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Novel immune‐type receptor genes
Abstract
Novel immune-type receptor (NITR) genes, which initially were identified in the Southern pufferfish (Spheroides nephelus), encode products which consist of an extracellular variable (V) and V-like C2 (V/C2) domain, a transmembrane region, and a cytoplasmic tail, which typically possesses an immunoreceptor tyrosine-based inhibition motif (ITIM). Multiple NITR genes have been identified in close, contiguous chromosomal linkage. The V regions of NITRs resemble prototypic forms defined for immunoglobulin (Ig) and T-cell antigen receptor (TCR), are present in multiple families and exhibit regionalized variation in sequence, which also occurs in Ig and TCR. Comparisons of exons encoding transmembrane and cytoplasmic regions of multiple NITRs suggest that exon shuffling has factored in the diversification of the NITR gene complex. Zebrafish (Danio rerio) NITRs exhibit many of these characteristics. NITRs that have been identified in additional species of bony fish demonstrate additional variation in the number of extracellular domains as well as in the presence of intramembranous charged residues, cytoplasmic tails and ITIMs. The presence in NITRs of V regions that are related closely to those found in Ig and TCR, as well as regulatory motifs and other structural features that are characteristic of immune inhibitory receptors encoded at the leukocyte receptor cluster, suggests that the NITRs are representative of an integral stage in the evolution of innate and adaptive immune function.
This research was supported by grants AI23338 to GWL and GM20231 to JAY from the National Institutes of Health as well as a grant from The Pediatric Cancer Foundation, Inc. to GWL.
... NITR clusters have been identified in all examined teleost lineages (Strong et al. 1999;Yoder et al. 2004;Desai et al. 2008;Yoder 2009;Ferraresso et al. 2009;Wcisel and Yoder 2016), and are expressed in lymphocytes (Yoder et al. 2010), including zebrafish NK-like cells (Carmona et al. 2017). NITRs have been implicated in allorecognition ) and, although their ligands remain unknown, are predicted to function as NK cell receptors (Litman et al. 2001;Yoder et al. 2010). Typically, NITRs possess two extracellular Ig domains, one membrane-distal variable (V)-like domain and one membrane-proximal intermediate (I)-like domain. ...
... We demonstrate that bowfin NITR Ig domains possess an architecture similar to other NITR V and I domains. Alignment-based comparisons between bowfin, gar, and teleost V domains (Fig. 8) reveal the presence of residues that are conserved across NITR, T cell receptor (TCR), and immunoglobulin Ig domains such as C 23 and C 104 (Litman et al. 2001;Yoder 2009). Moreover, NITR V domains display similar levels of diversity as DICP D1 domains, raising the possibility that bowfin NITRs bind a range of ligands using the V domain. ...
... The presence of J or J-like sequences in a single exon with a V or I domain is characteristic of teleost NITRs, and supports the proposal that NITRs either represent an ancient gene family that may have given rise to V(D) J recombination in the adaptive immune system, or an evolutionary novelty that arose as a consequence of the teleost genome duplication (Strong et al. 1999;Litman et al. 2001Litman et al. , 2003Yoder et al. 2004). The hypothesis that NITRs arose as a consequence of the TGD was supported by the lack of evidence for NITR homologs in cartilaginous fishes (Yoder et al. 2004). ...
Over 99% of ray-finned fishes (Actinopterygii) are teleosts, a clade that comprises half of all living vertebrate species that have diversified across virtually all fresh and saltwater ecosystems. This ecological breadth raises the question of how the immunogenetic diversity required to persist under heterogeneous pathogen pressures evolved. The teleost genome duplication (TGD) has been hypothesized as the evolutionary event that provided the substrate for rapid genomic evolution and innovation. However, studies of putative teleost-specific innate immune receptors have been largely limited to comparisons either among teleosts or between teleosts and distantly related vertebrate clades such as tetrapods. Here we describe and characterize the receptor diversity of two clustered innate immune gene families in the teleost sister lineage: Holostei (bowfin and gars). Using genomic and transcriptomic data, we provide a detailed investigation of the phylogenetic history and conserved synteny of gene clusters encoding diverse immunoglobulin domain-containing proteins (DICPs) and novel immune-type receptors (NITRs). These data demonstrate an ancient linkage of DICPs to the major histocompatibility complex (MHC) and reveal an evolutionary origin of NITR variable-joining (VJ) exons that predate the TGD by at least 50 million years. Further characterizing the receptor diversity of Holostean DICPs and NITRs illuminates a sequence diversity that rivals the diversity of these innate immune receptor families in many teleosts. Taken together, our findings provide important historical context for the evolution of these gene families that challenge prevailing expectations concerning the consequences of the TGD during actinopterygiian evolution.
... Eleven different distinct forms of NITR genes were identified that vary in the number of extracellular immunoglobulin domains, number and location of J-region-like motifs, presence of transmembrane regions , presence of charged residues in transmembrane regions, presence of cytoplasmic tails, and/or distribution of ITIM(s) within the cytoplasmic tails (). We have proposed that the NITRs that contain positively charged intramembrane residues correspond to activating receptors, although this has not been demonstrated functionally (Litman et al. 2001). A number of other families of leukocyte regulatory receptors (LRRs) possess equivalent charge relationships (Barten et al. 2001). ...
... It is presumed that the highly diversified LRRs arose earlier in phylogeny. The overall structural and organizational similarities between the NITRs identified in bony fish and certain members of the mammalian LRR suggest that some members are functionally related (Litman et al. 2001 ). Genetic mapping studies in zebrafish suggest that NITRs share conserved synteny with human LRC genes (Yoder et al. 2001 ); however, this synteny cannot be confirmed without more extensive sequencing of the zebrafish genome. ...
... It seems that the hallmark of NITRs, structural and functional variation, was recurrently achieved through both residue variation and exon shuffling (Litman, Hawke and Yoder, unpublished). Although the function of NITRs, as well as many KIRs and other inhibitory and activating LRRs, is not understood (Borges and Cosman 2000; Dietrich et al. 2000), the unequivocal V (and J) sequence present in NITRs, such as those described here, along with the observed patterns of sequence variation and apparent lack of somatic diversification suggest that NITRs function as innate receptor(s ) (Litman et al. 2001; Strong et al. 1999 ). We previously have suggested that one possible role for NITRs is that they represent the bony fish functional counterparts of the mammalian KIRs; in this case, specificity could be mediated through a V versus C2 interaction (Litman et al. 2001 ). ...
Novel immune-type receptor ( NITR) genes that encode two extracellular immunoglobulin domains and cytoplasmic immunoreceptor tyrosine-based inhibition motifs (ITIMs) have been described previously in three lineages of bony fish. In the current study, four ITIM-containing NITR cDNAs are identified in the rainbow trout ( Oncorhynchus mykiss), and their expression patterns and genomic complexity are characterized. The ITIM-containing NITR2 gene maps 1.3 cM from an ITIM-containing C-type lectin receptor ( TCL-2) on linkage group XXI. A comprehensive, phylogenetic analysis of NITRs from rainbow trout and three other major lineages of bony fish defines conserved families of NITRs and suggests an ancient lineage of distinct groups of genes. Several probable scenarios that explain the origins of variant forms of NITRs are described.
... However, the existence of the CTLD-type NK receptors (Group V) is still debatable (Panagos et al., 2006). It has been suggested that receptors which have structure features similar to the CTLD-type NK receptors are present in fish (Litman et al., 2001;Yoder et al., 2004). Interestingly, a vast group Fig. 6. ...
... of novel immune-type receptors (NITRs) which possess an extracellular Ig domain have also been reported in fish but cannot be assigned into any of the known mammalian immune receptor family (Litman et al., 2001;Yoder et al., 2004). It is believed that the C-type lectin receptors could have been extensively expanded in teleost fish (Petit et al., 2019). ...
Group II C-type lectin domain (CTLD) containing receptors belong to a large family of pattern recognition receptors which mainly act on the innate immunity. They are structurally related and consist of a cytoplasmic immunoreceptor tyrosine-based inhibitory motif (ITIM) and a single extracellular CTLD. Although they have been described in teleost fish, their involvement in immune responses is not well understood. In this study, four immune-related lectin-like receptors (termed CiILLR1 and CiILLR5-7), belonging to the group II CTLD receptors, were identified in grass carp (Ctenopharyngodon idella). They contain a short cytoplasmic tail and a single CTLD in the extracellular region. The CiILLR1 has a WxHxxxxxY motif similar to the WxHxxxxY motif which is required for the recognition of β-glucans by some of the group II CTLD containing lectins in mammals. Further, a modified QPD motif (EPD) known to be involved in binding to carbohydrate ligands is present in the CiILLR1, 5 and 6. However, CiILLR7 lacks these motifs. Expression analysis revealed that they were constitutively expressed in the head kidney and spleen. Moreover, CiILLR1, 5 and 6 could be up-regulated in the head kidney and spleen of fish after infection with Flavobacterium columnare and in the primary head kidney leukocytes by LPS and PHA. Expression of CiILLR1, CiILLR5 and CiILLR6 were mainly detected in the enriched lymphocytes whilst CiILLR7 was expressed in the enriched monocytes/macrophages. The results expand existing knowledge on the immune responses of the C-type lectin receptors in teleost fish.
