RAG1 Research Articles

Combination of 5-aminolevulinic acid and iron prevents skin fibrosis in murine sclerodermatous graft-versus-host disease.

Scleroderma or systemic sclerosis (SSc) is a clinically heterogeneous rheumatological autoimmune disease affecting the skin, internal organs and blood vessels. There is at present no effective treatment for this condition. Our study investigated the effects of 5-aminolevulinic acid (5-ALA), which is a precursor of haem synthesis, on graft-vs-host disease (GvHD)-induced SSc murine model. Lymphocytes were intravenously injected from donor mice (B10.D2) into recipient BALB/c mice (recombination-activating gene 2 (Rag-2)-null mice) deficient in mature T and B cells to induce sclerodermatous GvHD (scl-GvHD). To investigate the effect of 5-ALA on scl-GvHD, combination of 5-ALA and sodium ferrous citrate (SFC) was orally administered to the recipient mice for 9weeks. 5-ALA/SFC treatment significantly reduced progressive inflammation and fibrosis in the skin and ears. Furthermore, 5-ALA/SFC suppressed mRNA expression of transforming growth factor-β, type I collagen and inflammatory cytokines. These results indicate that the 5-ALA/SFC combination treatment has a protective effect against tissue fibrosis and inflammation in a murine scl-GvHD-induced skin and ear inflammation and fibrosis. Furthermore, the efficacy of 5-ALA/SFC suggests important implications of HO-1 protective activity in autoimmune diseases, and therefore, 5-ALA/SFC may have promising clinical applications. These findings suggested that the 5-ALA/SFC treatment may be the potential strategies for SSc.

Comparative utility of NRG and NRGS mice for the study of normal hematopoiesis, leukemogenesis, and therapeutic response

Cell-line-derived xenografts (CDXs) or patient-derived xenografts (PDXs) in immune-deficient mice have revolutionized our understanding of normal and malignant human hematopoiesis. Transgenic approaches further improved in vivo hematological research, allowing the development of human-cytokine-producing mice, which show superior human cell engraftment. The most popular mouse strains used in research, the NOG (NOD.Cg-PrkdcscidIl2rγtm1Sug/Jic) and the NSG (NOD/SCID-IL2Rγ-/-, NOD.Cg-PrkdcscidIl2rγtm1Wjl/SzJ) mouse, and their human-cytokine-producing (interleukin-3, granulocyte-macrophage colony-stimulating factor, and stem cell factor) counterparts (huNOG and NSGS), rely partly on a mutation in the DNA repair protein PRKDC, causing a severe combined immune deficiency (SCID) phenotype and rendering the mice less tolerant to DNA-damaging therapeutics, thereby limiting their usefulness in the investigation of novel acute myeloid leukemia (AML) therapeutics. NRG (NOD/RAG1/2-/-IL2Rγ-/-) mice show equivalent immune ablation through a defective recombination activation gene (RAG), leaving DNA damage repair intact, and human-cytokine-producing NRGS (NRG-SGM3) mice were generated, improving myeloid engraftment. Our findings indicate that unconditioned NRG and NRGS mice can harbor established AML CDXs and can tolerate aggressive induction chemotherapy at higher doses than NSG mice without overt toxicity. However, unconditioned NRGS mice developed less clinically relevant disease, with CDXs forming solid tumors throughout the body, whereas unconditioned NRG mice were incapable of efficiently supporting PDX or human hematopoietic stem cell engraftment. These findings emphasize the contextually dependent utility of each of these powerful new strains in the study of normal and malignant human hematopoiesis. Therefore, the choice of mouse strain cannot be random, but must be based on the experimental outcomes and questions to be addressed.

B lymphocyte–induced maturation protein 1 controls TH9 cell development, IL-9 production, and allergic inflammation

