Key Immune Genes Research Articles

Eda haplotypes in three-spined stickleback are associated with variation in immune gene expression

Haplotypes underlying local adaptation and speciation are predicted to have numerous phenotypic effects, but few genes involved have been identified, with much work to date concentrating on visible, morphological, phenotypes. The link between genes controlling these adaptive morphological phenotypes and the immune system has seldom been investigated, even though changes in the immune system could have profound adaptive consequences. The Eda gene in three-spined stickleback is one of the best studied major adaptation genes; it directly controls bony plate architecture and has been associated with additional aspects of adaptation to freshwater. Here, we exposed F2 hybrids, used to separate Eda genotype from genetic background, to contrasting conditions in semi-natural enclosures. We demonstrate an association between the Eda haplotype block and the expression pattern of key immune system genes. Furthermore, low plated fish grew less and experienced higher burdens of a common ectoparasite with fitness consequences. Little is currently known about the role of the immune system in facilitating adaptation to novel environments, but this study provides an indication of its potential importance.

Transcriptomic profiling analysis of tilapia (Oreochromis niloticus) following Streptococcus agalactiae challenge

Innate immune system is the primary defense mechanism against pathogen infection in teleost, which are living in pathogen-rich aquatic environment. It has been long hypothesized that the disease resistance in teleost are strongly correlated to the activities of innate immune genes. Tilapia is an important economical fish around the world, especially in China, where the production accounts for nearly half of the global production. Recently, S. agalactiae has become one of the most serious bacterial diseases in southern China, resulted in high cumulative mortality and economic loss to tilapia industry. Therefore, we sought here to characterize the expression profiles of tilapia against S. agalactiae infection at whole transcriptome level by RNA-seq technology. A total of 2822 genes were revealed significantly expressed in tilapia spleen with a general trend of induction. Notably, most of the genes were rapidly the most induced at the early timepoint. The significantly changed genes highlighted the function of pathogen attachment and recognition, antioxidant/apoptosis, cytoskeletal rearrangement, and immune activation. Collectively, the induced expression patterns suggested the strong ability of tilapia to rapidly recognize the invasive bacteria, and activation of downstream immune signaling pathways to clear the bacteria and prevent the tissue damage and bacteria triggered cell apoptosis. Our results heighted important roles of novel candidate genes which were often missed in previous tilapia studies. Further studies are needed to characterize the molecular relationships between key immune genes and disease resistance, and to identify the candidate genes for molecular-assistant selection of disease-resistant broodstock and evaluation of disease prevention and treatment measures.

Construction of an Ostrea edulis database from genomic and expressed sequence tags (ESTs) obtained from Bonamia ostreae infected haemocytes: Development of an immune-enriched oligo-microarray

The flat oyster, Ostrea edulis, is one of the main farmed oysters, not only in Europe but also in the United States and Canada. Bonamiosis due to the parasite Bonamia ostreae has been associated with high mortality episodes in this species. This parasite is an intracellular protozoan that infects haemocytes, the main cells involved in oyster defence. Due to the economical and ecological importance of flat oyster, genomic data are badly needed for genetic improvement of the species, but they are still very scarce. The objective of this study is to develop a sequence database, OedulisDB, with new genomic and transcriptomic resources, providing new data and convenient tools to improve our knowledge of the oyster's immune mechanisms. Transcriptomic and genomic sequences were obtained using 454 pyrosequencing and compiled into an O. edulis database, OedulisDB, consisting of two sets of 10,318 and 7159 unique sequences that represent the oyster's genome (WG) and de novo haemocyte transcriptome (HT), respectively. The flat oyster transcriptome was obtained from two strains (naïve and tolerant) challenged with B. ostreae, and from their corresponding non-challenged controls. Approximately 78.5% of 5619 HT unique sequences were successfully annotated by Blast search using public databases. A total of 984 sequences were identified as being related to immune response and several key immune genes were identified for the first time in flat oyster. Additionally, transcriptome information was used to design and validate the first oligo-microarray in flat oyster enriched with immune sequences from haemocytes. Our transcriptomic and genomic sequencing and subsequent annotation have largely increased the scarce resources available for this economically important species and have enabled us to develop an OedulisDB database and accompanying tools for gene expression analysis. This study represents the first attempt to characterize in depth the O. edulis haemocyte transcriptome in response to B. ostreae through massively sequencing and has aided to improve our knowledge of the immune mechanisms of flat oyster. The validated oligo-microarray and the establishment of a reference transcriptome will be useful for large-scale gene expression studies in this species.

