Cytokine Production Capacity Research Articles

Multi-Omics Integration Reveals Only Minor Long-Term Molecular and Functional Sequelae in Immune Cells of Individuals Recovered From COVID-19.

The majority of COVID-19 patients experience mild to moderate disease course and recover within a few weeks. An increasing number of studies characterized the long-term changes in the specific anti-SARS-CoV-2 immune responses, but how COVID-19 shapes the innate and heterologous adaptive immune system after recovery is less well known. To comprehensively investigate the post-SARS-CoV-2 infection sequelae on the immune system, we performed a multi-omics study by integrating single-cell RNA-sequencing, single-cell ATAC-sequencing, genome-wide DNA methylation profiling, and functional validation experiments in 14 convalescent COVID-19 and 15 healthy individuals. We showed that immune responses generally recover without major sequelae after COVID-19. However, subtle differences persist at the transcriptomic level in monocytes, with downregulation of the interferon pathway, while DNA methylation also displays minor changes in convalescent COVID-19 individuals. However, these differences did not affect the cytokine production capacity of PBMCs upon different bacterial, viral, and fungal stimuli, although baseline release of IL-1Ra and IFN-γ was higher in convalescent individuals. In conclusion, we propose that despite minor differences in epigenetic and transcriptional programs, the immune system of convalescent COVID-19 patients largely recovers to the homeostatic level of healthy individuals.

Protection against severe infant lower respiratory tract infections by immune training: Mechanistic studies

Results from recent clinical studies suggest potential efficacy of immune training (IT)-based approaches for protection against severe lower respiratory tract infections in infants, but underlying mechanisms are unclear. We used systems-level analyses to elucidate IT mechanisms in infants in a clinical trial setting. Pre- and posttreatment peripheral blood mononuclear cells from a placebo-controlled trial in which winter treatment with the IT agent OM85 reduced infant respiratory infection frequency and/or duration were stimulated for 24 hours with the virus/bacteria mimics polyinosinic:polycytidylic acid/lipopolysaccharide. Transcriptomic profiling via RNA sequencing, pathway and upstream regulator analyses, and systems-level gene coexpression network analyses were used sequentially to elucidate and compare responses in treatment and placebo groups. In contrast to subtle changes in antivirus-associated polyinosinic:polycytidylic acid response profiles, the bacterial lipopolysaccharide-triggered gene coexpression network responses exhibited OM85 treatment-associated upregulation of IFN signaling. This was accompanied by network rewiring resulting in increased coordination of TLR4 expression with IFN pathway-associated genes (especially master regulator IRF7); segregation of TNF and IFN-γ (which potentially synergize to exaggerate inflammatory sequelae) into separate expression modules; and reduced size/complexity of the main proinflammatory network module (containing, eg, IL-1,IL-6, and CCL3). Finally, we observed a reduced capacity for lipopolysaccharide-induced inflammatory cytokine (eg, IL-6 and TNF) production in the OM85 group. These changes are consistent with treatment-induced enhancement of bacterial pathogen detection/clearance capabilities concomitant with enhanced capacity to regulate ensuing inflammatory response intensity and duration. We posit that IT agents exemplified by OM85 potentially protect against severe lower respiratory tract infections in infants principally by effects on innate immune responses targeting the bacterial components of the mixed respiratory viral/bacterial infections that are characteristic of this age group.

Anti-thymocyte globulin-mediated immunosenescent alterations of T cells in kidney transplant patients.

Kidney transplant (KT) is the most effective treatment for end-stage renal disease. The immunosuppressant anti-thymocyte globulin (ATG) has been applied for induction therapy to reduce the risk of acute transplant rejection for patients at high immunological risk. Despite its putative role in replicative stress during immune reconstitution, the effects of ATG on T-cell immunosenescent changes remain to be understood. Phenotypic and functional features of senescent T cells were examined by flow cytometry in 116 healthy controls (HC) and 95 KT patients for comparative analysis according to ATG treatment and CMV reactivation. The TCR repertoire was analysed in peripheral blood mononuclear cells (PBMCs) of KT patients. T cells of KT patients treated with ATG (ATG+) show typical immunosenescent features, accumulation of CD28-, CD85j+ or CD57+ T cells, and imbalance of functional T-cell subsets, compared with untreated KT patients (ATG-). Plasma IL-15 and CMV-IgG levels were higher in KT patients than in HCs, and the IL-15 level positively correlated with the frequency of CD28- Tcells in KT patients. ATG+ patients had a higher prevalence of CMV reactivation, which is associated with an increased frequency of CD28- T cells. As a result, ATG+ patients had expanded CMV-specific T cells and decreased TCR diversity. However, proliferation, cytokine-producing capacity and polyfunctionality of T cells were preserved in ATG+ patients. Our findings suggest that ATG treatment contributes to the accumulation of senescent T cells, which may have lifelong clinical implications in KT patients. Thus, these patients require long-term and comprehensive immune monitoring.

