Epstein-Barr virus (EBV) is an important environmental risk factor in the development of several autoimmune conditions, with the mechanisms still to be fully elucidated. EBV primarily infects memory B cells, transitioning between lytic (active) and latent (dormant) phases of infection. Our group has previously proposed two molecular mechanisms linking EBV pathogenesis to autoimmunity: one indicates that EBV lytic switching contributes to systemic lupus erythematosus (SLE) pathogenesis, while another posits that latency III is more crucial in the development of multiple sclerosis (MS). In this study, we tested the proposed molecular model using a cohort of EBV-transformed lymphoblastoid cell lines derived from individuals with either SLE, MS or healthy controls. Measuring the expression levels of a panel of EBV genes, representing the different phases of the EBV lifecycle, we found compelling proof-of-concept evidence validating our proposed model. This discovery highlights promising signatures for further investigation, where the same approach can be explored across other EBV-associated immune conditions, deepening our understanding of the virus's lifecycle dysregulation in autoimmunity etiology and ultimately aiding in the design of new treatments.

Background: Microbial keratitis is one of the leading causes of blindness globally. An overactive immune response during an infection can exacerbate damage, causing corneal opacities and vision loss. This study aimed to identify the differentially expressed genes between corneal infection patients and healthy volunteers within the cornea and conjunctiva and elucidate the contributing pathways to these conditions' pathogenesis. Moreover, it compared the corneal and conjunctival transcriptomes in corneal-infected patients to cytokine levels in tears. Methods: Corneal and conjunctival swabs were collected from seven corneal infection patients and three healthy controls under topical anesthesia. RNA from seven corneal infection patients and three healthy volunteers were analyzed by RNA sequencing (RNA-Seq). Tear proteins were extracted from Schirmer strips via acetone precipitation from 38 cases of corneal infection and 14 healthy controls. The cytokines and chemokines IL-1 beta, IL-6, CXCL8 (IL-8), CX3CL1, IL-10, IL-12 (p70), IL-17A, and IL-23 were measured using an antibody bead assay. Results: A total of 512 genes were found to be differentially expressed in infected corneas compared to healthy corneas, with 508 being upregulated and four downregulated (fold-change (FC) <-2 or > 2 and adjusted p <0.01). For the conjunctiva, 477 were upregulated, and 3 were downregulated (FC <-3 or >= 3 and adjusted p <0.01). There was a significant overlap in cornea and conjunctiva gene expression in patients with corneal infections. The genes were predominantly associated with immune response, regulation of angiogenesis, and apoptotic signaling pathways. The most highly upregulated gene was CXCL8 (which codes for IL-8 protein). In patients with corneal infections, the concentration of IL-8 protein in tears was relatively higher in patients compared to healthy controls but did not show statistical significance. Conclusions: During corneal infection, many genes were upregulated, with most of them being associated with immune response, regulation of angiogenesis, and apoptotic signaling. The findings may facilitate the development of treatments for corneal infections that can dampen specific aspects of the immune response to reduce scarring and preserve sight.

Direct acting antiviral therapies rapidly clear chronic hepatitis C virus (HCV) infection and restore natural killer (NK) cell function. We investigated NK-cell memory formation following HCV clearance by examining NK-cell phenotype and responses from control and chronic HCV patients before and after therapy following sustained virologic response at 12 weeks post therapy (SVR12). NK-cell phenotype at SVR12 differed significantly from paired pretreatment samples, with an increase in maturation markers CD16, CD57, and KLRG1. HCV patients possessed stronger cytotoxic responses against HCV-infected cells as compared to healthy controls; a response that further increased following SVR12. The antigen-specific response was mediated by KLRG1(+) NK cells, as demonstrated by increased degranulation and proliferation in response to HCV antigen only. Our data suggest that KLRG1(+) HCV-specific memory NK cells develop following viral infection, providing insight into their role in HCV clearance and relevance with regard to vaccine design.

