Fish larvae are immediately exposed to microbes from hatching to maturation of their lymphoid organs, therefore effective innate mechanisms is very important for survival. However, the knowledge of the development of immune system in fish is limited and in demand now. In vertebrates, recombination-activating gene 1 (RAG-1) and immunoglobulin M (IgM) have been considered as very useful markers of the physiological maturity of the immune system. In this study, the expression of the both genes was assessed throughout the early developmental stages of olive flounder larvae (5-55 dph) and used as markers to follow the development of immune system. RAG-1 and IgM mRNA expression was detectable at 5 dph and remained so until 55 dph. These patterns of expression may suggest that the olive flounder start to develop its function around 5 dph. Tissue distribution was found that both genes mRNAs are only expressed in the immune-related organ such as spleen, kidney and gill. The early detection of IgM mRNA led to the investigation of its presence in oocytes. Both RAG-1 and IgM mRNA transcripts were detected in unfertilized oocytes, suggesting that they are maternally transferred. The biological significance of such a phenomenon remains to be investigated.

The hydrocarbonoclastic bacterium Acinetobacter venetianus RAG-1 has attracted substantial attention due to its powerful oil-degrading capabilities and its potential to play an important ecological role in the cleanup of alkanes. In this study, we compare the transcriptome of the strain RAG-1 grown in dodecane, the corresponding alkanol (dodecanol), and sodium acetate for the characterization of genes involved in dodecane uptake and utilization. Comparison of the transcriptional responses of RAG-1 grown on dodecane led to the identification of 1074 genes that were differentially expressed relative to sodium acetate. Of these, 622 genes were upregulated when grown in dodecane. The highly upregulated genes were involved in alkane catabolism, along with stress response. Our data suggest AlkMb to be primarily involved in dodecane oxidation. Transcriptional response of RAG-1 grown on dodecane relative to dodecanol also led to the identification of permease, outer membrane protein and thin fimbriae coding genes potentially involved in dodecane uptake. This study provides the first model for key genes involved in alkane uptake and metabolism in A. venetianus RAG-1.

Recombination-Activating Genes (RAG) 1 and 2 form the site specific recombinase that mediates V(D)J recombination, a process of DNA editing required for lymphocyte development and responsible for their diverse repertoire of antigen receptors. Mistargeted RAG activity associates with genome alteration and is responsible for various lymphoid tumors. Moreover several non-lymphoid tumors express RAG ectopically. A practical and powerful tool to perform quantitative assessment of RAG activity and to score putative RAG-Recognition signal sequences (RSS) is required in the fields of immunology, oncology, gene therapy, and development. Here we report the detailed characterization of a novel fluorescence-based reporter of RAG activity, named GFPi, a tool that allows measuring recombination efficiency (RE) by simple flow cytometry analysis. GFPi can be produced both as a plasmid for transient transfection experiments in cell lines or as a retrovirus for stable integration in the genome, thus supporting ex vivo and in vivo studies. The GFPi assay faithfully quantified endogenous and ectopic RAG activity as tested in genetically modified fibroblasts, tumor derived cell lines, developing pre-B cells, and hematopoietic cells. The GFPi assay also successfully ranked the RE of various RSS pairs, including bona fide RSS associated with V(D)J segments, artificial consensus sequences modified or not at specific nucleotides known to affect their efficiencies, or cryptic RSS involved in RAG-dependent activation of oncogenes. Our work validates the GFPi reporter as a practical quantitative tool for the study of RAG activity and RSS efficiencies. It should turn useful for the study of RAG-mediated V(D)J and aberrant rearrangements, lineage commitment, and vertebrate evolution.

