Corrigendum: Abnormalities of thymic stroma may contribute to immune dysregulation in murine models of leaky severe combined immunodeficiency.

[This corrects the article DOI: 10.3389/fimmu.2011.00015.].

Restoration of T and B Cell Differentiation after RAG1 Gene Transfer in Human RAG1 Defective Hematopoietic Stem Cells.

Recombinase-activating gene (RAG)-deficient SCID patients lack B and T lymphocytes due to the inability to rearrange immunoglobulin and T cell receptor genes. The two RAG genes act as a required dimer to initiate gene recombination. Gene therapy is a valid treatment alternative for RAG-SCID patients who lack a suitable bone marrow donor, but developing such therapy for RAG1/2 has proven challenging. Using a clinically approved lentiviral vector with a codon-optimized RAG1 gene, we report here preclinical studies using CD34+ cells from four RAG1-SCID patients. We used in vitro T cell developmental assays and in vivo assays in xenografted NSG mice. The RAG1-SCID patient CD34+ cells transduced with the RAG1 vector and transplanted into NSG mice led to restored human B and T cell development. Together with favorable safety data on integration sites, these results substantiate an ongoing phase I/II clinical trial for RAG1-SCID.

From variant of uncertain significance to likely pathogenic in two siblings with atypical RAG2 Deficiency: a case report and review of the literature.

BACKGROUND: Severe combined immunodeficiencies (SCIDs) are hereditary disorders characterized by impaired T and B cell function, resulting in significant immune system dysfunction. Recombination-activating gene (RAG) mutations account for a substantial proportion of SCID cases. Here, we present two sibling cases of SCID caused by a novel RAG2 gene mutation. CASE PRESENTATION: The index case was an 8-year-old boy who had a history of recurring infections. After a comprehensive immunological workup, the initial diagnosis of agammaglobulinemia was revised to combined immunodeficiency (CID). The patient underwent hematopoietic stem cell transplantation (HSCT) but succumbed to cytomegalovirus (CMV) infection. His brother, a 4-month-old boy, presented with CMV chorioretinitis. Leaky SCID was diagnosed based on genetic tests and immunological findings. The patient received appropriate treatment and was considered for HSCT. Both siblings had a homozygous RAG2 gene variant, with the first case classified as a variant of uncertain significance (VUS). The presence of the same mutation in the second brother, and the clinical phenotype, supports considering the mutation as likely pathogenic. CONCLUSIONS: This case report highlights a novel RAG2 gene mutation associated with CID. The classification of a VUS may evolve with accumulating evidence, and additional studies are warranted to establish its pathogenicity. Proper communication between genetic counselors and immunologists, accurate documentation of patient information, increased public awareness, and precise utilization of genetic techniques are essential for optimal patient management.

Insights into RAG Evolution from the Identification of "Missing Link" Family A RAGL Transposons.

A series of "molecular domestication" events are thought to have converted an invertebrate RAG-like (RAGL) transposase into the RAG1-RAG2 (RAG) recombinase, a critical enzyme for adaptive immunity in jawed vertebrates. The timing and order of these events are not well understood, in part because of a dearth of information regarding the invertebrate RAGL-A transposon family. In contrast to the abundant and divergent RAGL-B transposon family, RAGL-A most closely resembles RAG and is represented by a single orphan RAG1-like (RAG1L) gene in the genome of the hemichordate Ptychodera flava (PflRAG1L-A). Here, we provide evidence for the existence of complete RAGL-A transposons in the genomes of P. flava and several echinoderms. The predicted RAG1L-A and RAG2L-A proteins encoded by these transposons intermingle sequence features of jawed vertebrate RAG and RAGL-B transposases, leading to a prediction of DNA binding, catalytic, and transposition activities that are a hybrid of RAG and RAGL-B. Similarly, the terminal inverted repeats (TIRs) of the RAGL-A transposons combine features of both RAGL-B transposon TIRs and RAG recombination signal sequences. Unlike all previously described RAG2L proteins, RAG2L-A proteins contain an acidic hinge region, which we demonstrate is capable of efficiently inhibiting RAG-mediated transposition. Our findings provide evidence for a critical intermediate in RAG evolution and argue that certain adaptations thought to be specific to jawed vertebrates (e.g. the RAG2 acidic hinge) actually arose in invertebrates, thereby focusing attention on other adaptations as the pivotal steps in the completion of RAG domestication in jawed vertebrates.

