Vertebrate Immune System Research Articles

Genetic diversity and evolution of the MHC class II DRB gene in the Japanese marten, Martes melampus (Carnivora: Mustelidae)

High polymorphism in major histocompatibility complex (MHC) genes plays an essential role in the adaptive immune system of vertebrates for defense against various pathogens. To further understand the evolution of MHC in wildlife, partial sequences of exon 2 in MHC class II DRB genes were determined and analyzed for the Japanese marten (Martes melampus), which is endemic to Japan. From 26 individuals, 17 DRB alleles (Mame-DRBs) and four presumed pseudogenes were identified, some of them geographically widespread and others of limited distribution. Mixed-effect model evolution (MEME) and single breakpoint recombination (SBP) analyses indicated six amino acid positions under positive selection and one recombination site, respectively, that are implicated in maintaining the diversity of Mame-DRBs. In a Bayesian phylogenetic tree, Mame-DRBs did not group monophyletically, but were scattered among seven sub-clades, each containing DRBs from one or more other mustelid species. This trans-species polymorphism of Mame-DRBs has resulted from long-term balancing selection.

Knowing the Enemy: Inducible Defences in Freshwater Zooplankton

Phenotypic plasticity in defensive traits is an appropriate mechanism to cope with the variable hazard of a frequently changing predator spectrum. In the animal kingdom these so-called inducible defences cover the entire taxonomic range from protozoans to vertebrates. The inducible defensive traits range from behaviour, morphology, and life-history adaptations to the activation of specific immune systems in vertebrates. Inducible defences in prey species play important roles in the dynamics and functioning of food webs. Freshwater zooplankton show the most prominent examples of inducible defences triggered by chemical cues, so-called kairomones, released by predatory invertebrates and fish. The objective of this review is to highlight recent progress in research on inducible defences in freshwater zooplankton concerning behaviour, morphology, and life-history, as well as difficulties of studies conducted in a multipredator set up. Furthermore, we outline costs associated with the defences and discuss difficulties as well as the progress made in characterizing defence-inducing cues. Finally, we aim to indicate further possible routes in this field of research and provide a comprehensive table of inducible defences with respect to both prey and predator species.

V(D)J recombination, somatic hypermutation and class switch recombination of immunoglobulins: mechanism and regulation.

Immunoglobulins emerging from B lymphocytes and capable of recognizing almost all kinds of antigens owing to the extreme diversity of their antigen-binding portions, known as variable (V) regions, play an important role in immune responses. The exons encoding the V regions are known as V (variable), D (diversity), or J (joining) genes. V, D, J segments exist as multiple copy arrays on the chromosome. The recombination of the V(D)J gene is the key mechanism to produce antibody diversity. The recombinational process, including randomly choosing a pair of V, D, J segments, introducing double-strand breaks adjacent to each segment, deleting (or inverting in some cases) the intervening DNA and ligating the segments together, is defined as V(D)J recombination, which contributes to surprising immunoglobulin diversity in vertebrate immune systems. To enhance both the ability of immunoglobulins to recognize and bind to foreign antigens and the effector capacities of the expressed antibodies, naive B cells will undergo class switching recombination (CSR) and somatic hypermutation (SHM). However, the genetics mechanisms of V(D)J recombination, CSR and SHM are not clear. In this review, we summarize the major progress in mechanism studies of immunoglobulin V(D)J gene recombination and CSR as well as SHM, and their regulatory mechanisms.

Adaptive selection in the evolution of programmed cell death-1 and its ligands in vertebrates.

Programmed cell death-1 (PD-1) and its ligands, particularly PD-L1 and PD-L2, are the most important proteins responsible for signaling T-cell inhibition and arbitrating immune homeostasis and tolerance mechanisms. However, the adaptive evolution of these genes is poorly understood. In this study, we aligned protein-coding genes from vertebrate species to evaluate positive selection constraints and evolution in the PD1, PD-L1 and PD-L2 genes conserved across up to 166 vertebrate species, with an average of 55 species per gene. We determined that although the positive selection was obvious, an average of 5.3% of codons underwent positive selection in the three genes across vertebrate lineages, and increased positive selection pressure was detected in both the Ig-like domains and transmembrane domains of the proteins. Moreover, the PD1, PD-L1 and PD-L2 genes were highly expressed in almost all tissues of the selected species indicating a distinct expression pattern in different tissues among most species. Our study reveals that adaptive selection plays a key role in the evolution of PD1 and its ligands in the majority of vertebrate species, which is in agreement with the contribution of these residues to the mechanisms of pathogen identification and coevolution in the complexity and novelties of vertebrate immune systems.

