RAG Expression Research Articles

CCR7 Controls Thymus Recirculation, but Not Production and Emigration, of Foxp3(+) T Cells.

SummaryCurrent models of Foxp3+ regulatory T cell (Treg) development involve CCR7-mediated migration of thymocytes into the thymus medulla to enable essential interactions with medullary epithelium. However, increased Foxp3+ thymic Treg numbers in Ccr7−/− mice challenge this view, and the role of CCR7 in Treg development, emigration, and/or recirculation is unknown. Here, we have examined CCR7 and Rag2pGFP levels during Treg development and generated Rag2pGFPCcr7−/− mice to study its impact on the intrathymic Treg pool. We reveal surprising developmental heterogeneity in thymocytes described as Treg precursors, showing that they contain recirculating CCR6+CCR7−Rag2pGFP− T cells. Although CCR7 defines bona fide Rag2GFP+ Treg precursors, it is not required for Treg production and emigration. Rather, we show that lack of CCR7 renders the thymus more receptive to Treg thymus homing. Our study reveals a role for CCR7 in limiting Treg recirculation back to the thymus and enables separation of the mechanisms controlling Treg production and thymic recirculation.

Targeting Anti-Insulin B Cell Receptors Improves Receptor Editing in Type 1 Diabetes-Prone Mice.

Autoreactive B lymphocytes that commonly arise in the developing repertoire can be salvaged by receptor editing, a central tolerance mechanism that alters BCR specificity through continued L chain rearrangement. It is unknown whether autoantigens with weak cross-linking potential, such as insulin, elicit receptor editing, or whether this process is dysregulated in related autoimmunity. To resolve these issues, we developed an editing-competent model in which anti-insulin Vκ125 was targeted to the Igκ locus and paired with anti-insulin VH125Tg. Physiologic, circulating insulin increased RAG-2 expression and was associated with BCR replacement that eliminated autoantigen recognition in a proportion of developing anti-insulin B lymphocytes. The proportion of anti-insulin B cells that underwent receptor editing was reduced in the type 1 diabetes-prone NOD strain relative to a nonautoimmune strain. Resistance to editing was associated with increased surface IgM expression on immature (but not transitional or mature) anti-insulin B cells in the NOD strain. The actions of mAb123 on central tolerance were also investigated, because selective targeting of insulin-occupied BCR by mAb123 eliminates anti-insulin B lymphocytes and prevents type 1 diabetes. Autoantigen targeting by mAb123 increased RAG-2 expression and dramatically enhanced BCR replacement in newly developed B lymphocytes. Administering F(ab')2123 induced IgM downregulation and reduced the frequency of anti-insulin B lymphocytes within the polyclonal repertoire of VH125Tg/NOD mice, suggesting enhanced central tolerance by direct BCR interaction. These findings indicate that weak or faulty checkpoints for central tolerance can be overcome by autoantigen-specific immunomodulatory therapy.

NF-κB and AKT signaling prevent DNA damage in transformed pre-B cells by suppressing RAG1/2 expression and activity

AbstractIn developing lymphocytes, expression and activity of the recombination activation gene protein 1 (RAG1) and RAG2 endonuclease complex is tightly regulated to ensure ordered recombination of the immunoglobulin genes and to avoid genomic instability. Aberrant RAG activity has been implicated in the generation of secondary genetic events in human B-cell acute lymphoblastic leukemias (B-ALLs), illustrating the oncogenic potential of the RAG complex. Several layers of regulation prevent collateral genomic DNA damage by restricting RAG activity to the G1 phase of the cell cycle. In this study, we show a novel pathway that suppresses RAG expression in cycling-transformed mouse pre-B cells and human pre-B B-ALL cells that involves the negative regulation of FOXO1 by nuclear factor κB (NF-κB). Inhibition of NF-κB in cycling pre-B cells resulted in upregulation of RAG expression and recombination activity, which provoked RAG-dependent DNA damage. In agreement, we observe a negative correlation between NF-κB activity and the expression of RAG1, RAG2, and TdT in B-ALL patients. Our data suggest that targeting NF-κB in B-ALL increases the risk of RAG-dependent genomic instability.

