ABSTRACT
The fms-related tyrosine kinase 3 (Flt3) and its ligand (Flt3lg) are important regulators of hematopoiesis and dendritic cell (DC) homeostasis with unsettled coevolution. Gene synteny and deduced amino acid sequence analyses identified conserved flt3 gene orthologs across all jawed vertebrates. In contrast, flt3lg orthologs were not retrieved in ray-finned fish, and the gene locus exhibited more variability among species. Interestingly, duplicated flt3/flt3lg genes were maintained in the allotetraploid Xenopus laevis Comparison of modeled structures of X. laevis Flt3 and Flt3lg homoeologs with the related diploid Xenopus tropicalis and with humans indicated a higher conformational divergence between the homoeologous pairs than their respective counterparts. The distinctive developmental and tissue expression patterns of Flt3 and Flt3lg homoeologs in tadpoles and adult frogs suggest a subfunctionalization of these homoeologs. To characterize Flt3 cell surface expression, X. laevis-tagged rFlt3lg.S and rFlt3lg.L were produced. Both rFlt3lg.S and rFlt3lg.L bind in vitro Flt3.S and Flt3.L and can trigger Erk1/2 signaling, which is consistent with a partial overlapping function between homoeologs. In spleen, Flt3.S/L cell surface expression was detected on a fraction of B cells and a population of MHC class IIhigh/CD8+ leukocytes phenotypically similar to the recently described dual follicular/conventional DC-like XL cells. Our result suggests that 1) Flt3lg.S and Flt3lg.L are both involved in XL cell homeostasis and that 2) XL cells have hematopoietic origin. Furthermore, we detected surface expression of the macrophage/monocyte marker Csf1r.S on XL cells as in mammalian and chicken DCs, which points to a common evolutionary origin in vertebrate DCs.
Subject(s)
Dendritic Cells , Receptor Protein-Tyrosine Kinases , Animals , Dendritic Cells/metabolism , Humans , Ligands , Mammals , Monocytes , Receptor Protein-Tyrosine Kinases/metabolism , Xenopus laevis/genetics , fms-Like Tyrosine Kinase 3/metabolismABSTRACT
Innate-like T cells display characteristics of both innate lymphoid cells (ILCs) and mainstream αß T cells, leading to overlapping functions of innate-like T cells with both subsets. In this review, we show that although innate-like T cells are probably present in all vertebrates, their main characteristics are much better known in amphibians and mammals. Innate-like T cells encompass both γδ and αß T cells. In mammals, γδ TCRs likely coevolved with molecules of the butyrophilin family they interact with, whereas the semi-invariant TCRs of iNKT and mucosal-associated invariant T cells are evolutionarily locked with their restricting MH1b molecules, CD1d and MR1, respectively. The strong conservation of the Ag recognition systems of innate-like T cell subsets despite similar effector potentialities supports that each one fulfills nonredundant roles related to their Ag specificity.
Subject(s)
Mucosal-Associated Invariant T Cells , Animals , Immunity, Innate , Lymphocyte Count , Mammals , Receptors, Antigen, T-Cell , T-Lymphocyte SubsetsABSTRACT
Regeneration-competent vertebrates are considered to suppress inflammation faster than non-regenerating ones. Hence, understanding the cellular mechanisms affected by immune cells and inflammation can help develop strategies to promote tissue repair and regeneration. Here, we took advantage of naturally occurring tail regeneration-competent and -incompetent developmental stages of Xenopus tadpoles. We first establish the essential role of the myeloid lineage for tail regeneration in the regeneration-competent tadpoles. We then reveal that upon tail amputation there is a myeloid lineage-dependent change in amputation-induced apoptosis levels, which in turn promotes tissue remodelling, and ultimately leads to the relocalization of the regeneration-organizing cells responsible for progenitor proliferation. These cellular mechanisms failed to be executed in regeneration-incompetent tadpoles. We demonstrate that regeneration incompetency is characterized by inflammatory myeloid cells whereas regeneration competency is associated with reparative myeloid cells. Moreover, treatment of regeneration-incompetent tadpoles with immune-suppressing drugs restores myeloid lineage-controlled cellular mechanisms. Collectively, our work reveals the effects of differential activation of the myeloid lineage on the creation of a regeneration-permissive environment and could be further exploited to devise strategies for regenerative medicine purposes.