BACKGROUND & AIMS:Natural killer (NK) cells are primary innate effector cells that play an important role in the control of human viral infections. During chronic viral infection, NK cells undergo significant changes in phenotype, function and subset distribution, including the appearance of CD56-CD16+ (CD56-) NK cells, previously identified in chronic human immunodeficiency virus (HIV) and hepatitis C virus infection. However, the presence of CD56- NK cells in the pathogenesis of chronic hepatitis B (CHB) remains unknown.METHODS:Phenotype and function of CD56- NK cells from patients with CHB (n = 28) were assessed using flow cytometry and in vitro stimulation with HBV antigen.RESULTS:CHB patients had a higher frequency of CD56- NK cells compared to healthy controls in peripheral blood (6.2% vs 1.4%, P < .0001). Compared to CD56+ NK cells, CD56- NK cells had increased expression of inhibitory receptors, and reduced expression of activating receptors, as measured by MFI and qPCR. CD56- NK cells were less responsive to target cell and cytokine stimulation compared to their CD56+ counterparts. In addition, CD56- NK cells demonstrated defective dendritic cells (DCs) interactions resulting in reduced DCs maturation, lower expression of NK CD69 and impaired capacity of NK cells to eliminate immature DCs in co-culture studies. Finally, frequency of CD56- NK cells was positively correlated with serum HBV DNA levels.CONCLUSION:Chronic HBV infection induces the expansion of highly dysfunctional of CD56- NK cells that likely contribute to inefficient innate and adaptive antiviral immune response in chronic HBV infection.

Although genetic and epidemiological evidence indicates vitamin D insufficiency contributes to multiple sclerosis (MS), and serum levels of vitamin D increase on treatment with cholecalciferol, recent metanalyses indicate that this vitamin D form does not ameliorate disease. Genetic variation in genes regulating vitamin D, and regulated by vitamin D, affect MS risk. We evaluated if the expression of vitamin D responsive MS risk genes could be used to assess vitamin D response in immune cells. Peripheral blood mononuclear cells (PBMCs) were isolated from healthy controls and people with MS treated with dimethyl fumarate. We assayed changes in expression of vitamin D responsive MS risk (VDRMS) genes in response to treatment with 25 hydroxy vitamin D in the presence or absence of inflammatory stimuli. Expression of CYP24A1 and other VDRMS genes was significantly altered in PBMCs treated with vitamin D in the homeostatic and inflammatory models. Gene expression in MS samples had similar responses to controls, but lower initial expression of the risk genes. Vitamin D treatment abrogated these differences. Expression of CYP24A1 and other MS risk genes in blood immune cells indicate vitamin D response and could enable assessment of immunological response to vitamin D in clinical trials and on therapy.

Multiple Sclerosis (MS) is a complex immune-mediated disease of the central nervous system. Treatment is based on immunomodulation, including specifically targeting B cells. B cells are the main host for the Epstein-Barr Virus (EBV), which has been described as necessary for MS development. Over 200 genetic loci have been identified as increasing susceptibility to MS. Many MS risk genes have altered expression in EBV infected B cells, dependent on the risk genotype, and are themselves regulated by the EBV transcription factor EBNA2. Females are 2-3 times more likely to develop MS than males. We investigated if MS risk loci might mediate the gender imbalance in MS. From a large public dataset, we identified gender-specific associations with EBV traits, and MS risk SNP/gene pairs with gender differences in their associations with gene expression. Some of these genes also showed gender differences in correlation of gene expression level with Estrogen Receptor 2. To test if estrogens may drive these gender specific differences, we cultured EBV infected B cells (lymphoblastoid cell lines, LCLs), in medium depleted of serum to remove the effects of sex hormones as well as the estrogenic effect of phenol red, and then supplemented with estrogen (100 nM estradiol). Estradiol treatment altered MS risk gene expression, LCL proliferation rate, EBV DNA copy number and EBNA2 expression in a sex-dependent manner. Together, these data indicate that there are estrogen-mediated gender-specific differences in MS risk gene expression and EBV functions. This may in turn contribute to gender differences in host response to EBV and to MS susceptibility.

