The recombinase activating genes: architects of immune diversity during lymphocyte development.

The mature lymphocyte population of a healthy individual has the remarkable ability to recognise an immense variety of antigens. Instead of encoding a unique gene for each potential antigen receptor, evolution has used gene rearrangements, also known as variable, diversity, and joining gene segment (V(D)J) recombination. This process is critical for lymphocyte development and relies on recombination-activating genes-1 (RAG1) and RAG2, here collectively referred to as RAG. RAG serves as powerful genome editing tools for lymphocytes and is strictly regulated to prevent dysregulation. However, in the case of dysregulation, RAG has been implicated in cases of cancer, autoimmunity and severe combined immunodeficiency (SCID). This review examines functional protein domains and motifs of RAG, describes advances in our understanding of the function and (dys)regulation of RAG, discuss new therapeutic options, such as gene therapy, for RAG deficiencies, and explore in vitro and in vivo methods for determining RAG activity and target specificity.

Nociceptor neurons affect cancer immunosurveillance.

Solid tumours are innervated by nerve fibres that arise from the autonomic and sensory peripheral nervous systems1-5. Whether the neo-innervation of tumours by pain-initiating sensory neurons affects cancer immunosurveillance remains unclear. Here we show that melanoma cells interact with nociceptor neurons, leading to increases in their neurite outgrowth, responsiveness to noxious ligands and neuropeptide release. Calcitonin gene-related peptide (CGRP)-one such nociceptor-produced neuropeptide-directly increases the exhaustion of cytotoxic CD8+ T cells, which limits their capacity to eliminate melanoma. Genetic ablation of the TRPV1 lineage, local pharmacological silencing of nociceptors and antagonism of the CGRP receptor RAMP1 all reduced the exhaustion of tumour-infiltrating leukocytes and decreased the growth of tumours, nearly tripling the survival rate of mice that were inoculated with B16F10 melanoma cells. Conversely, CD8+ T cell exhaustion was rescued in sensory-neuron-depleted mice that were treated with local recombinant CGRP. As compared with wild-type CD8+ T cells, Ramp1-/- CD8+ T cells were protected against exhaustion when co-transplanted into tumour-bearing Rag1-deficient mice. Single-cell RNA sequencing of biopsies from patients with melanoma revealed that intratumoral RAMP1-expressing CD8+ T cells were more exhausted than their RAMP1-negative counterparts, whereas overexpression of RAMP1 correlated with a poorer clinical prognosis. Overall, our results suggest that reducing the release of CGRP from tumour-innervating nociceptors could be a strategy to improve anti-tumour immunity by eliminating the immunomodulatory effects of CGRP on cytotoxic CD8+ T cells.

Structural insights into the evolution of the RAG recombinase.

Adaptive immunity in jawed vertebrates relies on the assembly of antigen receptor genes by the recombination activating gene 1 (RAG1)-RAG2 (collectively RAG) recombinase in a reaction known as V(D)J recombination. Extensive biochemical and structural evidence indicates that RAG and V(D)J recombination evolved from the components of a RAG-like (RAGL) transposable element through a process known as transposon molecular domestication. This Review describes recent advances in our understanding of the functional and structural transitions that occurred during RAG evolution. We use the structures of RAG and RAGL enzymes to trace the evolutionary adaptations that yielded a RAG recombinase with exquisitely regulated cleavage activity and a multilayered array of mechanisms to suppress transposition. We describe how changes in modes of DNA binding, alterations in the dynamics of protein-DNA complexes, single amino acid mutations and a modular design likely enabled RAG family enzymes to survive and spread in the genomes of eukaryotes. These advances highlight the insight that can be gained from viewing evolution of vertebrate immunity through the lens of comparative genome analyses coupled with structural biology and biochemistry.

Genetic identification of species and natural hybridization determination based on mitochondrial DNA and nuclear DNA of genus Zacco in Korea.

Genus Zacco specimens collected in this study were classified genetically as five species, Zacco platypus, Z. temminckii, Z. koreanus and two unidentified species, using DNA barcoding analysis based on 655 bp of mitochondrial cytochrome c oxidase subunit I (COI) gene. Two of unidentified species (Z. sp.1 and Z. sp.2) were considered to be unrecorded or new species of genus Zacco according to genetic distances between Zacco species. In addition, we determined a natural hybrid based on polymorphic base at the diagnostic positions displayed on nuclear recombination activating gene 1 (RAG1) gene (965 bp), and estimated paternal and maternal species of natural hybrid comparing phylogenetic tree between COI and RAG1, and Z. sp.1♀ × Z. koreanus♂, Z. sp.2♀ × Z. koreanus♂ and Z. koreanus♀ × Z. sp.1♂ individuals were confirmed. The habitat of natural hybrids of Z. koreanus between Z. sp.1 and Z. sp.2 was identified as Geum and Yeongsan River, respectively. In our data, only F1 hybrid generation was identified; however, generations after F1 hybrid or backcross were not demonstrated.