Peptide Presentation to T Cells: Solving the Immunogenic Puzzle: Systems Immunology Profiling of Antigen Presentation for Prediction of CD8+ T Cell Immunogenicity.

The vertebrate immune system uses an impressive arsenal of mechanisms to combat harmful cellular states such as infection. One way is via cells delivering real-time snapshots of their protein content to the cell surface in the form of short peptides. Specialized immune cells (T cells) sample these peptides and assess whether they are foreign, warranting an action such as destruction of the infected cell. The delivery of peptides to the cell surface is termed antigen processing and presentation, and decades of research have provided unprecedented understanding of this process. However, predicting the capacity for a given peptide to be immunogenic-to elicit a T cell response-has remained both enigmatic and a long sought-after goal. In the era of big data, a point is being approached where the steps of antigen processing and presentation can be quantified and assessed against peptide immunogenicity in order to build predictive models. This review presents new findings in this area and contemplates challenges ahead.

Innate immunological memory: from plants to animals.

Immunological memory is defined by the ability of the host to recognise and mount a robust secondary response against a previously encountered pathogen. Classic immune memory is an evolutionary adaptation of the vertebrate immune system that has been attributed to adaptive lymphocytes, including T and B cells. In contrast, the innate immune system was known for its conserved, non-specific roles in rapid host defence, but historically was considered to be unable to generate memory. Recent studies have challenged our understanding of innate immunity and now provides a growing body of evidence for innate immune memory. However, in many species and in various cell types the underlying mechanisms of immune 'memory' formation remain poorly understood. The purpose of this review is to explore and summarise the emerging evidence for immunological 'memory' in plants, invertebrates, and vertebrates.

Integrative Analysis of Siglec-15 mRNA in Human Cancers Based on Data Mining.

Objective: Cancer is expected to be the leading cause of death worldwide within the 21st century and is the single most important obstacle to extending life expectancy. Unfortunately, the most effective approach to combating cancers remains a complex and unsolved problem. Siglec-15 is a member of the Siglec family and plays a conserved regulatory role in the immune system of vertebrates. Previous studies on Siglec-15 have focused on its function in osteoclast regulation. The purpose of this study was to explore the significance of Siglec-15 mRNA in human cancer mainly based on information obtained from online databases.Method: Data were collected from several online databases. Serial analysis of gene expression (SAGE) and Virtual Northern, UALCAN Database Analysis, Catalog of Somatic Mutations in Cancer (COSMIC) analysis, the cBio cancer genomics portal, Cancer Regulome tools and data, Kaplan-Meier Plotter Analysis and the UCSC Xena website were used to analyze the data.Results: Compared with normal tissues, Siglec-15 up-regulation was widely observed in tuomrs. Differences in Siglec-15 expression were associated with different prognoses. Siglec-15 mutations are widely observed in tumors and interact with different genes in different cancer types.Conclusion: Siglec-15 is a potential target for the expansion of cancer immunotherapy.

Identification of BoLA DRB3.2 Alleles Present in White Fulani and Muturu Cattle Breeds