MicroRNA-Related DNA Repair/Cell-Cycle Genes Independently Associated With Relapse After Radiation Therapy for Early Breast Cancer.

Local recurrence and distant failure after adjuvant radiation therapy for breast cancer remain significant clinical problems, incompletely predicted by conventional clinicopathologic markers. We had previously identified microRNA-139-5p and microRNA-1274a as key regulators of breast cancer radiation response in vitro. The purpose of this study was to investigate standard clinicopathologic markers of local recurrence in a contemporary series and to establish whether putative target genes of microRNAs involved in DNA repair and cell cycle control could better predict radiation therapy response in vivo. With institutional ethics board approval, local recurrence was measured in a contemporary, prospectively collected series of 458 patients treated with radiation therapy after breast-conserving surgery. Additionally, independent publicly available mRNA/microRNA microarray expression datasets totaling >1000 early-stage breast cancer patients, treated with adjuvant radiation therapy, with >10 years of follow-up, were analyzed. The expression of putative microRNA target biomarkers--TOP2A, POLQ, RAD54L, SKP2, PLK2, and RAG1--were correlated with standard clinicopathologic variables using 2-sided nonparametric tests, and to local/distant relapse and survival using Kaplan-Meier and Cox regression analysis. We found a low rate of isolated local recurrence (1.95%) in our modern series, and that few clinicopathologic variables (such as lymphovascular invasion) were significantly predictive. In multiple independent datasets (n>1000), however, high expression of RAD54L, TOP2A, POLQ, and SKP2 significantly correlated with local recurrence, survival, or both in univariate and multivariate analyses (P<.001). Low RAG1 expression significantly correlated with local recurrence (multivariate, P=.008). Additionally, RAD54L, SKP2, and PLK2 may be predictive, being prognostic in radiation therapy-treated patients but not in untreated matched control individuals (n=107; P<.05). Biomarkers of DNA repair and cell cycle control can identify patients at high risk of treatment failure in those receiving radiation therapy for early breast cancer in independent cohorts. These should be further investigated prospectively, especially TOP2A and SKP2, for which targeted therapies are available.

278. Successful Correction of Rag1 Severe Combined Immunedefciency at Clinically Relevant Vector Copy Numbers

RAG1 -SCID patients lack B- and T lymphocytes due to the inability to rearrange immunoglobulin (Ig) and T-cell receptor (TCR) genes. Gene therapy is a valid treatment alternative for RAG1-SCID patients lacking an appropriate bone marrow donor. However, developing such therapy for RAG1 has proven difficult. As proof-of-principle, we have previously shown with an SFFV promoter element that sufficient RAG1 expression could be achieved for correction at clinically acceptable vector copy numbers (VCN).In our current study, we tested clinically relevant lentiviral SIN vectors with different internal promoter elements (UCOE, PGK, MND, and UCOE-MND in tandem) to deliver human RAG1 sequences. In vitro assessment of promoter strength was done on transduced mouse lineage-depleted cells. The highest RAG1 expression per VCN was reached with the MND and the UCOE-MND containing vectors, followed by UCOE. The PGK displayed the lowest promoter strength, 3 to 10 times weaker than the other elements.Next, we used Rag1-/- mice as a preclinical model for RAG-SCID to assess the capacity of the various vectors to correct the SCID phenotype at very low to moderate VCN (0.01-0.2), where the VCN positively correlating with the RAG1 expression levels. All gene therapy-treated mice reconstituted their peripheral T cell compartment, however in animals that received a transplant with low RAG1 expression (n=9) this effect was limited to peripheral blood. When assessing phenotypic data from thymus 17 weeks after transplantation, only mice that received a transplant with high RAG1 expression, showed a thymocyte subset composition comparable to mice receiving wild type cells. This included a high percentage and number of double positive (DP) cells, assuring an ongoing supply of peripheral T cells. Mice receiving a transplant with low RAG1 expression levels, were mostly lacking the DP population.Similarly, when looking at developing B cells in the bone marrow, animals that were transplanted with cells with low RAG1 expression did not have a substantial number of surface IgM+ cells. This in contrast to animals that received cells with high RAG1 expression levels in which developing B cell subsets were readily detectable. These findings were paralleled in the peripheral blood, where the fraction of B220+ cells positively correlated with RAG1 expression in the transplant material.In the low RAG1 expression groups, we found that a number of mice developed skin rashes, while animals in the high RAG1 expression group as well as animals that received wild type cells or uncorrected Rag1-/- cells did not display any health problems. We are currently determining the TCR repertoire, since a limited repertoire could point to autoreactive T cells causing the rashes.To conclude, the level of B- and T cell development in primary lymphoid organs is directly correlated to the RAG1 expression levels in the transplant and we were able to obtain sufficient RAG1 expression for full T and B cell reconstitution even at a moderate, but clinically relevant, VCN.

