Immunoglobulin D enhances immune surveillance by activating antimicrobial, proinflammatory and B cell-stimulating programs in basophils.

Immunoglobulin D (IgD) is an enigmatic antibody isotype that mature B cells express together with IgM through alternative RNA splicing. Here we report active T cell-dependent and T cell-independent IgM-to-IgD class switching in B cells of the human upper respiratory mucosa. This process required activation-induced cytidine deaminase (AID) and generated local and circulating IgD-producing plasmablasts reactive to respiratory bacteria. Circulating IgD bound to basophils through a calcium-mobilizing receptor that induced antimicrobial, opsonizing, inflammatory and B cell-stimulating factors, including cathelicidin, interleukin 1 (IL-1), IL-4 and B cell-activating factor (BAFF), after IgD crosslinking. By showing dysregulation of IgD class-switched B cells and 'IgD-armed' basophils in autoinflammatory syndromes with periodic fever, our data indicate that IgD orchestrates an ancestral surveillance system at the interface between immunity and inflammation.

Distinct Host-Mycobacterial Pathogen Interactions between Resistant Adult and Tolerant Tadpole Life Stages of Xenopus laevis.

Mycobacterium marinum is a promiscuous pathogen infecting many vertebrates, including humans, whose persistent infections are problematic for aquaculture and public health. Among unsettled aspects of host-pathogen interactions, the respective roles of conventional and innate-like T (iT) cells in host defenses against M. marinum remain unclear. In this study, we developed an infection model system in the amphibian Xenopus laevis to study host responses to M. marinum at two distinct life stages, tadpole and adult. Adult frogs possess efficient conventional T cell-mediated immunity, whereas tadpoles predominantly rely on iT cells. We hypothesized that tadpoles are more susceptible and elicit weaker immune responses to M. marinum than adults. However, our results show that, although anti-M. marinum immune responses between tadpoles and adults are different, tadpoles are as resistant to M. marinum inoculation as adult frogs. M. marinum inoculation triggered a robust proinflammatory CD8+ T cell response in adults, whereas tadpoles elicited only a noninflammatory CD8 negative- and iT cell-mediated response. Furthermore, adult anti-M. marinum responses induced active granuloma formation with abundant T cell infiltration and were associated with significantly reduced M. marinum loads. This is reminiscent of local CD8+ T cell response in lung granulomas of human tuberculosis patients. In contrast, tadpoles rarely exhibited granulomas and tolerated persistent M. marinum accumulation. Gene expression profiling confirmed poor tadpole CD8+ T cell response, contrasting with the marked increase in transcript levels of the anti-M. marinum invariant TCR rearrangement (iVα45-Jα1.14) and of CD4. These data provide novel insights into the critical roles of iT cells in vertebrate antimycobacterial immune response and tolerance to pathogens.

Distinct MHC class I-like interacting invariant T cell lineage at the forefront of mycobacterial immunity uncovered in Xenopus.

The amphibian Xenopus laevis is to date the only species outside of mammals where a MHC class I-like (MHC-like) restricted innate-like (i) T cell subset (iVα6 T cells) reminiscent of CD1d-restricted iNKT cells has been identified and functionally characterized. This provides an attractive in vivo model to study the biological analogies and differences between mammalian iT cells and the evolutionarily antecedent Xenopus iT cell defense system. Here, we report the identification of a unique iT cell subset (Vα45-Jα1.14) requiring a distinct MHC-like molecule (mhc1b4.L or XNC4) for its development and function. We used two complementary reverse genetic approaches: RNA interference by transgenesis to impair expression of either XNC4 or the Vα45-Jα1.14 rearrangement, and CRISPR/Cas9-mediated disruption of the Jα1.14 gene segment. Both XNC4 deficiency that ablates iVα45T cell development and the direct disruption of the iVα45-Jα1.14 T cell receptor dramatically impairs tadpole resistance to Mycobacterium marinum (Mm) infection. The higher mortality of Mm-infected tadpoles deficient for iVα45T cells correlates with dysregulated expression responses of several immune genes. In contrast, iVα45-Jα1.14-deficient tadpoles remain fully competent against infection by the ranavirus FV3, which indicates a specialization of this unique iT cell subset toward mycobacterial rather than viral pathogens that involve iVα6 T cells. These data suggest that amphibians, which are evolutionarily separated from mammals by more than 350 My, have independently diversified a prominent and convergent immune surveillance system based on MHC-like interacting innate-like T cells.