... All current data suggest that such sequences (V-J or I-J) are always encoded within a single exon with no evidence for recombination. The one unifying feature of all NITR genes is that they all encode an authentic V domain [20]. The only known exception to this ''rule'' is found in alternatively spliced transcripts of zebrafish Nitr9 which remove part or all of the V domain [8,17]. ...
... The only known exception to this ''rule'' is found in alternatively spliced transcripts of zebrafish Nitr9 which remove part or all of the V domain [8,17]. NITR V domains possess highly conserved residues that are also conserved in T cell receptor (TCR) and immunoglobulin (Ig) V domains [20]; NITR V domains also include three hypervariable (HV) regions which correspond to the complementarity determining regions (CDRs) in Igs and TCRs (Fig. 3A) [8]. In addition, recent crystal structures of NITR V domains demonstrate that they are structurally similar to the V domains of antigen receptors (Fig. 3B) [21]. ...
Novel immune-type receptors (NITRs) are encoded by clusters of multigene families and have been identified in multiple bony fish species. All NITRs possess one extracellular immunoglobulin (Ig) domain of the variable (V) type and recent crystal structures of NITR V domains demonstrate their high degree of similarity to V domains of antigen receptors. Many NITRs possess a second extracellular Ig domain of the intermediate (I) type which helps differentiate NITRs from other V domain receptors. The majority of NITRs are type I transmembrane receptors; however, a small number are predicted to encode secreted proteins. Based on their sequence and structure, NITRs have been proposed to be "functional orthologs" of mammalian natural killer receptors (NKRs). Like NKRs, most NITRs possess short functional motifs permitting their classification as inhibitory or activating. NITRs lacking these motifs are functionally ambiguous. Inhibitory and activating NKRs utilize opposing signaling mechanisms to influence the response of NK cells to target cells; studies employing recombinant NITRs suggest that these signaling pathways are conserved between NKRs and NITRs. An analysis of all published NITR sequences demonstrates the conserved nature of multiple residues within the NITR Ig domains permitting the identification of NITR ESTs from salmon, cod, halibut, lake whitefish and stickleback species. Complete data sets of NITRs from the sequencing of the zebrafish and medaka genomes provide insight into the evolution of the NITRs within bony fish species. It is likely that all teleost species encode NITRs which function within innate immunity to regulate cell mediated cytotoxicity.
... In humans, mice, fruit flies (Drosophila melanogaster), nematodes (Caenorhabditis elegans) and several other species, high representation expressed sequence tag (EST) databases and evolving approaches for scanning genomic sequences have facilitated the identification of distantly related genes; however, this technique only can be used for species that have been analyzed by large-scale genomic experiments [5][6][7][8] . Polymerase chain reaction (PCR)-based approaches have been invaluable in defining genes of immunological relevance in both higher and lower vertebrate species; however, these approaches require two separate and appropriately spaced regions of conserved amino acid sequence identity [9][10][11][12][13][14][15] . In addition, the necessity of introducing sequence ambiguities in the design of oligonucleotide primers often results in high artifactual content, which can overwhelm efforts to amplify appropriate gene sequences. ...
... Defining the requisite structural features of V-type domains in relation to the more ancient I-type domains is essential to further interpretation of the VCBP genes. On the basis of crystallographic analyses and compilations of vertebrate V domains, V-frame determinant residues are comprised, in part, by a set of seven residues that are canonical to both V-and I-type Ig domains 11,22 : Gly 16 , Cys 23 , Trp 41 , Leu 89 (or other hydrophobic residue), Asp 98 , Tyr 102 and Cys 104 . An eighth canonical position, Arg 75 (with Lys or His seen less frequently at this position), Arg (Lys or His) 75 , is required along with Asp 98 for the formation of a salt bridge characteristic of both V and I domains 22 . ...
The evolutionary origin of adaptive immune receptors is not understood below the phylogenetic level of the jawed vertebrates. We describe here a strategy for the selective cloning of cDNAs encoding secreted or transmembrane proteins that uses a bacterial plasmid (Amptrap) with a defective beta-lactamase gene. This method requires knowledge of only a single target motif that corresponds to as few as three amino acids; it was validated with major histocompatibility complex genes from a cartilaginous fish. Using this approach, we identified families of genes encoding secreted proteins with two diversified immunoglobulin-like variable (V) domains and a chitin-binding domain in amphioxus, a protochordate. Thus, multigenic families encoding diversified V regions exist in a species lacking an adaptive immune response.
... Although NK cells have been functionally described from bony fish [52,53], numerous efforts to identify genes encoding KIRs or Ly49/Klra in fish have been unsuccessful [1]. Nevertheless, in the same way that humans use KIRs and mice use Ly49/Klras to mediate NK function, a candidate gene family that may facilitate mammalian-like NK cell function in fishes is the novel immune-type receptors (NITRs) gene family [54][55][56][57]. Similar to mammalian KIRs, NITRs are encoded in gene clusters, include inhibitory and activating forms, and display gene content variation [1,55,58]. ...
Following the draft sequence of the first human genome over 20 years ago, we have achieved unprecedented insights into the rules governing its evolution, often with direct translational relevance to specific diseases. However, staggering sequence complexity has also challenged the development of a more comprehensive understanding of human genome biology. In this context, interspecific genomic studies between humans and other animals have played a critical role in our efforts to decode human gene families. In this review, we focus on how the rapid surge of genome sequencing of both model and non-model organisms now provides a broader comparative framework poised to empower novel discoveries. We begin with a general overview of how comparative approaches are essential for understanding gene family evolution in the human genome, followed by a discussion of analyses of gene expression. We show how homology can provide insights into the genes and gene families associated with immune response, cancer biology, vision, chemosensation, and metabolism, by revealing similarity in processes among distant species. We then explain methodological tools that provide critical advances and show the limitations of common approaches. We conclude with a discussion of how these investigations position us to gain fundamental insights into the evolution of gene families among living organisms in general. We hope that our review catalyzes additional excitement and research on the emerging field of comparative genomics, while aiding the placement of the human genome into its existentially evolutionary context.
... In fact, a unique nitr gene on the whole genome was only found in this region. Nitr genes were initially identified in the Southern pufferfish (Spheroides nephelus) (Litman et al., 2001) and is only found in bony fish (Meng et al., 2014). It belongs to the immunoglobulin superfamily, encodes type I transmembrane proteins. ...
Yellow drum (Nibea albiflora) is one of the most important marine fish species in China. However, the natural outbreak of red head disease has seriously restricted the aquaculture development of yellow drum, and genetic improvement has become a potential strategy. In the research, 37 DNA pools (1066 samples) were generated with Pool-seq, which significantly reduces costs. In order to correct place factors and satisfy the requirement of multi-pool joint analysis, we propose and utilize three methods to locate disease-resistant regions with 33 DNA pools. The additive property of χ² distribution and log-likelihood ratio test of logit regression were firstly used for joint localization of disease-resistant regions; secondly, we take advantage of the modified G′ value to further narrow candidate regions. One red head disease-resistant region of 10.9 Mb–13.11 Mb on chr22 was determined. Forty-one genes (36 non-repetitive genes) were found in the region by functional gene annotation. Twelve non-repetitive genes were involved in the immune responses, where one nitr gene and several genes (i.e., tifa, alpk1, mapk1 and transcription factor p65) exist in NF-κB signaling pathway were identified, suggesting that NF-κB signaling pathway may be important in red head disease resistance.
... novel immune type receptors) or evolutionally conserved (e.g. channel catfish leukocyte immune-type receptors) or secretions of cytokines (20)(21)(22)(23). This further illustrates the complexity of cellular components in teleost immunity and a detailed characterization of immune cell subsets is required to further dissect specific roles of each cell type/subset, which can only be achieved when they are properly separated. ...
Akin to their mammalian counterparts, teleost fish possess a complex assortment of highly specialized immune cells that are capable of unleashing potent innate immune responses to eradicate or mitigate incoming pathogens, and also differentiate into memory lymphocytes to provide long-term protection. Investigations into specific roles and functions of fish immune cells depend on the precise separation of each cell type. Commonly used techniques, for example, density gradient centrifugation, rely on immune cells to have differing sizes or densities and thus fail to separate between similar cell types (e.g. T and B lymphocytes). Furthermore, a continuously growing database of teleost genomic information has revealed an inventory of cellular markers, indicating the possible presence of immune cell subsets in teleost fish. This further complicates the interpretation of results if subsets of immune cells are not properly separated. Consequently, monoclonal antibodies (mAbs) against specific cellular markers are required to precisely identify and separate novel subsets of immune cells in fish. In the field of fish immunology, mAbs are largely generated using the hybridoma technology, resulting in the development of mAbs against specific cellular markers in different fish species. Nevertheless, this technology suffers from being labour-intensive, time-consuming and most importantly, the inevitable loss of diversities of antibodies during the fusion of antibody-expressing B lymphocytes and myeloma cells. In light of this, the focus of this review is to discuss the potential applications of fluorescence-activated cell sorting and droplet-based microfluidics, two emerging technologies capable of screening and identifying antigen-specific B lymphocytes in a high-throughput manner, in promoting the development of valuable reagents for fish immunology studies. Our main goal is to encourage the incorporation of alternative technologies into the field of fish immunology to promote the production of specific antibodies in a high-throughput and cost-effective way, which could better allow for the precise separation of fish immune cells and also facilitate the identification of novel immune cell subsets in teleost fish.