Cattle production is an important aspect of animal agriculture. Disease predisposition in cattle can lead to lowered productivity and poor animal welfare. To select and breed for the fittest cattle population, it is expedient that we understand the genetic basis of disease tolerance/resistance. Major histocompatibility complex (MHC) is a vital component of the immune system in vertebrates. Its genes are crucial determinants for immune response and resistance to infectious diseases. The bovine MHC is referred to as Bovine Lymphocyte antigen (BoLA) with its most functional and highly variable region located in the exon 2 (BoLA-DRB3.2). Over 100 alleles of BoLA-DRB3.2 have been identified in cattle and many studies have associated polymorphism in this region with disease resistance/susceptibility. In this study, we investigated the polymorphic nature of BoLA-DRB3.2 in the White Fulani and Muturu cattle breeds using a single PCR-sequence based typing. We identified 26 and 25 alleles in White Fulani and Muturu breeds, respectively, with only six alleles being mutual in the two breeds. Some of the alleles identified in this study have been noted as markers for disease status in cattle. BoLA-DRB3*014:01:01, BoLA-DRB3*011:01, and BoLA-DRB3*008:01 alleles have been associated with Bovine leukemia virus (BLV) resistance in cattle. BoLA-DRB3*014:01:01, BoLA-DRB3*001:01, and BoLA-DRB3*011:01 were linked with mastitis resistance in Japanese Holstein cows. While no inference can be drawn in terms of association with disease status, this study confirms the highly polymorphic and diverse nature of BoLA-DRB3 in White Fulani and Muturu cattle breeds.

A self-marker-like protein governs hemocyte allorecognition in Halocynthia roretzi

BackgroundSelf-incompatibility, fusion/non-fusion reactions, and contact reactions (CRs) have all been identified as allorecognition phenomena in ascidians. CR is a reaction characteristic of the hemocytes of Halocynthia roretzi, whereby they release phenol oxidase (PO) upon contact with non-self hemocytes. Thus, these cells may represent a primitive form of the vertebrate immune system. In the present study, we focused on the CR of H. roretzi hemocytes and sought to identify self-marker proteins that distinguish between self and non-self cells.ResultsWe initially generated a CR-inducing monoclonal antibody against the complete hemocyte membrane-protein complement (mAb11B16B10). This antibody was identified based on the differential induction of PO activity in individual organisms. The level of PO activity induced by this antibody in individual ascidians was consistent with the observed CR-induced PO activity. mAb11B16B10 recognized a series of 12 spots corresponding to a 100-kDa protein, with differing isoelectric points (pIs). A comparison of the 2D electrophoresis gels of samples from CR-reactive/non-reactive individuals revealed that some spots in this series in hemocytes were common to the CR-non-inducible individuals, but not to CR-inducible individuals. We cloned the corresponding gene and named it Halocynthia roretzi self-marker-like protein-1 (HrSMLP1). This gene is similar to the glycoprotein DD3–3 found in Dictyostelium, and is conserved in invertebrates.ConclusionWe generated a CR-inducing monoclonal antibody (mAb11B16B10) that recognized a series of novel membrane proteins (HrSMLP1) in the hemocytes of H. roretzi. The combination of expressed spots of HrSMLP1 distinguishes non-self cells from self cells with respect to CR inducibility. Given that the HrSMLP1 gene is a single gene, it may represent a novel type of self-marker protein with a role in CR.

Complete loss of the MHC II pathway in an anglerfish, Lophius piscatorius.

Genome studies in fish provide evidence for the adaptability of the vertebrate immune system, revealing alternative immune strategies. The reported absence of the major compatibility complex (MHC) class II pathway components in certain species of pipefish (genus Syngnathus) and cod-like fishes (order Gadiformes) is of particular interest. The MHC II pathway is responsible for immunization and defence against extracellular threats through the presentation of exogenous peptides to T helper cells. Here, we demonstrate the absence of all genes encoding MHC II components (CD4, CD74 A/B, and both classical and non-classical MHC II α/β) in the genome of an anglerfish, Lophius piscatorius, indicating loss of the MHC II pathway. By contrast, it has previously been reported that another anglerfish, Antennarius striatus, retains all MHC II genes, placing the loss of MHC II in the Lophius clade to their most recent common ancestor. In the three taxa where MHC II loss has occurred, the gene loss has been restricted to four or five core MHC II components, suggesting that, in teleosts, only these genes have functions that are restricted to the MHC II pathway.

Inhibition of the Cytolytic Protein Perforin Prevents Rejection of Transplanted Bone Marrow Stem Cells in Vivo.