Origins of the RAG Transposome and the MHC.

How innate immunity gave rise to adaptive immunity in vertebrates remains unknown. We propose an evolutionary scenario beginning with pathogen-associated molecular pattern(s) (PAMPs) being presented by molecule(s) on one cell to specific receptor(s) on other cells, much like MHC molecules and T cell receptors (TCRs). In this model, mutations in MHC-like molecule(s) that bound new PAMP(s) would not be recognized by original TCR-like molecule(s), and new MHC-like gene(s) would be lost by neutral drift. Integrating recombination activating gene (RAG) transposon(s) in a TCR-like gene would result in greater recognition diversity, with new MHC-like variants recognized and selected, along with a new RAG/TCR-like system. MHC genes would be selected to present many peptides, through multigene families, allelic polymorphism, and peptide-binding promiscuity.

Analyses of RAG1 and RAG2 genes suggest different evolutionary rates in the Cetacea lineage.

V(D)J recombination is a process of somatic recombination catalyzed by proteins encoded by RAG1 and RAG2 genes, both restricted to the genome of jawed vertebrates. Their proteins constitute the enzymatic core of V(D)J recombination machinery and are crucial for jawed vertebrate adaptive immunity. Mammals possess great ecological diversity, and their complex evolutionary history associated with radiation to different environments presented many distinct pathogenic challenges from these different habitats. Cetaceans comprise a mammalian order of fully aquatic mammals that have arisen from a complete terrestrial ancestor and, accordingly, was confronted with challenges from changing environmental pathogens while they transitioned from land to sea. In this study we undertook molecular evolutionary analyses of RAG1 and RAG2 genes, exploring the possible role of natural selection acting on these genes focusing on the cetacean lineage. We performed phylogenetic reconstructions on IQ-TREE, together with selection analyses in the codeml program of the PAML package, and in the FITMODEL program for codon evolution and switching on both the RAG1 and RAG2 genes. Our findings demonstrate that RAG1 and RAG2 remained fairly conserved among tetrapods, with purifying selection acting on both genes, with evidence for a few punctuated shifts in nucleotide substitution rates of both genes along tetrapod evolution. We demonstrate differential evolution in the closely linked genes RAG1 and RAG2 specifically in cetaceans.

Comparative genetic stock structure in three species of commercially exploited Indo-Malay Carangidae (Teleosteii, Perciformes).

We examine genetic structuring in three commercially important species of the teleost family Carangidae from Malaysian waters: yellowtail scad Atule mate, bigeye scad Selar crumenophthalmus and yellowstripe scad Selaroides leptolepis, from the Indo-Malay Archipelago. In view of their distribution across contrasting habitats, we tested the hypothesis that pelagic species display less genetic divergence compared with demersal species, due to their potential to undertake long-distance migrations in oceanic waters. To evaluate population genetic structure, we sequenced two mitochondrial (mt)DNA [650 bp of cytochrome oxidase I (coI), 450 bp of control region (CR)] and one nuclear gene (910 bp of rag1) in each species. One hundred and eighty samples from four geographical regions within the Indo-Malay Archipelago including a population of yellowtail from Kuwait were examined. Findings revealed that the extent of genetic structuring among populations in the semi-pelagic and pelagic, yellowtail and bigeye were lower than demersal yellowstripe, consistent with the hypothesis that pelagic species display less genetic divergence compared with demersal species. The yellowtail phylogeny identified three distinct clades with bootstrap values of 86%-99% in mtDNA and 63%-67% in rag1. However, in bigeye, three clades were also observed from mtDNA data while only one clade was identified in rag1 dataset. In yellowstripe, the mtDNA tree was split into three closely related clades and two clades in rag1 tree with bootstraps value of 73%-99% and 56% respectively. However, no geographic structure appears in both mtDNA and rag1 datasets. Hierarchical molecular variance analysis (AMOVA), pair wise FST comparisons and the nearest-neighbour statistic (Snn ) showed significant genetic differences among Kuwait and Indo-Malay yellowtail. Within the Indo-Malay Archipelago itself, two distinct mitochondrial lineages were detected in yellowtail suggesting potential cryptic species. Findings suggests varying degrees of genetic structuring, key information relevant to management of exploited stocks, though more rapidly evolving genetic markers should be used in future to better delimit the nature and dynamics of putative stock boundaries.