Age-related alterations in immune responses to West Nile virus infection.

West Nile virus (WNV) is the most important causative agent of viral encephalitis worldwide and an important public health concern in the United States due to its high prevalence, severe disease, and the absence of effective treatments. Infection with WNV is mainly asymptomatic, but some individuals develop severe, possibly fatal, neurological disease. Individual host factors play a role in susceptibility to WNV infection, including genetic polymorphisms in key anti-viral immune genes, but age is the most well-defined risk factor for susceptibility to severe disease. Ageing is associated with distinct changes in immune cells and a decline in immune function leading to increased susceptibility to infection and reduced responses to vaccination. WNV is detected by pathogen recognition receptors including Toll-like receptors (TLRs), which show reduced expression and function in ageing. Neutrophils, monocyte/macrophages and dendritic cells, which first recognize and respond to infection, show age-related impairment of many functions relevant to anti-viral responses. Natural killer cells control many viral infections and show age-related changes in phenotype and functional responses. A role for the regulatory receptors Mertk and Axl in blood-brain barrier permeability and in facilitating viral uptake through phospholipid binding may be relevant for susceptibility to WNV, and age-related up-regulation of Axl has been noted previously in human dendritic cells. Understanding the specific immune parameters and mechanisms that influence susceptibility to symptomatic WNV may lead to a better understanding of increased susceptibility in elderly individuals and identify potential avenues for therapeutic approaches: an especially relevant goal, as the world's populating is ageing.

Altered Immune Cytokine Expression Associated with KoRV B Infection and Season in Captive Koalas.

Koala (Phascolarctos cinereus) populations are increasingly vulnerable and one of the main threats is chlamydial infection. Koala retrovirus (KoRV) has been proposed as an underlying cause of the koala’s susceptibility to infection with Chlamydia and high rates of lymphoid neoplasia; however, the regionally ubiquitous, endogenous nature of this virus suggests that KoRV A infection is not sufficient for immune suppression to occur. A recently discovered exogenous variant of KoRV, KoRV B, has several structural elements that cause increased pathogenicity in related retroviruses and was associated with lymphoid neoplasia in one study. The present study assesses whether KoRV B infection is associated with alterations in immune function. Cytokine gene expression by mitogen stimulated lymphocytes of KoRV B positive (n = 5–6) and negative (n = 6–7) captive koalas was evaluated by qPCR four times (April 2014-February 2015) to control for seasonal variation. Key immune genes in the Th1 pathway (IFNγ, TNFα), Th2 pathway (IL 10, IL4, IL6) and Th17 pathway (IL17A), along with CD4:CD8 ratio, were assessed. KoRV B positive koalas showed significantly increased up-regulation of IL17A and IL10 in three out of four sampling periods and IFNγ, IL6, IL4 and TNFα in two out of four. IL17A is an immune marker for chlamydial pathogenesis in the koala; increased expression of IL17A in KoRV B positive koalas, and concurrent immune dysregulation, may explain the differences in susceptibility to chlamydial infection and severity of disease seen between individuals and populations. There was also marked seasonal variation in up-regulation for most of the cytokines and the CD4:CD8 ratio. The up-regulation in both Th1 and Th2 cytokines mirrors changes associated with immune dysregulation in humans and felids as a result of retroviral infections. This is the first report of altered immune expression in koalas infected by an exogenous variant of KoRV and also the first report of seasonal variation in cytokine up-regulation and CD4:CD8 ratio in marsupials.