Pre-B cells suppress RAG expression in response to DNA double-strand breaks (HEM1P.225)

Abstract The ability of the Rag1/Rag2 (RAG) endonuclease to assemble antigen receptor (AgR) genes is essential for adaptive immunity. However, aberrant induction or repair of RAG-induced DNA double strand breaks (DSBs) can lead to oncogenic AgR translocations. We have previously shown that RAG-induced DSBs in pre-B cells activate the ATM kinase to prevent RAG cleavage of the homologous allele, and to inhibit expression of Rag1 and Rag2. These breaks also suppress expression of Gadd45α, which promotes Rag1 and Rag2 transcription. Since DSBs induced by ionizing radiation (IR) signal through ATM to increase Gadd45α expression in all other cell types including mature B cells, these results indicate that RAG DSBs and/or pre-B cells use unique mechanisms to regulate Gadd45α expression. We now demonstrate that IR-induced DSBs in pre-B cells signal through the ATM kinase to down-regulate Gadd45α expression. We also demonstrate that these IR DSBs signal ATM-dependent suppression of Rag1 and Rag2 transcription, without affecting stability of Rag1 or Rag2 mRNA. Because the presence of multiple DSBs in a cell greatly increases the risk of translocation, we hypothesize that pre-B cells have developed a unique developmental stage-specific DSB response to suppress the induction of RAG DSBs in the presence of other DSBs. Our ongoing studies will determine the role of this pre-B cell specific DSB response in suppressing AgR translocations and enforcing mono-allelic assembly of immunoglobulin genes.

Enhancement of rag1 and tdt expression in thymus after immunization.

Enhancement of rag1 and tdt expression in thymus after immunization.

The trenbolone acetate affects the immune system in rainbow trout, Oncorhynchus mykiss

In aquatic systems, the presence of endocrine-disrupting chemicals (EDC) can disrupt the reproductive function but also the immune system of wildlife. Some studies have investigated the effects of androgens on the fish immune parameters but the mechanisms by which the xenoandrogens alter the immunity are not well characterized. In order to test the effects of trenbolone acetate (TbA) on fish immune system, we exposed rainbow trout male juveniles during three weeks to TbA levels at 0.1 and 1μg/L. The present results suggest that TbA impacts, in a tissue-dependent manner, the rainbow trout immunity by affecting primarily the humoral immunity. Indeed, TbA inhibited lysozyme activity in plasma and liver and enhanced the alternative complement pathway activity (ACH50) in kidney. In plasma, the modulation of the complement system was time-dependent. The mRNA expression of genes encoding some cytokines such as renal TGF-β1, TNF-α in skin and hepatic IL-1β was also altered in fish exposed to TbA. Regarding the cellular immunity, no effect was observed on the leucocyte population. However, the expression of genes involved in the development and maturation of lymphoid cells (RAG-1 and RAG-2) was decreased in TbA-treated fish. Among those effects, we suggest that the modulation of RAG-1 and mucus apolipoprotein-A1 gene expression as well as plasma and hepatic lysozyme activities are mediated through the action of the androgen receptor. All combined, we conclude that trenbolone affects the rainbow trout immunity.

An anti-silencer- and SATB1-dependent chromatin hub regulates Rag1 and Rag2 gene expression during thymocyte development.