The transcriptional repressor B lymphocyte-induced maturation protein 1 (Blimp-1) has a key role in terminal differentiation in various T-cell subtypes. However, whether Blimp-1 regulates TH9 differentiation and its role in allergic inflammation are unknown. We aimed to investigate the role of Blimp-1 in TH9 differentiation and in the pathogenesis of allergic airway inflammation. Invitro TH9 differentiation, flow cytometry, ELISA, and real-time PCR were used to investigate the effects of Blimp-1 on TH9 polarization. T cell-specific Blimp-1-deficient mice, a model of allergic airway inflammation, and T-cell adoptive transfer to recombination-activating gene 1 (Rag-1)-/- mice were used to address the role of Blimp-1 in the pathogenesis of allergic inflammation. We found that Blimp-1 regulates TH9 differentiation because deleting Blimp-1 increased IL-9 production in CD4+ T cells invitro. In addition, we showed that in T cell-specific Blimp-1-deficient mice, deletion of Blimp-1 in T cells worsened airway disease, and this worsening was inhibited by IL-9 neutralization. In asthmatic patients CD4+ T cells in response to TGF-β plus IL-4 increased IL-9 expression and downregulated Blimp-1 expression compared with expression in healthy control subjects. Blimp-1 overexpression in human TH9 cells inhibited IL-9 expression. Blimp-1 is a pivotal negative regulator of TH9 differentiation and controls allergic inflammation.

Cryptic lineages and hybridization of the predaceous chub Parazacco spilurus (Actinopterygii, Cypriniformes, Xenocyprididae) in Hong Kong

Fine-scale genetic studies are essential for understanding population connectivity and formulating appropriate conservation measures for freshwater inhabitants. Despite their significance, such studies remain relatively limited in South China and Asia in general. We examined the genetic structure of the predaceous chub Parazacco spilurus in Hong Kong by incorporating genetic data from both mitochondrial (control region and cytochrome b oxidase gene) and nuclear (recombination-activating gene 1) DNA markers. We identified two highly divergent lineages having discrete distribution ranges with limited overlap near two reservoirs located in north-eastern (Plover Cove Reservoir) and south-eastern (Tai Tam Tuk Reservoir) Hong Kong. Each lineage is further divided into sublineages with geographical association. As such, gene flow is highly restricted at both sublineage and population levels. Despite the presence of sublineages, inter-lineage divergence is far greater than that divergence within lineage. It also exceeds the species divergence in closely related cyprinid genera, suggesting that the two lineages recovered potentially represent two distinct species. Yet genetic analyses based on the nuclear marker indicated a low degree of hybridization between the lineages at two closely situated localities. This study provides insights on the possible direction of conservation plans for P. spilurus in Hong Kong and South China.

Abstract 5507: RAG1 high expression associated with IKZF1 dysfunction in adult B-cell acute lymphoblastic leukemia

Abstract The recombination activating gene (RAG)-mediated recombination is the dominant mutational process and the predominant driver of oncogenic genomic rearrangement in acute lymphoblastic leukemia (ALL). This then leads to further leukemic clonal evolution. IKZF1 encodes a kruppel-like zinc finger protein, IKAROS that is essential for normal hematopoiesis and acts as a tumor suppressor in ALL. The genetic defects of a single IKZF1 allele are linked to the development of human ALL, characterized by an increased risk of relapses and poorer prognosis. We observed that RAG1 is significantly increased in subsets of B-ALL patients. High RAG1 expression correlates with high proliferation markers. IKAROS directly binds to the RAG1 promoter in B-ALL cells by quantitative chromatin-immunoprecipitation assay. IKAROS suppresses RAG1 promoter activity by luciferase reporter assay. Lentiviral IKAROS expression significantly suppresses RAG1 expression, but IKAROS shRNA promotes RAG1 expression in B-ALL cells. CK2 inhibitor by restoring IKAROS activity, significantly suppresses RAG1 expression in an IKAROS-dependent manner in B-ALL cells. RAG1 expression is significantly higher in patients with IKZF1 deletion, as compared to patients without IKZF1 deletion. Treatment with CK2 inhibitor also results in an increase in IKZF1 binding to the RAG1 promoter and suppression of RAG1 expression in primary B-ALL cells. Taken together, our results demonstrate that high expression of RAG1 correlates with high proliferation markers in B-ALL, and are the first to demonstrate that IKAROS directly suppresses RAG1 expression. Our data suggest RAG1 high expression works together with IKAROS dysfunction to drive oncogenesis of B-ALL, which have significance in an integrated prognostic model for adult ALL. Citation Format: zheng Ge, Qi Han, Jinlong Ma, Yan Gu, Huihui Song, Malika Kapadia, Sinisa Dovat, Chunhua Song. RAG1 high expression associated with IKZF1 dysfunction in adult B-cell acute lymphoblastic leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5507.