Subject(s)
Cell Lineage/physiology , Myeloid Cells/physiology , Regeneration/physiology , Tail/physiology , Xenopus laevis/physiology , Animals , Apoptosis/drug effects , Extracellular Matrix/metabolism , Gene Expression , Gene Expression Regulation, Developmental , Immunosuppressive Agents/pharmacology , Larva/physiology , Regeneration/drug effects , Regenerative Medicine/methodsABSTRACT
CellMiner Cross-Database (CellMinerCDB, discover.nci.nih.gov/cellminercdb) allows integration and analysis of molecular and pharmacological data within and across cancer cell line datasets from the National Cancer Institute (NCI), Broad Institute, Sanger/MGH and MD Anderson Cancer Center (MDACC). We present CellMinerCDB 1.2 with updates to datasets from NCI-60, Broad Cancer Cell Line Encyclopedia and Sanger/MGH, and the addition of new datasets, including NCI-ALMANAC drug combination, MDACC Cell Line Project proteomic, NCI-SCLC DNA copy number and methylation data, and Broad methylation, genetic dependency and metabolomic datasets. CellMinerCDB (v1.2) includes several improvements over the previously published version: (i) new and updated datasets; (ii) support for pattern comparisons and multivariate analyses across data sources; (iii) updated annotations with drug mechanism of action information and biologically relevant multigene signatures; (iv) analysis speedups via caching; (v) a new dataset download feature; (vi) improved visualization of subsets of multiple tissue types; (vii) breakdown of univariate associations by tissue type; and (viii) enhanced help information. The curation and common annotations (e.g. tissues of origin and identifiers) provided here across pharmacogenomic datasets increase the utility of the individual datasets to address multiple researcher question types, including data reproducibility, biomarker discovery and multivariate analysis of drug activity.
Subject(s)
Computational Biology/methods , Databases, Factual , Neoplasms/metabolism , Pharmacogenetics/methods , Proteomics/methods , Cell Line, Tumor , Data Curation/methods , Data Mining/methods , Drug Therapy/methods , Genomics/methods , Humans , Internet , Neoplasms/drug therapy , Neoplasms/geneticsABSTRACT
Ranaviruses such as frog virus 3 (FV3) are large double-stranded DNA (dsDNA) viruses causing emerging infectious diseases leading to extensive morbidity and mortality of amphibians and other ectothermic vertebrates worldwide. Among the hosts of FV3, some are highly susceptible, whereas others are resistant and asymptomatic carriers that can take part in disseminating the infectious virus. To date, the mechanisms involved in the processes of FV3 viral persistence associated with subclinical infection transitioning to lethal outbreaks remain unknown. Investigation in Xenopus laevis has revealed that in asymptomatic FV3 carrier animals, inflammation induced by heat-killed (HK) Escherichia coli stimulation can provoke the relapse of active infection. Since Toll-like receptors (TLRs) are critical for recognizing microbial molecular patterns, we investigated their possible involvement in inflammation-induced FV3 reactivation. Among the 10 different TLRs screened for changes in expression levels following FV3 infection and HK E. coli stimulation, only TLR5 and TLR22, both of which recognize bacterial products, showed differential expression, and only the TLR5 ligand flagellin was able to induce FV3 reactivation similarly to HK E. coli Furthermore, only the TLR5 ligand flagellin induced FV3 reactivation in peritoneal macrophages both in vitro and in vivo These data indicate that the TLR5 signaling pathway can trigger FV3 reactivation and suggest a role of secondary bacterial infections or microbiome alterations (stress or pollution) in initiating sudden deadly disease outbreaks in amphibian populations with detectable persistent asymptomatic ranavirus.IMPORTANCE This study in the amphibian Xenopus laevis provides new evidence of the critical role of macrophages in the persistence of ranaviruses in a quiescent state as well as in the reactivation of these pathogens into a virulent infection. Among the multiple microbial sensors expressed by macrophages, our data underscore the preponderant involvement of TLR5 stimulation in triggering the reactivation of quiescent FV3 in resident peritoneal macrophages, unveiling a mechanistic connection between the reactivation of persisting ranavirus infection and bacterial coinfection. This suggests a role for secondary bacterial infections or microbiome alterations (stress or pollution) in initiating sudden deadly disease outbreaks in amphibian populations with detectable persistent asymptomatic ranavirus.