Insights into RAG evolution from the identification of "missing link" family A RAGL transposons.

A series of "molecular domestication" events are thought to have converted an invertebrate RAG-like (RAGL) transposase into the RAG1-RAG2 (RAG) recombinase, a critical enzyme for adaptive immunity in jawed vertebrates. The timing and order of these events is not well understood, in part because of a dearth of information regarding the invertebrate RAGL-A transposon family. In contrast to the abundant and divergent RAGL-B transposon family, RAGL-A most closely resembles RAG and is represented by a single orphan RAG1-like (RAG1L) gene in the genome of the hemichordate Ptychodera flava (PflRAG1L-A). Here, we provide evidence for the existence of complete RAGL-A transposons in the genomes of P. flava and several echinoderms. The predicted RAG1L-A and RAG2L-A proteins encoded by these transposons intermingle sequence features of jawed vertebrate RAG and RAGL-B transposases, leading to a prediction of DNA binding, catalytic, and transposition activities that are a hybrid of RAG and RAGL-B. Similarly, the terminal inverted repeats (TIRs) of the RAGL-A transposons combine features of both RAGL-B transposon TIRs and RAG recombination signal sequences. Unlike all previously described RAG2L proteins, PflRAG2L-A and echinoderm RAG2L-A contain an acidic hinge region, which we demonstrate is capable of efficiently inhibiting RAG-mediated transposition. Our findings provide evidence for a critical intermediate in RAG evolution and argue that certain adaptations thought to be specific to jawed vertebrates (e.g., the RAG2 acidic hinge) actually arose in invertebrates, thereby focusing attention on other adaptations as the pivotal steps in the completion of RAG domestication in jawed vertebrates.

Detection of chromosomal alteration after infusion of gene-edited allogeneic CAR T cells.

A chromosome 14 inversion was found in a patient who developed bone marrow aplasia following treatment with allogeneic chimeric antigen receptor (CAR) Tcells containing gene edits made with transcription activator-like effector nucleases (TALEN). TALEN editing sites were not involved at either breakpoint. Recombination signal sequences (RSSs) were found suggesting recombination-activating gene (RAG)-mediated activity. The inversion represented a dominant clone detected in the context of decreasing absolute CAR Tcell and overall lymphocyte counts. The inversion was not associated with clinical consequences and wasnot detected in the drug product administered to this patient or in any drug product used in this or other trials using the same manufacturing processes. Neither was the inversion detected in this patient at earlier time points or in any other patient enrolled in this or other trials treated with this or other product lots. This case illustrates that spontaneous, possibly RAG-mediated, recombination events unrelated to gene editing can occur in adoptive cell therapy studies, emphasizes the need for ruling out off-target gene editing sites, and illustrates that other processes, such as spontaneous V(D)J recombination, can lead to chromosomal alterations in infused cells independent of gene editing.

'Cannibalism' of exogenous DNA sequences: The ancestral form of adaptive immunity which entails recognition of danger.

Adaptive immunity is a sophisticated form of immune response capable of retaining the molecular memory of a very great diversity of target antigens (epitopes) as non-self. It is capable of reactivating itself upon a second encounter with an immunoglobulin or T-cell receptor antigen-binding site with a known epitope that had previously primed the host immune system. It has long been considered that adaptive immunity is a highly evolved form of non-self recognition that appeared quite late in speciation and complemented a more generalist response called innate immunity. Innate immunity offers a relatively non-specific defense (although mediated by sensors that could specifically recognize virus or bacteria compounds) and which does not retain a memory of the danger. But this notion of recent acquisition of adaptive immunity is challenged by the fact that another form of specific recognition mechanisms already existed in prokaryotes that may be able to specifically auto-protect against external danger. This recognition mechanism can be considered a primitive form of specific (adaptive) non-self recognition. It is based on the fact that many archaea and bacteria use a genome editing system that confers the ability to appropriate viral DNA sequences allowing prokaryotes to prevent host damage through a mechanism very similar to adaptive immunity. This is indistinctly called, 'endogenization of foreign DNA' or 'viral DNA predation' or, more pictorially 'DNA cannibalism'. For several years evidence has been accumulating, highlighting the crucial role of endogenization of foreign DNA in the fundamental processes related to adaptive immunity and leading to a change in the dogma that adaptive immunity appeared late in speciation.