Phylogeography of the red-bellied lizard, Darevskia parvula in Turkey.

The groups of red-bellied lizards had a small distribution area in the Pontic zone. The several studies performed on these lizard groups are based on taxonomy and systematics. Although there were several taxonomic or systematic researches on some species of this group, the phylogeographical pattern and species disturbing boundaries of this group is still not clear. In the present study, we aimed to resolve the taxonomic and phylogenetic relationships of the red-bellied lizards in Turkey, based on two combined mitochondrial gene fragments and one protein-coding nuclear gene (rag1). Also, we evaluated ecological niches differentiations among red-bellied lizard groups. The mitochondrial DNA genes were found to be highly polymorphic in this group. One hundred and one variable nucleotide sites were detected on the combined gene sequences. According to phylogenetic trees based on the maximum likelihood (ML) and Bayesian inference (BI), the red-bellied lizards group have three species groups; Darevskia parvula, D. adjarica and unnamed Darevskia sp. (candidate species for Darevskia genus). This situation was supported by high bootstrap and posterior probability values in the trees of mitochondrial DNA gene fragments. However, no genetic variation was detected according to nuclear DNA (rag1) sequence. Because the species groups have no overlaps in terms of their ecological niches, ecological niche modelling (ENM) results revealed differences among the groups of D. parvula, D. adjarica, and unnamed Darevskia sp. Besides, we detected no geographical overlaps among three species groups, since there were geographical isolation zones among the species groups of red-bellied lizard.

RAG gene defects at the verge of immunodeficiency and immune dysregulation.

Mutations of the recombinase activating genes (RAG) in humans underlie a broad spectrum of clinical and immunological phenotypes that reflect different degrees of impairment of T- and B-cell development and alterations of mechanisms of central and peripheral tolerance. Recent studies have shown that this phenotypic heterogeneity correlates, albeit imperfectly, with different levels of recombination activity of the mutant RAG proteins. Furthermore, studies in patients and in newly developed animal models carrying hypomorphic RAG mutations have disclosed various mechanisms underlying immune dysregulation in this condition. Careful annotation of clinical outcome and immune reconstitution in RAG-deficient patients who have received hematopoietic stem cell transplantation has shown that progress has been made in the treatment of this disease, but new approaches remain to be tested to improve stem cell engraftment and durable immune reconstitution. Finally, initial attempts have been made to treat RAG deficiency with gene therapy.

A causal mechanism for childhood acute lymphoblastic leukaemia.

In this Review, I present evidence supporting a multifactorial causation of childhood acute lymphoblastic leukaemia (ALL), a major subtype of paediatric cancer. ALL evolves in two discrete steps. First, in utero initiation by fusion gene formation or hyperdiploidy generates a covert, pre-leukaemic clone. Second, in a small fraction of these cases, the postnatal acquisition of secondary genetic changes (primarily V(D)J recombination-activating protein (RAG) and activation-induced cytidine deaminase (AID)-driven copy number alterations in the case of ETS translocation variant 6 (ETV6)-runt-related transcription factor 1 (RUNX1)+ ALL) drives conversion to overt leukaemia. Epidemiological and modelling studies endorse a dual role for common infections. Microbial exposures earlier in life are protective but, in their absence, later infections trigger the critical secondary mutations. Risk is further modified by inherited genetics, chance and, probably, diet. Childhood ALL can be viewed as a paradoxical consequence of progress in modern societies, where behavioural changes have restrained early microbial exposure. This engenders an evolutionary mismatch between historical adaptations of the immune system and contemporary lifestyles. Childhood ALL may be a preventable cancer.

Characterization of an N-Terminal Non-Core Domain of RAG1 Gene Disrupted Syrian Hamster Model Generated by CRISPR Cas9.