Subject(s)
DNA Virus Infections/veterinary , Macrophages, Peritoneal/virology , Ranavirus/physiology , Toll-Like Receptor 5/metabolism , Virus Activation , Xenopus Proteins/metabolism , Xenopus laevis/virology , Animals , Carrier State , Cytokines/genetics , Cytokines/metabolism , DNA Virus Infections/virology , Escherichia coli/immunology , Flagellin/immunology , Gene Expression , Inflammation , Macrophages, Peritoneal/immunology , Macrophages, Peritoneal/metabolism , NLR Proteins/genetics , NLR Proteins/metabolism , Signal Transduction , Toll-Like Receptor 5/genetics , Toll-Like Receptors/genetics , Toll-Like Receptors/metabolism , Viral Load , Virus Latency , Xenopus Proteins/genetics , Xenopus laevis/immunologyABSTRACT
To explore the origins and consequences of tetraploidy in the African clawed frog, we sequenced the Xenopus laevis genome and compared it to the related diploid X. tropicalis genome. We characterize the allotetraploid origin of X. laevis by partitioning its genome into two homoeologous subgenomes, marked by distinct families of 'fossil' transposable elements. On the basis of the activity of these elements and the age of hundreds of unitary pseudogenes, we estimate that the two diploid progenitor species diverged around 34 million years ago (Ma) and combined to form an allotetraploid around 17-18 Ma. More than 56% of all genes were retained in two homoeologous copies. Protein function, gene expression, and the amount of conserved flanking sequence all correlate with retention rates. The subgenomes have evolved asymmetrically, with one chromosome set more often preserving the ancestral state and the other experiencing more gene loss, deletion, rearrangement, and reduced gene expression.
Subject(s)
Evolution, Molecular , Genome/genetics , Phylogeny , Tetraploidy , Xenopus laevis/genetics , Animals , Chromosomes/genetics , Conserved Sequence/genetics , DNA Transposable Elements/genetics , Diploidy , Female , Gene Deletion , Gene Expression Profiling , Karyotype , Molecular Sequence Annotation , Mutagenesis/genetics , Pseudogenes , Xenopus/geneticsABSTRACT
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.
Subject(s)
Disease Resistance/immunology , Host-Pathogen Interactions/immunology , Immune Tolerance , Larva/microbiology , Mycobacterium Infections, Nontuberculous/mortality , Mycobacterium marinum/immunology , Xenopus laevis/microbiology , Animals , CD8-Positive T-Lymphocytes/immunology , Disease Models, Animal , Disease Susceptibility/immunology , Gene Expression Profiling , Immunity, Cellular , Liver/microbiology , Liver/pathology , Mycobacterium Infections, Nontuberculous/microbiology , Mycobacterium marinum/genetics , RNA, Bacterial/genetics , Receptors, Antigen, T-Cell/immunology , Survival Rate , Xenopus laevis/growth & developmentABSTRACT
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.