Identification of novel signature genes attesting arsenic-induced immune alterations in adult zebrafish (Danio rerio)

Arsenic poisoning is a serious global issue. Apart from causing developmental and systemic toxicity, arsenic has recently been reported for its ability to hinder immune responses. The present study is designed to identify the global expression profile associated with arsenic–induced immune alterations at the organismic level. Adult zebrafish (Danio rerio) were exposed to 20, 40 and 80ppb of arsenic trioxide for 30days, sacrificed and global gene expression profile studied. Microarray data suggested 65 immune related genes were commonly affected in the three treatment regimens. The expression profile of key immune related genes (tlr1, nitr1f, nitr1c, crfb8, socs7, socs3b, abcb3/1, mch1uja, ifnγ1-2, cxcl12b and crlf1a) was validated by qPCR. Pathway analysis suggested the major involvement of JAK–STAT circuit in the process. The expression of these marker genes was also studied in arsenic exposed and bacteria (Aeromonas hydrophila) challenged zebrafish. Increase in bacterial colony forming units (CFU) coupled with gross histopathology of kidney in arsenic exposed-bacteria challenged fish suggested profound immuno-compromised condition. We propose that chronic arsenic exposure leads to hyperactivation of the immune system as a consequence when exposed to further stress (microbial) it induces immuno-suppression with pathological implications. The study provides a molecular snap shot for predicting arsenic immuno-toxicity.

Marek’s disease virus immunosuppression alters host cellular responses and immune gene expression in the skin of infected chickens

Marek’s disease virus (MDV), a highly cell-associated lymphotropic α-herpesvirus, is the causative agent of Marek’s disease (MD) in domestic chickens. MDV replicates in chicken lymphocytes and establishes a latent infection within CD4+ T cells. The latently infected CD4+ T cells carry the virus to visceral organs, peripheral nerves, and feather follicle epithelium (FFE). FFE is the only anatomical site where infectious enveloped cell-free virus particles are produced and disseminated into the environment. This study investigated the immunological responses and mechanism of viral-induced immunosuppression and immune evasion in the FFE. Strong viral replication and lack of a significant number of cytotoxic T lymphocytes (CTL) in the infected tissues was prominent. Although the overall gene expression pattern was suggestive of a Th1 type immune response, the expression levels of several key immune genes were down regulated in the infected tissues. The mechanism of MDV-induced immunosuppression appears to be through inhibition of CTL function due to down regulation of CD8 glycoprotein and/or blocking of CTL migration due to decrease expression of cell adhesion molecules.

The influence of trematode parasite burden on gene expression in a mammalian host.

BackgroundParasites can profoundly impact their hosts and are responsible for a plethora of debilitating diseases. To identify global changes in host gene expression related to parasite infection, we sequenced, assembled, and annotated the liver transcriptomes of Balb/cj mice infected with the trematode parasite Schistosoma mansoni and compared the results to uninfected mice. We used two different methodologies (i.e. de novo and reference guided) to evaluate the influence of parasite sequences on host transcriptome assembly.ResultsOur results demonstrate that the choice of assembly methodology significantly impacted the proportion of parasitic reads detected from the host library, yet the presence of non-target (xenobiotic) sequences did not create significant structural errors in the assembly. After removing parasite sequences from the mouse transcriptomes, we analyzed host gene expression under different parasite infection levels and observed significant differences in the associated immunologic and metabolic responses based on infection level. In particular, genes associated with T–helper type 1 (Th–1) and T–helper type 2 (Th–2) were up-regulated in infected mice whereas genes related to amino acid and carbohydrate metabolism were down-regulated in infected mice. These changes in gene expression scale with infection status and likely impact the evolutionary fitness of hosts.ConclusionsOverall, our data indicate that a) infected mice reduce the expression of key metabolic genes in direct proportion to their infection level; b) infected mice similarly increase the expression of key immune genes in response to infection; c) patterns of gene expression correspond to the pathological symptoms of schistosomiasis; and d) identifying and filtering out non-target sequences (xenobiotics) improves differential expression prediction. Our findings identify parasite targets for RNAi or other therapies and provide a better understanding of the pathology and host immune repertoire involved in response to S. mansoni infections.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-2950-5) contains supplementary material, which is available to authorized users.

Individual monitoring of immune responses in rainbow trout after cohabitation and intraperitoneal injection challenge with Yersinia ruckeri.