Perforin is a key effector protein in the vertebrate immune system and is secreted by cytotoxic T lymphocytes and natural killer cells to help eliminate virus-infected and transformed target cells. The ability to modulate perforin activity in vivo could be extremely useful, especially in the context of bone marrow stem cell transplantation where early rejection of immunologically mismatched grafts is driven by the recipient's natural killer cells, which overwhelmingly use perforin to kill their targets. Bone marrow stem cell transplantation is a potentially curative treatment for both malignant and nonmalignant disorders, but when the body recognizes the graft as foreign, it is rejected by this process, often with fatal consequences. Here we report optimization of a previously identified series of benzenesulfonamide-based perforin inhibitors for their physicochemical and pharmacokinetic properties, resulting in the identification of 16, the first reported small molecule able to prevent rejection of transplanted bone marrow stem cells in vivo by blocking perforin function.

Stop the global slaughter of sharks

The unsustainable slaughter of millions of sharks for their fins continues unabated even in EU waters, threatening the survival of several species. Meanwhile, new discoveries about the ecology of the top predators illustrate how much we still have to learn about the life we are about to destroy. Michael Gross reports.

Transposons to V(D)J Recombination: Evolution of the RAG Reaction

Evolutionarily, how RAG endonucleases in vertebrate immune systems could shed dangerous transposon-like propensities, and instead, support the organized assembly of antigen receptor variable domains, has been unclear. Recent structural work by Schatz and colleagues (Nature, 2019) identifies features of the RAG endonuclease deemed to be key in supporting this critical change in vertebrate advancement.

Abstract 4956: Oral antibiotics act as potent immunotherapeutic agents against pancreatic cancer

Abstract Background: Recent reports have elaborated on how the gut microbiota shape vertebrate immune system during ontogeny. Additionally, tumor-promoting inflammation and gut dysbiosis are often associated with multiple cancers including pancreatic ductal adenocarcinoma (PDAC). We sought to investigate the gut microbiota-immune relationship in mouse models of cancer. Methods: To investigate if cancer-associated dysbiosis modulates cancer per se, neonatal mice with unstable, dynamic gut microbiota (guests) were cohoused with either adult cancer-naive mice or adult pancreatic cancer-bearing KPC (KrasG12D/+;Trp53R172H/+;Pdx-1-Cre) mice. After 4 weeks of co-housing, guest mice were subcutaneously implanted with KPC PDAC and monitored for cancer progression. In a parallel series of experiments, tumor progression was compared between control mice and mice given oral antibiotics and also, between germ free mice and conventionalized mice. Later, oral antibiotics were combined with checkpoint inhibitors in preclinical trials of efficacy. Monoclonal antibodies and knockout strains of mice were used to elucidate mechanism. Pancreatic tumors were probed for bacteria. Results: Co-housing with KPC mice significantly increased cancer progression in guest mice compared to that in guest mice co-housed with non-cancer-bearing cagemates. Bowel sterilization dramatically reduced cancer burden in subcutaneous, metastatic, and orthotopic models of pancreatic cancer as well as in subcutaneous and metastatic models of melanoma. Similarly, germ free mice had decreased subcutaneous PDAC burden compared to littermate conventionalized mice. Surprisingly and in findings contradictory to previously published reports, oral antibiotics also potentiated the efficacy of checkpoint inhibitors in shrinking tumors. The tumor decreasing effect of antibiotics disappeared in immunodeficient Rag1-/- mice, wild type mice depleted of IL-17a but not in Tlr4-/- mice. Immunophenotyping further confirmed microbiota-mediated infiltration of immunosuppressive cells inside tumor and antibiotics-mediated activation of intratumoral Th1/Tc1 immunity. Interestingly, pancreatic cancer liver metastases revealed a significant presence of 16S rDNA resembling the gut flora and subcutaneous pancreatic cancer tissue grew viable bacteria on culture medium and this was prevented by oral antibiotics, thereby suggesting a gut-to-tumor translocation of bacteria. Conclusion: Our data suggest that gut microbiota-cancer cross talk is mediated by the immune system and that oral antibiotics may be repurposed as potent anti-cancer immunotherapeutic agents. Citation Format: Vrishketan Sethi, Saba Kurtom, Irina Fernandez, Maria Abreu, Sabita Roy, Sundaram Ramakrishnan, Ashok Saluja, Vikas Dudeja. Oral antibiotics act as potent immunotherapeutic agents against pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4956.