Subject(s)
Histocompatibility Antigens Class I/immunology , Immunity, Cellular , Mycobacterium Infections, Nontuberculous/immunology , Mycobacterium marinum/immunology , Receptors, Antigen, T-Cell, alpha-beta/immunology , T-Lymphocytes/immunology , Xenopus Proteins/immunology , Animals , Histocompatibility Antigens Class I/genetics , Larva/genetics , Larva/immunology , Mycobacterium Infections, Nontuberculous/genetics , Receptors, Antigen, T-Cell, alpha-beta/genetics , Xenopus Proteins/genetics , Xenopus laevisABSTRACT
Mycobacterium abscessus (Mab) is an emerging, nontuberculosis mycobacterium (NTM) that infects humans. Mab has two morphotypes, smooth (S) and rough (R), related to the production of glycopeptidolipid (GPL), that differ in pathogenesis. To further understand the pathogenicity of these morphotypes in vivo, the amphibian Xenopus laevis was used as an alternative animal model. Mab infections have been previously modeled in zebrafish embryos and mice, but Mab are cleared early from immunocompetent mice, preventing the study of chronic infection, and the zebrafish model cannot be used to model a pulmonary infection and T cell involvement. Here, we show that X. laevis tadpoles, which have lungs and T cells, can be used as a complementary model for persistent Mab infection and pathogenesis. Intraperitoneal (IP) inoculation of S and R Mab morphotypes disseminated to tadpole tissues including liver and lungs, persisting for up to 40 days without significant mortality. Furthermore, the R morphotype was more persistent, maintaining a higher bacterial load at 40 days postinoculation. In contrast, the intracardiac (IC) inoculation with S Mab induced significantly greater mortality than inoculation with the R Mab form. These data suggest that X. laevis tadpoles can serve as a useful comparative experimental organism to investigate pathogenesis and host resistance to M. abscessus.
Subject(s)
Disease Models, Animal , Mycobacterium abscessus/metabolism , Xenopus laevis/growth & development , Animals , Disease Resistance/immunology , Host-Pathogen Interactions , Humans , Larva/growth & development , Larva/immunology , Larva/microbiology , Liver/immunology , Liver/microbiology , Lung/immunology , Lung/microbiology , Mice, Inbred C57BL , Mycobacterium Infections, Nontuberculous/genetics , Mycobacterium Infections, Nontuberculous/microbiology , Mycobacterium abscessus/classification , Mycobacterium abscessus/pathogenicity , T-Lymphocytes/immunology , T-Lymphocytes/microbiology , Time Factors , Virulence , Xenopus laevis/immunology , Xenopus laevis/microbiologyABSTRACT
Populations of the eastern hellbender Cryptobranchus alleganiensis alleganiensis have been declining for decades, and emerging pathogens and pesticides are hypothesized to be contributing factors. However, few empirical studies have attempted to test the potential effects of these factors on hellbenders. We simultaneously exposed subadult hellbenders to environmentally relevant concentrations of either Batrachochytrium dendrobatidis (Bd) or a frog virus 3-like ranavirus (RV), a combination of the pathogens, or each pathogen following exposure to a glyphosate herbicide (Roundup). Additionally, we measured the ability of the skin mucosome to inactivate Bd and RV in growth assays. We found that mucosome significantly inactivated RV by an average of 40% but had no negative effects on Bd growth. All treatments that included RV exposure experienced reduced survival compared to controls, and the combination of RV and herbicide resulted in 100% mortality. Histopathology verified RV as the cause of mortality in all RV-exposed treatments. No animals were infected with Bd or died in the Bd-only treatment. Our results suggest that RV exposure may be a significant threat to the survival of subadult hellbenders and that Roundup exposure may potentially exacerbate this threat.
Subject(s)
DNA Virus Infections/veterinary , Glycine/analogs & derivatives , Herbicides/administration & dosage , Immunity, Innate , Mycoses/veterinary , Urodela/immunology , Animals , Batrachochytrium/physiology , DNA Virus Infections/virology , Glycine/administration & dosage , Mycoses/microbiology , Ranavirus/physiology , GlyphosateABSTRACT
BACKGROUND: Cytochrome P450 1B1 (CYP1B1) is mostly expressed in tumours and displays unusual properties. Its two polymorphic forms were differently associated with anticancer drug sensitivity. We decipher here the role of this polymorphism in anticancer drug efficacy in vitro, in vivo and in the clinical setting. METHODS: From head-and-neck squamous cell carcinoma cell lines not expressing CYP1B1, we generated isogenic derivatives expressing the two forms. Proliferation, invasiveness, stem cell characteristics, sensitivity to anticancer agents and transcriptome were analysed. Tumour growth and chemosensitivity were studied in vivo. A prospective clinical trial on 121 patients with advanced head-and-neck cancers was conducted, and a validation-retrospective study was conducted. RESULTS: Cell lines expressing the variant form displayed high rates of in vitro proliferation and invasiveness, stemness features and resistance to DNA-damaging agents. In vivo, tumours expressing the variant CYP1B1 had higher growth rates and were markedly drug-resistant. In the clinical study, overall survival was significantly associated with the genotypes, wild-type patients presenting a longer median survival (13.5 months) than the variant patients (6.3 months) (p = 0.0166). CONCLUSIONS: This frequent CYP1B1 polymorphism is crucial for cancer cell proliferation, migration, resistance to chemotherapy and stemness properties, and strongly influences head-and-neck cancer patients' survival.