Rag1 and Rag2 gene expression in CD4(+)CD8(+) double-positive (DP) thymocytes depends on the activity of a distant anti-silencer element (ASE) that counteracts the activity of an intergenic silencer. However, the mechanistic basis for ASE activity is unknown. Here, we show that the ASE physically interacts with the distant Rag1 and Rag2 gene promoters in DP thymocytes, bringing the two promoters together to form an active chromatin hub. Moreover, we show that the ASE functions as a classical enhancer that can potently activate these promoters in the absence of the silencer or other locus elements. In thymocytes lacking the chromatin organizer SATB1, we identified a partial defect in Tcra gene rearrangement that was associated with reduced expression of Rag1 and Rag2 at the DP stage. SATB1 binds to the ASE and Rag promoters, facilitating inclusion of Rag2 in the chromatin hub and the loading of RNA polymerase II to both the Rag1 and Rag2 promoters. Our results provide a novel framework for understanding ASE function and demonstrate a novel role for SATB1 as a regulator of Rag locus organization and gene expression in DP thymocytes.

Transcriptomic analysis and biomarkers (Rag1 and Igμ) for probing the immune system development in Pacific cod, Gadus macrocephalus

Mortality (>90%) is a big concern in larval rearing facilities of Pacific cod, Gadus macrocephalus, limiting its culture presently still in the experimental stages. Understanding the immune system development of G. macrocephalus is crucial to optimize the aquaculture of this species, to improve the use of economic resources and to avoid abuse of antibiotics. For the transcriptome analysis, using an Illumina sequencing platform, 61,775,698 raw reads were acquired. After a de novo assembly, 77,561 unigenes were obtained. We have classified functionally these transcripts by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG). 27 genes mainly related to hematopoietic or lymphoid organ development and somatic diversification of immune receptors have been reported for the first time in Pacific cod, and 14 Ig heavy chain (μ chain) locuses were assembled using Trinity. Based on our previous achievement, we have chosen Rag1 and Igμ as immune system development biomarkers. Full length cDNA of Rag1 and Igμ as biomarkers were obtained respectively using RACE PCR. Concerning Rag1, the deduced amino acid of Rag1 and protein immunodetection revealed a Rag1 isoform of 69 kDa, significantly different from other fish orthologs, such as Oncorhynchus mykiss (121 kDa). Phylogenetic analysis reveals a unique immune system for the Gadus genre, not exclusive for Atlantic cod, among vertebrates. Meanwhile, full length cDNA of Igμ included an ORF of 1710 bp and the deduced amino acid was composed of a leader peptide, a variable domain, CH1, CH2, Hinge, CH3, CH4 and C-terminus, which was in accordance with most teleost. Absolute quantification PCR revealed that significant expression of Rag1 appeared earlier than Igμ, 61 and 95 dph compared to 95 dph, respectively. Here we report the first transcriptomic analysis of G. macrocephalus as the starting point for genetic research on immune system development towards improving the Pacific cod aquaculture.

Physiological significance of Rag1 in retinal ganglion cell death.

Although the transcription factor, nuclear factor-κB (NF-κB) is known to regulate cell death and survival, its precise role in cell death within the central nervous system (CNS) remains unknown. We previously reported that mice with a homozygous deficiency for NF-κBp50 spontaneously developed optic neuropathy. We examined the expression and activation of pro-apoptotic factor(s) that mediate optic neuropathy in p50-/- mice. Recombination activating gene 1 (Rag1) is known to regulate the recombination of immunoglobulin V(D)J. Experiments with genetically engineered mice revealed the involvement of Rag1 expression in the apoptosis of Brn3a-positive retinal ganglion cells (RGCs), and also showed the specific effects of a p50-deficiency on the activation of Rag1 gene transcription. Furthermore, a genetic analysis of murine neuronal stem-like cells clarified the biological significance of Rag1 in N-methyl-D-aspartate (NMDA)-induced neuronal apoptosis. The apoptotic regulating factors, Bax, and cleaved caspase 3, 8, and 9 were detected in HEK293 cells expressing the external molecule of Rag1, and a human histological examination revealed the expression of Rag1 in RGCs. A recent study indicated that Rag1 played a role in optic neuropathy as a pro-apoptotic candidate in p50-/- mice. This result may lead to new therapeutic targets in optic neuropathy.