Although the causes of Multiple Sclerosis (MS) still remain largely unknown, multiple lines of evidence suggest that Epstein–Barr virus (EBV) infection may contribute to the development of MS. Here, we aimed to identify the potential contribution of EBV-encoded and host cellular miRNAs to MS pathogenesis. We identified differentially expressed host miRNAs in EBV infected B cells (LCLs) and putative host/EBV miRNA interactions with MS risk loci. We estimated the genotype effect of MS risk loci on the identified putative miRNA:mRNA interactions in silico. We found that the protective allele of MS risk SNP rs4808760 reduces the expression of hsa-mir-3188-3p. In addition, our analysis suggests that hsa-let-7b-5p may interact with ZC3HAV1 differently in LCLs compared to B cells. In vitro assays indicated that the protective allele of MS risk SNP rs10271373 increases ZC3HAV1 expression in LCLs, but not in B cells. The higher expression for the protective allele in LCLs is consistent with increased IFN response via ZC3HAV1 and so decreased immune evasion by EBV. Taken together, this provides evidence that EBV infection dysregulates the B cell miRNA machinery, including MS risk miRNAs, which may contribute to MS pathogenesis via interaction with MS risk genes either directly or indirectly.

Sepsis is associated with a dysregulated inflammatory response to infection. Despite the activation of inflammation, an immune suppression is often observed, predisposing patients to secondary infections. Therapies directed at restoration of immunity may be considered but should be guided by the immune status of the patients. In this paper, we described the use of a high-dimensional flow cytometry (HDCyto) panel to assess the immunophenotype of patients with sepsis. We then isolated peripheral blood mononuclear cells (PBMCs) from patients with septic shock and mimicked a secondary infection by stimulating PBMCs for 4 h in vitro with lipopolysaccharide (LPS) with or without prior exposure to either IFN-γ, or LAG-3Ig. We evaluated the response by means of flow cytometry and high-resolution clustering cum differential analysis and compared the results to PBMCs from healthy donors. We observed a heterogeneous immune response in septic patients and identified two major subgroups: one characterized by hypo-responsiveness (Hypo) and another one by hyper-responsiveness (Hyper). Hypo and Hyper groups showed significant differences in the production of cytokines/chemokine and surface human leukocyte antigen-DR (HLA-DR) expression in response to LPS stimulation, which were observed across all cell types. When pre-treated with either interferon gamma (IFN-γ) or lymphocyte-activation gene 3 (LAG)-3 recombinant fusion protein (LAG-3Ig) prior to LPS stimulation, cells from the Hypo group were shown to be more responsive to both immunostimulants than cells from the Hyper group. Our results demonstrate the importance of patient stratification based on their immune status prior to any immune therapies. Once sufficiently scaled, this approach may be useful for prescribing the right immune therapy for the right patient at the right time, the key to the success of any therapy.

Background The mechanisms linking UV radiation and vitamin D exposure to the risk of acquiring the latitude and critical period-dependent autoimmune disease, multiple sclerosis, is unclear. We examined the effect of vitamin D on DNA methylation and DNA methylation at vitamin D receptor binding sites in adult and paediatric myeloid cells. This was accomplished through differentiating CD34+ haematopoietic progenitors into CD14+ mononuclear phagocytes, in the presence and absence of calcitriol. Results Few DNA methylation changes occurred in cells treated with calcitriol. However, several VDR-binding sites demonstrated increased DNA methylation in cells of adult origin when compared to cells of paediatric origin. This phenomenon was not observed at other transcription factor binding sites. Genes associated with these sites were enriched for intracellular signalling and cell activation pathways involved in myeloid cell differentiation and adaptive immune system regulation. Conclusion These results suggest vitamin D exposure at critical periods during development may contribute to latitude-related differences in autoimmune disease incidence.