... Aside from Ig, the IgV domain is also present in several molecules of the innate immune system of jawed vertebrates. Of particular interest is the case of the novel immune-type receptors (NITRs), found in teleost fish [36,37]. NITR genes encode type I transmembrane receptors, which contain one or two extracellular Ig domains ( Figure 1F) and resemble molecules that serve as activating or inhibitory receptors. ...
The vertebrate immune system provides a powerful defense because of the ability to potentially recognize an unlimited number of pathogens. The antibody molecule, also termed immunoglobulin (Ig) is one of the major mediators of the immune response. It is built up from two types of Ig domains: the variable domain, which provides the capability to recognize and bind a potentially infinite range of foreign substances, and the constant domains, which exert the effector functions. In the last 20 years, advances in our understanding of the molecular mechanisms and structural features of antibody in mammals and in a variety of other organisms have uncovered the underlying principles and complexity of this fundamental molecule. One notable evolutionary topic is the origin and evolution of antibody. Many aspects have been clearly stated, but some others remain limited or obscure. By considering a wide range of prokaryotic and eukaryotic organisms through a literature survey about the topic, we have provided an integrated view of the emergence of antibodies in evolution and underlined the very ancient origins.
... All of the five Ig-domain containing multi-gene families iden- tified in teleost have been found that possess extensive poly- morphic and haplotypic variation (Haire et al., 2012;Kortum et al., 2014;Litman et al., 2001;Rodriguez-Nunez et al., 2016;Stafford et al., 2006;Stet et al., 2005;Yoder et al., 2008). In zebrafish, DICP diversity is mainly derived from the extensive polymorphic variation, haplotypic polymorphism and alternative mRNA pro- cessing ( Haire et al., 2012;Rodriguez-Nunez et al., 2016). ...
... To our knowledge, teleost NITRs are not related to any immunoregulatory receptor families in amphibians, birds, or mammals; but, they do participate in NK cell-mediated allorecognition, implying they are the functional equivalents of mammalian NK receptors (NKRs) [22,23]. Further information regarding teleost NITRs can be found in [24][25][26]. Alternatively, teleost leukocyte immune-type receptors (LITRs), which were originally identified in the channel catfish (Ictalurus punctatus), do share distant phylogenetic relationships with several families of mammalian IgSF members including FcRs, FcRLs, and various NKRs encoded within the leukocyte receptor complex (LRC) [27]. ...
Across vertebrates, innate immunity consists of a complex assortment of highly specialized cells capable of unleashing potent effector responses designed to destroy or mitigate foreign pathogens. The execution of various innate cellular behaviors such as phagocytosis, degranulation, or cell-mediated cytotoxicity are functionally indistinguishable when being performed by immune cells isolated from humans or teleost fishes; vertebrates that diverged from one another more than 450 million years ago. This suggests that vital components of the vertebrate innate defense machinery are conserved and investigating such processes in a range of model systems provides an important opportunity to identify fundamental features of vertebrate immunity. One characteristic that is highly conserved across vertebrate systems is that cellular immune responses are dependent on specialized immunoregulatory receptors that sense environmental stimuli and initiate intracellular cascades that can elicit appropriate effector responses. A wide variety of immunoregulatory receptor families have been extensively studied in mammals, and many have been identified as cell- and function-specific regulators of a range of innate responses. Although much less is known in fish, the growing database of genomic information has recently allowed for the identification of several immunoregulatory receptor gene families in teleosts. Many of these putative immunoregulatory receptors have yet to be assigned any specific role(s), and much of what is known has been based solely on structural and/or phylogenetic relationships with mammalian receptor families. As an attempt to address some of these shortcomings, this review will focus on our growing understanding of the functional roles played by specific members of the channel catfish (Ictalurus punctatus) leukocyte immune-type receptors (IpLITRs), which appear to be important regulators of several innate cellular responses via classical as well as unique biochemical signaling networks.
... Gallucci et al. [39] mentioned the efficacy of synergistic action between some terpenes on bacteria. Litman et al. [40] reported about the synergistic effect of linalool and eugenol on some fungi species. ...
With steadily growing of the microbial resistance against the classical antibiotics, searching new and effective natural antimicrobial agents become crucial. The study introduces new herbal extracts as antibacterial agents against both Gram-positive and Gram-negative bacteria. In vitro antibacterial activity of 15 weed plant species, collected from Aseer region, Saudi Arabia and their effective minimum inhibitory concentrations (MIC) were determined. The possible mode of action of the most effective plant extracts was investigated. Crude extracts of the investigated species were prepared using three organic solvents. Bacillus subtilis and Proteus vulgaris were used as test organisms to evaluate the antibacterial effect of the crude extracts and their (MIC). Examination with scanning electron microscopy of the treated bacteria as well as analysis of the plant extract using GC-MS were performed to explore the possible mode of action. Foeniculum vulgare, Xanthium spinosum, Abutilon pannosum, Solanum incanum, Forsskaolea tenacissima and Brassica deflexa were the most effective plants among the fifteenth tested species. Scan electron micrographs (SEM) revealed a clear morphological malformation in the bacterial shapes. This supports our hypothesis that extracts could alter the bacterial cell membrane permeability causing plasmolysis. GC-MS analysis of plant extracts proved the presence of aliphatic and aromatic compounds, whereas, the number and types of these compounds depended on the type of the organic solvent. The chloroform extracts of Xanthium spinosum, Abutilon pannosum are very promising antibacterial agents against both Gram-positive and Gram-negative bacteria. Further investigation is recommended to scale up and validate the application of the test plant extracts.
... Interaction partners of immunoglobulin-like domains are mainly other immunoglobulin-like domains, but sometimes they also associate with other domain types like fibronectin type III, cytokine receptor domains, EGF domains, C-type lectin and complement control protein domains (Barclay 2003). The structure of CEACAMs with the one aminoterminal Ig V -like domain followed by one or more Ig C2like domains is very typical for membrane proteins (Thomson, Krupey et al. 1969;Litman, Hawke et al. 2001). CEACAMs mediate cell-cell adhesion by homotypic binding to themselves or heterotypic binding to another member of the CEACAM family, also mediated by the CC´-FG-face of the Ig V -like domain (Benchimol, Fuks et al. 1989). ...
... These results were validated by RTqPCR with a Pearson's coefficient of 0.61 (MX in S3Fig). Novel immune-type receptor (nitr) genes belong to multigene families encoding transmembrane proteins containing immunoglobulin-like variable domains with a high degree of sequence variation [66, 67]. A maximum haplotype of 36 nitr zebrafish genes can be grouped into 12 families, including inhibitory (nitr1-8, having immune receptor tyrosine inhibition motifs ITIM) and activating (nitr5, nitr7a, nitr9, nitr10, nitr11a, nitr12) receptors [68]. ...
Since adaptive features such as memory were discovered in mammalian innate immunity,
interest in the immunological status of primitive vertebrates after infections has grown. In
this context, we used zebrafish (Danio rerio), a primitive vertebrate species suited to molecular
and genetic studies to explore transcriptional memories of the immune system in longterm
survivors of viral haemorrhagic septicemia virus infections. Immune-gene targeted
microarrays designed in-house, multipath genes, gene set enrichment, and leading-edge
analysis, reveal unexpected consistent correlations between the viral-survivor phenotype
and several innate multigene families. Thus, here we describe in survivors of infections the
upregulation of the multigene family of proteasome subunit macropains, zebrafish-specific
novel gene sets, mitogen activated protein kinases, and epidermal growth factor. We also
describe the downregulation of the multigene families of c-reactive proteins, myxovirusinduced
proteins and novel immunoglobulin-type receptors. The strength of those immunological
memories was reflected by the exceptional similarity of the transcriptional profiles of
survivors before and after re-infection compared with primary infected fish. On the other
hand, the high levels of neutralizing antibodies in the blood plasma of survivors contrasted
with the depletion of transcripts specific for most cell types present in lymphoid organs.
Therefore, long-term survivors maintained unexpected molecular/cellular memories of previous
viral encounters by modulating the expression levels of innate multigene families as
well as having specific adaptive antibodies. The implications of the so-called "trained immunity"
for future research in this field are also discussed.
... Novel immune-type receptor (nitr) genes belong to multigene families encoding transmembrane proteins containing immunoglobulin-like variable domains with a high degree of sequence variation [66,67]. A maximum haplotype of 36 nitr zebrafish genes can be grouped into 12 families, including inhibitory (nitr1-8, having immune receptor tyrosine inhibition motifs ITIM) and activating (nitr5, nitr7a, nitr9, nitr10, nitr11a, nitr12) receptors [68]. ...
Since adaptive features such as memory were discovered in mammalian innate immunity, interest in the immunological status of primitive vertebrates after infections has grown. In this context, we used zebrafish (Danio rerio), a primitive vertebrate species suited to molecular and genetic studies to explore transcriptional memories of the immune system in long-term survivors of viral haemorrhagic septicemia virus infections. Immune-gene targeted microarrays designed in-house, multipath genes, gene set enrichment, and leading-edge analysis, reveal unexpected consistent correlations between the viral-survivor phenotype and several innate multigene families. Thus, here we describe in survivors of infections the upregulation of the multigene family of proteasome subunit macropains, zebrafish-specific novel gene sets, mitogen activated protein kinases, and epidermal growth factor. We also describe the downregulation of the multigene families of c-reactive proteins, myxovirus-induced proteins and novel immunoglobulin-type receptors. The strength of those immuno-logical memories was reflected by the exceptional similarity of the transcriptional profiles of survivors before and after re-infection compared with primary infected fish. On the other hand, the high levels of neutralizing antibodies in the blood plasma of survivors contrasted with the depletion of transcripts specific for most cell types present in lymphoid organs. Therefore, long-term survivors maintained unexpected molecular/cellular memories of previous viral encounters by modulating the expression levels of innate multigene families as well as having specific adaptive antibodies. The implications of the so-called "trained immu-nity" for future research in this field are also discussed.