Subject(s)
Cytochrome P-450 CYP1B1/genetics , Head and Neck Neoplasms/genetics , Head and Neck Neoplasms/pathology , Neoplastic Stem Cells/pathology , Squamous Cell Carcinoma of Head and Neck/genetics , Squamous Cell Carcinoma of Head and Neck/pathology , Animals , Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Cell Growth Processes/physiology , Cell Line, Tumor , Cell Movement/physiology , Cetuximab/administration & dosage , DNA Methylation , Female , Head and Neck Neoplasms/drug therapy , Head and Neck Neoplasms/enzymology , Heterografts , Humans , Male , Mice , Mice, Inbred NOD , Mice, SCID , Neoplasm Invasiveness , Neoplastic Stem Cells/enzymology , Promoter Regions, Genetic , Squamous Cell Carcinoma of Head and Neck/drug therapy , Squamous Cell Carcinoma of Head and Neck/enzymologyABSTRACT
The Chinese sturgeon (Acipenser sinensis) is one of the critically endangered aquatic species in China. It is also among the oldest extant actinopterygian fish species. To advance the characterization of the Chinese sturgeon immune system, we identified the gene encoding the macrophage migration inhibitory factor (MIF), a multifunctional cytokine that contributes to both innate and adaptive immune responses. Molecular and phylogenic analysis indicates the Chinese sturgeon (cs) MIF share a high degree of structural conservation with other MIF sequences and is closely related to other bony fish MIF. At steady state, cs-mif gene is expressed at relatively high levels in the brain, and to a lesser but significant level in liver, spleen, kidney, gut and skin. The spatial expression patterns determined by in situ hybridization indicates a preferential distribution of cs-mif transcripts in the cerebral cortex, the gut epithelium, hematopoietic tissues of kidney, spleen and liver parenchyma, and skin epidermis. Marked increase of cs-mif gene expression was induced by lipopolysaccharide (LPS) stimulation and Aeromonas hydrophila infection in all tested tissues. Furthermore, higher cs-mif transcript levels were detected in the liver, spleen, kidney, gut and skin during stress response resulting from hyperthermia. These results are not only consistent with the expected role of cs-mif gene in innate immunity but also suggest a potential role of this gene in stress response to hyperthermia in the Chinese sturgeon.
Subject(s)
Fish Diseases/immunology , Fishes/genetics , Fishes/immunology , Gene Expression Regulation/immunology , Immunity, Innate/genetics , Macrophage Migration-Inhibitory Factors/genetics , Macrophage Migration-Inhibitory Factors/immunology , Aeromonas hydrophila/physiology , Amino Acid Sequence , Animals , Base Sequence , Fish Proteins/chemistry , Fish Proteins/genetics , Fish Proteins/immunology , Gene Expression Profiling/veterinary , Gram-Negative Bacterial Infections/immunology , Gram-Negative Bacterial Infections/veterinary , Lipopolysaccharides/pharmacology , Macrophage Migration-Inhibitory Factors/chemistry , Phylogeny , Sequence Alignment/veterinaryABSTRACT
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.