Cryptic biodiversity in the freshwater fishes of the Kimberley endemism hotspot, northwestern Australia

The prevalence of unrecognised cryptic species impairs biodiversity estimates, clouds biological research and hinders conservation planning. As the rate of cryptic species detection increases globally, research is needed to determine how frequent cryptic species are, whether they are more common in given management regions, and whether these patterns are consistent across taxonomic groups. The Kimberley region in remote northwestern Australia harbours some of the most speciose, and morphologically and functionally diverse, endemic animal and plant communities on the continent. The rugged and changeable landscape also appears to contain a large proportion of cryptic terrestrial species, raising the question of whether similar patterns are also found among aquatic taxa, which have yet to be studied using integrative systematic approaches. If true, then the actual levels of aquatic biodiversity are yet to be fully realised. Here we conducted a molecular assessment of where species boundaries may exist in the Kimberley regions’ most speciose freshwater fish family, the Terapontidae (grunters), with a combined morphological assessment of the regions’ most speciose terapontid genus, Syncomistes. Assessment of nuclear markers (54 allozyme loci), sequence data (mitochondrial cytochrome b (cytb); nuclear recombination activation gene one (RAG1)) and 31 meristic and 36 morphometric characters provides evidence for 13 new candidate species across three different genera. Many of these candidate species are narrow range endemics. Our findings raise several questions about the evolutionary origin of the Kimberley’s endemic fish fauna and highlight the likelihood that freshwater fish species diversity in the Kimberley is severely under-represented by current systematic frameworks, with significant implications for ecological research, conservation and management.

Development of a Fluorescence in Situ Hybridization Probe for Detecting IKZF1 Deletion Mutations in Patients with Acute Lymphoblastic Leukemia

Intragenic deletion of IKZF1 is a recurrent genomic alteration in acute lymphoblastic leukemia. The deletions are mediated by illegitimate variable(diversity)joining recombination via cryptic recombination signal sequences (RSSs). We developed a fluorescence in situ hybridization (FISH) probe set that can detect any type of IKZF1 deletion, including the commonly deleted exon 4 to 7 region. The probe set consists of a designed probe for the commonly deleted region (Cy3; red) and a bacterial artificial chromosomes clone probe for detecting the 3' flanking region (Spectrum Green). Intact IKZF1 showed a fusion signal, and the deleted allele showed loss of the red signal (0R1G1F). The FISH probes worked correctly for human leukemic cell lines and clinical samples. One case showed an atypical break-apart signal (1R1G1F). Inverse PCR of the case revealed rearrangement of the excised IKZF1 fragment into a legitimate RSS site at Ig κ on chromosome 2, suggesting a pathogenic role of this recombination-activating gene 1/2-mediated event. In this study, we established FISH probe detecting IKZF1 deletion in a quick, quantitative, and cost-effective manner, and the results provided a novel insight into B-cell receptor editing by rearrangement of a cryptic RSS-mediated genomic fragment in acute lymphoblastic leukemia pathology.

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

Mutations in recombination-activating gene (RAG) 1 and RAG2 are associated with a broad range of clinical and immunologic phenotypes in human subjects. 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. 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. 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. 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.

Telomerase Mediates Lymphocyte Proliferation but Not the Atherosclerosis-Suppressive Potential of Regulatory T-Cells.

Atherosclerosis is an age-related disease characterized by systemic oxidative stress and low-grade inflammation. The role of telomerase and telomere length in atherogenesis remains contentious. Short telomeres of peripheral leukocytes are predictive for coronary artery disease. Conversely, attenuated telomerase has been demonstrated to be protective for atherosclerosis. Hence, a potential causative role of telomerase in atherogenesis is critically debated. In this study, we used multiple mouse models to investigate the regulation of telomerase under oxidative stress as well as its impact on atherogenesis in vitro and in vivo. Using primary lymphocytes and myeloid cell cultures, we demonstrate that cultivation under hyperoxic conditions induced oxidative stress resulting in chronic activation of CD4+ cells and significantly reduced CD4+ T-cell proliferation. The latter was telomerase dependent because oxidative stress had no effect on the proliferation of primary lymphocytes isolated from telomerase knockout mice. In contrast, myeloid cell proliferation was unaffected by oxidative stress nor reliant on telomerase. Telomerase reverse transcriptase deficiency had no effect on regulatory T-cell (Treg) numbers in vivo or suppressive function ex vivo. Adoptive transfer of telomerase reverse transcriptase-/- Tregs into Rag2-/- ApoE-/- (recombination activating gene 2/apolipoprotein E) double knockout mice demonstrated that telomerase function was not required for the ability of Tregs to protect against atherosclerosis. However, telomere length was critical for Treg function. Telomerase contributes to lymphocyte proliferation but plays no major role in Treg function, provided that telomere length is not critically short. We suggest that oxidative stress may contribute to atherosclerosis via suppression of telomerase and acceleration of telomere attrition in Tregs.