Antimicrobial Late Cornified Envelope Proteins: The Psoriasis Risk Factor Deletion of LCE3B/C Genes Affects Microbiota Composition

Late cornified envelope proteins are predominantly expressed in the skin and other cornified epithelia. On the basis of sequence similarity, this 18-member homologous gene family has been subdivided into six groups. The LCE3 proteins have been the focus of dermatological research because the combined deletion of LCE3B and LCE3C genes (LCE3B/C-del) is a risk factor for psoriasis. We previously reported that LCE3B/C-del increases the expression of the LCE3A gene and that LCE3 proteins exert antibacterial activity. In this study, we analyzed the antimicrobial properties of other family members and the role of LCE3B/C-del in the modulation of microbiota composition of the skin and oral cavity. Differences in killing efficiency and specificity between the late cornified envelope proteins and their target microbes were found, and the amino acid content rather than the order of the well-conserved central domain of the LCE3A protein was found responsible for its antibacterial activity. Invivo, LCE3B/C-del correlated with a higher beta-diversity in the skin and oral microbiota. From these results, we conclude that all late cornified envelope proteins possess antimicrobial activity. Tissue-specific and genotype-dependent antimicrobial protein profiles impact skin and oral microbiota composition, which could direct toward LCE3B/C-del‒associated dysbiosis and a possible role for microbiota in the pathophysiology of psoriasis.

Extracellular Lactate Acts as a Metabolic Checkpoint and Shapes Monocyte Function Time Dependently.

Elevated blood lactate levels are frequently found in critically ill patients and thought to result from tissue hypoperfusion and cellular oxygen shortage. Considering the close relationship between immune cell function and intracellular metabolism, lactate is more than a glycolytic waste molecule but able to regulate the immune response. Our aim was to elucidate the temporal and mechanistic effect of extracellular lactate on monocytes. To this end, primary human monocytes and the human monocytic cell line MonoMac6 were stimulated with various toll-like-receptor agonists after priming with Na-L-lactate under constant pH conditions. As readout, cytokine production was measured, real-time assessment of intracellular energy pathways was performed, and intracellular metabolite concentrations were determined. Irrespective of the immunogenic stimulus, short-term Na-lactate-priming strongly reduced cytokine production capacity. Lactate and hexoses accumulated intracellularly and, together with a decreased glycolytic flux, indicate a lactate-triggered impairment of glycolysis. To counteract intracellular hyperglycemia, glucose is shunted into the branching polyol pathway, leading to sorbitol accumulation. In contrast, long-term priming with Na-L-lactate induced cellular adaption and abolished the suppressive effect. This lactate tolerance is characterized by a decreased cellular respiration due to a reduced complex-I activity. Our results indicate that exogenous lactate shapes monocyte function by altering the intracellular energy metabolism and acts as a metabolic checkpoint of monocyte activation.

Repurposing Bi-Specific Chimeric Antigen Receptor (CAR) Approach to Enhance CAR T Cell Activity Against Low Antigen Density Tumors