Retrotransposons are genetic elements capable of their own propagation and insertion into the human genome. Because of their mutagenic potential, retrotransposons are heavily suppressed by mechanisms including DNA methylation. Increased age is associated with decreasing DNA methylation of the LINE-1 retrotransposon and may partially explain the predisposition towards malignancy with advancing age. Vitamin D has been investigated for its effects on DNA methylation at LINE-1 elements with mixed results. This study hypothesised that LINE-1 DNA methylation is altered by vitamin D exposure and age. Using whole genome bisulfite sequencing of adult and newborn haematopoietic progenitors, DNA methylation at LINE-1 elements was not found to vary between cells cultured with or without calcitriol, both in adults and newborns. In contrast, several LINE-1 regions were found to be differentially methylated between adults and children, but these were not uniformly hypermethylated in paediatric cells. The results of this study suggest that at least in haematopoietic cells, vitamin D does not appear to affect LINE-1 methylation.### Competing Interest StatementThe authors have declared no competing interest.

OBJECTIVE:Vaccination against hepatitis B virus (HBV) confers protection from subsequent infection through immunological memory that is traditionally considered the domain of the adaptive immune system. This view has been challenged following the identification of antigen-specific memory natural killer cells (mNKs) in mice and non-human primates. While the presence of mNKs has been suggested in humans based on the expansion of NK cells following pathogen exposure, evidence regarding antigen-specificity is lacking. Here, we demonstrate the existence of HBV-specific mNKs in humans after vaccination and in chronic HBV infection.DESIGN:NK cell responses were evaluated by flow cytometry and ELISA following challenge with HBV antigens in HBV vaccinated, non-vaccinated and chronic HBV-infected individuals.RESULTS:NK cells from vaccinated subjects demonstrated higher cytotoxic and proliferative responses against autologous hepatitis B surface antigen (HBsAg)-pulsed monocyte-derived dendritic cells (moDCs) compared with unvaccinated subjects. Moreover, NK cell lysis of HBsAg-pulsed moDCs was significantly higher than that of hepatitis B core antigen (HBcAg)-pulsed moDCs (non-vaccine antigen) or tumour necrosis factor α-activated moDCs in a NKG2D-dependent manner. The mNKs response was mediated by CD56dim NK cells coexpressing CD57, CD69 and KLRG1. Further, mNKs from chronic hepatitis B patients exhibited greater degranulation against HBcAg-pulsed moDCs compared with unvaccinated or vaccinated patients. Notably, mNK activity was negatively correlated with HBV DNA levels.CONCLUSIONS:Our data support the presence of a mature mNKs following HBV antigen exposure either through vaccination or infection. Harnessing these antigen specific, functionally active mNKs provides an opportunity to develop novel treatments targeting HBV in chronic infection.