Impacts of the MHC class I-like XNC10 and innate-like T cells on tumor tolerance and rejection in the amphibian Xenopus.

The conditions that lead to antitumor or protumor functions of natural killer T (NKT) cells against mammalian tumors are only partially understood. Therefore, insights into the evolutionary conservation of NKT and their analogs-innate-like T (iT) cells-may reveal factors that contribute to tumor eradication. As such, we investigated the amphibian Xenopus laevis iT cells and interacting MHC class I-like (XNC or mhc1b.L) genes against ff-2 thymic lymphoid tumors. Upon ff-2 intraperitoneal transplantation into syngeneic tadpoles, two iT cell subsets iVα6 and iVα22, characterized by an invariant T-cell receptor α chain rearrangement (Vα6-Jα1.43 and Vα22-Jα1.32 respectively), were recruited to the peritoneum, concomitant with a decreased level of these transcripts in the spleen and thymus. To address the hypothesize that different iT cell subsets have distinct, possibly opposing, roles upon ff-2 tumor challenge, we determined whether ff-2 tumor growth could be manipulated by impairing Vα6 iT cells or by deleting their restricting element, the XNC gene, XNC10 (mhc1b10.1.L), on ff-2 tumors. Accordingly, the in vivo depletion of Vα6 iT cells using XNC10-tetramers enhanced tumor growth, indicating Vα6 iT cell-mediated antitumor activities. However, XNC10-deficient transgenic tadpoles that also lack Vα6 iT cells were resistant to ff-2 tumors, uncovering a potential new function of XNC10 besides Vα6 iT cell development. Furthermore, the CRISPR/Cas9-mediated knockout of XNC10 in ff-2 tumors broke the immune tolerance. Together, our findings demonstrate the relevance of XNC10/iT cell axis in controlling Xenopus tumor tolerance or rejection.

Exploring the functions of nonclassical MHC class Ib genes in Xenopus laevis by the CRISPR/Cas9 system.

A large family of highly related and clustered Xenopus nonclassical MHC class Ib (XNC) genes influences Xenopus laevis immunity and potentially other physiological functions. Using RNA interference (RNAi) technology, we previously demonstrated that one of XNC genes, XNC10.1, is critical for the development and function of a specialized innate T (iT) cell population. However, RNAi limitation such as a variable and unstable degree of gene silencing in F0 and F1 generations is hampering a thorough functional analysis of XNC10.1 and other XNC genes. To overcome this obstacle, we adapted the CRISPR/Cas9-mediated gene editing technique for XNC genes. We efficiently and specifically generated single gene knockouts of XNC10.1, XNC11, and XNC1 as well as double gene knockouts of XNC10.1 and XNC11 in X. laevis. In single XNC10.1 knockout X. laevis tadpoles, the absence of XNC10.1 and Vα6-Jα1.43 invariant T cell receptor rearrangement transcripts indicated XNC10.1 loss-of-function and deficiency in Vα6-Jα1.43 iT cells. Notably, targeting XNC10.1 did not affect neighboring XNC genes exhibiting high sequence similarity. Furthermore, XNC1 gene disruption induced mortality during developmental stage 47, suggesting some non-immune but essential function of this gene. These data demonstrate that the CRISPR/Cas9 system can be successfully adapted for genetic analysis in F0 generation of X. laevis.

Nonclassical MHC-Restricted Invariant Vα6 T Cells Are Critical for Efficient Early Innate Antiviral Immunity in the Amphibian Xenopus laevis.