... The nitr genes are members of a highly diversified, multigene family of novel immune type receptor found in teleosts. Nitr genes do not rearrange like immune receptors but show structural similarities to both the mammalian T-cell or Ig-like receptors [37,38]. Loss of these genes could be relevant to one facet of progression, which is to avoid immune surveillance, consistent with a critical role of immune regulation in human melanoma [39]. ...
The need for scalable strategies to probe the biological consequences of candidate cancer genes has never been more pressing. The zebrafish, with its capacity for high-throughput transgenesis, in vivo imaging and chemical/genetic screening, has ideal features for undertaking this task. Unique biological insights from zebrafish have already led to the identification of novel oncogenic drivers and small molecules being used to treat the human cancer. This review summarizes the recent main findings and describes pertinent areas where the zebrafish can greatly contribute to our understanding of cancer biology and treatment.
... The relationship between D2 domains does not consistently mirror the relationships between D1 domains, e.g., the D2 domains of Pigrl1.4 and Pigrl2.1 group together, but the D1 domains from these genes are distinctly different (Fig. 3). Recombination and exon swapping between innate immune receptor genes has been described previously (Litman et al. 2001). ...
The polymeric immunoglobulin (Ig) receptor (pIgR) is an integral transmembrane glycoprotein that plays an important role in the mammalian immune response by transporting soluble polymeric Igs across mucosal epithelial cells. Single pIgR genes, which are expressed in lymphoid organs including mucosal tissues, have been identified in several teleost species. A single pigr gene has been identified on zebrafish chromosome 2 along with a large multigene family consisting of 29 pigr-like (PIGRL) genes. Full-length transcripts from ten different PIGRL genes that encode secreted and putative inhibitory membrane-bound receptors have been characterized. Although PIGRL and pigr transcripts are detected in immune tissues, only PIGRL transcripts can be detected in lymphoid and myeloid cells. In contrast to pIgR which binds Igs, certain PIGRL proteins bind phospholipids. PIGRL transcript levels are increased after infection with Streptococcus iniae, suggesting a role for PIGRL genes during bacterial challenge. Transcript levels of PIGRL genes are decreased after infection with Snakehead rhabdovirus, suggesting that viral infection may suppress PIGRL function.
... The nitr genes are members of a highly diversified, multigene family of novel immune type receptor found in teleosts. Nitr genes do not rearrange like immune receptors but show structural similarities to both the mammalian T-cell or Ig-like receptors [37,38]. Loss of these genes could be relevant to one facet of progression, which is to avoid immune surveillance, consistent with a critical role of immune regulation in human melanoma [39]. ...
Melanoma is the most deadly form of skin cancer. Expression of oncogenic BRAF or NRAS, which are frequently mutated in human melanomas, promote the formation of nevi but are not sufficient for tumorigenesis. Even with germline mutated p53, these engineered melanomas present with variable onset and pathology, implicating additional somatic mutations in a multi-hit tumorigenic process.
To decipher the genetics of these melanomas, we sequence the protein coding exons of 53 primary melanomas generated from several BRAFV600E or NRASQ61K driven transgenic zebrafish lines. We find that engineered zebrafish melanomas show an overall low mutation burden, which has a strong, inverse association with the number of initiating germline drivers. Although tumors reveal distinct mutation spectrums, they show mostly C > T transitions without UV light exposure, and enrichment of mutations in melanogenesis, p53 and MAPK signaling. Importantly, a recurrent amplification occurring with pre-configured drivers BRAFV600E and p53-/- suggests a novel path of BRAF cooperativity through the protein kinase A pathway.
This is the first analysis of a melanoma mutational landscape in the absence of UV light, where tumors manifest with remarkably low mutation burden and high heterogeneity. Genotype specific amplification of protein kinase A in cooperation with BRAF and p53 mutation suggests the involvement of melanogenesis in these tumors. This work is important for defining the spectrum of events in BRAF or NRAS driven melanoma in the absence of UV light, and for informed exploitation of models such as transgenic zebrafish to better understand mechanisms leading to human melanoma formation.
... Data collected so far demonstrate that NITRs represent an extraordinarily divergent and rapidly evolving gene family. Different NITR profiles and a remarkable diversity of gene content were found across teleost species, suggesting that independent, lineage-specific tandem gene duplications and family expansions are the most frequent mode for the NITR gene complex evolution (Litman et al. 2001; Yoder 2009). The functional significance of such lineage-specific gene expansions remains to be further investigated. ...
In the last decade, a new gene family encoding non-rearranging receptors, called novel immune-type recep-tors (NITRs), has been discovered in teleost fish. NITRs belong to the immunoglobulin superfamily and represent an extraordinarily divergent and rapidly evolving gene com-plex. Genomic analysis of a region spanning 270 kb led to the discovery of a NITR gene cluster in the European sea bass (Dicentrarchus labrax). In total, 27 NITR genes and three putative pseudogenes, organised in a tandemly arrayed cluster, were identified. Sea bass NITR genes maintain the three major genomic organisations that appear to be essentially conserved among fish species along with new features presumably involving processes of intron loss, exon deletion and acquisition of new exons. Comparative and evolutionary analyses suggest that these receptors have evolved following a "birth-and-death" model of gene evolution in which duplication events together with lineage-specific gain and loss of individual members contributed to the rapid diversification of individual gene families. In this study, we demonstrate that species-specific gene expansions provide the raw material for diversifying, positive Darwinian selection favouring the evolution of a highly diverse array of molecules.
... To further explore the similarity between CDS7 and members of the Ig superfamily, we constructed multiple sequence alignments between domains I-III and canonical V and I-set Ig-like domains. V and I-set Ig-like domains are characterized by a series of 8 beta-strands, labeled A, B, C, C', D, E, F, and G. V-set domains are longer than I-set domains due to a longer C' strand and an additional beta-strand (C " ) located between C' and D. Sequence profiles of V and I-set domains share a common set of frame residues, including a nearly invariant tryptophan in beta-strand C and a pair of cysteines forming a disulphide bridge between strands B and F [2, 3]. We found domains I-III to match the common V/I-set frame residues at many positions, but that the conserved Trp was replaced by Met or Phe residues in domains I and III, respectively (Figure S4). ...
BLASTP searches with the CDS7 protein returned significant alignments to many proteins containing tandem immunoglobulin-like (Ig-like) domains. Domains I, II, and III each aligned to full or partial Ig-like domains from a diversity of molecules. These included vertebrate neurotrimin (e = 0.001, 28% identity), limbic-system associated membrane protein (e = 0.19, 27% identity), and opiod binding protein/cell adhesion molecule (e = 0.006, 29% identity), all of which are members of the IgLON family of neural cell adhesion molecules characterized GPI linkages and three tandem Ig-like domains (1). Alignments were also obtained to a wasp homolog of lachesin, a Drosophila cell-surface protein similar to the IgLON family. Searches against PFAM and the conserved domain database at NCBI with the sequences of each CDS7 domain returned hits to Ig-like domains with e-values ranging from 0.009 to 3.3. (Table S2). Since distant protein homologies are often better detected by methods sensitive to tertiary structure, we also used four protein structure prediction programs to evaluate domains I- III. A majority of these algorithms predicted Ig-like folds for domains I-III with strong statistical confidence (Table S2). In all of these searches, domain I was consistently most similar to V-set Ig-like domains, while domains II and III were most similar to I-set Ig- like domains. To further explore the similarity between CDS7 and members of the Ig superfamily, we constructed multiple sequence alignments between domains I-III and canonical V and I-set Ig-like domains. V and I-set Ig-like domains are characterized by a series of 8 beta-strands, labeled A, B, C, C', D, E, F, and G. V-set domains are longer than I-set domains due to a longer C' strand and an additional beta-strand (C") located between C' and D. Sequence profiles of V and I-set domains share a common set of frame residues, including a nearly invariant tryptophan in beta-strand C and a pair of cysteines forming a disulphide bridge between strands B and F (2, 3). We found domains I-III to match the common V/I-set frame residues at many positions, but that the conserved Trp was replaced by Met or Phe residues in domains I and III, respectively (Figure S4). Although the conserved cysteine residues were absent from all of the CDS7 domains, the disulphide bridge is not necessary for the fold (4) and several cysteines were replaced by small hydrophobic residues similar to those found in other Ig-like domains
... Recently, a FcRg-like molecule containing a single ITAM has been identified in the common carp, a representative teleosts [22]. Litman and colleagues have described a large family of channel catfish and zebrafish cell surface receptors (NITR), which may be subdivided into inhibitory and activating members [23,24]. While some of the family members bear ITIM in the cytoplasmic tails, others have positively charged residues in the transmembrane regions suggesting their association with unknown ITAM-containing subunits. ...