The accumulating evidence demonstrates that Syrian hamsters have advantages as models for various diseases. To develop a Syrian hamster (Mesocricetus auratus) model of human immunodeficiency caused by RAG1 gene mutations, we employed the CRISPR/Cas9 system and introduced an 86-nucleotide frameshift deletion in the hamster RAG1 gene encoding part of the N-terminal non-core domain of RAG1. Histological and immunohistochemical analyses demonstrated that these hamsters (referred herein as RAG1-86nt hamsters) had atrophic spleen and thymus, and developed significantly less white pulp and were almost completely devoid of splenic lymphoid follicles. The RAG1-nt86 hamsters had barely detectable CD3⁺ and CD4⁺ T cells. The expression of B and T lymphocyte-specific genes (CD3γ and CD4 for T cell-specific) and (CD22 and FCMR for B cell-specific) was dramatically reduced, whereas the expression of macrophage-specific (CD68) and natural killer (NK) cell-specific (CD94 and KLRG1) marker genes was increased in the spleen of RAG1-nt86 hamsters compared to wildtype hamsters. Interestingly, despite the impaired development of B and T lymphocytes, the RAG1-86nt hamsters still developed neutralizing antibodies against human adenovirus type C6 (HAdV-C6) upon intranasal infection and were capable of clearing the infectious viruses, albeit with slower kinetics. Therefore, the RAG1-86nt hamster reported herein (similar to the hypomorphic RAG1 mutations in humans that cause Omenn syndrome), may provide a useful model for studying the pathogenesis of the specific RAG1-mutation-induced human immunodeficiency, the host immune response to adenovirus infection and other pathogens as well as for evaluation of cell and gene therapies for treatment of this subset of RAG1 mutation patients.

Unmasking the complexity of species identification in Australasian flying-foxes.

Pteropus (flying-foxes) are a speciose group of non-echolocating large bats, with five extant Australian species and 24 additional species distributed amongst the Pacific Islands. In 2015, an injured flying-fox with unusual facial markings was found in Sydney, Australia, following severe and widespread storms. Based on an initial assessment, the individual belonged to Pteropus but could not be readily identified to species. As a consequence, four hypotheses for its identification/origin were posited: the specimen represented (1) an undescribed Australian species; or (2) a morphological variant of a recognised Australian species; or (3) a hybrid individual; or (4) a vagrant from the nearby Southwest Pacific Islands. We used a combination of morphological and both mitochondrial- and nuclear DNA-based identification methods to assess these hypotheses. Based on the results, we propose that this morphologically unique Pteropus most likely represents an unusual P. alecto (black flying-fox) potentially resulting from introgression from another Pteropus species. Unexpectedly, this individual, and the addition of reference sequence data from newly vouchered specimens, revealed a previously unreported P. alecto mitochondrial DNA lineage. This lineage was distinct from currently available haplotypes. It also suggests long-term hybridisation commonly occurs between P. alecto and P. conspicillatus (spectacled flying-fox). This highlights the importance of extensive reference data, and the inclusion of multiple vouchered specimens for each species to encompass both intraspecific and interspecific variation to provide accurate and robust species identification. Moreover, our additional reference data further demonstrates the complexity of Pteropus species relationships, including hybridisation, and potential intraspecific biogeographical structure that may impact on their management and conservation.

[Clinical characteristics of human recombination activating gene 1 mutations in 8 immunodeficiency patients with diverse phenotypes].

Objective: To investigate the clinical characteristics of 8 immunodeficiency cases caused by human recombination activating gene 1 (RAG1) mutations, and to explore the relationship among genotypes, clinical manifestations and immunophenotypes. Methods: Clinical data were collected and analyzed from patients with RAG1 mutations who visited the Department of Clinical Immunology, Children's Hospital of Fudan University between October 2013 and June 2017. The data included clinical manifestations, immunophenotypes and genotypes. Results: A total of 8 patients were diagnosed with RAG1 deficiency (6 boys and 2 girls). The minimum age of onset was 2 months, and the maximum age was 4 months. The minimum age of diagnosis was 2 months, and the maximum age was 13 years. Four patients had a family history of infant death due to severe infections. Two cases were born to the same consanguineous parents. All cases had recurrent infections, including involvement of respiratory tract (8 cases), digestive tract (6 cases), urinary tract (1 case), and central nervous system (1 case). The pathogens of infection included bacteria, viruses and fungi. Rotavirus was found in 3 cases, cytomegalovirus (CMV) in 5 cases, bacillus Calmette-Guérin adverse reaction in 2 cases (1 of whom had a positive acid-fast smear from lymph node puncture fluid), fungal infection in 3 cases. One case had multiple nodular space-occupying lesions in lungs and abdominal cavity complicated with multiple bone destruction. The peripheral blood lymphocyte counts of all patients ranged between 0.1 ×10(9)/L and 3.3×10(9)/L (median, 0.65×10(9)/L). Eosinophilia was found in 3 cases (range, (0.48-1.69) ×10(9)/L). The patients were classified according to immunophenotype as severe combined immunodeficiency phenotype (4 cases), leaky severe combined immunodeficiency (2 cases), Omenn syndrome (1 case) and combined immunodeficiency (1 case) . Decreased serum IgG levels were found in 3 cases, increased serum IgM levels in 3 cases, increased serum IgE levels in 5 cases. RAG1 homozygous mutations were detected in 5 cases and RAG1 compound heterozygous mutations in 3 cases. Two novel mutations and six previously reported mutations were identified. Three cases were successfully treated with hematopoietic stem cell transplantation. Four cases died due to infections, and the 13 year-old patient was still under follow-up in the outpatient clinic. Conclusions: Different RAG1 gene mutations can lead to diverse clinical presentations and immune phenotypes. Clinicians should pay attention to the family history of infant death with severe infection. In that situation, immunological evaluation and gene detection should be performed as early as possible.