Yersinia ruckeri, the causative agent of enteric red mouth disease (ERM), is a widely studied pathogen in disease models using rainbow trout. This infection model, mostly based on intraperitoneally injection or bath immersion challenges, has an impact on both components (innate and adaptive) of the fish immune system. Although there has been much attention in studying its host-pathogen interactions, there is still a lack of knowledge regarding the impact of a cohabitation challenge. To tackle this we used a newly established non-lethal sampling method (by withdrawing a small amount of blood) in rainbow trout which allowed the individual immune monitoring before (non-infected) and after infection with Yersinia ruckeri either by intraperitoneal (i.p.) injection or by cohabitation (cohab). A range of key immune genes were monitored during the infection by real-time PCR, and results were compared between the two infection routes. Results indicated that inflammatory (IL-1β1 and IL-8) cytokines and certain antimicrobial peptides (cathelicidins) revealed a different pattern of expression between the two infected groups (i.p. vs cohab), in comparison to adaptive immune cytokines (IL-22, IFN-γ and IL-4/13A) and β-defensins. This suggests a different involvement of distinct immune markers according to the infection model, and the importance of using a cohabitation challenge as a more natural disease model that likely simulates what would occur in the environment.

MicroRNAs as regulatory elements in immune system logic.

MicroRNAs (miRNAs) are crucial post-transcriptional regulators of haematopoietic cell fate decisions. They act by negatively regulating the expression of key immune development genes, thus contributing important logic elements to the regulatory circuitry. Deletion studies have made it increasingly apparent that they confer robustness to immune cell development, especially under conditions of environmental stress such as infectious challenge and ageing. Aberrant expression of certain miRNAs can lead to pathological consequences, such as autoimmunity and haematological cancers. In this Review, we discuss the mechanisms by which several miRNAs influence immune development and buffer normal haematopoietic output, first at the level of haematopoietic stem cells, then in innate and adaptive immune cells. We then discuss the pathological consequences of dysregulation of these miRNAs.

The role of epigenetics in tuberculosis infection.

Epigenetic mechanisms are pivotal in regulating gene expression during cellular response to extracellular stimuli. Bacterial infections have a profound effect on the host epigenome, which triggers susceptibility to diseases. Recent studies suggest that Mycobacterium tuberculosis (Mtb) can alter the host epigenome to modulate the transcriptional machinery and plays a major role in immunomodulation of the host immune response. However, the mechanism of epigenetic alterations during Mtb infection has not yet been fully understood. Thus, Mtb-induced epigenetic changes may affect the host cell by either activation or suppression of key immune genes involved in immune response or pathogen persistence. In this review, we discuss the principles of epigenetics, recent advances in Mtb-induced alterations in the host epigenetic landscape and their role in the host immune response.

Long-term tryptophan supplementation decreased the welfare and innate immune status of pikeperch juveniles

It has been demonstrated that short-term supplementation of L-tryptophan (TRP) can mitigate the primary neuroendocrine response to stress in some fish species, but such stress reduction was reported to be both doseand context-dependent (Basic et al, 2013; Machado et al, 2015). So, the TRP responses may be species related or depend on the stress levels experienced by the fish. Since percid fish were reported to be more stress responsiveness than common aquaculture species such as rainbow trout (Jentoft et al, 2005), this study aimed (1) to determine to what extent a long-term dietary TRP mitigate the physiological response of pikeperch (Sander lucioperca) to emersion stress, and (2) to characterize the related immune status. Pikeperch juveniles of 10 g received four experimental diets: (CT) = control groups without any stress and any feed TRP supplement, (CTs) = control groups submitted to emersion stress but without any feed TRP supplement, 3TRPs = groups receiving 3-time TRP diet and submitted to emersion stress, 6TRPs = groups receiving 6-time TRP diet and submitted to emersion stress. Various organs were sampled on D7, D37 and D91 of TRP supplementation for evaluation of physiological and immune responses; samplings were done one hour after the emersion challenge stress. Specific growth rate (SGR) as well as food conversion rate (FCR) were also checked. Emersion stress induced a significant increase in plasma cortisol both after a single stress or repeated stress challenges. Dietary TRP significantly decreased cortisol levels in a dose related manner both after a single or repeated stress; but plasma glucose level was only affected after a single stress but not after long-term feeding. The two stress indicators measured showed that pikeperch displayed higher stress responsiveness as already demonstrated for European perch in comparison to salmonids (Jentoft et al, 2005). The reduction in physiological stress status by dietary TRP was associated to a significant decrease in plasma lysosomal activity, especially on D91; indicating negative interaction with the innate immune pathways. Growth rate was slowed by the longterm dietary TRP in association to an increase in FCR values. The available results indicate that long-term TRP supplementation has negative impact on the overall welfare status of pikeperch. Other analyses are ongoing and more results concerning keybrain neurotransmitters such as serotonin, dopamine and their metabolites concentrations as well as on the expressions of key-immune genes (C3-1, TNF-α, IL-1β, etc) will be discussed.