Using a multi-gene approach to infer the complicated phylogeny and evolutionary history of lorises (Order Primates: Family Lorisidae)

Extensive phylogenetic studies have found robust phylogenies are modeled by using a multi-gene approach and sampling from the majority of the taxa of interest. Yet, molecular studies focused on the lorises, a cryptic primate family, have often relied on one gene, or just mitochondrial DNA, and many were unable to include all four genera in the analyses, resulting in inconclusive phylogenies. Past phylogenetic loris studies resulted in lorises being monophyletic, paraphyletic, or an unresolvable trichotomy with the closely related galagos. The purpose of our study is to improve our understanding of loris phylogeny and evolutionary history by using a multi-gene approach. We used the mitochondrial genes cytochrome b, and cytochrome c oxidase subunit 1, along with a nuclear intron (recombination activating gene 2) and nuclear exon (the melanocortin 1 receptor). Maximum Likelihood and Bayesian phylogenetic analyses were conducted based on data from each locus, as well as on the concatenated sequences. The robust, concatenated results found lorises to be a monophyletic family (Lorisidae) (PP ≥ 0.99) with two distinct subfamilies: the African Perodictinae (PP ≥ 0.99) and the Asian Lorisinae (PP ≥ 0.99). Additionally, from these analyses all four genera were all recovered as monophyletic (PP ≥ 0.99). Some of our single-gene analyses recovered monophyly, but many had discordances, with some showing paraphyly or a deep-trichotomy. Bayesian partitioned analyses inferred the most recent common ancestors of lorises emerged ∼42 ± 6 million years ago (mya), the Asian Lorisinae separated ∼30 ± 9 mya, and Perodictinae arose ∼26 ± 10 mya. These times fit well with known historical tectonic shifts of the area, as well as with the sparse loris fossil record. Additionally, our results agree with previous multi-gene studies on Lorisidae which found lorises to be monophyletic and arising ∼40 mya (Perelman et al., 2011; Pozzi et al., 2014). By taking a multi-gene approach, we were able to recover a well-supported, monophyletic loris phylogeny and inferred the evolutionary history of this cryptic family.

359 - Unsuspected role of Interleukin-7 produced by hematopoietic cells to prevent autoimmunity in Recombination-activating gene (RAG)-deficient mice

359 - Unsuspected role of Interleukin-7 produced by hematopoietic cells to prevent autoimmunity in Recombination-activating gene (RAG)-deficient mice

The Mitochondrial‐targeted Antioxidant MitoQ Attenuates LV Dysfunction and Gene Expression Related to Oxidative Stress in Cardiomyocyte‐specific GPER KO Female Mice