Subject(s)
Histocompatibility Antigens Class I/metabolism , Natural Killer T-Cells/immunology , T-Lymphocyte Subsets/immunology , Thymus Neoplasms/immunology , Tumor Escape/immunology , Xenopus Proteins/metabolism , Animals , Animals, Genetically Modified , Cell Line, Tumor/transplantation , Disease Models, Animal , Gene Knockout Techniques , Histocompatibility Antigens Class I/genetics , Histocompatibility Antigens Class I/immunology , Humans , Larva , Natural Killer T-Cells/metabolism , Receptors, Antigen, T-Cell, alpha-beta/immunology , Receptors, Antigen, T-Cell, alpha-beta/metabolism , T-Lymphocyte Subsets/metabolism , Thymus Neoplasms/pathology , Xenopus Proteins/immunology , Xenopus laevisABSTRACT
Although the amphibian Xenopus laevis produces antibodies as diversified as those from mammals in the primary repertoire, antibody affinity maturation after immunization is relatively poor and has been associated with a poor B cell selection of AID-mediated hypermutations and lack of germinal centers in the spleen, the only secondary lymphoid organ of this amphibian. In this issue of the European Journal of Immunology, Neely et al., [Eur. J. Immunol. 2018. 48: 430-440] have unveiled the role of distinctive dendritic cell (DC) subset, XL cells, which have the capacity to acquire and retain native antigens for B cell maturation. The complementary evidence presented by this study (immunohistology, tracing antigen complexes, flow cytometry analysis and gene expression profiles of sorted XL cells) provides novel fundamental insights into a major evolutionary step in functional and cellular specialization of DC and follicular dendritic cells (FDCs) in regulating B cell responses.
Subject(s)
Dendritic Cells, Follicular , Germinal Center , Animals , B-Lymphocytes , Dendritic Cells , SpleenABSTRACT
Cancers impose a significant health and economic burden. By harnessing the immune system, current immunotherapies have revolutionized the treatment against human cancers and potentially offer a long-term cure. Among others, innate-like T (iT) cells, including natural killer T cells, are promising candidates for immunotherapies. Unlike conventional T cells, iT cells regulate multiple immune processes and express an invariant T cell receptor that is shared among different individuals. However, the conditions that activate the pro- and antitumor functions of iT cells are partially understood. These gaps in knowledge hamper the use of iT cell in clinics. It might be beneficial to examine the roles of iT cells in an alternative animal model - the amphibian Xenopus whose immune system shares many similarities to that of mammals. Here, we review the iT cell biology in the context of mammalian cancers and discuss the challenges currently found in the field. Next, we introduce the advantages of Xenopus as a model to investigate the role of iT cells and interacting major histocompatibility complex (MHC) class I-like molecules in tumor immunity. In Xenopus, 2 specific iT cell subsets, Vα6 and Vα22 iT cells, recognize and fight tumor cells. Furthermore, our recent data reveal the complex functions of the Xenopus MHC class I-like (XNC) gene XNC10 in tumor immune responses. By utilizing reverse genetics, transgenesis, and MHC tetramers, we have a unique opportunity to uncover the relevance of XNC genes and iT cell in Xenopus tumor immunity.
Subject(s)
Biological Evolution , Disease Models, Animal , Histocompatibility Antigens Class I/immunology , Natural Killer T-Cells/immunology , Neoplasms/immunology , Animals , Histocompatibility Antigens Class I/genetics , Humans , Receptors, Antigen, T-Cell/immunology , Xenopus laevis/genetics , Xenopus laevis/immunologyABSTRACT
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.
Subject(s)
CRISPR-Cas Systems , Genes, MHC Class I , Histocompatibility Antigens Class I/genetics , Xenopus Proteins/genetics , Xenopus laevis/genetics , Animals , Animals, Inbred Strains , Base Sequence , Chromosome Mapping , Embryo, Nonmammalian , Gene Knockout Techniques , Histocompatibility Antigens Class I/immunology , Larva , Microinjections , Multigene Family , Mutation , Protein Domains , RNA, Guide, Kinetoplastida/genetics , Reverse Genetics , Sequence Alignment , Sequence Homology, Nucleic Acid , Xenopus/genetics , Xenopus/immunology , Xenopus Proteins/immunology , Xenopus laevis/growth & development , Xenopus laevis/immunologyABSTRACT
Tail regression is one of the most prominent transformations observed during anuran metamorphosis. A tadpole tail that is twice as long as the tadpole trunk nearly disappears within 3 days in Xenopus tropicalis. Several years ago, it was proposed that this phenomenon is driven by an immunological rejection of larval-skin-specific antigens, Ouro proteins. We generated ouro-knockout tadpoles using the TALEN method to reexamine this immunological rejection model. Both the ouro1- and ouro2-knockout tadpoles expressed a very low level of mRNA transcribed from a targeted ouro gene, an undetectable level of Ouro protein encoded by a target gene and a scarcely detectable level of the other Ouro protein from the untargeted ouro gene in tail skin. Furthermore, congenital athymic frogs were produced by Foxn1 gene modification. Flow cytometry analysis showed that mutant frogs lacked splenic CD8(+) T cells, which play a major role in cytotoxic reaction. Furthermore, T-cell-dependent skin allograft rejection was dramatically impaired in mutant frogs. None of the knockout tadpoles showed any significant delay in the process of tail shortening during the climax of metamorphosis, which shows that Ouro proteins are not essential to tail regression at least in Xenopus tropicalis and argues against the immunological rejection model.