Characterization of neurophysiological and immune responses of pikeperch juveniles to major stress factors under intensive culture conditions

Pikeperch Sander lucioperca has been identified as a valuable candidate species for diversification of European freshwater aquaculture but the development of its culture is still limited by a high mortality rate or impairment in growth rate during the juvenile stage. A low welfare status may be related to high stress responsiveness since the rearing conditions for pikeperch are not yet optimized; and it has been shown that percid species are more sensitive to aquaculture stressors than salmonids (Jentoft et al, 2005). It has been also shown that exposure of the latter species to handling, emersion, and moderate hypoxia may impair significantly growth related parameters and various physiological pathways including changes in some key immune gene proteins (Strand et al, 2007; Douxfils et al, 2014). The objectives of this study were (1) to determine the main stressful husbandry and environmental conditions for pikeperch juveniles, and (2) to characterize the relative physiological and immune responses. Eight factors considered as relevant for the welfare of pikeperch were compared in two modalities using a fractional multifactorial design (2) in order to account for the interactions between the tested factors. So, each experimental unit represented a combination of sixteen variants including grading, stocking density (15 vs 30 kg/m), food type (floating vs sinking), light intensity (10 vs 100 lux or spectrum (red vs white), short vs long photoperiod, dissolved oxygen (60 vs 90%), temperature (21 vs 26°C). After 63 days of stress challenges, growth rate was significantly affected by the type of food with the highest values for fish receiving sinking food, while survival rate was improved by low intensity and red light. The latter light characteristics were also associated to a low stress response in terms of plasma cortisol and glucose but various interactions were observed between light intensity and some husbandry factors. Moreover, the highest values for the expression of glucocorticoid receptor-1 were observed for fish reared at 26°C and 90% dissolved oxygen. A decrease in the stress response was observed after 2 months, and this was associated to an improvement in the immune status in terms of lysozyme and ACH50 activities. The available results indicate that low light intensity and red spectrum in combination with appropriate food type may improve the welfare of pikeperch but other endpoints are still under analysis such as brain neurotransmitter responses (serotonin,, etc) and expressions of key-immune genes (C3-1, TNF-α, IL-1β, etc) for final recommendations.

Genomic landscape of the individual host response and outcomes in sepsis: a prospective cohort study