BACKGROUNDChimeric antigen receptor (CAR) T cell therapy has revolutionized the treatment of relapsed/refractory B-cell malignancies, as highlighted by high complete remission rates and FDA approval of CD19-specific CAR T cell products. However, depth and duration of remission are limited by antigen loss/downregulation on tumors, as observed in clinical trials using CAR T cells targeting the CD19 or CD22 in leukemia and lymphoma, BCMA in multiple myeloma, and EGFRvIII in glioblastoma. This observation forms the basis of current efforts to develop multi-targeting CAR T cells to prevent antigen-negative escape. Antigen density is an important factor modulating CAR T cell response, since antigen expression below a certain threshold fails to trigger the full range of T cell functions. Given that signal strength induced upon antigen encounter determines CAR T cell activity, we hypothesized that simultaneous targeting of two dimly-expressed antigens will result in enhanced CAR T cell signaling and anti-tumor function, approaching that seen in response to one highly-expressed antigen. This is important given the heterogeneity of antigen expression in various cancers. Therefore, the bi-specific CAR T cells currently being developed to prevent antigen-negative escape could also be used to enhance efficacy against low antigen density (LAD) tumors. Results from this study will provide a novel rationale for using multi-specific CAR T cells and illuminate the mechanisms of successful CAR T cell therapy.METHODSLentivirus transduction was performed to generate CAR T cells from healthy human T cells, using second generation 4-1BBz CARs specific for either human CD19 or CD22, or both in cis, herein referred to as CAR19, CAR22, or CAR19/22, respectively (Figure 1A). For in vitro functional characterization, we performed co-culture assay of T cells and B cell leukemia cell line NALM6, which is known to express high levels of both CD19 and CD22. To assess T cell function against LAD tumor cells, primary patients' B-ALL samples expressing low antigen density in comparison to the NALM6 cell line were used (Figure 1B). CAR T cell anti-tumor potency was determined by assessing CAR T cell cytotoxicity and cytokine production. For in vivo therapeutic study, primary patients' B-ALL samples with dimly expressed CD19 and CD22 were used to evaluate and compare the therapeutic efficacy of mono- versus bi-specific CAR T cells. Additionally, we generated a LAD tumor model by deleting the highly expressed CD19 and CD22 from the ALL cell line NALM6 using CRISPR/Cas9, transducing the now antigen-negative cell line with CD19 and CD22, followed by single cell cloning to generate a cell line expressing low antigen density for both the CD19 and CD22. We engrafted tumor cells in NSG mice, followed by administration of CAR19, CAR22, CAR19/22 or untransduced T cells. Therapeutic efficacy was assessed by measuring tumor burden using either flow cytometry or bioluminescent imaging.RESULTSCytotoxicity assay revealed that the bi-specific CAR19/22 T cells killed tumor cells more rapidly than CAR19 or CAR22 T cells. Further, compared to mono-specific CAR T cells, the bi-specific CAR19/22 T cells produced significantly more pro-inflammatory cytokines including IL-2 and IFNg, in response to stimulation with LAD primary samples or NALM6 cells. This increased cytokine-producing capacity compared to mono-specific CAR T cells was maintained following repeated antigen stimulation when in vitro exhaustion assay was performed. In vivo, enhanced tumor elimination was observed in mice receiving bi-specific CAR19/22 T cells compared to either of the mono-specific CAR T cells, in both low antigen density primary ALL and NALM6 tumor models. This translated to increased survival rates seen in mice treated with the bi-specific CAR19/22 T cells (Figure 1C-D).CONCLUSIONSHere we showed that bi-specific CAR19/22 T cells are superior to mono-specific CAR19 or CAR22 T cells, not only against LAD tumors but also tumor cells expressing high antigen density, NALM6. This was demonstrated by their enhanced cytokine-producing function, cytotoxic capacity, and therapeutic efficacy in vivo. Results from this study provide a novel rationale for repurposing multi-specific CAR T cells as a strategy to improve efficacy against LAD tumors, in addition to the recognized benefit of reducing antigen-negative escape. [Display omitted] DisclosuresShestova: Hemogenyx Pharmaceuticals LLC: Research Funding. Grupp: Novartis, Roche, GSK, Humanigen, CBMG, Eureka, and Janssen/JnJ: Consultancy; Novartis, Kite, Vertex, and Servier: Research Funding; Novartis, Adaptimmune, TCR2, Cellectis, Juno, Vertex, Allogene and Cabaletta: Other: Study steering committees or scientific advisory boards; Jazz Pharmaceuticals: Consultancy, Other: Steering committee, Research Funding. Ruella: viTToria biotherapeutics: Research Funding; Novartis: Patents & Royalties; BMS, BAYER, GSK: Consultancy; AbClon: Consultancy, Research Funding; Tmunity: Patents & Royalties. Gill: Novartis: Other: licensed intellectual property, Research Funding; Interius Biotherapeutics: Current holder of stock options in a privately-held company, Research Funding; Carisma Therapeutics: Current holder of stock options in a privately-held company, Research Funding.