Role of Microbes in B-Cell Lymphopoiesis and Early Ig Repertoire Development

Progenitor (pro-) and precursor (pre-) B cells express the recombination-activating gene (RAG1/RAG2) endonuclease, which initiates the V(D)J recombination reaction that assembles Immunoglobulin heavy (IgH) and light (IgL) chain variable region exons from germline gene segments. Expression of productively assembled IgH μ chains and IgL (Igκ or Igλ) chains generates IgM molecules that form the B-cell antigen binding receptor (BCR) with a diverse primary repertoire of binding specificities. As the newly assembled and expressed IgM interacts with local antigens, RAG expression can continue, allowing continued Igκ V(D)J recombination that can replace the previously assembled VκJκ exon with one that generates a new specificity. This "receptor editing" process provides an antigen-mediated regulatory checkpoint that helps shape the pre-immune BCR repertoire. As the principal location of early B-cell development, the bone marrow antigenic environment is the main force affecting receptor editing. However, the extent to which self-antigens outside of the bone marrow — as well as environmental antigens — may shape the primary Ig repertoire is not known. Here we show that early B-cell development takes place in peripheral sites, including the small intestinal mucosa, in young mice. Cells in these sites co-express RAG with cytoplasmic Igκ and Igμ, indicating active receptor editing; and, correspondingly, they have significant differences in Vκ usage compared to RAG2-expressing bone marrow B-lineage cells and other RAG2-expressing B-lineage cells from other peripheral sites. In addition, the luminal microbial environment appears to also affect Igλ/Igκ ratios. We conclude that early B-cell development can occur in the periphery, including the intestinal mucosa, where commensal microbes may influence BCR diversification and gut pre-immune Ig repertoires. DisclosuresNo relevant conflicts of interest to declare.

Mechanisms of Clonal Evolution of Pre-Leukemic Clones in Childhood Pre-B Acute Lymphoblastic Leukemia

Background and hypothesis: Childhood pre-B acute lymphoblastic leukemia (ALL) can frequently be retraced to a pre-leukemic clone carrying a prenatally acquired genetic lesion (e.g. ETV6-RUNX1gene rearrangement). After birth, pre-leukemic clones can acquire secondary mutations and, hence, evolve towards overt leukemia. While this concept is well established, the mechanism(s) driving clonal evolution are not known. Epidemiological findings hint to a role of delayed childhood infections and chronic inflammation as etiologic factors of childhood ALL, but do not illuminate mechanism of clonal evolution of pre-leukemic cells. In this study, we demonstrate that cooperation between the AID cytosine deaminase and the RAG1/RAG2 V(D)J recombinase promotes acquisition of secondary genetic lesions that promote progress of pre-leukemic B cell precursors towards full-blown leukemia.Results: The enzymatic activity of RAG1/RAG2 (VDJ recombination) and AID (somatic hypermutation, class-switch recombination) are strictly segregated to early and late stages of B cell development, respectively. While RAG1 and RAG2 are actively expressed at stages of early B cell development (bone marrow and fetal liver) that give rise to pre-B ALL, little is known about the function of AID in early B-lymphopoesis. As the involvement of AID in pre-B leukemic clonal evolution is incumbent on its expression during early stages of B-lymphopoesis, we tested CD19+ pre-B cells isolated from human bone marrow (BM) for indicators of AID activity, namely, somatic hypermutation (SHM) and class switch recombination (CSR). Interestingly, most pre-B cell clones carry rearranged Ig VH region genes that are mutated at low levels (average mutation frequency 26 x 10-3 bp). Likewise, pre-B cells isolated from fetal liver tissues (three donors; 10-19 weeks of gestation) carried Ig VH region genes mutated at low levels (average mutation frequency 14 x 10-3 bp). In addition, about one third of fetal liver pre-B cells had undergone CSR to Cγ3, Cγ1 and Cα regions. These findings highlight the previously unknown function of AID in two important sites of early human B-lymphopoesis. Based on these results, we hypothesized that a specific B cell subset during early pro- and pre-B cell differentiation can concomitantly express both AID and the RAGs and, hence, would be particularly susceptible to clonal evolution of cells that carry a pre-leukemic lesion.Our subsequent studies identified late pre-B cells (Fraction D) as a natural subset of increased genetic vulnerability. Late pre-B cells downregulate IL7 receptor/Stat5 signaling, which enables expression of RAG1 and RAG2 and immunoglobulin light chain gene rearrangement. Loss of IL7 receptor/Stat5 signaling also removes an important safeguard against premature expression of AID. Therefore, late pre-B cells are poised to express AID at high levels in response to inflammatory stimuli (e.g. LPS) in concurrence with RAG1 and RAG2. Studying clonal evolution of patient-derived pre-B ALL cells, we found evidence for concomitant AID and RAG1/RAG2 activity. Further studying a genetic mouse model for pre-leukemic pre-B cells carrying ETV6-RUNX1, we found that repeated exposure to LPS can cause overt leukemia but not in the absence of either AID or Rag1. Additionally, whole exome sequencing of human B cell clones that were engineered to express AID, RAG1/RAG2 alone or in combination revealed that concurrent expression of AID with RAG1/RAG2 dramatically increased the frequency of structural chromosomal lesions.Conclusion: Consistent with epidemiological findings on the etiology of childhood ALL, we conclude that reduced cytokine signaling (here, IL7R) in late pre-B cells renders pre-leukemic clones distinctively vulnerable to genetic lesions that can be acquired in the context of repeated exposure to inflammatory stimuli (e.g. chronic and recurrent infections during childhood). Our results support a role for AID and RAGs cooperation for the generation of secondary lesions in leukemia subgroups that require additional leukemogenic events, and therefore, provide the genetic and molecular basis to support the Delayed Infections Hypothesis for leukemia progression in children. DisclosuresNo relevant conflicts of interest to declare.