The genes encoding FcRγ and TCRζ homologs were identified using a bioinformatic approach in the amphibian Xenopus laevis. Deduced amino acid sequence of Xenopus TCRζ is highly similar to the mammalian and avian counterparts, whereas that of FcRγ differs by the presence of an additional ITAM-like motif. The presence of the negatively charged residue in the transmembrane regions of both subunits suggests their ability to serve as signal transducing modules in complex with activating receptors. The short extracellular regions contain characteristic cysteine residues responsible for dimerization in the mammalian subunits. According to Southern blot analysis, Xenopus laevis may possess two non-allelic genes for each subunit. Northern blots revealed FcRγ transcripts of two sizes differentially expressed in thymus, spleen, intestine, liver and kidney. TCRζ mRNA was predominantly expressed in the thymus and spleen. These data indicate that the amphibian immune system employs activating receptor complexes arranged in a mammalian-like way.
... In humans, they belong to the killer immunoglobulin (KIR) or C-type lectin membrane (NKG2/CD94) receptors with either activating (ITAM) or inhibitory (ITIM) intracellular motifs. In fish, orthologs to human KIR and NKG2/CD94 gene receptors have been identified and named novel immune-type receptor (NITR) and KLR, respectively (Litman et al., 2001;Yoder 2004). Functional characterization of these receptors will help to elucidate the innate cytotoxic populations in fish, their regulation and role in disease. ...
... We have described variable (V) region-containing transmembrane receptors (novel immunetype receptors [NITRs]) in zebrafish and other bony fish [4]. NITRs are the most complex family of V region-containing immune-type receptors described thus far outside of immunoglobulin (Ig) and T cell antigen receptors (TCRs) [5]. NITRs function in allogeneic recognition in a manner akin to activating/inhibitory NK receptors [6]. ...
A heretofore-unrecognized multigene family encoding diverse immunoglobulin (Ig) domain-containing proteins (DICPs) was identified in the zebrafish genome. Twenty-nine distinct loci mapping to three chromosomal regions encode receptor-type structures possessing two classes of Ig ectodomains (D1 and D2). The sequence and number of Ig domains, transmembrane regions and signaling motifs vary between DICPs. Interindividual polymorphism and alternative RNA processing contribute to DICP diversity. Molecular models indicate that most D1 domains are of the variable (V) type; D2 domains are Ig-like. Sequence differences between D1 domains are concentrated in hypervariable regions on the front sheet strands of the Ig fold. Recombinant DICP Ig domains bind lipids, a property shared by mammalian CD300 and TREM family members. These findings suggest that novel multigene families encoding diversified immune receptors have arisen in different vertebrate lineages and affect parallel patterns of ligand recognition that potentially impact species-specific advantages.
... Approximately half of these IgSF proteins contain two Ig domains; an N-proximal V-type followed by a C-proximal C2- type Ig domain [5]. In teleost fish, this type of organization can be found in, for example, the NITR family [32,40]. The observed SITR Ig domain organisation may be unusual but is not unique to carp and seems a conserved feature of the SITR-related genes found in the zebrafish genome (unpublished data). ...
The innate immune system relies upon a wide range of germ-line encoded receptors including a large number of immunoglobulin superfamily (IgSF) receptors. Different Ig-like immune receptor families have been reported in mammals, birds, amphibians and fish. Most innate immune receptors of the IgSF are type I transmembrane proteins containing one or more extracellular Ig-like domains and their regulation of effector functions is mediated intracellularly by distinct stimulatory or inhibitory pathways.
Carp SITR was found in a substracted cDNA repertoire from carp macrophages, enriched for genes up-regulated in response to the protozoan parasite Trypanoplasma borreli. Carp SITR is a type I protein with two extracellular Ig domains in a unique organisation of a N-proximal V/C2 (or I-) type and a C-proximal V-type Ig domain, devoid of a transmembrane domain or any intracytoplasmic signalling motif. The carp SITR C-proximal V-type Ig domain, in particular, has a close sequence similarity and conserved structural characteristics to the mammalian CD300 molecules. By generating an anti-SITR antibody we could show that SITR protein expression was restricted to cells of the myeloid lineage. Carp SITR is abundantly expressed in macrophages and is secreted upon in vitro stimulation with the protozoan parasite T. borreli. Secretion of SITR protein during in vivo T. borreli infection suggests a role for this IgSF receptor in the host response to this protozoan parasite. Overexpression of carp SITR in mouse macrophages and knock-down of SITR protein expression in carp macrophages, using morpholino antisense technology, provided evidence for the involvement of carp SITR in the parasite-induced NO production.
We report the structural and functional characterization of a novel soluble immune-type receptor (SITR) in a teleost fish and propose a role for carp SITR in the NO-mediated response to a protozoan parasite.
... Markers 194s1 and 194c28 represent the alr1 interval limits. been recognized in ascidians [4,24252627. Given the finding that both placozoan and cnidarian genomes have been shown to display surprisingly large tracks of synteny with chordates282930, the question of whether the Hydractinia alr-containing chromosomal intervals display synteny with vertebrate IgSF gene complexes is of immediate interest. ...
Allorecognition, the ability to discriminate between self and nonself, is ubiquitous among colonial metazoans and widespread among aclonal taxa. Genetic models for the study of allorecognition have been developed in the jawed vertebrates, invertebrate chordate Botryllus, and cnidarian Hydractinia. In Botryllus, two genes contribute to the histocompatibility response, FuHC and fester. In the cnidarian Hydractinia, one of the two known allorecognition loci, alr2, has been isolated, and a second linked locus, alr1, has been mapped to the same chromosomal region, called the allorecognition complex (ARC). Here we isolate alr1 by positional cloning and report it to encode a transmembrane receptor protein with two hypervariable extracellular regions similar to immunoglobulin (Ig)-like domains. Variation in the extracellular domain largely predicts fusibility within and between laboratory strains and wild-type isolates. alr1 was found embedded in a family of immunoglobulin superfamily (IgSF)-like genes, thus establishing that the ARC histocompatibility complex is an invertebrate IgSF-like gene complex.
... Data collected so far demonstrate that NITRs represent an extraordinarily divergent and rapidly evolving gene family. Different NITR profiles and a remarkable diversity of gene content were found across teleost species, suggesting that independent, lineage-specific tandem gene duplications and family expansions are the most frequent mode for the NITR gene complex evolution (Litman et al. 2001; Yoder 2009). The functional significance of such lineage-specific gene expansions remains to be further investigated. ...
In the last decade, a new gene family encoding non-rearranging receptors, called novel immune-type receptors (NITRs), has been discovered in teleost fish. NITRs belong to the immunoglobulin superfamily and represent an extraordinarily divergent and rapidly evolving gene complex. Genomic analysis of a region spanning 270 kb led to the discovery of a NITR gene cluster in the European sea bass (Dicentrarchus labrax). In total, 27 NITR genes and three putative pseudogenes, organised in a tandemly arrayed cluster, were identified. Sea bass NITR genes maintain the three major genomic organisations that appear to be essentially conserved among fish species along with new features presumably involving processes of intron loss, exon deletion and acquisition of new exons. Comparative and evolutionary analyses suggest that these receptors have evolved following a “birth-and-death” model of gene evolution in which duplication events together with lineage-specific gain and loss of individual members contributed to the rapid diversification of individual gene families. In this study, we demonstrate that species-specific gene expansions provide the raw material for diversifying, positive Darwinian selection favouring the evolution of a highly diverse array of molecules.
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... Together with the previously demonstrated type III interferon response, these results show that the counterparts of the mammalian viral and bacterial interferon-dependent defence functions are in place in zebrafish embryos, and suggest that zebrafish IFN-1 and IFN-2 are functionally equivalent to mammalian IFN-. mammals, but significant divergence has been observed as well, both in the way the adaptive immune system creates diversity, and among the innate immune recognition and effector components that are involved in direct interactions with pathogens (Traver et al., 2003; Meijer et al., 2004; Jault et al., 2004; Litman et al., 2001; Yoder et al., 2004; van der Aa et al., 2009; Stein et al., 2007). One set of fast-evolving components that have a central role in immune defence are the interferons and their receptors, of which only a small subset has been studied in the zebrafish. ...
The zebrafish genome contains ten genes that encode class II cytokine-like peptides, of which the two that are related most closely to mammalian interferon gamma (IFN-gamma) were named IFN-gamma1 and IFN-gamma2. Although the zebrafish has become a popular model system to study immune mechanisms, and although interferons are central regulators of immunity, which zebrafish cytokines correspond functionally to mammalian IFN-gamma has not been established. We used zebrafish embryos to assay the functions of IFN-gamma1 and IFN-gamma2, and have identified a subset of zebrafish homologs of the mammalian IFN-responsive genes as IFN-gamma targets in the zebrafish embryo: these genes are upregulated in response to raised levels of either IFN-gamma1 or IFN-gamma2. Infection studies using two different pathogens show that IFN-gamma signalling is required for resistance against bacterial infections in the young embryo and that the levels of IFN-gamma need to be regulated tightly: raising IFN-gamma levels sensitizes fish embryos against bacterial infection. Embryos injected with high doses of Escherichia coli are able to clear the bacteria within a day, and the gamma-interferons are necessary for this defence reaction. The protective response to Yersinia ruckeri, a natural fish pathogen that is lethal at low doses, also requires IFN-gamma. As in the induction of target genes, the two interferons act at least partly redundantly. Together with the previously demonstrated type III interferon response, these results show that the counterparts of the mammalian viral and bacterial interferon-dependent defence functions are in place in zebrafish embryos, and suggest that zebrafish IFN-gamma1 and IFN-gamma2 are functionally equivalent to mammalian IFN-gamma.