Subject(s)
Keratins/metabolism , Metamorphosis, Biological/genetics , Xenopus Proteins/metabolism , Animals , CD8-Positive T-Lymphocytes/metabolism , Forkhead Transcription Factors/genetics , Forkhead Transcription Factors/metabolism , Gene Expression Regulation, Developmental , Keratins/genetics , Thymus Gland/cytology , Thymus Gland/metabolism , Xenopus , Xenopus Proteins/geneticsABSTRACT
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.
Subject(s)
Amphibian Proteins/immunology , DNA Virus Infections/immunology , DNA Virus Infections/veterinary , Histocompatibility Antigens Class I/immunology , Immunity, Innate , Ranavirus/immunology , T-Lymphocytes/immunology , Amphibian Proteins/antagonists & inhibitors , Amphibian Proteins/genetics , Animals , Cell Movement , DNA Virus Infections/pathology , DNA Virus Infections/virology , Female , Gene Expression , Histocompatibility Antigens Class I/genetics , Immunophenotyping , Macrophages/immunology , Macrophages/pathology , Macrophages/virology , Natural Killer T-Cells/immunology , Natural Killer T-Cells/pathology , Natural Killer T-Cells/virology , Peritoneum/immunology , Peritoneum/pathology , Peritoneum/virology , Protein Multimerization , RNA Interference , RNA, Small Interfering/genetics , RNA, Small Interfering/immunology , Signal Transduction , Spleen/immunology , Spleen/pathology , Spleen/virology , T-Lymphocytes/pathology , T-Lymphocytes/virology , Xenopus laevisABSTRACT
A major goal in regenerative medicine is to identify therapies to facilitate our body׳s innate abilities to repair and regenerate following injury, disease or aging. In the past decade it has become apparent that the innate immune system is able to affect the speed and quality of the regenerative response through mechanisms that are not entirely clear. For this reason there has been a resurgent interest in investigating the role of inflammation during tissue repair and regeneration. Remarkably, there have only been a handful of such studies using organisms with high regenerative capacity. Here we perform a study of the inflammatory response following injury in Xenopus larvae, which are able to achieve scarless wound healing and to regenerate appendages, as a preamble into understanding the role that inflammation plays during tissue repair and regeneration in this organism. We characterized the morphology and migratory behavior of granulocytes and macrophages following sterile and infected wounding regimes, using various transgenic lines that labeled different types of myeloid lineages, including granulocytes and macrophages. Using this approach we found that the inflammatory response following injury and infection in Xenopus larvae is very similar to that seen in humans, suggesting that this model provides an easily tractable and medically relevant system to investigate inflammation following injury and infection in vivo.
Subject(s)
Bacterial Infections/complications , Inflammation/etiology , Inflammation/pathology , Wounds and Injuries/complications , Animals , Animals, Genetically Modified , Bacterial Infections/pathology , Cell Movement , Disease Models, Animal , Humans , Microscopy, Fluorescence , Microscopy, Video , Myeloid Cells/pathology , Myeloid Cells/physiology , Regeneration , Wounds and Injuries/pathology , Xenopus laevis/genetics , Xenopus laevis/growth & development , Xenopus laevis/physiologyABSTRACT
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.