Rag2 Deficiency Enhances Susceptibility to Systemic Mouse Adenovirus Type 1 Infection.

INTRODUCTION: Recombination-activating gene (Rag) 1 and Rag2, which are essential in V(D)J recombination, play a crucial role in B- and T-cell maturation. METHOD: We investigated the effects of Rag2 deficiency in clustered regularly interspaced short palindromic repeats/Cas9-mediated FVB-Rag2 knockout (KO) and wild-type (WT) mice infected with mouse adenovirus type 1 (MAV-1) via the intranasal route. RESULTS: MAV-1 infection caused more severe histopathological changes in FVB-Rag2 KO mice than in WT mice. FVB-Rag2 KO mice exhibited moderate to severe inflammation on day 4 and severe inflammation on day 8 post infection. In contrast, WT mice showed mild inflammation on day 4 and mild to severe inflammation on day 8 post infection, including interstitial pneumonia and inflammatory cell infiltration in the lungs and liver. Viral loads in the spleen and kidneys were significantly higher in FVB-Rag2 KO mice than in WT mice on day 8 post infection. Levels of cytokines and chemokines, including macrophage inflammatory protein-1α, induced protein 10, interferon (IFN)-α, IFN-γ, and tumor necrosis factor alpha, were upregulated in the spleens of FVB-Rag2 KO mice compared with those of WT mice. The upregulation of several cytokines occurred concurrently with the histopathological changes. MAV-1 infection induced more severe systemic infection in FVB-Rag2 KO mice than in WT mice. CONCLUSION: In mice, Rag2 deficiency induces inflammatory cell recruitment via the upregulation of cytokine and chemokine levels. The MAV-1 infection model can be utilized to assess the efficacy and safety of therapeutic agents for human adenoviral diseases.

A novel homozygous RAG1 mutation is associated with severe combined immunodeficiency and neurological presentations.

INTRODUCTION AND OBJECTIVES: Severe combined immunodeficiency (SCID) is a subset of primary immunodeficiency diseases caused by a hereditary deficiency of the adaptive immune system. Mutation in recombination activating gene (RAG) is known as the underlying genetic cause of SCID. RAG protein plays a pivotal role in V(D)J recombination which is the main process to assemble lymphocyte antigen receptors during T- and B-cell development. The patients are characterized by recurrent infections, failure to thrive, chronic diarrhea, and fever, in early infancy. Herein, we present a case of SCID with rare neurological manifestations affected by a mutation in RAG1. PATIENTS AND METHODS: The patient was a 15-month-old infant born to a consanguineous family. She was presented with neurological abnormalities including facial nerve palsy, seizure, and decreased consciousness. Next-generation sequencing (NGS)-based primary immunodeficiency disease (PID)-gene panel screen and Sanger sequencing were performed to identify the genetic mutation. RESULTS: We found a novel homozygous missense mutation in RAG1, c.1210C>T,p.Arg404Trp, which was predicted to be deleterious (combined annotation dependent depletion, CADD score of 27.4). Both parents were heterozygous carriers for this mutation. According to her laboratory data, both T cell and B cell numbers were decreased and the patient was diagnosed as RAG1- SCID. CONCLUSIONS: SCID is a pediatric emergency with a variety of manifestations in infants. Therefore, accurate diagnosis importantly in the case of rare manifestations must be considered in these patients. Our findings point toward the importance of genetic assessment for early diagnosis and timely treatment of this disorder.

Cisplatin induces differentiation in teratomas derived from pluripotent stem cells.

