 were inversely correlated with MAIT cell frequency in both tumors and blood. MAIT cells were higher in the tumors of node-negative patients, but were not significantly associated with other clinical parameters. This study demonstrates that OAC tumors are infiltrated by MAIT cells, a type of CD8 T cell featuring immune checkpoint expression and cytotoxic potential. These findings may have implications for immunotherapy and immune scoring approaches