Nonclassical MHC class Ib-restricted invariant T (iT) cell subsets are attracting interest because of their potential to regulate immune responses against various pathogens. The biological relevance and evolutionary conservation of iT cells have recently been strengthened by the identification of iT cells (invariant Vα6 [iVα6]) restricted by the nonclassical MHC class Ib molecule XNC10 in the amphibian Xenopus laevis. These iVα6 T cells are functionally similar to mammalian CD1d-restricted invariant NKT cells. Using the amphibian pathogen frog virus 3 (FV3) in combination with XNC10 tetramers and RNA interference loss of function by transgenesis, we show that XNC10-restricted iVα6 T cells are critical for early antiviral immunity in adult X. laevis. Within hours following i.p. FV3 infection, iVα6 T cells were specifically recruited from the spleen into the peritoneum. XNC10 deficiency and concomitant lack of iVα6 T cells resulted in less effective antiviral and macrophage antimicrobial responses, which led to impaired viral clearance, increased viral dissemination, and more pronounced FV3-induced kidney damage. Together, these findings imply that X. laevis XNC10-restricted iVα6 T cells play important roles in the early anti-FV3 response and that, as has been suggested for mammalian invariant NKT cells, they may serve as immune regulators polarizing macrophage effector functions toward more effective antiviral states.

Semi-solid tumor model in Xenopus laevis/gilli cloned tadpoles for intravital study of neovascularization, immune cells and melanophore infiltration.

Tumors have the ability to grow as a self-sustaining entity within the body. This autonomy is in part accomplished by the tumor cells ability to induce the formation of new blood vessels (angiogenesis) and by controlling cell trafficking inside the tumor mass. These abilities greatly reduce the efficacy of many cancer therapies and pose challenges for the development of more effective cancer treatments. Hence, there is a need for animal models suitable for direct microscopy observation of blood vessel formation and cell trafficking, especially during early stages of tumor establishment. Here, we have developed a reliable and cost effective tumor model system in tadpoles of the amphibian Xenopus laevis. Tadpoles are ideally suited for direct microscopy observation because of their small size and transparency. Using the thymic lymphoid tumor line 15/0 derived from, and transplantable into, the X. laevis/gilli isogenic clone LG-15, we have adapted a system that consists in transplanting 15/0 tumor cells embedded into rat collagen under the dorsal skin of LG-15 tadpole recipients. This system recapitulates many facets of mammalian tumorigenesis and permits real time visualization of the active formation of the tumor microenvironment induced by 15/0 tumor cells including neovascularization, collagen rearrangements as well as infiltration of immune cells and melanophores.

Evolution of innate-like T cells and their selection by MHC class I-like molecules.

Until recently, major histocompatibility complex (MHC) class I-like-restricted innate-like αßT (iT) cells expressing an invariant or semi-invariant T cell receptor (TCR) repertoire were thought to be a recent evolutionary acquisition restricted to mammals. However, molecular and functional studies in Xenopus laevis have demonstrated that iT cells, defined as MHC class I-like-restricted innate-like αßT cells with a semi-invariant TCR, are evolutionarily conserved and prominent from early development in amphibians. As these iT cells lack the specificity conferred by conventional αß TCRs, it is generally considered that they are specialized to recognize conserved antigens equivalent to pathogen-associated molecular patterns. Thus, one advantage offered by the MHC class I-like iT cell-based recognition system is that it can be adapted to a common pathogen and function on the basis of a relatively small number of T cells. Although iT cells have only been functionally described in mammals and amphibians, the identification of non-classical MHC/MHC class I-like genes in other groups of endothermic and ectothermic vertebrates suggests that iT cells have a broader phylogenetic distribution than previously envisioned. In this review, we discuss the possible role of iT cells during the emergence of the jawed vertebrate adaptive immune system.

Nonclassical MHC class I-dependent invariant T cells are evolutionarily conserved and prominent from early development in amphibians.

Human and murine MHC nonclassical class Ib-restricted invariant T (iT) cell subsets, such as invariant natural killer T cells (iNKT) and mucosal-associated invariant T cells, have specialized functions early in immune responses, especially in modulating subsequent adaptive immune responses. Here, we characterize a prominent iT population in the amphibian Xenopus laevis and show the requirement of the class Ib molecule, Xenopus nonclassical gene 10, in its differentiation and function. Using Xenopus nonclassical gene 10 tetramers and RNAi loss of function by transgenesis, we identified a large class Ib-dependent CD8(-)/CD4(-) iT subset in unmanipulated frogs and tadpoles. This population is critical for antiviral immunity during early larval stages when classical MHC class Ia function is suboptimal. Furthermore, in young tadpoles with low class Ia expression, deep sequencing revealed additional preponderant invariant T cell receptor (TCR)α rearrangements, implying other iT cell subsets and a predominant selection process mediated by other class Ib molecules. The restriction and requirement of class Ib molecules for development and antiviral immunity of a mammalian iNKT or mucosal-associated invariant T cell counterpart in the amphibian Xenopus show the importance of iT cells in the emergence and evolution of the adaptive immune system.