INTRODUCTION: Currently, embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) can be induced to differentiate at the cellular level but not to form mature tissues or organs suitable for transplantation. ESCs/iPSCs form immature teratomas after injection into immunodeficient mice. In humans, immature teratomas often transform into fully differentiated mature teratomas after administration of anticancer agents. METHODS: We first investigated the ability of cisplatin to induce changes in mouse ESCs/iPSCs in vitro. Next, we designed experiments to analyze ESC/iPSC-derived immature teratoma tissue in vivo after treatment of cisplatin. Groups of six mice carrying ESC- or iPSC-derived teratomas were given either low or high dose intraperitoneal injection of cisplatin, while the control group received saline for 4 weeks. RESULTS: Treatment of ESC/iPSC cultures with cisplatin for 3 days caused a dose-related decrease in cell numbers without inducing any morphological changes to the cells. ESC/iPSC-derived teratomas showed lower growth rates with a significantly higher mature components ratio in a concentration dependent manner after cisplatin treatment (P < 0.05); however, immunohistochemical analyses demonstrated a significantly reduced PCNA labelling index and an increase in an apoptosis marker on immature neural components (P < 0.05) along with emergence of h-Caldesmon+ mature smooth muscle cells in treated mice. Moreover, newly differentiated components not found in the control group, such as mature adipose tissue, cartilage, and pancreas, as well as striated muscle, salivary glands, gastric mucosa with fundic glands, and hair follicles emerged. The identities of these components were confirmed by immunostaining for specific markers. CONCLUSIONS: Cisplatin has the ability to reduce immature components in ESC/iPSC-derived teratomas, presumably through apoptosis, and also to induce them to differentiate.

Structural basis for the activation and suppression of transposition during evolution of the RAG recombinase.

Jawed vertebrate adaptive immunity relies on the RAG1/RAG2 (RAG) recombinase, a domesticated transposase, for assembly of antigen receptor genes. Using an integration-activated form of RAG1 with methionine at residue 848 and cryo-electron microscopy, we determined structures that capture RAG engaged with transposon ends and U-shaped target DNA prior to integration (the target capture complex) and two forms of the RAG strand transfer complex that differ based on whether target site DNA is annealed or dynamic. Target site DNA base unstacking, flipping, and melting by RAG1 methionine 848 explain how this residue activates transposition, how RAG can stabilize sharp bends in target DNA, and why replacement of residue 848 by arginine during RAG domestication led to suppression of transposition activity. RAG2 extends a jawed vertebrate-specific loop to interact with target site DNA, and functional assays demonstrate that this loop represents another evolutionary adaptation acquired during RAG domestication to inhibit transposition. Our findings identify mechanistic principles of the final step in cut-and-paste transposition and the molecular and structural logic underlying the transformation of RAG from transposase to recombinase.

TNF-alpha inhibition ameliorates HDV-induced liver damage in a mouse model of acute severe infection.

BACKGROUND & AIMS: HDV infection induces the most severe form of human viral hepatitis. However, the specific reasons for the severity of the disease remain unknown. Recently, we developed an HDV replication mouse model in which, for the first time, liver damage was detected. METHODS: HDV and HBV replication-competent genomes and HDV antigens were delivered to mouse hepatocytes using adeno-associated vectors (AAVs). Aminotransferase elevation, liver histopathology, and hepatocyte death were evaluated and the immune infiltrate was characterized. Liver transcriptomic analysis was performed. Mice deficient for different cellular and molecular components of the immune system, as well as depletion and inhibition studies, were employed to elucidate the causes of HDV-mediated liver damage. RESULTS: AAV-mediated HBV/HDV coinfection caused hepatocyte necrosis and apoptosis. Activated T lymphocytes, natural killer cells, and proinflammatory macrophages accounted for the majority of the inflammatory infiltrate. However, depletion studies and the use of different knockout mice indicated that neither T cells, natural killer cells nor macrophages were necessary for HDV-induced liver damage. Transcriptomic analysis revealed a strong activation of type I and II interferon (IFN) and tumor necrosis factor (TNF)-α pathways in HBV/HDV-coinfected mice. While the absence of IFN signaling had no effect, the use of a TNF-α antagonist resulted in a significant reduction of HDV-associated liver injury. Furthermore, hepatic expression of HDAg resulted in the induction of severe liver damage, which was T cell- and TNF-α-independent. CONCLUSIONS: Both host (TNF-α) and viral (HDV antigens) factors play a relevant role in HDV-induced liver damage. Importantly, pharmacological inhibition of TNF-α may offer an attractive strategy to aid control of HDV-induced acute liver damage. LAY SUMMARY: Chronic hepatitis delta constitutes the most severe form of viral hepatitis. There is limited data on the mechanism involved in hepatitis delta virus (HDV)-induced liver pathology. Our data indicate that a cytokine (TNF-α) and HDV antigens play a relevant role in HDV-induced liver damage.