Adipose tissue induces trained innate immunity in patients with obesity

Abstract Introduction Obesity is the most prevalent modifiable risk factor for atherosclerotic cardiovascular disease and is characterized as a chronic inflammatory disease. Cells of the innate immune system, especially monocytes and macrophages, play a pivotal role in the various stages of atherosclerosis, although it still remains elusive why the strong inflammatory response persists in time. We recently showed that cells of the innate immune system, such as monocytes, can adopt a long-term immunological memory. Upon brief stimulation with atherogenic stimuli, monocytes demonstrate an enhanced long-term pro-inflammatory and pro-atherogenic phenotype. This is termed trained immunity and is mediated via epigenetic and metabolic reprogramming. The clinical relevance of these findings was verified in patients with symptomatic atherosclerosis, in which circulating monocytes showed a trained immune phenotype. Purpose As various adipose tissue-related particles, including pro-inflammatory cytokines and fatty acids, are capable of inducing trained immunity in vitro, we hypothesized that adipose tissue from obese subjects might induce training in peripheral monocytes, thereby contributing to the increased risk of atherosclerotic CVD in these patients. In line with this hypothesis, it is unclear whether chronic inflammation sustains after a previous period of obesity despite significant weight loss. Methods We obtained blood from 25 patients with obesity before and 6 months after bariatric surgery. Monocyte subsets and activation phenotype were studied using flow cytometry. Cytokine production capacity of isolated PBMCs was studied after ex vivo stimulation with several infectious and metabolic stimuli and we characterized isolated monocytes using transcriptomics. Next, we obtained visceral (VAT) and subcutaneous adipose tissue (SAT) biopsies from 10 patients. Using our established in vitro model for trained immunity, we co-incubated healthy human monocytes with the adipose tissue biopsies for 24 hours in a trans-well set-up. After 24 hours, the adipose tissue was removed and monocytes were rested. On day 6, the cells were re-stimulated for 24 hours with a second stimulus and cytokine production and the transcriptome of the macrophages was analyzed. Results Both SAT and VAT obtained from patients with obesity can induce a long-term memory in healthy human monocytes, as demonstrated by an increased cytokine production capacity 6 days after co-incubation. Interestingly, VAT induced a higher cytokine response compared to SAT. Analysis of the inflammatory phenotype of peripheral cells before and after bariatric surgery is currently ongoing. Conclusions Adipose tissue-secreted metabolites, particularly secreted by VAT, have the potential to induce persistent innate immune cell activation. Our further analyses will show whether the secretion of these molecules and the activation of the innate immune system persists upon weight loss. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Dutch Heart Foundation

Functional and transcriptional differences in monocytes from children with obesity compared to children of healthy weight

Abstract Background and aim Cardiometabolic risk accrues across the entire life course and childhood is a key epoch for effective prevention. Obesity in childhood is the most prevalent modifiable risk factor for later cardiovascular disease (CVD). Inflammatory biomarkers and innate immune capacity are increased in adults with obesity, but childhood data are scarce. We aimed to investigate (i) innate immune cell activation in children with and without obesity; and (ii) whether weight loss impacts the innate immune inflammatory phenotype. Methods The innate immune phenotype of Peripheral Blood Mononuclear Cells (PBMCs) from 31 children with obesity (BMI z-score>2.5) and 22 children of healthy weight (−1.5≤BMIz≤1.5, sex, age and pubertal stage matched) was characterized by high dimensional flow cytometry, ex vivo stimulation assays with subsequent 27-plex cytokine measurements, and transcriptome analysis using RNA sequencing (Figure 1). Children with obesity participated to the Royal Children's Hospital Weight Management Service (median 5 years) and at follow-up, PBMCs were obtained again as well as anthropometric data and subclinical cardiovascular phenotypes. Results Flow cytometric analysis showed marked differences in cell composition between children with obesity and children of healthy weight. Specifically, children with obesity have significant changes in monocyte subsets and an increased expression of monocyte activation markers. Upon stimulation, monocytes of children with obesity show an increased cytokine production capacity. Finally, transcriptomic analysis shows significant differences between monocytes from obese children and healthy controls. Effects of weight loss on these immune parameters and correlations with preclinical CVD phenotypes are currently being analysed. Conclusions Monocytes from children with obesity have a pro-inflammatory phenotype compared to children of normal weight. Heightened inflammation may contribute to increased CVD risk later in life and may offer opportunities for early intervention. Funding Acknowledgement Type of funding sources: Other. Main funding source(s): Dutch Scientific Organisation (NWO) - Rubicon grant to S.B. Dutch Heart Foundation - CVON IN CONTROL II to N.P.R. and D.B. Figure 1. Schematical overview of study

Characteristics of mucosal-associated invariant T cells and their roles in immune diseases.

Mucosal-associated invariant T (MAIT) cells are a subset of innate-like T cells that express a semi-invariant T-cell receptor and are restricted by the major histocompatibility complex class I-related molecule 1 (MR1). MAIT cells recognize biosynthetic derivatives of the riboflavin synthesis pathway present in microbes. MAIT cells have attracted increased interest related to various immune responses because of their unique features including their abundance in humans, non-peptidic antigens and ability to respond to antigenic and non-antigenic stimuli. The numbers of circulating MAIT cells are decreased in many immune diseases such as multiple sclerosis, systemic lupus erythematosus and inflammatory bowel diseases. However, the remaining MAIT cells have an increased cytokine-producing capacity and activated status, which are related to disease activity. Additionally, MAIT cells have been observed at sites of inflammation including the kidneys, synovial fluid and intestinal mucosa. These findings suggest their involvement in the pathogenesis of immune diseases. In this mini-review, we summarize the recent findings of MAIT cells in human immune diseases and animal models, and discuss their role and potential as a therapeutic target.

Evolution of cytokine production capacity in ancient and modern European populations.