An imbalanced immune system has long been known to influence a variety of mood disorders including anxiety, obsessive-compulsive disorders and depression. In this study, we sought to model the impact of an immunocompromised state on these emotional behaviors using RAG-1⁻/⁻ mice, which lack T and B cells. We also investigated the relative contribution of CD4⁺ or CD8⁺ T cells to these manifestations using RAG-1⁻/⁻/OT-II and RAG-1⁻/⁻/OT-I transgenic mice, respectively. Our results show that RAG-1⁻/⁻ mice present a significant increase in digging and marble-burying activities compared with wild-type mice. Surprisingly, these anxiety-like behaviors were significantly reverted in RAG-1⁻/⁻/OT-II but not RAG-1⁻/⁻/OT-I transgenic mice. Immunodepletion experiments with anti-CD4 or anti-CD8 in C57/BL6 mice or repopulation studies in RAG-1⁻/⁻ mice did not reproduce these findings. Microarray analysis of the brain of RAG-1⁻/⁻ and RAG-1⁻/⁻/OT-II mice revealed a significantly different gene fingerprint, with the latter being more similar to wild-type mice than the former. Further analysis revealed nine main signaling pathways as being significantly modulated in RAG-1⁻/⁻ compared with wild-type mice. Taken together, these results suggest that life-long rather than transient immunodeficient conditions influence the emotional behaviors in mice. Most interestingly, these effects seem to correlate with a specific absence of CD4⁺ rather than CD8⁺ T cells. Validation of these findings in man might provide new clues on the mechanism by which early life immune modulation might impact mood response in adults and provide a further link between immune and emotional well-being.

The origin of the recombination-activating genes (RAGs) is considered to be a foundation hallmark for adaptive immunity, characterised by the presence of antigen receptor genes that provide the ability to recognise and respond to specific peptide antigens. In vertebrates, a diverse repertoire of antigen-specific receptors, T cell receptors and immunoglobulins is generated by V(D)J recombination performed by the RAG-1 and RAG-2 protein complex. RAG homologues were identified in many jawed vertebrates. Despite their crucial importance, no homologues have been found in jawless vertebrates and invertebrates. This paper focuses on the RAG homologues in humans and other vertebrates for which the genome is completely sequenced, and also discusses the main contribution of the use of RAG homologues in phylogenetics and vertebrate evolution. Since mutations in both genes cause a spectrum of severe combined immunodeficiencies, including the Omenn syndrome (OS), these topics are discussed in detail. Finally, the relevance to genomic diversity and implications to immunomics are addressed. The search for homologues could enlighten us about the evolutionary processes that shaped the adaptive immune system. Understanding the diversity of the adaptive immune system is crucially important for the design and development of new therapies to modulate the immune responses in humans and/or animal models.

V(D)J (V, variable; D, diversity; J, joining) combination of immunoglobulin (Ig) genes established in premature B cells has been thought to be conserved throughout differentiation at mature stages. However, germinal center (GC) B cells have been shown to reexpress recombination-activating gene (RAG)-1 and RAG-2 proteins in immunized mice. Here, we present several lines of evidence indicating that RAG proteins thus induced are functional as the V(D)J recombinase. DNA excision product reflecting Vlambda1 to Jlambda1 rearrangement was generated in parallel with the expression of RAG genes in mature mouse B cells that were activated in vitro with LPS and IL-4. Similar lambda chain gene rearrangement was observed in the draining lymph node of immunized mice. Further, B cells that underwent lambda gene rearrangement were shown by in situ PCR to be localized within GCs. Thus, secondary rearrangement of Ig genes (receptor editing) can occur in mature B cells.

Somatic hypermutation and isotype switch recombination occur in germinal center B cells, are linked to transcription, and are similarly affected by deficiency in MutS homologue (MSH)2. Class-switch recombination is abrogated by disruption of genes encoding components of the catalytic subunit of DNA-dependent protein kinase (DNA-PK(cs))/Ku complex and likely involves nonhomologous end joining (NHEJ). That somatic hypermutation might also be associated with end joining is suggested by its association with the creation of deletions, duplications, and sites accessible to terminal transferase. However, a requirement for NHEJ in the mutation process has not been demonstrated. Here we show that somatic mutation in mice deficient in NHEJ can be tested by introduction of rearranged immunoglobulin and T cell receptor transgenes: the transgene combination not only permits reconstitution of peripheral lymphoid compartments but also allows formation of germinal centers, despite the wholly monoclonal nature of the lymphocyte antigen receptors in these animals. Using this strategy, we confirm that somatic hypermutation like class-switching can occur in the absence of recombination-activating gene (RAG)1 but show that the two processes differ in that hypermutation can proceed essentially unaffected by deficiency in DNA-PK(cs) activity.