Recombination-activating gene 1 and 2 (RAG1 and RAG2) in flounder (Paralichthys olivaceus).

During the development of B and T lymphocytes, Ig and TCR variable region genes are assembled from germline V, D, and J gene segments by a site-specific recombination reaction known as V(D)J recombination. The process of somatic V(D)J recombination, mediated by the recombination-activating gene (RAG) products, is the most significant characteristic of adaptive immunity in jawed vertebrates. Flounder (Paralichthys olivaceus) RAG1 and RAG2 were isolated by Genome Walker and RT-PCR, and their expression patterns were analysed by RT-PCR and in situ hybridization on sections. RAG1 spans over 7.0 kb, containing 4 exons and 3 introns, and the full-length ORF is 3207 bp, encoding a peptide of 1068 amino acids. The first exon lies in the 5'-UTR, which is an alternative exon. RAG2 full-length ORF is 1062 bp, encodes a peptide of 533 amino acids, and lacks introns in the coding region. In 6-month old flounders, the expression of RAG1 and RAG2 was essentially restricted to the pronephros (head kidney) and mesonephros (truck kidney). Additionally, both of them were mainly expressed in the thymus. These results revealed that the thymus and kidney most likely serve as the primary lymphoid tissues in the flounder.

Physiological significance of recombination‐activating gene 1 in neuronal death, especially optic neuropathy

Although the transcription factor nuclear factor-κB (NF-κB) is known to regulate cell death and survival, its precise role in cell death within the central nervous system remains unknown. We previously reported that mice with a homozygous deficiency for NF-κBp50 spontaneously develop optic neuropathy. The aim of the present study was to demonstrate the expression and activation of the proapoptotic factor(s) that mediate optic neuropathy in p50-deficient mice. Recombination-activating gene (Rag) 1 is known to activate the recombination of immunoglobulin V(D)J. In this study, experiments with genetically engineered mice revealed the involvement of Rag1 expression in apoptosis of Brn3a-positive retinal ganglion cells, and also demonstrated the specific effect of p50 deficiency on the activation of Rag1 gene transcription. Furthermore, genetic analysis of murine neuronal stem-like cells clarified the biological significance of Rag1 in N-methyl-D-aspartate-induced neuronal apoptosis. We also detected the apoptosis-regulating factors Bax and cleaved caspase 3, 8 and 9 in HEK293 cells transfected-molecule of Rag1, and a human histological examination revealed the expression of Rag1 in retinal ganglion cells. The results of the present study indicate that Rag1 plays a role in optic neuropathy as a proapoptotic candidate in p50-deficient mice. This finding may lead to new therapeutic targets in optic neuropathy.