Arthritis in Two Patients With Partial Recombination Activating Gene Deficiency.

Autoimmunity is becoming an increasingly recognized complication in patients with primary immunodeficiencies (PIDs), including a variety of combined immune deficiencies such as Recombination Activating Gene (RAG) defects. The approach to treating autoimmunity in PID patients is complex, requiring a balance between immunosuppression and susceptibility to infection. Inflammatory arthritis is a feature of immune dysregulation in many PIDs, and the optimal treatment may differ from first line therapies that usually consist of disease-modifying anti rheumatic drugs (DMARDs). An example of mechanism-based therapy of arthritis in PID uses blockade of IL-6 signaling with tocilizumab for patients with STAT 3 gain-of-function (GOF) mutation and augmented IL-6 pathway. Herein, we describe two PID cases with arthritis who were found to have defects in RAG. One patient with refractory inflammatory arthritis experienced remarkable improvement in symptoms with tocilizumab therapy. Arthritis can be a clinical feature of immune dysregulation in RAG deficiency, and tocilizumab therapy has been suggested to have utility in treatment of arthritis in RAG deficiency.

The RAG2 gene of yellow catfish (Tachysurus fulvidraco) and its immune response against Edwardsiella ictaluri infection.

Recombination-activating gene 2 (rag 2) allies with recombination-activating gene 1 (rag 1) and regulates the V(D)J recombination of immunoglobulin (Ig) and T-cell receptor (TCR) genes. Being a key player in the adaptive immune response of vertebrates, functional characterization of rag 2 from yellow catfish is beneficial for understanding the biological response towards the pathogens. In this report, we have cloned and characterized the rag 2 gene of yellow catfish, and a particular pattern of expression was analysed in the major tissues of yellow catfish. The results showed that the open reading frame (ORF) of yellow catfish rag 2 was 1596 bp in length, which encodes a peptide of 531 amino acids. The multiple sequence alignment and phylogenetic analysis of rag 2 of yellow catfish with other species showed the conserved regions and the classical taxonomic evolution among the different vertebrate species. The qRT-PCR and Western blot results revealed that rag 2 transcripts and proteins were present in various tissues of yellow catfish with relatively high expression in the tissues of the thymus, head-kidney, and spleen. The systematic distribution analysis of the rag 2 expression by immunohistochemistry (IHC) using the rabbit polyclonal antibody, exposed relatively high expression in head kidney, spleen and thymus tissues after infected with Edwardsiella ictaluri. Moreover, the temporal expression of rag 2 and pro-inflammatory cytokines (IL-1ß and TNF-α) were significantly upregulated at different time points in the specific lymphoid tissues of yellow catfish following E. ictaluri infection. Our findings suggest that rag 2 potentially exhibited the immunological response in primary lymphoid tissues of yellow catfish against bacterial infection. This study will provide an essential source about rag 2 gene and its relationship with the inflammatory cytokines during infection.

Outcomes and Treatment Strategies for Autoimmunity and Hyperinflammation in Patients with RAG Deficiency.