... The same biochemical pathway is used by lectin-like inhibitory CD94-NKG2 receptors, which are found in both rodents and humans. In addition, ITIM-containing inhibitory receptors are found in many cell types and across several vertebrate species 66,67 . These observations suggest that these inhibitory pathways are evolutionarily ancient. ...
The signaling pathways that regulate B and T lymphocytes are remarkably conserved between humans and mice. However, recent evidence suggests that the pathways regulating natural killer (NK) cell activation may actually differ between these two species. We discuss the controversies in the field and propose that this divergence could be deceptive: despite some clear differences between human and mouse NK cell receptors, the many ways of activating NK cells and their functions may well be conserved.
... The second point is exemplified by the recent report of a series of mutations in rag1 1 that were identified by direct sequencing of randomly mutagenized animals [8]. The third point is illustrated by the novel immune-type receptor genes (nitrs) and is particularly relevant when considering the evolution of immune-related genes and the relationships between adaptive and innate immune receptors [6,9]. Although these features of the zebrafish system are significant, it is neither likely nor necessarily relevant that studies in zebrafish will approach the current levels of functional immunological investigation that are conducted as a matter of routine in mammals. ...
Two decades of research have established the zebrafish (Danio rerio) as a significant model system for studying vertebrate development and gene structure-function relationships. Recent advances in mutation screening, the creation of genomic resources, including the Zebrafish Genome Project and the development of efficient transgenesis procedures, make this model increasingly attractive for immunological study.
Discus Symphysodon spp. employs an unusual parental care behavior where fry feed on parental skin mucus after hatching. Studies on discus immunoglobulin superfamily (IgSF) especially during parental care are scarce. Here, a total of 518 IgSF members were identified based on discus genome and clustered into 12 groups, unevenly distributing on 30 linkage groups. A total of 92 pairs of tandem duplication and 40 pairs of segmental duplication that underwent purifying selection were identified. IgSF genes expressed differentially in discus skin during different care stages and between male and female parents. Specifically, the transcription of btn1a1, similar with mammalian lactation, increased after spawning, reached a peak when fry started biting on parents' skin mucus, and then decreased. The expression of btn2a1 and other immune members, e.g., nect4, fcl5 and cd22, were up-regulated when fry stopped biting on mucus. These results suggest the expression differentiation of IgSF genes in skin of discus fish during parental care.
For over half a century, deciphering the origins of the genomic loci that form the jawed vertebrate adaptive immune response has been a major topic in comparative immunogenetics. Vertebrate adaptive immunity relies on an extensive and highly diverse repertoire of tandem arrays of variable (V), diversity (D), and joining (J) gene segments that recombine to produce different immunoglobulin (Ig) and T cell receptor (TCR) genes. The current consensus is that a recombination-activating gene (RAG)-like transposon invaded an exon of an ancient innate immune VJ-bearing receptor, giving rise to the extant diversity of Ig and TCR loci across jawed vertebrates. However, a model for the evolutionary relationships between extant non-recombining innate immune receptors and the V(D)J receptors of the jawed vertebrate adaptive immune system has only recently begun to come into focus. In this review, we provide an overview of non-recombining VJ genes, including CD8β, CD79b, natural cytotoxicity receptor 3 (NCR3/NKp30), putative remnants of an antigen receptor precursor (PRARPs), and the multigene family of signal-regulatory proteins (SIRPs), that play a wide range of roles in immune function. We then focus in detail on the VJ-containing novel immune-type receptors (NITRs) from ray-finned fishes, as recent work has indicated that these genes are at least 50 million years older than originally thought. We conclude by providing a conceptual model of the evolutionary origins and phylogenetic distribution of known VJ-containing innate immune receptors, highlighting opportunities for future comparative research that are empowered by this emerging evolutionary perspective.
Antigen processing and presentation by major histocompatibility complex (MHC) molecules is a cornerstone in vertebrate immunity. Like mammals, teleosts possess both classical MHC class I and multiple families of divergent MHC class I genes. However, while certain mammalian MHC class I-like molecules have proven to be integral in immune regulation against a broad array of pathogens, the biological relevance of the different MHC class I lineages in fish remains elusive. This work focuses on MHC class I L lineage genes and reveals unique regulatory patterns of six genes (Sasa-lia, Sasa-lda, Sasa-lca, Sasa-lga, Sasa-lha, and Sasa-lfa) in antimicrobial immunity of Atlantic salmon (Salmo salar L.). Using two separate in vivo challenge models with different kinetics and immune pathologies combined with in vitro stimulation using viral and bacterial TLR ligands, we show that de novo synthesis of different L lineage genes is distinctly regulated in response to various microbial stimuli. Prior to the onset of classical MHC class I gene expression, lia was rapidly and systemically induced in vivo by the single-stranded (ss) RNA virus salmonid alpha virus 3 (SAV3) but not in response to the intracellular bacterium Piscirickettsia salmonis. In contrast, lga expression was upregulated in response to both viral and bacterial stimuli. A role for distinct MHC class I L-lineage genes in anti-microbial immunity in salmon was further substantiated by a marked upregulation of lia and lga gene expression in response to type I IFNa stimulation in vitro. Comparably, lha showed no transcriptional induction in response to IFNa stimulation but was strongly induced in response to a variety of viral and bacterial TLR ligands. In sharp contrast, lda showed no response to viral or bacterial challenge. Similarly, induction of lca, which is predominantly expressed in primary and secondary lymphoid tissues, was marginal with the exception of a strong and transient upregulation in pancreas following SAV3 challenge Together, these findings suggest that certain Atlantic salmon MHC class I L lineage genes play important and divergent roles in early anti-microbial response and that their regulation, in response to different activation signals, represents a system for selectively promoting the expression of distinct non-classical MHC class I genes in response to different types of immune challenges.
Teleost genomes encode multiple multigene families of immunoglobulin domain-containing innate immune receptors (IIIRs) with unknown function and no clear mammalian orthologs. However, the genomic organization of IIIR gene clusters and the structural and signaling motifs of the proteins they encode are similar to those of mammalian innate immune receptor families such as the killer cell immunoglobulin-like receptors (KIRs), leukocyte immunoglobulin-like receptors (LILRs), Fc receptors, triggering receptors expressed on myeloid cells (TREMs) and CD300s. Teleost IIIRs include novel immune-type receptors (NITRs); diverse immunoglobulin domain containing proteins (DICPs); polymeric immunoglobulin receptor-like proteins (PIGRLs); novel immunoglobulin-like transcripts (NILTs) and leukocyte immune-type receptors (LITRs). The accumulation of genomic sequence data has revealed that IIIR gene clusters in zebrafish display haplotypic and gene content variation. This intraspecific genetic variation, as well as significant interspecific variation, frequently confounds the identification of definitive orthologous IIIR sequences between teleost species. Nevertheless, by defining which teleost lineages encode (and do not encode) different IIIR families, predictions can be made about the presence (or absence) of specific IIIR families in each teleost lineage. It is anticipated that further investigations into available genomic resources and the sequencing of a variety of multiple teleost genomes will identify additional IIIR families and permit the modeling of the evolutionary origins of IIIRs.
This chapter describes the diverse forms of immunoglobulin (Ig) structures found in modern representatives of phylogenetically ancient species and attempts to describe systematic trends in Ig phylogeny. It emphasis on defining those features of structural diversification that potentially are most significant for interpreting the function and genetic regulation of the Ig genes as antigen binding receptors. Further the chapter discusses the recent efforts to describe candidate Ig-like molecules, as well as the more widespread use of genomics technology, and holds considerable promise for elucidating both the distant phylogenetic origins of the diversified Ig-like genes and the mechanisms that have diversified complex families of humoral immune receptors. The efficient transcription of all vertebrate Ig loci that have been examined experimentally depends not only on promoters, but also on B cell–specific transcriptional enhancers, which not only function in Ig gene transcription, but also factor in V(D)J recombination, class switching, and somatic hypermutation..