Signal transduction pathways guided by cellular receptors commonly exhibit low-level constitutive signaling in a continuous, ligand-independent manner. The dynamic equilibrium of positive and negative regulators establishes such a tonic signal. Ligand-independent signaling by the precursors of mature antigen receptors regulates development of B and T lymphocytes. Here we describe a basal signal that controls gene expression profiles in the Jurkat T cell line and mouse thymocytes. Using DNA microarrays and Northern blots to analyze unstimulated cells, we demonstrate that expression of a cluster of genes, including RAG-1 and RAG-2, is repressed by constitutive signals requiring the adapter molecules LAT and SLP-76. This TCR-like pathway results in constitutive low-level activity of Erk and Abl kinases. Inhibition of Abl by the drug STI-571 or inhibition of signaling events upstream of Erk increases RAG-1 expression. Our data suggest that physiologic gene expression programs depend upon tonic activity of signaling pathways independent of receptor ligation.

The recombination-activating genes (RAG-1 and RAG-2) encode a V(D)J recombinase responsible for rearrangements of antigen-receptor genes during T and B cell development, and RAG expression is known to correlate strictly with the process of rearrangement. In contrast to RAG-1, the expression of RAG-2 was not previously detected during any other stage of lymphopoiesis or in any other normal tissue. Here we report that the CpG island-associated promoter of the NWC gene (the third evolutionarily conserved gene in the RAG locus), which is located in the second intron of RAG-2, has bidirectional activity and is responsible for the detectable transcription of RAG-2 in some non-lymphoid tissues. We also identify evolutionarily conserved promoter fragments responsible for this bidirectional activity, and show that it is activated by transcription factor ZFP143. The possible implications of our findings are briefly discussed.

The recombination-activating genes (RAG) 1 and 2 are indispensable for diversifying the primary B cell receptor repertoire and pruning self-reactive clones via receptor editing in the bone marrow; however, the impact of RAG1/RAG2 on peripheral tolerance is unknown. Partial RAG deficiency (pRD) manifesting with late-onset immune dysregulation represents an 'experiment of nature' to explore this conundrum. By studying B cell development and subset-specific repertoires in pRD, we demonstrate that reduced RAG activity impinges on peripheral tolerance through the generation of a restricted primary B cell repertoire, persistent antigenic stimulation and an inflammatory milieu with elevated B cell-activating factor. This unique environment gradually provokes profound B cell dysregulation with widespread activation, remarkable extrafollicular maturation and persistence, expansion and somatic diversification of self-reactive clones. Through the model of pRD, we reveal a RAG-dependent 'domino effect' that impacts stringency of tolerance and B cell fate in the periphery.

In order to better understand the development and function of γδ T cells in Xenopus frogs, it is necessary to determine where and when γδ T cells are found in Xenopus tissues. This study examined the expression of TCR genes, focused primarily on TCR γ, in tissues of adult and larval Xenopus laevis and provide new data about the expression pattern of these different TCR genes in this anuran amphibian. TCR gene expression was detected by RT-PCR in adult frog tissues including the thymus, spleen, skin, intestine, lung, and liver, but not the testes. TCR γ and β genes were detected in the larval (tadpole) tail and intestine. The absence of RAG-1 expression in these larval tissues is consistent with differentiation of the T cells in the thymus. Together, these data provide evidence that migration of these cells from the thymus likely occurs relatively early in larval development. These studies provide a necessary foundation for future studies of the functions of γδ T cells in amphibians, which are placed at an intermediate position flanked by fishes on one end and mammals and chickens on the other.

The many binding studies of monoclonal immunoglobulin (Ig) produced by plasmacytomas have found no universally common binding properties, but instead, groups of plasmacytomas with specific antigen-binding activities to haptens such as phosphorylcholine, dextrans, fructofuranans, or dinitrophenyl. Subsequently, it was found that plasmacytomas with similar binding chain specificities not only expressed the same idiotype, but rearranged the same light (V(L)) and heavy (V(H)) variable region genes to express a characteristic monoclonal antibody. In this study, we have examined by enzyme-linked immunosorbent assay five antibodies secreted by silicone-induced mouse plasmacytomas using a broader panel of antigens including actin, myosin, tubulin, single-stranded DNA, and double-stranded DNA. We have determined the Ig heavy and light chain V gene usage in these same plasmacytomas at the DNA and RNA level. Our studies reveal: (a) antibodies secreted by plasmacytomas bind to different antigens in a manner similar to that observed for natural autoantibodies; (b) the expressed Ig heavy genes are restricted in V gene usage to the V(H)-J558 family; and (c) secondary rearrangements occur at the light chain level with at least three plasmacytomas expressing both kappa and lambda light chain genes. These results suggest that plasmacytomas use a restricted population of B cells that may still be undergoing rearrangement, thereby bypassing the allelic exclusion normally associated with expression of antibody genes.