Background: Natural Killer (NK) cells are an innate immune cell type that has somewhat been overlooked in the context of systemic lupus erythematosus (SLE). SLE patients display a reduced number of NK cells with an activated phenotype and increased capacity to produce IFN-γ, decreased antibody-dependent cellular cytotoxicity (ADCC), and altered natural cytotoxicity (1). NK cell activation is determined by the integration of input from a myriad of activating and inhibitory receptors. Previously, using Nanostring® gene expression technologies, we found our SLE cohort showed decreased gene expression of a number of these receptors (KLRC2, KLRC1, KLRB1, KLRF1, KLRG1, PRF1 and IL2RB) leading us to explore NK cells in SLE in more depth. Objectives: Our aim was to develop a high-dimensional flow cytometry panel to characterise dysregulation of NK cell in SLE, with particular reference to the activating and inhibitory receptors found to be dysregulated in SLE at the gene expression level. Methods: Markers for NK panel were selected to include canonical phenotyping/functional molecules of NK cells with a particular emphasis on receptors found to be lower in our SLE cohort’s gene expression findings. NK panel was designed to minimise spectral overlap, expression and co-expression of markers was taken into consideration. Antibodies were titrated, and voltages optimised to achieve the best separation index for each of the antibodies. The 24-marker panel was run on 52 SLE patients of various disease manifestations, treatments and disease severity. 20 healthy controls were also run for comparison. Results: A 24-marker flow cytometry panel including 19 NK cell antigens was optimised, including basic phenotype (CD3/CD56/CD16/NKp46) and NK differentiation markers (CD57/CD94), activating and inhibitory receptors (NKG2A/NKG2C/NKG2D), costimulatory receptors (CD244/CD226), transcription factors (Eomes/Tbet) and effector molecules (granzyme/perforin). Immunophenotypic high-parameter analysis of SLE and control samples is in progress and results will be presented. Conclusion: Our development of a high-dimensional immunophenotypic panel allows identification of changes in NK cells in SLE including antigen expression levels, subset percentages and potentially of novel subsets. This panel will be used to investigate NK cell changes with disease course/activity, therapeutic response, and to discover potential drug targets for SLE. References: [1]Spada R, Rojas JM, Barber DF. Recent findings on the role of natural killer cells in the pathogenesis of systemic lupus erythematosus. J Leukocyte Biol. 2015;98(4):479-487. doi:10.1189/jlb.4ru0315-081rr Acknowledgments: Westmead Institute for Medical Research Genomics Facility Westmead Institute for Medical Research Flow Cytometry Facility Staff Specialists’ TESL and Trust Fund Committee Disclosure of Interests: None declared

Background & Aims: Natural killer (NK) cells are known to exert strong antiviral activity. Killer cell lectin-like receptor subfamily G member 1 (KLRG1) is expressed by terminally differentiated NK cells and KLRG1-expressing lymphocytes are known to expand following chronic viral infections. We aimed to elucidate the previously unknown role of KLRG1 in the pathogenesis of chronic hepatitis B (CHB). Methods: KLRG1+ NK cells were taken from the blood and liver of healthy individuals and patients with CHB. The phenotype and function of these cells was assessed using flow cytometry and in vitro stimulation. Results: Patients with CHB had a higher frequency of KLRG1+ NK cells compared to healthy controls (blood 13.4 vs. 2.3%, p <0.0001 and liver 23.4 vs. 2.6%, p <0.01). KLRG1+ NK cells were less responsive to K562 and cytokine stimulation, but demonstrated enhanced cytotoxicity (9.0 vs. 4.8%, p <0.05) and IFN-gamma release (8.0 vs. 1.5%, p <0.05) via antibody dependent cellular cytotoxicity compared to their KLRG1- counterparts. KLRG1+ NK cells possessed a mature phenotype, demonstrating stronger cytolytic activity and IFN-gamma secretion against hepatic stellate cells (HSCs) than KLRG1- NK cells. Moreover, KLRG1+ NK cells more effectively induced primary HSC apoptosis in a TRAIL-dependent manner. Increased KLRG1+ NK cell frequency in the liver and blood was associated with lower fibrosis stage (F0/F1) in patients with CHB. Finally, the expression of CD44, degranulation and IFN-gamma production were all increased in KLRG1+ NK cells following stimulation with osteopontin, the CD44 ligand, suggesting that HSC-derived osteopontin may cause KLRG1+ NK cell activation. Conclusions: KLRG1+ NK cells likely play an antifibrotic role during the natural course of CHB infection. Harnessing this antifibrotic function may provide a novel therapeutic approach to treat liver fibrosis in patients with CHB. Lay summary: Individuals that are chronically infected with hepatitis B virus (HBV) possess an increased number of immune cells, called natural killer (NK) cells expressing the surface marker KLRG1 in the blood and liver. Here, we demonstrate that these specific NK cells are able to kill activated stellate cells in the liver. Because activated stellate cells contribute to liver scarring, i.e. fibrosis, and subsequent liver dysfunction in individuals with chronic HBV infection, KLRG1+ NK cells are a novel immune cell type that can limit liver scarring. (C) 2019 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