Evolution of nonclassical MHC-dependent invariant T cells.

TCR-mediated specific recognition of antigenic peptides in the context of classical MHC molecules is a cornerstone of adaptive immunity of jawed vertebrate. Ancillary to these interactions, the T cell repertoire also includes unconventional T cells that recognize endogenous and/or exogenous antigens in a classical MHC-unrestricted manner. Among these, the mammalian nonclassical MHC class I-restricted invariant T cell (iT) subsets, such as iNKT and MAIT cells, are now believed to be integral to immune response initiation as well as in orchestrating subsequent adaptive immunity. Until recently the evolutionary origins of these cells were unknown. Here we review our current understanding of a nonclassical MHC class I-restricted iT cell population in the amphibian Xenopus laevis. Parallels with the mammalian iNKT and MAIT cells underline the crucial biological roles of these evolutionarily ancient immune subsets.

A critical role of non-classical MHC in tumor immune evasion in the amphibian Xenopus model.

Non-classical class Ib (class Ib) genes are found in all jawed vertebrates, including the amphibian Xenopus, which possesses at least 20 distinct Xenopus non-classical class Ib genes (XNCs). As an immune evasion strategy, tumors often downregulate surface expression of classical major histocompatibility complex class Ia molecules. In contrast, cancers commonly express class Ib molecules, presenting an alternative for tumor immune recognition. We characterized a novel XNC, XNC10, functionally similar to CD1d from a class Ia-deficient thymic lymphoid tumor (15/0), which grows aggressively in Xenopus LG-15 cloned animals. To investigate the roles of XNC10 in antitumor immunity, we generated stable 15/0-transfectants with silenced XNC10 mRNA and protein expression. Notably, XNC10 silencing resulted in acute tumor rejection by naturally class Ia-deficient syngeneic tadpoles, with greater potency of rejection in tumors with more efficient XNC10 knockdown. In vivo killing assays shows that the rejection of XNC10-deficient tumors is due to a cell-mediated cytotoxic immune response elicited by the tadpole host. Importantly, priming enhances XNC10-deficient tumor rejection. Flow cytometry reveals that XNC10-deficient tumor rejection is associated with an accumulation of XNC10-restricted invariant T cells and conventional CD8 T cells as well as other leukocytes. Similarly, semisolid tumor grafts in tadpoles also exhibit leukocytes infiltration. These findings suggest that XNC10 allows the 15/0-tumor to escape immune recognition and class Ia-independent cytotoxicity, thus emphasizing the critical roles of class Ibs in tumor immunity.

A prominent role for invariant T cells in the amphibian Xenopus laevis tadpoles.

Invariant T (iT) cells expressing an invariant or semi-invariant T cell receptor (TCR) repertoire have gained attention in recent years because of their potential as specialized regulators of immune function. These iT cells are typically restricted by nonclassical MHC class I molecules (e.g., CD1d and MR1) and undergo differentiation pathways distinct from conventional T cells. While the benefit of a limited TCR repertoire may appear counterintuitive in regard to the advantage of the diversified repertoire of conventional T cells allowing for exquisite specificity to antigens, the full biological importance and evolutionary conservation of iT cells are just starting to emerge. It is generally considered that iT cells are specialized to recognize conserved antigens equivalent to pathogen-associated molecular pattern. Until recently, little was known about the evolution of iT cells. The identification of class Ib and class I-like genes in nonmammalian vertebrates, despite the heterogeneity and variable numbers of these genes among species, suggests that iT cells are also present in ectothermic vertebrates. Indeed, recent studies in the amphibian Xenopus have revealed a drastic overrepresentation of several invariant TCRs in tadpoles and identified a prominent nonclassical MHC class I-restricted iT cell subset critical for tadpole antiviral immunity. This suggests an important and perhaps even dominant role of multiple nonclassical MHC class I-restricted iT cell populations in tadpoles and, by extension, other aquatic vertebrates with rapid external development that are under pressure to produce a functional lymphocyte repertoire with small numbers of cells.