Mouse germinal center (GC) B cells have been shown to undergo secondary V(D)J (V, variable; D, diversity; J, joining) recombination (receptor editing) mediated by the reexpressed products of recombination activating gene (RAG)-1 and RAG-2. We show here that interleukin (IL)-7 as well as IL-4 was effective in inducing functional RAG products in mouse IgD+ B cells activated via CD40 in vitro. Blocking of the IL-7 receptor (IL-7R) by injecting an anti- IL-7R monoclonal antibody resulted in a marked suppression of the reexpression of RAG-2 and subsequent V(D)J recombination in the draining lymph node of immunized mice, whereas RAG-2 expression was not impaired in immunized IL-4-deficient mice. Further, these peripheral B cells activated in vitro or in vivo were found to express IL-7R. These findings indicate a novel role for IL-7 and IL-7R in inducing receptor editing in GC B cells.

Mice lacking the interleukin 7 receptor (IL-7R) generate alpha/beta T cells at a detectable but greatly reduced rate, but gamma/delta T cells are completely absent. The special role of IL-7R signaling in gamma/delta T cell development has remained unclear. IL-7Ralpha(-/-) mice exhibit a paucity of gamma gene rearrangements. This striking observation can be explained by a defect in T cell receptor (TCR)-gamma gene rearrangement, a defect in TCR-gamma gene transcription leading to death of gamma/delta lineage cells, and/or a requirement for IL-7R in commitment of cells to the gamma/delta lineage. To determine the role of IL-7R signaling in gamma/delta T cell development, we examined transcription of a prerearranged TCR-gamma transgene in IL-7Ralpha(-/-) mice, as well as the effects of IL-7 on transcription of endogenous, rearranged TCR-gamma genes in alpha/beta lineage cells. The results demonstrate that IL-7R-mediated signals are necessary for the normal expression of rearranged TCR-gamma genes. Equally significant, the results show that the poor expression of TCR-gamma genes in IL-7Ralpha(-/-) mice is responsible for the selective deficit in gamma/delta cells in these mice, since a high copy TCR-gamma transgene exhibited sufficient residual expression in IL-7Ralpha(-/-) mice to drive gamma/delta cell development. The results indicate that the absence of gamma/delta T cells in IL-7Ralpha(-/-) mice is due to insufficient TCR-gamma gene expression.

The transcription factors encoded by the E2A and early B cell factor (EBF) genes are required for the proper development of B lymphocytes. However, the absence of B lineage cells in E2A- and EBF-deficient mice has made it difficult to determine the function or relationship between these proteins. We report the identification of a novel model system in which the role of E2A and EBF in the regulation of multiple B lineage traits can be studied. We found that the conversion of 70Z/3 pre-B lymphocytes to cells with a macrophage-like phenotype is associated with the loss of E2A and EBF. Moreover, we show that ectopic expression of the E2A protein E12 in this macrophage line results in the induction of many B lineage genes, including EBF, IL7Ralpha, lambda5, and Rag-1, and the ability to induce kappa light chain in response to mitogen. Activation of EBF may be one of the critical functions of E12 in regulating the B lineage phenotype since expression of EBF alone leads to the activation of a subset of E12-inducible traits. Our data demonstrate that, in the context of this macrophage line, E12 induces expression of EBF and together these transcription factors coordinately regulate numerous B lineage-associated genes.