SummaryBackgroundEffective targeted therapy for sepsis requires an understanding of the heterogeneity in the individual host response to infection. We investigated this heterogeneity by defining interindividual variation in the transcriptome of patients with sepsis and related this to outcome and genetic diversity.MethodsWe assayed peripheral blood leucocyte global gene expression for a prospective discovery cohort of 265 adult patients admitted to UK intensive care units with sepsis due to community-acquired pneumonia and evidence of organ dysfunction. We then validated our findings in a replication cohort consisting of a further 106 patients. We mapped genomic determinants of variation in gene transcription between patients as expression quantitative trait loci (eQTL).FindingsWe discovered that following admission to intensive care, transcriptomic analysis of peripheral blood leucocytes defines two distinct sepsis response signatures (SRS1 and SRS2). The presence of SRS1 (detected in 108 [41%] patients in discovery cohort) identifies individuals with an immunosuppressed phenotype that included features of endotoxin tolerance, T-cell exhaustion, and downregulation of human leucocyte antigen (HLA) class II. SRS1 was associated with higher 14 day mortality than was SRS2 (discovery cohort hazard ratio (HR) 2·4, 95% CI 1·3–4·5, p=0·005; validation cohort HR 2·8, 95% CI 1·5–5·1, p=0·0007). We found that a predictive set of seven genes enabled the classification of patients as SRS1 or SRS2. We identified cis-acting and trans-acting eQTL for key immune and metabolic response genes and sepsis response networks. Sepsis eQTL were enriched in endotoxin-induced epigenetic marks and modulated the individual host response to sepsis, including effects specific to SRS group. We identified regulatory genetic variants involving key mediators of gene networks implicated in the hypoxic response and the switch to glycolysis that occurs in sepsis, including HIF1α and mTOR, and mediators of endotoxin tolerance, T-cell activation, and viral defence.InterpretationOur integrated genomics approach advances understanding of heterogeneity in sepsis by defining subgroups of patients with different immune response states and prognoses, as well as revealing the role of underlying genetic variation. Our findings provide new insights into the pathogenesis of sepsis and create opportunities for a precision medicine approach to enable targeted therapeutic intervention to improve sepsis outcomes.FundingEuropean Commission, Medical Research Council (UK), and the Wellcome Trust.

Streptococcus uberis strains isolated from the bovine mammary gland evade immune recognition by mammary epithelial cells, but not of macrophages.

Streptococcus uberis is frequently isolated from the mammary gland of dairy cattle. Infection with some strains can induce mild subclinical inflammation whilst others induce severe inflammation and clinical mastitis. We compared here the inflammatory response of primary cultures of bovine mammary epithelial cells (pbMEC) towards S. uberis strains collected from clinical or subclinical cases (seven strains each) of mastitis with the strong response elicited by Escherichia coli. Neither heat inactivated nor live S. uberis induced the expression of 10 key immune genes (including TNF, IL1B, IL6). The widely used virulent strain 0140J and the avirulent strain, EF20 elicited similar responses; as did mutants defective in capsule (hasA) or biofilm formation (sub0538 and sub0539). Streptococcus uberis failed to activate NF-κB in pbMEC or TLR2 in HEK293 cells, indicating that S. uberis particles did not induce any TLR-signaling in MEC. However, preparations of lipoteichoic acid (LTA) from two strains strongly induced immune gene expression and activated NF-κB in pbMEC, without the involvement of TLR2. The immune-stimulatory LTA must be arranged in the intact S. uberis such that it is unrecognizable by the relevant pathogen receptors of the MEC. The absence of immune recognition is specific for MEC, since the same S. uberis preparations strongly induced immune gene expression and NF-κB activity in the murine macrophage model cell RAW264.7. Hence, the sluggish immune response of MEC and not of professional immune cells to this pathogen may aid establishment of the often encountered belated and subclinical phenotype of S. uberis mastitis.Electronic supplementary materialThe online version of this article (doi:10.1186/s13567-015-0287-8) contains supplementary material, which is available to authorized users.

Evaluation of the transcriptional status of host cytokines and viral genes in the trachea of vaccinated and non-vaccinated chickens after challenge with the infectious laryngotracheitis virus

ABSTRACTInfectious laryngotracheitis is a highly contagious disease of chickens responsible for significant economic losses for the poultry industry worldwide. The disease is caused by Gallid herpesvirus-1 (GaHV-1) commonly known as the infectious laryngotracheitis virus. Although characterized by their potential to regain virulence, chicken embryo origin (CEO) vaccines are the most effective vaccines against laryngotracheitis as they significantly reduce the replication of challenge virus in the trachea and conjunctiva. Knowledge on the nature of protective immunity elicited by CEO vaccines is very limited. Therefore, elucidating the origin of the immune responses elicited by CEO vaccination is relevant for development of safer control strategies. In this study the transcription levels of key host immune genes (IFN-γ, IFN-β, IL-1β, IL-6, IL-8, IL-18) and viral genes (ICP4, ICP27, UL46, UL49), as well as viral genome loads in trachea were quantified at 6 and 12 hours post-challenge of CEO vaccinated and non-vaccinated chickens. Immediately after challenge a significant increase in IFN-γ gene expression was followed by a significant reduction in viral replication. In contrast to the rapid induction of IFN-γ, expression of the pro-inflammatory cytokines (IL-1β, IL-6, IL-8) and type I IFN β was either slightly reduced or remained at basal levels. These suggest that the former cytokines may not play important roles during immediate early responses induced by ILTV challenge in either vaccinated or non-vaccinated chickens. Overall, these results suggest that the rapid expression of IFN-γ may induce pathways of antiviral responses necessary for blocking early virus replication.