As our ancestors migrated throughout different continents, natural selection increased the presence of alleles advantageous in the new environments. Heritable variations that alter the susceptibility to diseases vary with the historical period, the virulence of the infections, and their geographical spread. In this study we built polygenic scores for heritable traits that influence the genetic adaptation in the production of cytokines and immune-mediated disorders, including infectious, inflammatory, and autoimmune diseases, and applied them to the genomes of several ancient European populations. We observed that the advent of the Neolithic was a turning point for immune-mediated traits in Europeans, favoring those alleles linked with the development of tolerance against intracellular pathogens and promoting inflammatory responses against extracellular microbes. These evolutionary patterns are also associated with an increased presence of traits related to inflammatory and auto-immune diseases.

Targeted knockdown of Tim3 by short hairpin RNAs improves the function of anti-mesothelin CAR T cells

T-cell immunoglobulin mucin 3 (Tim3) is an immune checkpoint receptor that plays a central role in chimeric antigen receptor (CAR) T cell exhaustion within the tumor microenvironment. This study was aimed to evaluate the effects of targeted-knockdown of Tim3 on the antitumor function of anti-mesothelin (MSLN)-CAR T cells. To knockdown Tim3 expression, three different shRNA sequences specific to different segments of the human Tim3 gene were designed and co-inserted with an anti-MSLN-CAR transgene into lentiviral vectors. To investigate the efficacy of Tim3 targeting in T cells, expression of Tim3 was assessed before and after antigen stimulation. Afterwards, cytotoxic effects, proliferative response and cytokine production of MSLN-CAR T cells and Tim3-targeted MSLN-CAR T cells were analyzed. Our results showed that activation of T cells and MSLN-CAR T cells led to up-regulation of Tim3. Tim3 knockdown significantly decreased its expression in different groups of MSLN-CAR T cells. Tim3 knockdown significantly improved cytotoxic function, cytokine production and proliferation capacity of MSLN-CAR T cells. Our findings indicate that targeted knockdown of Tim3 allows tumor-infiltrating CAR T cells that would otherwise be inactivated to continue to expand and carry out effector functions, thereby altering the tumor microenvironment from immunosuppressive to immunosupportive via mitigated Tim3 signaling.

An epigenetic and transcriptomic signature of immune tolerance in human monocytes through multi-omics integration

BackgroundThe plasticity of monocytes enables them to exert multiple roles during an immune response, including promoting immune tolerance. How monocytes alter their functions to convey immune tolerance in the context of lower respiratory tract infections in humans is not well understood. Here, we sought to identify epigenetic and transcriptomic features of cytokine production capacity in circulating monocytes during community-acquired pneumonia (CAP).MethodsCirculating CD14+ monocytes were obtained from the blood of CAP patients included in a longitudinal, observational cohort study, on hospitalization (acute stage, n=75), and from the same patients after a 1-month follow-up (recovery stage, n=56). Age and sex-matched non-infectious participants were included as controls (n=41). Ex vivo cytokine production after lipopolysaccharide (LPS) exposure was assessed by multiplex assay. Transcriptomes of circulating monocytes were generated by RNA-sequencing, and DNA methylation levels in the same monocytes were measured by reduced representation bisulfite sequencing. Data were integrated by fitting projection-to-latent-structure models, and signatures derived by partial least squares discrimination.ResultsMonocytes captured during the acute stage exhibited impaired TNF, IL-1β, IL-6, and IL-10 production after ex vivo stimulation with LPS, relative to controls. IL-6 production was not resolved in recovery monocytes. Multivariate analysis of RNA-sequencing data identified 2938 significantly altered RNA transcripts in acute-stage monocytes (fold expression ≤−1.5 or ≥1.5; adjusted p ≤ 0.01), relative to controls. Comparing DNA methylation levels in circulating monocytes of CAP patients to controls revealed minimal differences, specifically in DNAse hypersensitive sites (HS) of acute-stage monocytes. Data integration identified a cholesterol biosynthesis gene signature and DNAse HS axis of IL-1β and IL-10 production (R2 =0.51).ConclusionsCirculating monocytes obtained from CAP patients during the acute stage exhibited impaired cytokine production capacities, indicative of reprogramming to a state of immune tolerance, which was not fully resolved after 1 month. Our split-sample study showed that 51% of the immune tolerance phenotype can be explained, at least in part, by coordinated shifts in cholesterol biosynthesis gene expression and DNAse HS methylation levels. A multi-scale model identified an epigenetic and transcriptomic signature of immune tolerance in monocytes, with implications for future interventions in immunosuppression.Trial registrationNCT number NCT02928367

An Explorative Study on Monocyte Reprogramming in the Context of Periodontitis In Vitro and In Vivo.