Transcriptional characterization of the T cell population within the salmonid interbranchial lymphoid tissue.

Previously, our group has shown that the interbranchial lymphoid tissue (ILT) is a distinct structure largely consisting of T cells embedded in a meshwork of epithelial cells, with no direct resemblance to previously described lymphoid tissues. In this study, we aim to focus on the T cell population and the possibility of the ILT being a thymus analog. By characterizing structural responsiveness to Ag challenge, the presence of recombination activating genes, and different T cell-related transcripts, we attempt to further approach the immunological function of the ILT in salmonid gills. In addition to eight healthy individuals, a group of eight infectious salmon anemia virus-challenged fish were included to observe T cell responses related to infection. The results showed reduced size of ILT in the infected group, no expression of RAG-1 and -2, and a high degree of T cell diversity within the ILT. Taking into account that the ILT can be regarded as a strategically located T cell reservoir and possibly an evolutionary forerunner of mammalian MALTs right at the border to the external environment, the alteration in transcription observed may likely represent a shift in the T cell population to optimize local gill defense mechanisms.

The Ataxia Telangiectasia Mutated and Cyclin D3 Proteins Cooperate To Help Enforce TCRβ and IgH Allelic Exclusion

Coordination of V rearrangements between loci on homologous chromosomes is critical for Ig and TCR allelic exclusion. The Ataxia Telangietasia mutated (ATM) protein kinase promotes DNA repair and activates checkpoints to suppress aberrant Ig and TCR rearrangements. In response to RAG cleavage of Igκ loci, ATM inhibits RAG expression and suppresses further Vκ-to-Jκ rearrangements to enforce Igκ allelic exclusion. Because V recombination between alleles is more strictly regulated for TCRβ and IgH loci, we evaluated the ability of ATM to restrict biallelic expression and V-to-DJ recombination of TCRβ and IgH genes. We detected greater frequencies of lymphocytes with biallelic expression or aberrant V-to-DJ rearrangement of TCRβ or IgH loci in mice lacking ATM. A preassembled DJβ complex that decreases the number of TCRβ rearrangements needed for a productive TCRβ gene further increased frequencies of ATM-deficient cells with biallelic TCRβ expression. IgH and TCRβ proteins drive proliferation of prolymphocytes through cyclin D3 (Ccnd3), which also inhibits VH transcription. We show that inactivation of Ccnd3 leads to increased frequencies of lymphocytes with biallelic expression of IgH or TCRβ genes. We also show that Ccnd3 inactivation cooperates with ATM deficiency to increase the frequencies of cells with biallelic TCRβ or IgH expression while decreasing the frequency of ATM-deficient lymphocytes with aberrant V-to-DJ recombination. Our data demonstrate that core components of the DNA damage response and cell cycle machinery cooperate to help enforce IgH and TCRβ allelic exclusion and indicate that control of V-to-DJ rearrangements between alleles is important to maintain genomic stability.

Rictor is required for early B cell development in bone marrow.