Genome wide association studies have identified > 200 susceptibility loci accounting for much of the heritability of multiple sclerosis (MS). Epstein-Barr virus (EBV), a memory B cell tropic virus, has been identified as necessary but not sufficient for development of MS. The molecular and immunological basis for this has not been established. Infected B cell proliferation is driven by signalling through the EBV produced cell surface protein LMP1, a homologue of the MS risk gene CD40. We have investigated transcriptomes of B cells and EBV-infected B cells at Latency III (LCLs) and identified MS risk genes with altered expression on infection and with expression levels associated with the MS risk genotype (LCLeQTLs). The association of LCLeQTL genomic burden with EBV phenotypes in vitro and in vivo was examined. The risk genotype effect on LCL proliferation with CD40 stimulation was assessed. These LCLeQTL MS risk SNP:gene pairs (47 identified) were over-represented in genes dysregulated between B and LCLs (p < 1.53 × 10−4), and as target loci of the EBV transcription factor EBNA2 (p < 3.17 × 10−16). Overall genetic burden of LCLeQTLs was associated with some EBV phenotypes but not others. Stimulation of the CD40 pathway by CD40L reduced LCL proliferation (p < 0.001), dependent on CD40 and TRAF3 MS risk genotypes. Both CD40 and TRAF3 risk SNPs are in binding sites for the EBV transcription factor EBNA2, with expression of each correlated with EBNA2 expression dependent on genotype. These data indicate targeting EBV may be of therapeutic benefit in MS.

Epidemiological, molecular and genetic studies have indicated that high serum vitamin D levels are associated with lower risk of several autoimmune diseases. The vitamin D receptor (VDR) binding sites in monocytes and dendritic cells (DCs) are more common in risk genes for diseases with latitude dependence than in risk genes for other diseases. The transcription factor genes Zinc finger MIZ domain-containing protein 1 (ZMIZ1) and interferon regulatory factor 8 (IRF8)-risk genes for many of these diseases-have VDR binding peaks co-incident with the risk single nucleotide polymorphisms (SNPs). We show these genes are responsive to vitamin D: ZMIZ1 expression increased and IRF8 expression decreased, and this response was affected by genotype in different cell subsets. The IL10/IL12 ratio in tolerogenic DCs increased with vitamin D. These data indicate that vitamin D regulation of ZMIZ1 and IRF8 in DCs and monocytes contribute to latitude-dependent autoimmune disease risk.

Genome wide association studies have identified >200 susceptibility loci accounting for much of the heritability of Multiple Sclerosis (MS). Epstein Barr virus (EBV), a memory B cell tropic virus, has been identified as necessary but not sufficient for development of MS, with evidence for disease causation. The molecular and immunological basis for this has not been established. LCL proliferation is driven by signalling through the EBV produced cell surface protein LMP1, a homologue of the MS risk gene CD40. We show that the CD40 ligand, CD40L, potentially through competitive signalling with LMP1, reduces LCL proliferation (p<0.001). The MS risk variants of the LMP1 signalling inhibitor, TRAF3, had altered expression in B cells and LCLs. Both CD40 and TRAF3 risk SNPs are in binding sites for the EBV transcription factor EBNA2. We have investigated transcriptomes of B cells and EBV infected B cells at Latency III (LCLs) and identified 47 MS risk genes with altered expression, associated with the risk genotype. Overall these MS risk SNPs were overrepresented in target loci of the EBV transcription factor EBNA2 (p<10 −16 ), in genes dysregulated between B and LCLs (p<10 −5 ), and as targets for EBV miRNAs (p<10 −4 ). The risk gene ZC3HAV1 is the putative target for multiple EBV miRNAs. It amplifies the interferon response, and was shown to have reduced expression in LCLs for the risk allele. These data indicate targeting EBV EBNA2, miRNAs, and MS risk genes on the LMP1/LMP2 pathways, and the pathways themselves, may be of therapeutic benefit in MS.