IntroductionLow‐grade systemic inflammation associates with both postmenopause and the development of heart failure (HF). Given that markers of inflammation and oxidative stress predict incident HF, inflammation may have a direct effect on the estrogen‐deficient myocardium, possibly through increased oxidative stress. In ovariectomized (OVX) models of aging and hypertension, we've shown that estradiol (E2) or activation of the G‐protein coupled estrogen receptor, GPER, by its specific agonist G1, improves diastolic function and limits left ventricular (LV) remodeling. Also, diastolic function decreased and relative wall thickness increased in female mice with cardiomyocyte (CM) specific GPER deletion (GPER KO), with corresponding CM transcriptome alterations in mitochondrial and inflammatory response genes when compared to age‐matched wild type (WT) littermates. To corroborate a potential antioxidant defense role for GPER in the female heart, we determined the efficacy of a mitochondrial‐specific antioxidant, mitoquinone (MitoQ), on ameliorating cardiac dysfunction and the expression of oxidative stress‐related genes in LVs from GPER KO mice.Methods15 GPER KO and 14 WT littermates (6 mo) were randomized to receive either 8 weeks of oral treatment with MitoQ (KO‐MQ: n=8; WT‐MQ: n=7) or its vehicle (KO‐V: n=7; WT‐V: n=7). Tail cuff blood pressure and echocardiograms were obtained prior to sacrifice. LV samples (3 mice/group) were processed using the RT‐qPCR/Reverse transcriptase‐quantitative Polymerase Chain Reaction method for expression of 84 genes related to oxidative stress and oxidative defense. Genes that were 3‐times higher (hyper‐expression), or 3‐times lower (hypo‐expression), than genes from WT‐V were used for comparison. Data were analyzed by ANOVA (significance P<0.05).ResultsGPER knockdown resulted in reductions in fractional shortening (%FS) and myocardial relaxation (e′) and increases in filling pressure (E/e′), without altering blood pressure. MitoQ lessened the dysfunctional cardiac phenotype in KO, but did not modify the normal functioning WT heart (Fig 1). MitoQ‐mediated improvements in cardiac function occurred together with a blunting of oxidative stress genes. Twenty‐one genes relating to oxidative stress were hyper‐expressed in KO‐V hearts while only 5 genes met the criteria for over‐expression in KO‐MQ hearts (Fig 2). Of the 5 concurrently expressed genes, MitoQ blunted over‐expression by 24 to 48% when compared to vehicle. Only hearts from KO‐V exhibited hyper‐expression of Epx (Eosinophil peroxidase), Hspa1a (Heat shock 70), Rag2 (Recombination activating gene 2), Txn1 (Thioredoxin), Nox1 (NADPH oxidase 1), Gpx3 (Glutathione peroxidase 3), and Apoe (Apolipoprotein) genes. The antioxidant gene for glutathione reductase, Gstk, was under‐expressed in KO‐V but not in KO‐MQ hearts.ConclusionThese data show that mitochondria‐derived oxidative stress has an important role in the dysfunction from GPER KO and that mitochondrial targeted anti‐oxidants may be promising strategies to limit or defend against cardiac oxidative stress to preserve cardiac function/structure after E2 loss/GPER deactivation from surgery or menopause.Support or Funding InformationNIH AG033727 (LG); NIH HL051952 (LG and CMF)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Redox‐Driven Lymphocyte Inflammation Sensitizes Mice to Psychological Stress‐Mediated Hypertension

Post‐traumatic stress disorder (PTSD) is a debilitating psychological disease associated with increased activation of the sympathetic nervous system and norepinephrine (NE) outflow. PTSD increases the risk of cardiovascular diseases like hypertension by over 50%, but the mechanisms controlling this predisposition remain unclear. It is now well‐accepted that hypertension is regulated in part due to immune system‐driven inflammation, and our laboratory has previously reported that exposure to exogenous NE can modulate levels of T‐lymphocyte pro‐inflammatory cytokines (i.e. IL‐17A and IL‐6). Due to this, we hypothesized that the increased sympathoexcitation observed in PTSD drives pro‐inflammatory cytokine production from lymphocytes, and this inflammation leads to a susceptibility to hypertension. Using a mouse model of PTSD‐like psychological stress, we have demonstrated a two‐stage hypertensive phenotype. First, a modest but sustained increase in mean arterial pressure immediately following the stress induction (+9.8 mmHg, p=0.029 vs. controls). Second, a sensitization to low‐dose angiotensin II (AngII) after termination of the stress induction, which resulted in a more pronounced increase in blood pressure (+17.9 mmHg, p=0.002 vs. controls). To assess the physiological changes after psychological stress that may contribute to this increased blood pressure, we first assessed the activation status of the sympathetic nervous system. Interestingly, stressed mice demonstrated no appreciable change in circulating NE content, but splenic NE content was increased 2.5 fold vs. controls (p=0.019). Moreover, splenic tyrosine hydroxylase was increased 4.1 fold over control spleens (p=0.0009), inferring sustained sympathetic activation to this secondary lymphoid organ. Due to this, we next measured an array of circulating pro‐inflammatory cytokines, and observed increases in IL‐17A (+3.1 fold, p=0.011), IL‐22 (+1.9 fold, p=0.034), IL‐6 (+4.8 fold, p=0.063), and TNFα (+1.8 fold, p=0.021) following stress induction, which suggests a lymphocyte‐driven pro‐inflammatory cascade occurring in psychologically stressed animals. We have previously demonstrated that exogenous NE may modulate pro‐inflammatory cytokine production in T‐lymphocytes via alterations in redox signaling, thus, we assessed the redox status of splenocytes in stressed and control animals. Splenic lymphocytes from stressed mice demonstrated increases in both dihydroethidium (+2.1 fold, p=0.012) and MitoSOX Red (+2.2 fold, p=0.004) fluorescence, signifying a potentially novel role for immune activation via reactive oxygen species in vivo. Last, to assess the causal role of lymphocytes in driving the predisposition to hypertension during psychological stress, we utilized recombination activating gene 2 (Rag2) knock‐out animals, which are devoid of mature lymphocytes. Preliminary studies exposing these animals to psychological stress demonstrated attenuated cardiovascular and inflammatory alterations compared to wild‐type controls, which suggests lymphocytes play a direct role in regulating the stress‐induced pro‐inflammatory environment leading to increased blood pressure. Overall, our data suggest the potential for a new paradigm involving redox control of lymphocytes in the regulation of psychological stress‐driven hypertension.Support or Funding InformationThis work supported by NIH R00HL123471 and the Mary G. and George W. White Fund for Medical ResearchThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