Identification and characterization of TCRγ and TCRδ chains in channel catfish, Ictalurus punctatus.

Channel catfish, Ictalurus punctatus, T cell receptors (TCR) γ and δ were identified by mining of expressed sequence tag databases, and full-length sequences were obtained by 5'-RACE and RT-PCR protocols. cDNAs for each of these TCR chains encode typical variable (V), diversity (D), joining (J), and constant (C) regions. Three TCRγ V families, seven TCRγ J sequences, and three TCRγ C sequences were identified from sequencing of cDNA. Primer walking on bacterial artificial chromosomes (BACs) confirmed that the TRG locus contained seven TRGJ segments and indicated that the locus consists of (Vγ3-Jγ6-Cγ2)-(Vγ1n-Jγ7-Cγ3)-(Vγ2-Jγ5-Jγ4-Jγ3-Jγ2-Jγ1-Cγ1). In comparison for TCRδ, two V families, four TCRδ D sequences, one TCRδ J sequence, and one TCRδ C sequence were identified by cDNA sequencing. Importantly, the finding that some catfish TCRδ cDNAs contain TCR Vα-D-Jδ rearrangements and some TCRα cDNAs contain Vδ-Jα rearrangements strongly implies that the catfish TRA and TRD loci are linked. Finally, primer walking on BACs and Southern blotting suggest that catfish have four TRDD gene segments and a single TRDJ and TRDC gene. As in most vertebrates, all three reading frames of each of the catfish TRDD segments can be used in functional rearrangements, and more than one TRDD segment can be used in a single rearrangement. As expected, catfish TCRδ CDR3 regions are longer and more diverse than TCRγ CDR3 regions, and as a group they utilize more nucleotide additions and contain more nucleotide deletions than catfish TCRγ rearrangements.

Unusual evolutionary conservation and further species-specific adaptations of a large family of nonclassical MHC class Ib genes across different degrees of genome ploidy in the amphibian subfamily Xenopodinae.

Nonclassical MHC class Ib (class Ib) genes are a family of highly diverse and rapidly evolving genes wherein gene numbers, organization, and expression markedly differ even among closely related species rendering class Ib phylogeny difficult to establish. Whereas among mammals there are few unambiguous class Ib gene orthologs, different amphibian species belonging to the anuran subfamily Xenopodinae exhibit an unusually high degree of conservation among multiple class Ib gene lineages. Comparative genomic analysis of class Ib gene loci of two divergent (~65 million years) Xenopodinae subfamily members Xenopus laevis (allotetraploid) and Xenopus tropicalis (diploid) shows that both species possess a large cluster of class Ib genes denoted as Xenopus/Silurana nonclassical (XNC/SNC). Our study reveals two distinct phylogenetic patterns among these genes: some gene lineages display a high degree of flexibility, as demonstrated by species-specific expansion and contractions, whereas other class Ib gene lineages have been maintained as monogenic subfamilies with very few changes in their nucleotide sequence across divergent species. In this second category, we further investigated the XNC/SNC10 gene lineage that in X. laevis is required for the development of a distinct semi-invariant T cell population. We report compelling evidence of the remarkable high degree of conservation of this gene lineage that is present in all 12 species of the Xenopodinae examined, including species with different degrees of ploidy ranging from 2, 4, 8 to 12 N. This suggests that the critical role of XNC10 during early T cell development is conserved in amphibians.

Identification of two IgD+ B cell populations in channel catfish, Ictalurus punctatus.