AimsPeriodontitis is an independent risk factor for cardiovascular disease, but the mechanistic link is not fully understood. In atherosclerotic cardiovascular disease, monocytes can adopt a persistent hyperresponsive phenotype, termed trained immunity. We hypothesized that periodontitis-associated bacteria can induce trained immunity in monocytes, which subsequently accelerate atherosclerosis development.Materials and MethodsWe combined in vitro experiments on human primary monocytes and in vivo techniques in patients with periodontitis to test this hypothesis. Adherent peripheral blood mononuclear cells (PBMCs) were transiently exposed in vitro to Porphyromonas gingivalis for 24 hours, and restimulated with lipopolysaccharide (LPS) or Pam3CysK4 (P3C) six days later, to measure interleukin-6 (IL-6) and tumor necrosis factor α (TNFα) production. In an exploratory observational study, patients with severe periodontitis (63 ± 6 years, n=14) and control subjects with no-to-mild periodontitis (54 ± 10 years, n=14) underwent venipuncture and 2’-deoxy-2’-[18F]fluoro-D-glucose positron-emission-tomography ([18F]FDG PET/CT) scanning.ResultsWhen adherent peripheral blood mononuclear cells (PBMCs) were transiently exposed in vitro to Porphyromonas gingivalis for 24 hours, and restimulated with LPS or P3C six days later, IL-6 and TNFα production was significantly increased (TNFα/P3C, p<0.01). Circulating leukocytes, IL-6 and interleukin-1 receptor antagonist (IL-1Ra) concentrations were generally higher in patients compared to controls (leukocytes: p<0.01; IL-6: p=0.08; IL-1Ra: p=0.10). Cytokine production capacity in PBMCs after 24h stimulation revealed no differences between groups. [18F]FDG PET/CT imaging showed a trend for increased [18F]FDG-uptake in the periodontium [mean standard uptake value (SUVmean), p=0.11] and in femur bone marrow (SUVmean, p=0.06), but no differences were observed for vascular inflammation. Positive correlations between severity of periodontitis, measured by The Dutch Periodontal Screening Index and pocket depth, with circulating inflammatory markers and tissue inflammation were found.ConclusionsP. gingivalis induces long-term activation of human monocytes in vitro (trained immunity). Patients with severe periodontitis did have signs of increased systemic inflammation and hematopoietic tissue activation. However, their circulating monocytes did not show a hyperresponsive phenotype. Together we suggest that trained immunity might contribute to local periodontal inflammation which warrants further investigation.

Postnatal inflammation in ApoE-/- mice is associated with immune training and atherosclerosis.

Preterm birth is associated with increased risk of cardiovascular disease (CVD). This may reflect a legacy of inflammatory exposures such as chorioamnionitis which complicate pregnancies delivering preterm, or recurrent early-life infections, which are common in preterm infants. We previously reported that experimental chorioamnionitis followed by postnatal inflammation has additive and deleterious effects on atherosclerosis in ApoE-/- mice. Here, we aimed to investigate whether innate immune training is a contributory inflammatory mechanism in this murine model of atherosclerosis. Bone marrow-derived macrophages and peritoneal macrophages were isolated from 13-week-old ApoE-/- mice, previously exposed to prenatal intra-amniotic (experimental choriomanionitis) and/or repeated postnatal (peritoneal) lipopolysaccharide (LPS). Innate immune responses were assessed by cytokine responses following ex vivo stimulation with toll-like receptor (TLR) agonists (LPS, Pam3Cys) and RPMI for 24-h. Bone marrow progenitor populations were studied using flow cytometric analysis. Following postnatal LPS exposure, bone marrow-derived macrophages and peritoneal macrophages produced more pro-inflammatory cytokines following TLR stimulation than those from saline-treated controls, characteristic of a trained phenotype. Cytokine production ex vivo correlated with atherosclerosis severity in vivo. Prenatal LPS did not affect cytokine production capacity. Combined prenatal and postnatal LPS exposure was associated with a reduction in populations of myeloid progenitor cells in the bone marrow. Postnatal inflammation results in a trained phenotype in atherosclerosis-prone mice that is not enhanced by prenatal inflammation. If analogous mechanisms occur in humans, then there may be novel early life opportunities to reduce CVD risk in infants with early life infections.

The Roles of Kidney-Resident ILC2 in Renal Inflammation and Fibrosis.

Innate lymphoid cells (ILCs) are a recently discovered lymphocyte population with high cytokine productive capacity. Type-2 ILCs (ILC2s) are the most studied, and they exert a rapid type-2 immune response to eliminate helminth infections. Massive and sustainable ILC2 activation induces allergic tissue inflammation, so it is important to maintain correct ILC2 activity for immune homeostasis. The ILC2-activating cytokine IL-33 is released from epithelial cells upon tissue damage, and it is upregulated in various kidney disease mouse models and in kidney disease patients. Various kidney diseases eventually lead to renal fibrosis, which is a common pathway leading to end-stage renal disease and is a chronic kidney disease symptom. The progression of renal fibrosis is affected by the innate immune system, including renal-resident ILC2s; however, the roles of ILC2s in renal fibrosis are not well understood. In this review, we summarize renal ILC2 function and characterization in various kidney diseases and highlight the known and potential contributions of ILC2s to kidney fibrosis.