Molecular characterization, constitutive expression and GTP binding mechanism of Cirrhinus mrigala (Hamilton, 1822) Myxovirus resistance (Mx) protein

Myxovirus resistance (Mx) proteins represents the subclass of the dynamin superfamily of large Guanosine triphosphates (GTPases), play esential role in intracellular vesicle trafficking, endocytosis, organelle homeostasis and mitochondria distribution. These proteins are key players of the vertebrate immune system, induced by type-I and type-III interferons (IFN) of infected host and inhibit viral replication by sequestering its nucleoprotein. In the present study, we report the sequencing and characterization of Cirrhinus mrigala Mx protein (CmMx) for the first time and observed its constitutive expression in different tissues for a period of fourteen days. The synthetic peptide, LSGVALPRGTGI, was dissolved in PBS and injected into a rabbit and the antibody raised against CmMx was used to study the level of its expression. The full length of the CmMx cDNA is 2244 bp with a molecular mass of 70.9 kDa and a predicted isoelectric point of 8.25. The 627 amino acids polypeptide formed of three main functional domains: N-terminal GTPase domain (GD), a middle domain (MD) and GTPase effector domain (GED) with carboxy terminal leucine zipper motif. The 3D models of CmMx protein was modeled based on available close structural homologs and further validated through molecular dynamics (MD) simulations. MD study revealed the importance of G-domain responsible for recognition of GTP, which perfectly corroborate with earlier studies. MM/PBSA binding free energy analysis displayed that van der Waals and electrostatic energy were the key driving force behind molecular recognition of GTP by CmMx protein. The results from this study will illuminate more lights into the ongoing research on myxovirus resistance protein and its role in inhibition of viral replication in other eukaryotic system as well.

Multiplicative fitness, rapid haplotype discovery, and fitness decay explain evolution of human MHC

The major histocompatibility complex (MHC) is a central component of the vertebrate immune system and hence evolves in the regime of a host-pathogen evolutionary race. The MHC is associated with quantitative traits which directly affect fitness and are subject to selection pressure. The evolution of haplotypes at the MHC HLA (HLA) locus is generally thought to be governed by selection for increased diversity that is manifested in overdominance and/or negative frequency-dependent selection (FDS). However, recently, a model combining purifying selection on haplotypes and balancing selection on alleles has been proposed. We compare the predictions of several population dynamics models of haplotype frequency evolution to the distributions derived from 6.59-million-donor HLA typings from the National Marrow Donor Program registry. We show that models that combine a multiplicative fitness function, extremely high haplotype discovery rates, and exponential fitness decay over time produce the best fit to the data for most of the analyzed populations. In contrast, overdominance is not supported, and population substructure does not explain the observed haplotype frequencies. Furthermore, there is no evidence of negative FDS. Thus, multiplicative fitness, rapid haplotype discovery, and rapid fitness decay appear to be the major factors shaping the HLA haplotype frequency distribution in the human population.

RNase L facilitates the repair of DNA double-strand breaks through the nonhomologous end-joining pathway.

RNA molecules have been found to play important roles in DNA double-strand break (DSB) repair, but the exact underlying mechanism remains unclear. Here, we aimed to clarify the function of RNase L, an important ribonuclease in the immune system of vertebrates, in DSB repair. Knockdown of RNase L reduces cell survival after induction of DSBs by ionizing radiation or camptothecin and causes a significant decrease in DSB repair, as evidenced by an increase in the extent of phosphorylation of histone H2AX on Ser139 (γH2AX) and γH2AX nuclear foci formation. Thus, our findings indicate that RNase L interacts with the core factors involved in DNA end joining, such as XRCC4 and Lig4, and facilitates DSB repair through the nonhomologous end-joining pathway.