Channel catfish Ictalurus punctatus express two Ig isotypes: IgM and IgD. Although catfish IgM has been extensively studied at the functional and structural levels, much less is known about IgD. In this study, IgM(+)/IgD(+) and IgM(-)/IgD(+) catfish B cell populations were identified through the use of anti-IgM and anti-IgD mAbs. Catfish IgM(+)/IgD(+) B cells are small and agranular. In contrast, IgM(-)/IgD(+) B cells are larger and exhibit a plasmablast morphology. The use of cell sorting, flow cytometry, and RT-PCR demonstrated that IgD(+) B cell expression varies among individuals. For example, some catfish have <5% IgM(-)/IgD(+) B cells in their PBLs, whereas in others the IgM(-)/IgD(+) B cell population can represent as much as 72%. Furthermore, IgD expressed by IgM(-)/IgD(+) B cells preferentially associates with IgL σ. Comparatively, IgM(+)/IgD(+) B cells can express any of the four catfish IgL isotypes. Also, transfection studies show that IgD functions as a typical BCR, because Igδ-chains associate with CD79a and CD79b molecules, and all membrane IgD transcripts from sorted IgM(-)/IgD(+) B cells contain viable VDJ rearrangements, with no bias in family member usage. Interestingly, all secreted IgD transcripts from IgM(+)/IgD(+) and IgM(-)/IgD(+) B cells were V-less and began with a leader spliced to Cδ1. Importantly, transfection of catfish clonal B cells demonstrated that this leader mediated IgD secretion. Together, these findings imply that catfish IgM(-)/IgD(+) B cells likely expand in response to certain pathogens and that the catfish IgD Fc-region, as has been suggested for human IgD, may function as a pattern recognition molecule.

Channel catfish CD8α and CD8ß co-receptors: characterization, expression and polymorphism.

In this study we report the identification and characterization of channel catfish, Ictalurus punctatus CD8α and CD8ß genes. Both genes encode predicted proteins containing a leader, a immunoglobulin superfamily V domain, a stalk/hinge region, a transmembrane region and a positively charged cytoplasmic tail (CYT) containing the conserved teleost C-X-H motif. Catfish CD8α and CD8ß are encoded as single copy genes and as in other vertebrates exhibit a conserved head to tail synteny; the CD8ß gene is found 14.1kb upstream of the CD8α gene. Both CD8α and CD8ß transcripts showed a low degree of polymorphism. Finally, as determined by q-PCR both CD8α and CD8ß are expressed in various catfish lymphoid tissues with the highest expression observed in thymus from 2 month old catfish-fry. In the future these results will provide the basis for evaluating the role of CD8(+) CTL and other CD8-bearing cells in response to immunization or infection in the catfish.

Profiling the T Cell Receptor Alpha/Delta Locus in Salmonids.

In jawed vertebrates, two major T cell populations have been characterized. They are defined as α/ß or γ/δ T cells, based on the expressed T cell receptor. Salmonids (family Salmonidae) include two key teleost species for aquaculture, rainbow trout (Oncorhynchus mykiss) and Atlantic salmon (Salmo salar) which constitute important models for fish immunology and important targets for vaccine development. The growing interest to decipher the dynamics of adaptive immune responses against pathogens or vaccines has resulted in recent efforts to sequence the immunoglobulin (IG) or antibodies and T cell receptor (TR) repertoire in these species. In this context, establishing a comprehensive and coherent locus annotation is the fundamental basis for the analysis of high-throughput repertoire sequencing data. We therefore decided to revisit the description and annotation of TRA/TRD locus in Atlantic salmon and two strains of rainbow trout (Swanson and Arlee) using the now available high-quality genome assemblies. Phylogenetic analysis of functional TRA/TRD V genes from these three genomes led to the definition of 25 subgroups shared by both species, some with particular feature. A total of 128 TRAJ genes were identified in Salmo, the majority with a close counterpart in Oncorhynchus. Analysis of expressed TRA repertoire indicates that most TRAV gene subgroups are expressed at mucosal and systemic level. The present work on TRA/TRD locus annotation along with the analysis of TRA repertoire sequencing data show the feasibility and advantages of a common salmonid TRA/TRD nomenclature that allows an accurate annotation and analysis of high-throughput sequencing results, across salmonid T cell subsets.

Piscine Orthoreovirus (PRV)-3, but Not PRV-2, Cross-Protects against PRV-1 and Heart and Skeletal Muscle Inflammation in Atlantic Salmon.