This chapter discusses the immune system of a Zebrafish and its development. It considers the major elements of adaptive immunity (for example, B and T lymphocytes and their antigen-specific receptors), the MHC, and some of the cells and molecules of innate immunity. On the early development of lymphoid cells and organs, it includes an initial section presenting an overview of vertebrate and zebrafish hematopoiesis. It also includes discussion of research directed at identifying mutants in developmental pathways, which has been a focus in zebrafish research. In the final section of the chapter, it discusses the mutant phenotypes in zebrafish that resemble human disease syndromes. Zebrafish mutations that appear to be models for human diseases have been reviewed. The ease of identifying defects in red blood cells (RBC) formation has facilitated the identification of a large number of mutants in erythropoiesis. Only one zebrafish mutation is known to cause a specific defect in adaptive immunity, the rag1 mutation. The mutant, a potential model for severe combined immunodeficiency, was not identified by a traditional phenotypic screen, however it was screened by mutated genomes for sequence changes in the rag1 gene. The zebrafish has an increasing use in drug discovery. Thousands of chemicals can be screened for their effects on living embryos. In conclusion, chemical screens can complement genetic screens in the dissection of developmental pathways, and in providing models of human disease
Novel immune-type receptor (NITR) genes belong to the immunoglobulin superfamily and are encoded by clusters of multigene families. NITRs encode type I transmembrane proteins and are only found in teleosts. In the current study, total 21 NITR genes are identified from miiuy croaker (Miichthys miiuy) and named as MmNITR1 to MmNITR21. Miiuy croaker NITR genes that encoded one or two extracellular immunoglobulin (Ig) domains, a transmembrane (TM) region, an immunoreceptor tyrosine-based inhibitor motif (ITIM) in the cytoplasmic (Cyt) region. The majority of MmNITRs possess cytoplasmic ITIM that can be classified as inhibitory receptors. However, a smaller number of NITRs (MmNITR8, MmNITR15 and MmNITR16) can be classified as activating receptors by the lack of cytoplasmic ITIMs and presence of a positively charged residue within their transmembrane domain. As typical inhibitory receptors, MmNITR1, MmNITR2 and MmNITR3 have different characteristics of the structure. In MmNITR1 gene, variable (V) and intermediate (I) domains are encoded by two separate exons. In contrast to MmNITR1, MmNITR3 gene encode V and I domains in a single exon. And MmNITR2 gene is characterized by the presence of only one Ig-like (V-type) extracellular domain and lack of J or J-like motifs. Also MmNITR2 gene displays an additional exon which is 48bp long between the V domain and the TM region. Two and four potential N-link giycosylation sites (N-X-S/T) are present in the extracellular Ig domains. Real-time RT-PCR results showed that upon induction with Vibrio anguillarum, NITR gene expressions were induced by bacteria in kidney, liver and spleen. Meanwhile, NITRs are also primarily detected in different tissues. Phylogenetic analyses of NITR V domains indicate that MmNITR1 and MmNITR2 are more similar than MmNITR3.
Five large multigene families encoding innate-type immune receptors that are comprised of immunoglobulin domains have been identified in bony fish, of which four do not possess definable mammalian orthologs. The members of some of the multigene families exhibit unusually extensive patterns of divergence and the individual family members demonstrate marked variation in interspecific comparisons. As a group, the gene families reveal striking differences in domain type and content, mechanisms of intracellular signaling, basic structural features, haplotype and allelic variation and ligand binding. The potential functional roles of these innate immune receptors, their relationships to immune genes in higher vertebrate species and the basis for their adaptive evolution are of broad interest. Ongoing investigations are expected to provide new insight into alternative mechanisms of immunity.
The immunoglobulin superfamily (IgSF) is a large group of cell surface proteins that include various immunoregulatory receptors such as novel immune type receptors (NITRs), which are a family of diversified proteins found exclusively in bony fish. In this study, we identified and analyzed an IgSF protein, SoIgSF1, from red drum (Sciaenops ocellatus). SoIgSF1 is composed of 225 amino acid residues and moderately related to teleost NITRs. In silico analysis indicated that SoIgSF1 is a type I transmembrane glycoprotein and contains an N-terminal signal peptide sequence, a single extracellular immunoglobulin V domain, a transmembrane region, and a cytoplasmic region. However, unlike most NITRs, the cytoplasmic region of SoIgSF1 exhibits no consensus inhibitory or stimulatory signaling sequences but has two tyrosine-containing motifs that conform to the right-half sequence of the immunoreceptor tyrosine-based inhibitory motif (ITIM). Quantitative real time RT-PCR analysis showed that SoIgSF1 expression occurred mainly in immune organs and was drastically induced by viral and bacterial infection. Immunofluorescence microscopy indicated that viral infection of head kidney (HK) leukocytes induced surface expression of SoIgSF1, which was able to interact with antibodies against recombinant SoIgSF1. Antibody cross-linking of SoIgSF1 on HK leukocytes inhibited the expression of immune relevant genes and promoted viral and bacterial infection. Taken together, these results indicate that SoIgSF1, though lacking canonical intracellular signaling motifs, is involved in regulation of host immune response during pathogen infection possibly by functioning as a negative signaling receptor through a novel mechanism.
In all animals innate immunity is the first line of immune defense from invading pathogens. The prototypical innate cellular responses such as phagocytosis, degranulation, and cellular cytotoxicity are elicited by leukocytes in a diverse range of animals including fish, amphibians, birds and mammals reinforcing the importance of such primordial defense mechanisms. In mammals, these responses are intricately controlled and coordinated at the cellular level by distinct subsets of immunoregulatory receptors. Many of these surface proteins belong to the immunoglobulin superfamily and in mammals elaborate immunoregulatory receptor networks play a major role in the control of infectious diseases. Recent examination of teleost immunity has begun to further illustrate the complexities of these receptor networks in lower vertebrates. However, little is known about the mechanisms that control how immunoregulatory receptors influence cellular decision making in ectothermic vertebrates. This review focuses on several families of recently discovered immunoglobulin superfamily members in fish that share structural, phylogenetic and in some cases functional relationships with mammalian immunoregulatory receptors. Further characterization of these teleost innate immune receptor families will provide detailed information regarding the conservation and importance of innate immune defense strategies throughout vertebrate evolution.
München, Univ., Diss, 2004 (Nicht für den Austausch).
Immunoglobulin superfamily (IgSF) proteins are known for their abilities to specifically recognize and adhere to cells. In this paper, we predicted the presence of 133 IgSF proteins in the silkworm (Bombyx mori) genome. Comparison with similar proteins in other model organisms (Caenorhabditis elegans, Drosophila melanogaster, Anopheles gambiae, Apis mellifera and Homo sapiens) indicated that IgSF proteins are conserved but have rapidly evolved from worms to human beings. However, these proteins are well conserved amongst insects. Silkworm microarray-based expression data showed tissue expression of 57 IgSF genes and microbe-induced differential expression of 37 genes. Based on the expression data, we can conclude that the silkworm IgSF is active.
Recent genomic studies have led to an inventory of the genes encoding their immune receptors. Some have been submitted to great diversification by duplication in the germline. In some families like LRR and IgSF several species of vertebrates and invertebrates diversify further the receptors by individual somatic processes demanding cellular control and selection that can lead to the genesis of adaptive systems. At all levels many convergences reveal even better than conservations the intensity of pressure exerted on the immune systems to diversify their immunoreceptors in front of the changing environment.
185/333 genes and transcripts from the purple sea urchin, Strongylocentrotus purpuratus, predict high levels of amino acid diversity within the encoded proteins. Based on their expression patterns, 185/333 proteins appear to be involved in immune responses. In the present study, one- and two-dimensional Western blots show that 185/333 proteins exhibit high levels of molecular diversity within and between individual sea urchins. The molecular masses of 185/333-positive bands or spots range from 30 to 250 kDa with a broad array of isoelectric points. The observed molecular masses are higher than those predicted from mRNAs, suggesting that 185/333 proteins form strong associations with other molecules or with each other. Some sea urchins expressed >200 distinct 185/333 proteins, and each animal had a unique suite of the proteins that differed from all other individuals. When sea urchins were challenged in vivo with pathogen-associated molecular patterns (PAMPs; bacterial LPS and peptidoglycan), the expression of 185/333 proteins increased. More importantly, different suites of 185/333 proteins were expressed in response to different PAMPs. This suggests that the expression of 185/333 proteins can be tailored toward different PAMPs in a form of pathogen-specific immune response.
The use of probiotic microorganisms in aquaculture is gaining a lot of interest. Gnotobiotic model systems are required in order to fully understand the effects and modes-of-action of these microorganisms, as the native microbial communities present in non-sterile animals can lead to false conclusions. In this study, a gnotobiotic sea bass larvae (Dicentrarchus labrax) test system was developed. In order to obtain bacteria-free animals, the eggs were disinfected with glutaraldehyde and subsequently incubated in a solution of rifampicin and ampicillin. Axenity was confirmed using culture-dependent and -independent techniques. The gnotobiotic larvae were fed axenic Artemia sp. from 7 days after hatching onwards. In the challenge test, one of the three opportunistic pathogens, Aeromonas hydrophila, Listonella anguillarum serovar O1 and O2a, was added to the model system via the water and encapsulated in Artemia sp. Only serovar O2a led to increased mortality in the sea bass larvae. The presented gnotobiotic model can be used for research on, among others, reciprocal metabolic effects between microorganisms and the host (e.g. as measured by gene expression), immunostimulants, pharmacological research and the histological development of the gastrointestinal tract and growth of larvae.
Natural killer (NK) activity has been examined in birds for over 30 years, but evidence that avian NK activity plays crucial roles in disease is only suggestive. In chickens, NK activity is mediated by TCR0 cells in the intestinal epithelium, but elsewhere subsets of alphabeta and gammadelta T cells (NKT cells) may be more important. There are few lectin-like NK receptor genes, located in the genomic region syntenic with the natural killer complex (NKC) as well as the major histocompatibility complex (MHC). In contrast, a huge number of Ig-like receptor genes are located in a region syntenic with the leukocyte receptor complex (LRC).