The development of early B cells, which are generated from hematopoietic stem cells (HSCs) in a series of well-characterized stages in bone marrow (BM), represents a paradigm for terminal differentiation processes. Akt is primarily regulated by phosphorylation at Thr308 by PDK1 and at Ser473 by mTORC2, and Akt signaling plays a key role in hematopoiesis. However, the role of mTORC2 in the development of early B cells remains poorly understood. In this study, we investigated the functional role of mTORC2 by specifically deleting an integral component, Rictor, in a hematopoietic system. We demonstrated that the deletion of Rictor induced an aberrant increase in the FoxO1 and Rag-1 proteins in BM B cells and that this increase was accompanied by a significant decrease in the abundance of B cells in the peripheral blood (PB) and the spleen, suggesting impaired development of early B cells in adult mouse BM. A BM transplantation assay revealed that the B cell differentiation defect induced by Rictor deletion was not affected by the BM microenvironment, thus indicating a cell-intrinsic mechanism. Furthermore, the knockdown of FoxO1 in Rictor-deleted HSCs and hematopoietic progenitor cells (HPCs) promoted the maturation of B cells in the BM of recipient mice. In addition, we revealed that treatment with rapamycin (an mTORC1 inhibitor) aggravated the deficiency in B cell development in the PB and BM. Taken together, our results provide further evidence that Rictor regulates the development of early B cells in a cell-intrinsic manner by modifying the expression of FoxO1 and Rag-1.

Functions of IDH1 and its mutation in the regulation of developmental hematopoiesis in zebrafish

Isocitrate dehydrogenase 1 mutation (IDH1R132) was recently identified in AML by next generation sequencing, occurring in 10% of AML with normal cytogenetics. The cancer-associated mutation may result in novel enzymatic activity whereby a-ketoglutarate is converted to 2-hydroxyglutarate (2-HG). The latter may inhibit the function of a-ketoglutarate-dependent dioxygenases, e.g. TET2 and PHDs and result in DNA and histone hypermethylation and hence leukemia initiation. In this study, we examined the role of zebrafish idh1 (zidh1) and the phenotypes of human IDH1R132H expression in embryonic hematopoiesis. The amino acid sequence of zidh1 was remarkably similar to human IDH1. z1dh1 gene was expressed around the AGM region at 36 hpf and in the CHT at 48 hpf. Morpholino knockdown of zidh1 significantly increased gene expression associated with early myeloid progenitor (pu.1) and decreased those of late myelomonocytic differentiation (l-plastin, mpeg1 and mpo) at 18 hpf. Definitive hematopoiesis was also reduced, characterized by down-regulation of c-myb expression along the ventral wall of dorsal aorta at 36 hpf and CHT at 48 and 96 hpf and decreased rag1 expression at 96 hpf. To validate our observations, we constructed a TALEN pair targeting exon 1 of zidh1 and injected the TALEN mRNAs into 1-cell embryos, which showed high mutation frequency (more than 95%) of targeted indels by sequencing. These injected embryos also demonstrated similar hematopoietic phenotypes with that of the zidh1 morphants. Microinjection of cDNA encoding human IDH1R132H and zebrafish idh1R146H but not wild-type IDH1 at 1 cell-stage significantly increased pu.1, l-plastin, mpeg1 and mpo expression and 2-HG level at 24 hpf. The effects could be inhibited by AGI-5198, a specific inhibitor of human IDH1R132H. Overexpression of zebrafish tet2 mRNA could partially rescue the hematopoietic phenotypes of increased myeloid expression by human IDH1R132H. Co-overexpression of human IDH1R132H and FLT3-ITD on zebrafish significantly increased myelopoiesis in the embryos. The observations underscored a hitherto undescribed role of idh1 in zebrafish hematopoiesis and the potential of zebrafish in modeling human AML.

Immune parameters in the intestine of wild and reared unvaccinated and vaccinated Atlantic salmon (Salmo salar L.)

Forming a barrier to the outside world, the gut mucosa faces the challenge of absorbing nutrients and fluids while initiating immune reactions towards potential pathogens. As a continuation to our previous publication focusing on the regional intestinal morphology in wild caught post smolt and spawning Atlantic salmon, we here investigate selected immune parameters and compare wild, reared unvaccinated and vaccinated post smolts.We observed highest transcript levels for most immune-related genes in vaccinated post smolts followed by reared unvaccinated and finally wild post smolts, indicating that farming conditions like commercial feed and vaccination might contribute to a more alerted immune system in the gut. In all groups, higher levels of immune transcripts were observed in the second segment of mid-intestine and in the posterior segment. In the life stages and conditions investigated here, we found no indication of a previously suggested population of intestinal T cells expressing MHC class II nor RAG1 expression.