Appropriate PI3K signals generated by the antigen receptor are essential to promote B cell development. Regulation of recombination activating gene (RAG)-1 and RAG-2 expression is one key process that is mediated by PI3K to ensure developmental progression and selection. When PI3K signals are too high or too low, expression of RAGs does not turn off and B cell development is impaired or blocked. Yet, the mechanism which tunes PI3K activity to control RAG expression during B cell development in the bone marrow is unknown. Recently we showed that a c-Myc/miR17-92/PTEN axis regulates PI3K activity for positive and negative selection of immature B cells. Here, we show that the c-Myc/miR17-92/PTEN axis tunes PI3K activity to control the expression of RAGs in proB cells. Using different genetically engineered mouse models we show that impaired function of the c-Myc/miR17-92/PTEN axis alters the PI3K/Akt/Foxo1 pathway to result in dis-regulated expression of RAG and a block in B cell development. Studies using 38c-13 B lymphoma cells, where RAGs are constitutively expressed, suggest that this regulatory effect is mediated post-translationally through Foxo1.

The Recombination Activating Genes (RAG) 1/2 are important for the development and function of T and B cells. Loss of RAG1/2 function results in severe combined immunodeficiency (SCID), which could lead to early death. We studied the prevalence of RAG1/2 mutations in ten SCID patients in Egypt. We identified two novel homozygous nonsense mutations in RAG1, a novel homozygous deletion, and a previously reported homozygous missense mutation from four patients, as well as two homozygous mutations in RAG2 from the same patient. Prenatal diagnosis performed in the mother of a patient with RAG1 deficiency determined that the fetus was heterozygous for the same mutation. This represents the first report on RAG1/2 mutations in SCID patients in Egypt. The early diagnosis dramatically affects the outcome of the disease by allowing bone marrow transplantation at an early age, and providing prenatal diagnosis and genetic counseling for families with a history of SCID.

Severe respiratory syncytial virus (RSV) infection is a major cause of morbidity and mortality in infants <2 years-old. Here we describe that high-fiber diet protects mice from RSV infection. This effect was dependent on intestinal microbiota and production of acetate. Oral administration of acetate mediated interferon-β (IFN-β) response by increasing expression of interferon-stimulated genes in the lung. These effects were associated with reduction of viral load and pulmonary inflammation in RSV-infected mice. Type 1 IFN signaling via the IFN-1 receptor (IFNAR) was essential for acetate antiviral activity in pulmonary epithelial cell lines and for the acetate protective effect in RSV-infected mice. Activation of Gpr43 in pulmonary epithelial cells reduced virus-induced cytotoxicity and promoted antiviral effects through IFN-β response. The effect of acetate on RSV infection was abolished in Gpr43-/- mice. Our findings reveal antiviral effects of acetate involving IFN-β in lung epithelial cells and engagement of GPR43 and IFNAR.

The transcriptional repressor Blimp-1 promotes the differentiation of CD8(+) T cells into short-lived effector cells (SLECs) that express the lectin-like receptor KLRG-1, but how it operates remains poorly defined. Here we show that Blimp-1 bound to and repressed the promoter of the gene encoding the DNA-binding inhibitor Id3 in SLECs. Repression of Id3 by Blimp-1 was dispensable for SLEC development but limited the ability of SLECs to persist as memory cells. Enforced expression of Id3 was sufficient to restore SLEC survival and enhanced recall responses. Id3 function was mediated in part through inhibition of the transcriptional activity of E2A and induction of genes regulating genome stability. Our findings identify the Blimp-1-Id3-E2A axis as a key molecular switch that determines whether effector CD8(+) T cells are programmed to die or enter the memory pool.

Evolutionarily conserved, "natural" (n)IgM is broadly reactive to both self and foreign antigens. Its selective deficiency leads to increases in autoimmune diseases and infections. In mice, nIgM is secreted independent of microbial exposure to bone marrow (BM) and spleen B-1 cell-derived plasma cells (B-1PC), generating the majority of nIgM, or by B-1 cells that remain non-terminally differentiated (B-1sec). Thus, it has been assumed that the nIgM repertoire is broadly reflective of the repertoire of body cavity B-1 cells. Studies here reveal, however, that B-1PC generate a distinct, oligoclonal nIgM repertoire, characterized by short CDR3 variable immunoglobulin heavy chain regions, 7-8 amino acids in length, some public, many arising from convergent rearrangements, while specificities previously associated with nIgM were generated by a population of IgM-secreting B-1 (B-1sec). BM, but not spleen B-1PC, or B-1sec also required the presence of TCRαβ CD4 T cells for their development from fetal precursors. Together, the studies identify important previously unknown characteristics of the nIgM pool.