TCR deep sequencing of transgenic RAG-1-deficient mice reveals endogenous TCR recombination: a cause for caution.

The utility of T-cell receptor (TCR) transgenic mice in medical research has been considerable, with applications ranging from basic biology all the way to translational and clinical investigations. Crossing of TCR transgenic mice with either recombination-activating gene (RAG)-1 or RAG-2 knockouts is frequently used to generate mice with a monoclonal T-cell repertoire. However, low level productive TCR rearrangement has been reported in RAG-deficient mice expressing transgenic TCRs. Using deep sequencing, we set out to directly examine and quantify the presence of these endogenous TCRs. Our demonstration that functional nontransgenic TCRs are present in nonmanipulated mice has wide reaching ramifications worthy of critical consideration.

A cold-blooded view of adaptive immunity.

The adaptive immune system arose 500 million years ago in ectothermic (cold-blooded) vertebrates. Classically, the adaptive immune system has been defined by the presence of lymphocytes expressing recombination-activating gene (RAG)-dependent antigen receptors and the MHC. These features are found in all jawed vertebrates, including cartilaginous and bony fish, amphibians and reptiles and are most likely also found in the oldest class of jawed vertebrates, the extinct placoderms. However, with the discovery of an adaptive immune system in jawless fish based on an entirely different set of antigen receptors - the variable lymphocyte receptors - the divergence of T and B cells, and perhaps innate-like lymphocytes, goes back to the origin of all vertebrates. This Review explores how recent developments in comparative immunology have furthered our understanding of the origins and function of the adaptive immune system.

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

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

Prevalence and clinical challenges among adults with primary immunodeficiency and recombination-activating gene deficiency

Prevalence and clinical challenges among adults with primary immunodeficiency and recombination-activating gene deficiency

Expression of immunoglobulin G in human podocytes, and its role in cell viability and adhesion.

Podocyte injury occurs during the initiation and development of numerous forms of glomerular disease, and antibodies targeting podocytes have become a biomarker for diagnosis and monitoring treatment response. Accumulating evidence has suggested that immunoglobulin (Ig) is expressed in non-B lineage cells, including epithelial cancer cells, myeloid cells and several types of normal cells. The main aim of the present study was to ascertain the expression of IgG in human podocytes and to determine its potential role in cellular bioactivity. The present study detected positive staining for IgG heavy chain (Igγ) and its subtype γ4, and the light chains κ and λ in the cytoplasm or on the membrane by immunofluorescence. In addition, positive bands were detected for Igγ, γ1, γ3, γ4, κ and λ in the lysates of a podocyte cell line by western blotting. Mass spectrometry confirmed IgG1 as an intact tetramer in the culture supernatant. Constant region transcripts of Igγ, γ1, γ3, γ4, κ and λ were identified by reverse transcription-polymerase chain reaction, and DNA sequencing of these transcripts revealed 96–99% similarity with Ig mRNAs in the National Center for Biotechnology Information database. Compared with the diverse gene rearrangements from B cell-derived Ig, podocyte-derived Ig exhibited conservative V(D)J patterns in the variable regions of Igγ and κ chains. Furthermore, the present study investigated the mechanism underlying IgG production in these cells by examining the expression of recombination activating gene (RAG)1, RAG2 and activation-induced cytidine deaminase. The expression levels of these proteins suggested that podocyte-derived Ig and traditional Ig may be generated in a similar manner. Furthermore, small interfering RNA-mediated downregulation of IgG expression reduced podocyte viability and adhesive capabilities. These findings suggested that IgG is expressed in podocytes and that this expression may be associated with podocyte function. Due to its potential biological and clinical significance, this phenomenon warrants further investigation.