Naïve B cells reduce fungal dissemination in Cryptococcus neoformans infected Rag1-/- mice.

IgM and B-1 cell deficient mice exhibit early C. neoformans dissemination from lungs to brain, but a definitive role for B cells in conferring resistance to C. neoformans dissemination has not been established. To address this question, we developed an intranasal (i.n.) C. neoformans infection model in B and T cell deficient Rag1-/- mice and found they also exhibit earlier fungal dissemination and higher brain CFU than wild-type C57Bl/6 (wild-type) mice. To probe the effect of B cells on fungal dissemination, Rag1-/- mice were given splenic (intravenously) or peritoneal (intraperitoneally) B cells from wild-type mice and infected i.n. with C. neoformans 7 d later. Mice that received B cells had lung histopathology resembling wild type mice 14 d post-infection, and B-1, not B-2 or T cells in their lungs, and serum and lung IgM and IgG 21 d post-infection. Lung CFU were comparable in wild-type, Rag1-/-, and Rag1-/- mice that received B cells 21 d post-infection, but brain CFU were significantly lower in mice that received B cells than Rag1-/- mice that did not. To determine if natural antibody can promote immunity in our model, we measured alveolar macrophage phagocytosis of C. neoformans in Rag1-/- mice treated with naive wild-type IgM-sufficient or sIgM-/- IgM-deficient sera before infection. Compared to IgM-deficient sera, IgM-sufficient sera significantly increased phagocytosis. Our data establish B cells are able to reduce early C. neoformans dissemination in mice and suggest natural IgM may be a key mediator of early antifungal immunity in the lungs.

The evolutionary conservation of the bidirectional activity of the NWC gene promoter in jawed vertebrates and the domestication of the RAG transposon.

The RAG-1 and RAG-2 genes form a recombinase complex that is indispensable for V(D)J recombination, which generates the diversity of immunoglobulins and T-cell receptors. It is widely accepted that the presence of RAGs in the genomes of jawed vertebrates and other lineages is a result of the horizontal transfer of a mobile genetic element. While a substantial amount of evidence has been gathered that clarifies the nature of the RAG transposon, far less attention has been paid to the genomic site of its integration in various host organisms. In all genomes of the jawed vertebrates that have been studied to date, the RAG genes are located in close proximity to the NWC gene. We have previously shown that the promoter of the murine NWC genes exhibits a bidirectional activity, which may have facilitated the integration and survival of the RAG transposon in the host genome. In this study, we characterise the promoters of the NWC homologues that are present in the representatives of other jawed vertebrates (H. sapiens, X. tropicalis and D. rerio). We show that the features that are characteristic for promoters as the hosts of a successful transposon integration (in terms of the arrangement, bidirectional and constitutive activity and the involvement of the Zfp143 transcription factor in the promoter regulation) are evolutionarily conserved, which indicates that the presence of RAG genes in jawed vertebrates is a direct result of a successful transposon integration into the NWC locus.

Impact of Immune Deficiency on Remodeling of Maternal Resistance Vasculature 4 Weeks Postpartum in Mice.