Contrasting functional responses of resident Kupffer cells and recruited liver macrophages to irradiation and liver X receptor stimulation.

In the murine liver, there are two major macrophage populations, namely resident Kupffer cells (resKCs) with phagocytic activity and recruited macrophages (recMφs) with cytokine-producing capacity. This study was performed to clarify the functional differences between these two populations, focusing on their susceptibility to radiation and response to stimulation via liver X receptors (LXRs), which are implicated in cholesterol metabolism and immune regulation. Liver mononuclear cells (MNCs) were obtained from C57BL/6 (WT) mice with or without 2 Gy irradiation, and the phagocytic activity against Escherichia coli (E. coli) as well as TNF-α production were compared between the two macrophage populations. To assess LXR functions, phagocytosis, TNF-α production, and endocytosis of acetylated low-density lipoprotein (LDL) were compared after synthetic LXR ligand stimulation. Furthermore, LXRα/β knockout (KO) mice and LXRα KO mice were compared with WT mice. Irradiation decreased intracellular TNF-α production by recMφs but did not affect the phagocytic activity of resKCs. In vitro LXR stimulation enhanced E. coli phagocytosis by resKCs but decreased E. coli-stimulated TNF-α production by recMφs. Phagocytic activity and acetylated LDL endocytosis were decreased in both LXRα/β KO mice and LXRα KO mice, with serum TNF-α levels after E. coli injection in the former being higher than those in WT mice. In conclusion, resKCs and recMφs exhibited different functional features in response to radiation and LXR stimulation, highlighting their distinct roles liver immunity and lipid metabolism.

Seasonal and Nonseasonal Longitudinal Variation of Immune Function.

Different components of the immune response show large variability between individuals, but they also vary within the same individual because of host and environmental factors. In this study, we report an extensive analysis of the immune characteristics of 56 individuals over four timepoints in 1 single year as part of the Human Functional Genomics Project. We characterized 102 cell subsets using flow cytometry; quantified production of eight cytokines and two chemokines in response to 20 metabolic, bacterial, fungal, and viral stimuli; and measured circulating markers of inflammation. Taking advantage of the longitudinal sampling, both seasonal and nonseasonal sources of variability were studied. The circulating markers of inflammation IL-18, IL-18 binding protein, and resistin displayed clear seasonal variability, whereas the strongest effect was observed for α-1 antitrypsin. Cytokine production capacity also showed strong seasonal changes, especially after stimulation with the influenza virus, Borrelia burgdorferi, and Escherichia coli Furthermore, we observed moderate seasonality effects on immune cell counts, especially in several CD4+/CD8+ T cell subpopulations. Age of the volunteers was an important factor influencing IFN-γ and IL-22 production, which matched the strong impact of age on several T cell subsets. Finally, on average, genetics accounted for almost 50% of the interindividual variance not already explained by age, sex, and body mass index, although this varies strongly for different parameters. In conclusion, seasonality is an important environmental factor that influences immune responses, in addition to specific genetic and nongenetic host factors, and this may well explain the seasonal variation in the incidence and severity of immune-mediated diseases.

Features of interleukin-19 production in patients with atherosclerosis

Objective. To study the features of local and systemic production of interleukin-19 in patients with atherosclerosis.Material and Methods. The study comprised a total of 46 patients (26 women and 20 men) treated for arterial hypertension in the therapeutic department of Republican Clinical Hospital named after G.Y. Remishevskaya. The mean age of subjects was 63.4 ± 3.2 years. The control group included 40 patients (23 women and 17 men aged 44.7 ± 5.5 years) who did not have atherosclerosis. Samples of atherosclerotic plaques and venous blood were examined. Atherosclerotic plaques were obtained by endarterectomy and then subjected to homogenization followed by enzymatic hydrolysis for 1 h at 37 °C with collagenase IV in the presence of proteinase III inhibitors. The serum levels of cytokines (in the control group and in patients with atherosclerosis) and in the atherosclerotic plaque homogenate (in patients with atherosclerosis) were determined by ELISA. To assess the cytokine-producing capacity of blood leukocytes and white blood cells isolated from atherosclerotic plaques, spontaneous and phytohemagglutinin (PHA)-induced cytokine production was determined when the cells were cultured in RPMI-1640.Results. The serum levels of IL-19 did not significantly differ between the patients with atherosclerosis and the control group. A statistically significant two-fold increase in the spontaneous expression of IL-19 by blood leukocytes was observed in the group of patients with atherosclerosis in comparison with the control group. When comparing the contents of IL-19 in blood serum and atherosclerotic plaque homogenate in patients with atherosclerosis, no statistically significant differences were found (p = 0.182). The level of PHA-induced IL-19 production by the atherosclerotic plaque white blood cells was significantly lower than that of blood leucocytes.Conclusion. The study showed that the reserve capacity for IL-19 synthesis in the atherosclerotic plaque white blood cells decreases leading to the progression of inflammation. The obtained results suggest that IL-19 plays the anti-atherogenic role and its production is involved in the maintaining the mechanisms for down-regulation of inflammation in atherosclerotic plaques.