Heart and skeletal muscle inflammation (HSMI), caused by infection with Piscine orthoreovirus-1 (PRV-1), is a common disease in farmed Atlantic salmon (Salmo salar). Both an inactivated whole virus vaccine and a DNA vaccine have previously been tested experimentally against HSMI and demonstrated to give partial but not full protection. To understand the mechanisms involved in protection against HSMI and evaluate the potential of live attenuated vaccine strategies, we set up a cross-protection experiment using PRV genotypes not associated with disease development in Atlantic salmon. The three known genotypes of PRV differ in their preference of salmonid host species. The main target species for PRV-1 is Atlantic salmon. Coho salmon (Oncorhynchus kisutch) is the target species for PRV-2, where the infection may induce erythrocytic inclusion body syndrome (EIBS). PRV-3 is associated with heart pathology and anemia in rainbow trout, but brown trout (S. trutta) is the likely natural main host species. Here, we tested if primary infection with PRV-2 or PRV-3 in Atlantic salmon could induce protection against secondary PRV-1 infection, in comparison with an adjuvanted, inactivated PRV-1 vaccine. Viral kinetics, production of cross-reactive antibodies, and protection against HSMI were studied. PRV-3, and to a low extent PRV-2, induced antibodies cross-reacting with the PRV-1 σ1 protein, whereas no specific antibodies were detected after vaccination with inactivated PRV-1. Ten weeks after immunization, the fish were challenged through cohabitation with PRV-1-infected shedder fish. A primary PRV-3 infection completely blocked PRV-1 infection, while PRV-2 only reduced PRV-1 infection levels and the severity of HSMI pathology in a few individuals. This study indicates that infection with non-pathogenic, replicating PRV could be a future strategy to protect farmed salmon from HSMI.

Identification of Igsigma and Iglambda in channel catfish, Ictalurus punctatus, and Iglambda in Atlantic cod, Gadus morhua.

Immunoglobulin light (IGL) chain genes encoding sigma and lambda from channel catfish, Ictalurus punctatus, and lambda from Atlantic cod, Gadus morhua, were identified by mining of expressed sequence tag databases, 5'-RACE and RT-PCR protocols. cDNAs for each of these IGL chains encode typical variable (V), joining (J), and constant (C) regions and Southern blot analyses and genomic sequencing show that genes encoding these isotypes, like other teleost IGL genes, are found in a cluster organization of one or two V gene segments, followed by single J and C gene segments, all in the same transcriptional orientation. However, unlike the teleost kappa genes, genes encoding catfish sigma and lambda are few in number and the two isotypes are each encoded by only two clusters. Similarly, Atlantic cod lambda genes are predicted to be encoded by two or three clusters. As expected, sequence and phylogenetic analyses comparisons demonstrate that catfish Vsigma and Csigma genes are most similar to Vsigma and Csigma genes of other ectothermic vertebrates. Although catfish and Atlantic cod Vlambda genes cluster with other vertebrate Vlambda genes, their Clambda sequences cluster in a distinct group separate from other vertebrate IGL C sequences. However, support for classifying these sequences as lambda, is their V and J recombination signal sequence (RSS) organization. The catfish and Atlantic cod genes have typical lambda-like RSS with the Vlambda RSS consisting of heptamer-23 bp spacer-nonamer and the Jlambda RSS consisting of heptamer-12 bp spacer-nonamer. This is the first report demonstrating the presence of Iglambda in teleosts.

Critical Role of an MHC Class I-Like/Innate-Like T Cell Immune Surveillance System in Host Defense against Ranavirus (Frog Virus 3) Infection.

Besides the central role of classical Major Histocompatibility Complex (MHC) class Ia-restricted conventional Cluster of Differentiation 8 (CD8) T cells in antiviral host immune response, the amphibian Xenopuslaevis critically rely on MHC class I-like (mhc1b10.1.L or XNC10)-restricted innate-like (i)T cells (iVα6 T cells) to control infection by the ranavirus Frog virus 3 (FV3). To complement and extend our previous reverse genetic studies showing that iVα6 T cells are required for tadpole survival, as well as for timely and effective adult viral clearance, we examined the conditions and kinetics of iVα6 T cell response against FV3. Using a FV3 knock-out (KO) growth-defective mutant, we found that upregulation of the XNC10 restricting class I-like gene and the rapid recruitment of iVα6 T cells depend on detectable viral replication and productive FV3 infection. In addition, by in vivo depletion with XNC10 tetramers, we demonstrated the direct antiviral effector function of iVα6 T cells. Notably, the transitory iV6 T cell defect delayed innate interferon and cytokine gene response, resulting in long-lasting negative inability to control FV3 infection. These findings suggest that in Xenopus and likely other amphibians, an immune surveillance system based on the early activation of iT cells by non-polymorphic MHC class-I like molecules is important for efficient antiviral immune response.