Pregnancy manifests changes in the vascular and immune systems that persist postpartum (PP), have important implications for future pregnancies, and may modify responses to cardiovascular stress in late life. The association between immune and vascular function and the generation or progression of cardiovascular disease beg the question of whether altered immunity modifies pregnancy-induced changes in the vasculature. Our objective was to compare changes in the function and remodeling of systemic resistance vessels 4 weeks PP in normal C57BL/6 (B6), and immunodeficient mice recombinase 1-deficient/B6 ( Rag1-/-). Immune deficiency did not change the responsiveness to acetylcholine (ACh) and phenylephrine at baseline but decreased arterial distensibility and increased stiffness PP. Adoptive transfer of CD8 T cells into Rag1-/- mice decreased the response to ACh while increasing distensibility and wall thickness. When compared to PP Rag1-/-, vessels from PP CD4-deficient mice, which have B cells and CD8 T cells, but no CD4 cells, show increased distensibility and decreased responsiveness to ACh in a pattern similar to that seen in Rag1-/- given CD8 T cells prior to mating. These studies suggest a key role for T cell, particularly CD8 T cell, associated factors in the PP remodeling of maternal resistance vessels.

Of the multiple mechanisms leading to type 1 diabetes, T cell receptor revision may play a prominent role (is type 1 diabetes more than a single disease?).

A single determinant factor for autoimmunity does not exist; disease development probably involves contributions from genetics, the environment and immune dysfunction. Type 1 diabetes is no exception. Genomewide-associated studies (GWAS) analysis in T1D has proved disappointing in revealing contributors to disease prediction; the only reliable marker has been human leucocyte antigen (HLA). Specific HLAs include DR3/DR4/DQ2/DQ8, for example. Because HLA molecules present antigen to T cells, it is reasonable that certain HLA molecules have a higher affinity to present self-antigen. Recent studies have shown that additional polymorphisms in HLA that are restricted to autoimmune conditions are further contributory. A caveat is that not all individuals with the appropriate 'pro-autoimmune' HLA develop an autoimmune disease. Another crucial component is autoaggressive T cells. Finding a biomarker to discriminate autoaggressive T cells has been elusive. However, a subset of CD4 helper cells that express the CD40 receptor have been described as becoming pathogenic. An interesting function of CD40 on T cells is to induce the recombination-activating gene (RAG)1/RAG2 T cell receptor recombination machinery. This observation is contrary to immunology paradigms that changes in TCR molecules cannot take place outside the thymic microenvironment. Alteration in TCR, called TCR revision, not only occurs, but may help to account for the development of autoaggressive T cells. Another interesting facet is that type 1 diabetes (T1D) may be more than a single disease; that is, multiple cellular components contribute uniquely, but result ultimately in the same clinical outcome, T1D. This review considers the process of T cell maturation and how that could favor auto-aggressive T cell development in T1D. The potential contribution of TCR revision to autoimmunity is also considered.

Anaplastic large cell lymphoma arises in thymocytes and requires transient TCR expression for thymic egress.

Anaplastic large cell lymphoma (ALCL) is a peripheral T-cell lymphoma presenting mostly in children and young adults. The natural progression of this disease is largely unknown as is the identity of its true cell of origin. Here we present a model of peripheral ALCL pathogenesis where the malignancy is initiated in early thymocytes, before T-cell receptor (TCR) ß-rearrangement, which is bypassed in CD4/NPM-ALK transgenic mice following Notch1 expression. However, we find that a TCR is required for thymic egress and development of peripheral murine tumours, yet this TCR must be downregulated for T-cell lymphomagenesis. In keeping with this, clonal TCR rearrangements in human ALCL are predominantly in-frame, but often aberrant, with clonal TCRα but no comparable clonal TCRß rearrangement, yielding events that would not normally be permissive for survival during thymic development. Children affected by ALCL may thus harbour thymic lymphoma-initiating cells capable of seeding relapse after chemotherapy.

Zebrafish Nk-lysins: First insights about their cellular and functional diversification.

Nk-lysins are antimicrobial proteins produced by cytotoxic T lymphocytes and natural killer cells with a broad antimicrobial spectrum (including bacteria, fungi and parasites). Nevertheless, the implication of these proteins in the protection against viral infections is still poorly understood. In this work, four different Nk-lysin genes (nkla, nklb, nklc and nkld) were identified in the zebrafish genome. That means that zebrafish is the species with the higher repertoire of Nk-lysin genes described so far. The differential expression pattern of the Nk-lysins in several tissues, during ontogeny, among the different kidney cell populations, as well as between Rag1(-/-) and Rag1(+/+) individuals, could suggest a certain specialization of different cell types in the production of different Nk-lysin. Moreover, only two of these genes (nkla and nkld) were significantly up-regulated after viral infection, and this observation could be also a consequence of a functional diversification of the zebrafish Nk-lysins.