BACKGROUND: Although autoimmunity and hyperinflammation secondary to recombination activating gene (RAG) deficiency have been associated with delayed diagnosis and even death, our current understanding is limited primarily to small case series. OBJECTIVE: Understand the frequency, severity, and treatment responsiveness of autoimmunity and hyperinflammation in RAG deficiency. METHODS: In reviewing the literature and our own database, we identified 85 patients with RAG deficiency, reported between 2001 and 2016, and compiled the largest case series to date of 63 patients with prominent autoimmune and/or hyperinflammatory pathology. RESULTS: Diagnosis of RAG deficiency was delayed a median of 5 years from the first clinical signs of immune dysregulation. Most patients (55.6%) presented with more than 1 autoimmune or hyperinflammatory complication, with the most common etiologies being cytopenias (84.1%), granulomas (23.8%), and inflammatory skin disorders (19.0%). Infections, including live viral vaccinations, closely preceded the onset of autoimmunity in 28.6% of cases. Autoimmune cytopenias had early onset (median, 1.9, 2.1, and 2.6 years for autoimmune hemolytic anemia, immune thrombocytopenia, and autoimmune neutropenia, respectively) and were refractory to intravenous immunoglobulin, steroids, and rituximab in most cases (64.7%, 73.7%, and 71.4% for autoimmune hemolytic anemia, immune thrombocytopenia, and autoimmune neutropenia, respectively). Evans syndrome specifically was associated with lack of response to first-line therapy. Treatment-refractory autoimmunity/hyperinflammation prompted hematopoietic stem cell transplantation in 20 patients. CONCLUSIONS: Autoimmunity/hyperinflammation can be a presenting sign of RAG deficiency and should prompt further evaluation. Multilineage cytopenias are often refractory to immunosuppressive treatment and may require hematopoietic cell transplantation for definitive management.

Recombination activity of human recombination-activating gene 2 (RAG2) mutations and correlation with clinical phenotype.

BACKGROUND: Mutations in recombination-activating gene (RAG) 1 and RAG2 are associated with a broad range of clinical and immunologic phenotypes in human subjects. OBJECTIVE: Using a flow cytometry-based assay, we aimed to measure the recombinase activity of naturally occurring RAG2 mutant proteins and to correlate our results with the severity of the clinical and immunologic phenotype. METHODS: Abelson virus-transformed Rag2-/- pro-B cells engineered to contain an inverted green fluorescent protein (GFP) cassette flanked by recombination signal sequences were transduced with retroviruses encoding either wild-type or 41 naturally occurring RAG2 variants. Bicistronic vectors were used to introduce compound heterozygous RAG2 variants. The percentage of GFP-expressing cells was evaluated by using flow cytometry, and high-throughput sequencing was used to analyze rearrangements at the endogenous immunoglobulin heavy chain (Igh) locus. RESULTS: The RAG2 variants showed a wide range of recombination activity. Mutations associated with severe combined immunodeficiency and Omenn syndrome had significantly lower activity than those detected in patients with less severe clinical presentations. Four variants (P253R, F386L, N474S, and M502V) previously thought to be pathogenic were found to have wild-type levels of activity. Use of bicistronic vectors permitted us to assess more carefully the effect of compound heterozygous mutations, with good correlation between GFP expression and the number and diversity of Igh rearrangements. CONCLUSIONS: Our data support genotype-phenotype correlation in the setting of RAG2 deficiency. The assay described can be used to define the possible disease-causing role of novel RAG2 variants and might help predict the severity of the clinical phenotype.

The c-Myc/miR17-92/PTEN Axis Tunes PI3K Activity to Control Expression of Recombination Activating Genes in Early B Cell Development.

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.

iRAGu: A Novel Inducible and Reversible Mouse Model for Ubiquitous Recombinase Activity.