The kinetics of cellular and humoral immune responses of common carp to presporogonic development of the myxozoan Sphaerospora molnari

BackgroundSphaerospora molnari is a myxozoan parasite causing skin and gill sphaerosporosis in common carp (Cyprinus carpio) in central Europe. For most myxozoans, little is known about the early development and the expansion of the infection in the fish host, prior to spore formation. A major reason for this lack of information is the absence of laboratory model organisms, whose life-cycle stages are available throughout the year.ResultsWe have established a laboratory infection model for early proliferative stages of myxozoans, based on separation and intraperitoneal injection of motile and dividing S. molnari stages isolated from the blood of carp. In the present study we characterize the kinetics of the presporogonic development of S. molnari, while analyzing cellular host responses, cytokine and systemic immunoglobulin expression, over a 63-day period. Our study shows activation of innate immune responses followed by B cell-mediated immune responses. We observed rapid parasite efflux from the peritoneal cavity (< 40 hours), an initial covert infection period with a moderate proinflammatory response for about 1–2 weeks, followed by a period of parasite multiplication in the blood which peaked at 28 days post-infection (dpi) and was associated with a massive lymphocyte response. Our data further revealed a switch to a massive anti-inflammatory response (up to 1456-fold expression of il-10), a strong increase in the expression of IgM transcripts and increased number of IgM+ B lymphocytes, which produce specific antibodies for the elimination of most of the parasites from the fish at 35 dpi. However, despite the presence of these antibodies, S. molnari invades the liver 42 dpi, where an increase in parasite cell number and indistinguishable outer cell membranes are indicative of effective exploitation and disguise mechanisms. From 49 dpi onwards, the acute infection changes to a chronic one, with low parasite numbers remaining in the fish.ConclusionsTo our knowledge, this is the first time myxozoan early development and immune modulation mechanisms have been analyzed along with innate and adaptive immune responses of its fish host, in a controlled laboratory system. Our study adds important information on host–parasite interaction and co-evolutionary adaptation of early metazoans (Cnidaria) with basic vertebrate (fish) immune systems and the evolution of host adaptation and parasite immune evasion strategies.

Identifying antibodies and T-cell receptors with known specificity in repertoire sequencing data

Abstract The adaptive immune system in vertebrates has evolved to recognize an immense number of pathogens. B and T cells play important role in the antigen recognition by expressing a substantially diverse repertoire of antigen-specific receptors. The recent advances in the high-throughput immune repertoire sequencing has led to a tremendous increase in the antibody and T-cell receptor information. Identification of specific epitopes recognized by the receptors is vital in analyzing the repertoire sequencing data. Here, we present a tool that examines data from an immune receptor repertoire experiment, and identifies receptors (or highly related variants) that are present in the Immune Epitope Database (IEDB). The IEDB is a free resource which compiles experiments characterizing immune epitopes. IEDB also captures the epitope-specific immune receptor sequences and groups them based on their epitope specificity. IEDB currently has information on over 26,000 receptor groups. Users can provide receptor repertoire sequence data in multiple formats, and specify different CDR sequence identity cutoffs to restrict or broaden the receptor search. We expect that this tool will provide new insights into B and T cell responses captured in the receptor repertoire experiments by identifying specific epitopes, and facilitate development of new strategies for monitoring and treating of infectious, allergic and autoimmune diseases.

The Drosophila melanogaster antimicrobial peptides Mtk-1 and Mtk-2 are active against the malarial parasite Plasmodium falciparum.

Antimicrobial peptides (AMPs) are important components of the vertebrate and invertebrate innate immune systems. Although AMPs are widely recognized for their broad-spectrum activity against bacteria, fungi, and viruses, their activity against protozoan parasites has not been investigated in detail. In this study, we tested 10 AMPs from three different insect species: the greater wax moth Galleria mellonella (cecropin A-D), the fruit fly Drosophila melanogaster (drosocin, Mtk-1 and Mtk-2), and the blow fly Lucilia sericata (LSerPRP-2, LSerPRP-3 and stomoxyn). We tested each AMP against the protozoan parasite Plasmodium falciparum which is responsible for the most severe form of malaria in humans. We also evaluated the impact of these insect AMPs on mouse and pig erythrocytes. Whereas all AMPs showed low hemolytic effects towards mouse and pig erythrocytes, only D. melanogaster Mtk-1 and Mtk-2 significantly inhibited the growth of P. falciparum at low concentrations. Mtk-1 and Mtk-2 could therefore be considered as leads for the development of antiparasitic drugs targeting the clinically important asexual blood stage of P. falciparum.