Abstract LB158: Implications of Targeting Novel Pathways in Exhausted T Cells for Immuno-Oncology Therapeutic Intervention

Abstract The induction of CD8+ T cell exhaustion, driven by the tumor microenvironment, is thought to be a major mechanism by which tumors can escape immune surveillance and are able to persist and progress. T cell exhaustion is typically characterized by the expression of co-inhibitory markers such as PD-1, LAG-3 and TIM-3 and a reduced capacity for pro-inflammatory cytokine production. Whilst treatment with therapeutics targeted at checkpoint inhibitors, such as Nivolumab (anti-PD-1), have shown great promise in the treatment of a subset of individuals and tumor types, resistance to these approaches is prevalent. As such, novel approaches are required in order to provide alternative options for the rescue of T cell exhaustion and cancer treatment. Here we describe the characteristics of an in vitro human T cell exhaustion assay whereby in vitro stimulated T cells phenotypically and functionally recapitulate the exhausted T cells found within the tumor microenvironment and where exhaustion can be partially rescued by treatment with anti-PD-1. Furthermore, we demonstrate the effect of treatment with an inhibitor of a novel target identified in exhausted T cells on T cell function; both through analysis of pro-inflammatory cytokine production and the ability to kill tumor cells. These assays and approaches enable investigation into the ability of compounds to influence reversal of T cell exhaustion where anti-PD-1 treatment does not fully reverse the exhausted phenotype and offers the ability to test combination therapy approaches. Citation Format: Rhoanne McPherson, Francis Acklam, Joanne Hay, Mark Barbour, Courtney Grant, Preeti Singh, Adriana Gambardella, Justyna Rzepecka, Stephen Anderton, Hayley Gooding. Implications of Targeting Novel Pathways in Exhausted T Cells for Immuno-Oncology Therapeutic Intervention [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB158.

Abstract 115: Neutrophil extracellular traps express PD-L1 which promotes T cell exhaustion in the tumor microenvironment

Abstract Neutrophil extracellular traps (NETs) have recently been implicated in the growth of tumors, however their impact on immune cells within the tumor microenvironment (TME) remain incompletely understood. We hypothesize that NETs can directly interact with infiltrating T cells to promote an immunosuppressive TME. We performed liver Ischemia/Reperfusion (I/R) in an established colorectal cancer metastasis model to induce NET formation in the TME. In this NET-rich TME we discovered elevated levels of phenotypically and functionally exhausted T cells. We demonstrated that the majority of CD4 and CD8 T cells are not effector cells, but rather express multiple inhibitory receptors and are highly dysfunctional, with reduced cytokine production and proliferative capacity. This effect was limited to the T cells present the the NET-rich TME in comparison to T cells isolated from the spleen. In vitro, NETs contained the immunosuppressive ligand PD-L1 responsible for T cell exhaustion; an effect abrogated by using PD-L1 KO NETs. This exhausted state was directly linked to mitochondrial dysfunction in the T cells. Furthermore, NETs were targeted in vivo by treating mice with DNAse or anti-PD-L1 at time of I/R. Treatment caused decreased metastatic burden, decreased NET formation and higher levels of functioning non-exhausted T cells. Conversely, injecting NETs in tumors led to acceleration of tumor growth and T cell exhaustion in the TME. In addition, we measured sPDL-1 and MPO-DNA, a NET marker, in the serum of patients undergoing surgery for colorectal liver metastases resection. High levels of both markers were significantly associated with decreased disease free survival. Neutrophils isolated from patients after surgery were primed to form NETs that caused a similar exhaustion and dysfunction of human CD4 and CD8 T cells. Our data thus reveal that NETs have the capability of suppressing T cell responses and promoting tumor growth. This mechanism of T cell suppression by NETs represents a viable target for sustaining immune competence within the TME. Citation Format: Christof Kaltenmeier, Hamza Yazdani, Samer Tohme. Neutrophil extracellular traps express PD-L1 which promotes T cell exhaustion in the tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 115.