A Single-Cell Gene-Expression Profile Reveals Inter-Cellular Heterogeneity within Human Monocyte Subsets.

Human monocytes are a heterogeneous cell population classified into three different subsets: Classical CD14++CD16-, intermediate CD14++CD16+, and non-classical CD14+CD16++ monocytes. These subsets are distinguished by their differential expression of CD14 and CD16, and unique gene expression profile. So far, the variation in inter-cellular gene expression within the monocyte subsets is largely unknown. In this study, the cellular variation within each human monocyte subset from a single healthy donor was described by using a novel single-cell PCR gene-expression analysis tool. We investigated 86 different genes mainly encoding cell surface markers, and proteins involved in immune regulation. Within the three human monocyte subsets, our descriptive findings show multimodal expression of key immune response genes, such as CD40, NFⱪB1, RELA, TLR4, TLR8 and TLR9. Furthermore, we discovered one subgroup of cells within the classical monocytes, which showed alterations of 22 genes e.g. IRF8, CD40, CSF1R, NFⱪB1, RELA and TNF. Additionally one subgroup within the intermediate and non-classical monocytes also displayed distinct gene signatures by altered expression of 8 and 6 genes, respectively. Hence the three monocyte subsets can be further subdivided according to activation status and differentiation, independently of the traditional classification based on cell surface markers. Demonstrating the use and the ability to discover cell heterogeneity within defined populations of human monocytes is of great importance, and can be useful in unravelling inter-cellular variation in leukocyte populations, identifying subpopulations involved in disease pathogenesis and help tailor new therapies.

The T Cell Receptor (TCR) Repertoire Is a Key Determinant of the Tumour Microenvironment (TME) in Diffuse Large B Cell Lymphoma (DLBCL)