Forest Dormouse ( Dryomys nitedula ) populations in southern Italy belong to a deeply divergent evolutionary lineage: implications for taxonomy and conservation

The Forest Dormouse (Dryomys nitedula) is a small rodent with a wide, albeit severely fragmented distribution, ranging from central Europe to central Asia. Within the Italian region, D. nitedula populations are restricted to forested mountain areas of two largely disconnected regions, the eastern Alps and the Calabria region, where two distinct subspecies (D. nitedula intermedius and D. nitedula aspromontis, respectively) have been described on the basis of phenotypic characters (i.e., fur colour). Here we analysed D. nitedula samples from both regions, to investigate patterns of genetic divergence and phylogenetic relationship among these two populations. Genetic variation was studied at the level of one mitochondrial (cytochrome b gene) and three nuclear gene fragments (exon1 of the interstitial retinoid-binding protein, exon 10 of the growth hormone receptor, and recombination activating gene 1). Phylogenetic analyses were performed using Maximum Likelihood and Bayesian inference methods. D. n. aspromontis and D. n. intermedius were found to be reciprocally monophyletic in all the phylogenetic analyses, and the genetic divergence observed between them at the mitochondrial CYTB gene was conspicuous (HKY: 0.044) when compared to previously observed values among many sister species of rodents. Our results clearly show that D. nitedula aspromontis is a deeply divergent, narrow endemic evolutionary lineage, and its conservation needs should be carefully evaluated in the near future. Moreover, such deep genetic divergence, together with phenotypic differentiation between D. n. intermedius and D. n. aspromontis, suggest that D. nitedula populations in southern Italy might belong to a distinct, previously unrecognized species.

Mast cells regulate CD4+ T-cell differentiation in the absence of antigen presentation

Given their unique capacity for antigen uptake, processing, and presentation, antigen-presenting cells (APCs) are critical for initiating and regulating innate and adaptive immune responses. We have previously shown the role of nicotinamide adenine dinucleotide (NAD+) in T-cell differentiation independently of the cytokine milieu, whereas the precise mechanisms remained unknown. The objective of this study is to further dissect the mechanism of actions of NAD+ and determine the effect of APCs on NAD+-mediated T-cell activation. Isolated dendritic cells and bone marrow-derived mast cells (MCs) were used to characterize the mechanisms of action of NAD+ on CD4+ T-cell fate invitro. Furthermore, NAD+-mediated CD4+ T-cell differentiation was investigated invivo by using wild-type C57BL/6, MC-/-, MHC class II-/-, Wiskott-Aldrich syndrome protein (WASP)-/-, 5C.C7 recombination-activating gene 2 (Rag2)-/-, and CD11b-DTR transgenic mice. Finally, we tested the physiologic effect of NAD+ on the systemic immune response in the context of Listeria monocytogenes infection. Our invivo and invitro findings indicate that after NAD+ administration, MCs exclusively promote CD4+ T-cell differentiation, both in the absence of antigen and independently of major APCs. Moreover, we found that MCs mediated CD4+ T-cell differentiation independently of MHC II and T-cell receptor signaling machinery. More importantly, although treatment with NAD+ resulted in decreased MHC II expression on CD11c+ cells, MC-mediated CD4+ T-cell differentiation rendered mice resistant to administration of lethal doses of L monocytogenes. Collectively, our study unravels a novel cellular and molecular pathway that regulates innate and adaptive immunity through MCs exclusively and underscores the therapeutic potential of NAD+ in the context of primary immunodeficiencies and antimicrobial resistance.