Since an immunoreceptor tyrosine-based inhibition motif (ITIM) was first identified in the intracytoplasmic domain of Fc gammaRIIB, ITIMs have been found in a large number of inhibitory molecules that were shown to negatively regulate cell activation. Due to their wide tissue distribution and to the variety of their extracellular ligands, ITIM-containing molecules are involved in the control of a large spectrum of biological functions, mostly but not exclusively related to immunity. On the basis of sequence comparison, ITIMs were structurally defined as 6-amino acid sequences containing a tyrosine (Y) with loosely conserved N-terminal (Y-2) and C-terminal (Y+3) residues. Molecular analysis of signaling events demonstrated that when coaggregated with activating receptors, ITIMs are phosphorylated by Src-family tyrosine kinases, which enables them to recruit Src homology 2 domain-containing phosphatases that antagonize activation signals. Because ITIM-dependent negative regulation seems to be a fundamental regulatory mechanism, both in rodents and in humans, and because it can be used either as a target or as a powerful tool in various diseases, we undertook (i) a genome-wide search of potential novel ITIM-containing molecules in humans, mice, frogs, birds, and flies and (ii) a comparative analysis of potential ITIMs in major animal phyla, from mammals to protozoa. We found a surprisingly high number of potential ITIM-containing molecules, having a great diversity of extracellular domains, and being expressed by a variety of immune and non-immune cells. ITIMs could be traced back to the most primitive metazoa. The genes that encode ITIM-containing molecules that belong to the immunoglobulin superfamily or to the C-lectin family seem to derive from a common set of ancestor genes and to have dramatically expanded and diverged in Gnathostomata (from fish to mammals).
Immune inhibitory receptor genes that encode a variable (V) region, a unique V-like C2 (V/C2) domain, a transmembrane region, and a cytoplasmic tail containing immunoreceptor tyrosine-based inhibition motifs (ITIMs) have been described previously in two lineages of bony fish. In the present study, eleven related genes encoding distinct structural forms have been identified in Ictalurus punctatus (channel catfish), a well characterized immunological model system that represents a third independent bony fish lineage. Each of the different genes encodes an N-terminal V region but differs in the number of extracellular Ig domains, number and location of joining (J) region-like motifs, presence of transmembrane regions, presence of charged residues in transmembrane regions, presence of cytoplasmic tails, and/or distribution of ITIM(s) within the cytoplasmic tails. Variation in the numbers of genomic copies of the different gene types, their patterns of expression, and relative levels of expression in mixed leukocyte cultures (MLC) is reported. V region-containing immune-type genes constitute a far more complex family than recognized originally and include individual members that might function in inhibitory or, potentially activatory manners.
The genome of the pufferfish, Fugu rubripes (Fugu) is compact. With a similar gene complement to mammals and a genome size of just 400 Mb, gene density is high averaging one every 6-7 kb. Initial characterization of this genome has shown that although genes are much smaller and more densely spaced, their intron/exon structure is conserved with the resulting introns being small. There is little repetitive DNA in the genome and this greatly facilitates comparative genomic studies. The coding content of genes is highly conserved as are critical regulatory elements of some genes. Other DNA is not, however, and this allows the identification of homologous coding sequence between Fugu and mammalian genes. Although the genome of Fugu is 7.5 times smaller than the human genome, not all genes are reduced proportionately. Some regions of the genome show conserved synteny with mammalian genomes, although at the present time only short physical distances have been examined. The structure of the genome is also being studied. Initial data suggest that this may be different to that found in mammals. It is not clear that the same kind of isochore structure is present in this early vertebrate genome. Patterns of methylation may be different resulting in a different distribution of CpG islands. An attempt is being made to centralize both resources and data from the genome of Fugu so that everything may be integrated into a single, publicly accessible database which in turn, may be integrated with databases from other organisms.
The presence and expression of killer inhibitory receptor (KIR) and CD94:NKG2 genes from 68 donors were analyzed using molecular typing techniques. The genes encoding CD94:NKG2 receptors were present in each person, but KIR gene possession varied. Most individuals expressed inhibitory KIR for the three well-defined HLA-B and -C ligands, but noninhibitory KIR genes were more variable. Twenty different KIR phenotypes were defined. Two groups of KIR haplotypes were distinguished and occurred at relatively even frequency. Group A KIR haplotypes consist of six genes: the main inhibitory KIR, one noninhibitory KIR, and a structurally divergent KIR. Allelic polymorphism within five KIR genes was detected. Group B comprises more noninhibitory KIR genes and contains at least one additional gene not represented in group A. The KIR locus therefore appears to be polygenic and polymorphic within the human population.
This review addresses issues related to the evolution of the complex multigene families of antigen binding receptors that function in adaptive immunity. Advances in molecular genetic technology now permit the study of immunoglobulin (Ig) and T cell receptor (TCR) genes in many species that are not commonly studied yet represent critical branch points in vertebrate phylogeny. Both Ig and TCR genes have been defined in most of the major lineages of jawed vertebrates, including the cartilaginous fishes, which represent the most phylogenetically divergent jawed vertebrate group relative to the mammals. Ig genes in cartilaginous fish are encoded by multiple individual loci that each contain rearranging segmental elements and constant regions. In some loci, segmental elements are joined in the germline, i.e. they do not undergo genetic rearrangement. Other major differences in Ig gene organization and the mechanisms of somatic diversification have occurred throughout vertebrate evolution. However, relating these changes to adaptive immune function in lower vertebrates is challenging. TCR genes exhibit greater sequence diversity in individual segmental elements than is found in Ig genes but have undergone fewer changes in gene organization, isotype diversity, and mechanisms of diversification. As of yet, homologous forms of antigen binding receptors have not been identified in jawless vertebrates; however, acquisition of large amounts of structural data for the antigen binding receptors that are found in a variety of jawed vertebrates has defined shared characteristics that provide unique insight into the distant origins of the rearranging gene systems and their relationships to both adaptive and innate recognition processes.
The number of V-alpha and V-beta sequences of T cell receptors now available allows a meaningful analysis of their variability profiles. Variability plots were derived using a modified form of Wu and Kabat's algorithm: variability is not computed as a proportion of the number of different residues occurring at a position, but rather proportionally to the physicochemical differences between the different residues. Results show that the classical hypervariable regions occurring in immunoglobulins also occur in T cell receptors at equivalent positions. Contrary to immunoglobulins the framework of Tcr V regions displays many relatively variable regions and positions. This phenomenon can be connected with the genetic organization of V genes of T cell receptors which seem to avoid any framework homogenization and the resulting gene conversion. More importantly an additional hypervariable region was detected in V-beta but not in V-alpha. This fourth hypervariable region is located between the second and the D hypervariable CDR. The predicted three-dimensional location of this additional hypervariable region is compatible with a possible role in antigen recognition and therefore also in positive and/or negative selection. Furthermore our data suggest that this fourth hypervariable region is involved in the recognition of superantigens like bacterial enterotoxins. Indeed this additional hypervariable region is not detected when variability is derived using an alignment of the V-beta subgroups stimulated by one toxin of S. aureus. Finally we propose a new and simple molecular model to explain alloreactivity as crossreactivity between the universe of shapes (isomers of conformation) of different MHC haplotypes.
The immunoglobulin superfamily (IgSF) is an extensively diversified multigene family whose members share a common structural feature, the Ig fold. Members of the Ig/T-cell antigen receptor (TCR) subset of the IgSF mediate antigen-specific recognition in adaptive immune responses. Antigen-binding receptors belonging to this subset are present in all species of jawed vertebrates. To explore whether there are additional structurally related but otherwise distinct members of this subset, we have developed a technique termed the short-primer polymerase chain reaction (PCR) that targets structurally conserved short motifs in the Ig fold. Large-scale sequencing efforts and recent advances in information biotechnology, including "electronic PCR," provide additional computational means to implement similarly directed searches within databases. The use of these approaches has led to the discoveries of Ig/TCR homologues in a variety of phylogenetically diverse organisms, a diversified family of novel immune-type receptor genes, as well as a novel human IgSF member. The potential of random sequencing efforts and virtual screening of databases is described in the context of two novel genes in bony fish. The various methodologies that are discussed and the examples shown provide means for further investigating, and/or elucidating novel, IgSF receptors as well as components of pathways that are involved in immune responses in both traditional and nontraditional model systems.
Although natural killer (NK) cells are known to preferentially kill cells that lack major histocompatibility complex class I antigens, we do not know what signals the attack of these targets. Several membrane receptors have recently been implicated in this process and include molecules with immunoreceptor tyrosine-based activation motifs (ITAM) and motifs that bind phosphoinositide-3 kinase (PI3K). Evidence is emerging that NK cells may use a combination of several receptors and signaling pathways to protect the host against infection and possibly against malignancies.
In addition to being an excellent model system for studying vertebrate development, the zebrafish has become a great tool for gene discovery by mutational analysis. The recent availability of the zebrafish EST database and radiation hybrid mapping panels has dramatically expanded the framework for genomic research in this species. Developing comparative maps of the zebrafish and human genomes is of particular importance for zebrafish mutagenesis studies in which human orthologs are sought for zebrafish genes. However, only partial cDNA sequences are determined routinely for mapped ESTs, leaving the identity of the EST in question. It previously had been reported that zebrafish linkage group 7 shares conserved synteny with human chromosome 11q13. In an effort to further define this relationship, five full-length zebrafish cDNAs, fth1, slc3a2, prkri, cd81, and pc, as well as one putative human gene, DBX were identified and their map positions ascertained. These six genes, along with men1, fgf3 and cycd1 define two regions of conserved synteny between linkage group 7 and 11q13.
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