Background: We have recently demonstrated that an 'immune score' is strongly and independently prognostic in de novo DLBCL treated with R-CHOP immuno-chemotherapy. The score quantifies the relative composition of immune effectors (T cells) and checkpoints (e.g. PD-1 axis molecules and M2 macrophages), as a measure of net anti-tumoral immunity within the TME. It is also known that a diverse TCR repertoire is a hallmark of a robust anti-HIV T cell immune response; conversely in metastatic melanoma treated with anti-PD-1 checkpoint blockade, narrow more clonal TCR repertoires are associated with favorable response. The relationship between the intra-tumoral TCR repertoire and the TME in DLBCL following R-CHOP immuno-chemotherapy is unknown.MethodsHigh-throughput unbiased TCR β chain sequencing was performed on 116 nodal tissues (101 de novo DLBCL patients treated with R-CHOP with long-term follow-up including 8 EBV+DLBCL; and 15 age/gender matched healthy lymph nodes). Outcomes included measurement of productive uniques (a measure of the number of functional T cells with a distinct TCR rearrangement or 'richness'); entropy (a measure of TCR 'diversity'), 'clonality' (a measure of clonal expansions) and the 'maximal frequency' of the most highly expressed clone within tumor biopsies. Results were compared to digital quantification (by nanoString) of key immune effector and checkpoint genes within the TME, the immune score, malignant cell-of-origin (COO), R-IPI and patient survival.Results:First we compared the TCR repertoire in lymphomatous and healthy nodes. There was a marked increase in clonality, reduced diversity and high maximal frequency within DLBCL nodes relative to healthy nodal tissue (both p<0.0001), consistent with an abnormally narrow TCR repertoire of antigen-specific T cells. Next, we tested the relationship between TCR and the TME. Notably, there was modest (r=0.3-0.7) but highly significant (all p<0.001) positive correlations between both richness and diversity (but not clonality) with CD3/CD4/CD8 T cells, and a range of immune checkpoints including PD-L1, PD-L2, LAG-3, CSF-1 and TIM-3. These findings are strongly suggestive of an adaptive immune response, in which malignant B cells influence (i.e. 'adapt') the TME in an attempt to counter an effective anti-lymphoma T-cell response that is in part influenced by the breadth of the TCR repertoire. Then we investigated the TCR repertoire in the context of prognosis and overall survival (OS) following R-CHOP. There were no correlations between COO or R-IPI with any TCR parameter. However, the presence of a high maximal frequency in the tumour biopsy was associated with significantly inferior 5 year OS of 59% compared to 81% in patients without a high maximal frequency (p=0.03, Figure 1). As expected, the immune score stratified patients into highly disparate outcomes: high-score 5-year overall survival 96% versus 42% for low-score (p<0.0001). Interestingly, there were significant differences in the TCR repertoire between the two groups. There was a significant increase for both richness and diversity in high immune score lymphoma patients (p=0.015 and p=0.018 respectively). In keeping, clonality was not increased in high-immune score patients. The only samples associated with increased T cell clonality were those patients with very high levels of intratumoral EBV, potentially reflecting the latent viral antigens expressed by this lymphoma. In the group of patients with poor prognosis (5 year OS 59%), defined by high maximal frequency, the immune score stratified two groups with very different outcomes (5 year OS 90% vs. 30%, p=0.003).Conclusions:These findings indicate the TCR repertoire as a key parameter of the TME that the malignant B cell attempts to narrow. A broad TCR repertoire is associated with a good prognostic immune score (i.e. increased T cells relative to PD-1 axis molecules and M2 macrophages checkpoints) after R-CHOP immunoÐchemotherapy, whereas a more clonal T cell response is associated with significantly inferior outcome. [Display omitted] DisclosuresHamm:Adaptive Biotech: Employment.

Mapping toll-like receptor signaling pathway genes of Zhikong scallop (Chlamys farreri) with FISH

Toll-like receptor (TLR) signaling pathway plays a pivotal role in the innate immune system. Studies on TLR signaling pathway genes in Zhikong scallop (Chlamys farreri) have mainly focused on sequence analysis and expression profiling, no research has been carried out on their localization. The chromosomal position of TLR signaling pathway genes can be valuable for assemblying scallop genome and analysizing gene regulatory networks. In the present study, five key TLR signaling pathway genes (CfTLR, CfMyd88, CfTRAF6, CfNFκB, and CfIκB) containing bacterial artificial chromosomes (BACs) were isolated and physically mapped through fluorescence in situ hybridization on five non-homologous chromosome pairs, showing a similar distribution to another five model species. The isolation and mapping of these key immune genes of C. farreri will aid to the research on innate immunity, assignment of interested genes to chromosomes, and integration of physical, linkage and cytogenetic maps of this species.

Protein kinase C in the immune system: from signalling to chromatin regulation.

Protein kinase C (PKC) form a key family of enzymes involved in signalling pathways that specifically phosphorylates substrates at serine/threonine residues. Phosphorylation by PKC is important in regulating a variety of cellular events such as cell proliferation and the regulation of gene expression. In the immune system, PKCs are involved in regulating signal transduction pathways important for both innate and adaptive immunity, ultimately resulting in the expression of key immune genes. PKCs act as mediators during immune cell signalling through the immunological synapse. PKCs are traditionally known to be cytoplasmic signal transducers and are well embedded in the signalling pathways of cells to mediate the cells' response to a stimulus from the plasma membrane to the nucleus. PKCs are also found to transduce signals within the nucleus, a process that is distinct from the cytoplasmic signalling pathway. There is now growing evidence suggesting that PKC can directly regulate gene expression programmes through a non-traditional role as nuclear kinases. In this review, we will focus on the role of PKCs as key cytoplasmic signal transducers in immune cell signalling, as well as its role in nuclear signal transduction. We will also highlight recent evidence for its newly discovered regulatory role in the nucleus as a chromatin-associated kinase.