Developing lymphocytes express the recombination activating genes (RAGs) 1 and 2 products that form a site specific recombinase complex (RAG), introducing double strand DNA breaks (DSBs) at recombination signal sequences (RSSs) flanking the V, D, and J gene segments in the antigen receptor loci. The subsequent steps in the reaction consist in the ligation of DSBs by ubiquitous enzymes of the non-homologous end joining DNA repair pathway. This mutagenesis process is responsible for the generation of the very large clonal diversity of T and B lymphocytes, itself allowing the recognition of a virtually open-ended antigenic universe. Sequences resembling RSS are found at high frequency all over the genome, and involved in RAG mediated illegitimate recombination and translocations. Hence, natural and induced ectopic activity of RAG is a threat to the genome only recently underscored. Here, we report and characterize a novel mouse transgenic system for which ubiquitous expression of the recombinase is inducible. In this system, the RAG1 protein is constitutively expressed and functional, while the RAG2 protein, coupled to the estrogen receptor, becomes functionally active upon 4-hydroxytamoxifen (TAM) administration. We describe two transgenic lines. The first one, when introgressed into an endogenous Rag2-/- genetic background is faithfully recapitulating lymphocyte development, repertoire dynamics and cryptic rearrangements, in a TAM-dependent manner. In this model, deprivation of TAM is followed by lymphocyte development arrest, evidencing the reversibility of the system. The second transgenic line is leaky, as the transgenes promote lymphocyte differentiation in absence of TAM treatment. Upon TAM-induction defects in lymphocytes composition and global health reveals the deleterious effect of uncontrolled RAG activity. Overall, this novel transgenic model provides a tool where RAG activity can be specifically manipulated to assess the dynamics of lymphocyte differentiation and the challenges imposed by the recombinase on the vertebrate genome.

Recombination activating gene-2null severe combined immunodeficient pigs and mice engraft human induced pluripotent stem cells differently.

This study comparatively investigated the transcriptional, physiological, and phenotypic differences of the immune disorder between severe combined immunodeficient (SCID) mouse and pig models. We discovered that the recombination activating gene-2 (Rag-2) SCID mice, but not RAG-2 SCID pigs, showed intense, infrequent, and mild cluster of CD3+-, CD4+-, and CD8+ signals respectively, suggesting that distinct species-specific effects exist. Furthermore, the expression of six relevant genes (NFATC1, CD79B, CD2, BLNK, FOXO1, and CD40) was more downregulated than that in the Rag-2 SCID mice, which provides a partial rationale for the death of T/B cells in the lymphoid organs of RAG-2 SCID pigs but not in Rag-2 SCID mice. Further, NK cell maturation-related gene expression was significantly lower in RAG-2 SCID pigs than in Rag-2 SCID mice. Consistently, the RAG-2 SCID pigs, but not Rag-2 SCID mice, developed human induced pluripotent stem cell-derived teratomas that were the same as those of perforin/Rag-2 SCID mice. Therefore, these unexpected findings indicate the superiority of RAG-2 SCID pigs over Rag-2 SCID mice as a suitable model for investigating human diseases.

The Complex Interplay between DNA Injury and Repair in Enzymatically Induced Mutagenesis and DNA Damage in B Lymphocytes.

Lymphocytes are endowed with unique and specialized enzymatic mutagenic properties that allow them to diversify their antigen receptors, which are crucial sensors for pathogens and mediators of adaptive immunity. During lymphocyte development, the antigen receptors expressed by B and T lymphocytes are assembled in an antigen-independent fashion by ordered variable gene segment recombinations (V(D)J recombination), which is a highly ordered and regulated process that requires the recombination activating gene products 1 & 2 (RAG1, RAG2). Upon activation by antigen, B lymphocytes undergo additional diversifications of their immunoglobulin B-cell receptors. Enzymatically induced somatic hypermutation (SHM) and immunoglobulin class switch recombination (CSR) improves the affinity for antigen and shape the effector function of the humoral immune response, respectively. The activation-induced cytidine deaminase (AID) enzyme is crucial for both SHM and CSR. These processes have evolved to both utilize as well as evade different DNA repair and DNA damage response pathways. The delicate balance between enzymatic mutagenesis and DNA repair is crucial for effective immune responses and the maintenance of genomic integrity. Not surprisingly, disturbances in this balance are at the basis of lymphoid malignancies by provoking the formation of oncogenic mutations and chromosomal aberrations. In this review, we discuss recent mechanistic insight into the regulation of RAG1/2 and AID expression and activity in lymphocytes and the complex interplay between these mutagenic enzymes and DNA repair and DNA damage response pathways, focusing on the base excision repair and mismatch repair pathways. We discuss how disturbances of this interplay induce genomic instability and contribute to oncogenesis.