Recombinase-activating Gene-1 Research Articles

Folate-Appended Hydroxypropyl-β-Cyclodextrin Induces Autophagic Cell Death in Acute Myeloid Leukemia Cells

Acute myeloid leukemia (AML) is a heterogenous myeloid neoplasm that remains challenging to treat. Because intensive conventional chemotherapy reduces survival rates in elderly patients, drugs with lower toxicity and fewer side effects are needed urgently. 2-Hydroxypropyl-β-cyclodextrin (HP-β-CyD) is used clinically as a pharmaceutical excipient for poorly water-soluble drugs. Previously, we showed that HP-β-CyD exerts antitumor activity by disrupting cholesterol homeostasis. Recently, we developed folate-conjugated HP-β-CyD (FA-HP-β-CyD) and demonstrated its potential as a new antitumor agent that induces not only apoptosis, but also autophagic cell death; however, we do not know whether FA-HP-β-CyD exerts these effects against AML. Here, we investigated the effects of FA-HP-β-CyD on folate receptor (FR)-expressing AML cells. We found that the cytotoxic activity of FA-HP-β-CyD against AML cells was stronger than that of HP-β-CyD. Also, FA-HP-CyD induced the formation of autophagosomes in AML cell lines. FA-HP-β-CyD increased the inhibitory effects of cytarabine and a BCL-2-selective inhibitor, Venetoclax, which are commonly used treat elderly AML patients. Notably, FA-HP-β-CyD suppressed the proliferation of AML cells in BALB/c nude recombinase-activating gene-2 (Rag-2)/Janus kinase 3 (Jak3) double-deficient mice with AML. These results suggest that FA-HP-β-CyD acts as a potent anticancer agent for AML chemotherapy by regulating autophagy.

Limited Expansion of Human Hepatocytes in FAH/RAG2-Deficient Swine.

The mammalian liver's regenerative ability has led researchers to engineer animals as incubators for expansion of human hepatocytes. The expansion properties of human hepatocytes in immunodeficient mice are well known. However, little has been reported about larger animals that are more scalable and practical for clinical purposes. Therefore, we engineered immunodeficient swine to support expansion of human hepatocytes and identify barriers to their clinical application. Immunodeficient swine were engineered by knockout of the recombinase-activating gene 2 (RAG2) and fumarylacetoacetate hydrolase (FAH). Immature human hepatocytes (ihHCs) were injected into fetal swine by intrauterine cell transplantation (IUCT) at day 40 of gestation. Human albumin was measured as a marker of engraftment. Cytotoxicity against ihHCs was measured in transplanted piglets and control swine. We initially detected higher levels of human albumin in cord blood of newborn FAH/RAG2-deficient (FR) pigs compared with immunocompetent controls (196.26 ng/dL vs. 39.29 ng/dL, p = 0.008), indicating successful engraftment of ihHCs after IUCT and adaptive immunity in the fetus. Although rare hepatocytes staining positive for human albumin were observed, levels of human albumin did not rise after birth, but declined, suggesting rejection of xenografted ihHCs. Cytotoxicity against ihHCs increased after birth by 3.8% (95% CI: [2.1%–5.4%], p < 0.001) and inversely correlated with declining levels of human albumin (p = 2.1 × 10−5, R2 = 0.17). Circulating numbers of T cells and B cells were negligible in FR pigs. However, circulating natural killer (NK) cells exerted cytotoxicity against ihHCs. NK cell activity was lower in immunodeficient piglets after IUCT than in naive controls (30.4% vs. 40.1%, p = 0.011, 95% CI for difference [2.7%–16.7%]). In conclusion, ihHCs were successfully engrafted in FR swine after IUCT. NK cells were a significant barrier to expansion of hepatocytes. New approaches are needed to overcome this hurdle and allow large-scale expansion of human hepatocytes in immunodeficient swine.Impact statementThere is currently a need for robust expansion of human hepatocytes. We describe an immunodeficient swine model into which we engrafted immature human hepatocytes (ihHCs). We identified the mechanism of the eventual graft rejection by the intact NK cell population, which has not been previously shown to have a significant role in xenograft rejection. By both improving engraftment and reducing NK cell-mediated cytotoxicity toward the graft through intrauterine cell transfer, we confirmed the presence of residual adaptive immunity in this model of immunodeficiency and the ability to induce hyposensitization in the NK cell population by taking advantage of the fetal microenvironment.

Investigating the Variation of TREC/KREC in Combined Immunodeficiencies

T-cell receptor excision circles (TREC)/Kappa-deleting recombination excision circles (KREC) assay has been recently recognized for detecting patients with primary (T- and/or B-cell) immunodeficiency (PID). We aimed to investigate the alterations of these biomarkers in some combined immunodeficiency patients compared to the healthy controls in different age groups. TREC and KREC were assessed in a total of 82 PID patients, most of them with exact genetic diagnosis (3 months to 42 years); using quantitative real-time-polymerase chain reaction (PCR). Patients had a final diagnosis of common variable immunodeficiency (n=23), ataxia-telangiectasia (AT) (n=17), hyper-IgE syndrome (HIES) (7 with DOCK8 deficiency, 4 with signal transducer and activator of transcription 3 (STAT3) deficiency, and 8 children with unknown genetic defects), Wiskott-Aldrich syndrome (WAS) (n=20), purine nucleoside phosphorylase (PNP)deficiency(n=1), dedicator of cytokinesis2 (DOCK2) deficiency (n=1), recombinase activating gene1 (RAG1) deficiency (n=1). Very low to zero amounts of TREC and/or KREC were detected in 14 out of 23 cases of common variable immunodeficiency (CVID), 14 out of 17 cases of AT, 8 out of 20 cases of WAS, 6 out of 7 cases of DOCK8-deficiency patients, 4 out of 8 cases of HIES with unknown genetic defects and all patients with defects in DOCK2, PNP, and RAG1. STAT3-deficient patients were normal for both biomarkers. All patients showed a significant difference in both markers compared to age-matched healthy controls. Our findings highlight that apart from severe types of T/B cell defects, this assay can also be used for early diagnosis the patients with late-onset of disease and even PIDs without a positive family history.

Preclinical Development of Autologous Hematopoietic Stem Cell-Based Gene Therapy for Immune Deficiencies: A Journey from Mouse Cage to Bed Side

Recent clinical trials using patient’s own corrected hematopoietic stem cells (HSCs), such as for primary immunodeficiencies (Adenosine deaminase (ADA) deficiency, X-linked Severe Combined Immunodeficiency (SCID), X-linked chronic granulomatous disease (CGD), Wiskott–Aldrich Syndrome (WAS)), have yielded promising results in the clinic; endorsing gene therapy to become standard therapy for a number of diseases. However, the journey to achieve such a successful therapy is not easy, and several challenges have to be overcome. In this review, we will address several different challenges in the development of gene therapy for immune deficiencies using our own experience with Recombinase-activating gene 1 (RAG1) SCID as an example. We will discuss product development (targeting of the therapeutic cells and choice of a suitable vector and delivery method), the proof-of-concept (in vitro and in vivo efficacy, toxicology, and safety), and the final release steps to the clinic (scaling up, good manufacturing practice (GMP) procedures/protocols and regulatory hurdles).

Successful Preclinical Development of Gene Therapy for Recombinase-Activating Gene-1-Deficient SCID

Recombinase-activating gene-1 (RAG1)-deficient severe combined immunodeficiency (SCID) patients lack B and T lymphocytes due to the inability to rearrange immunoglobulin and T cell receptor genes. Gene therapy is an alternative for those RAG1-SCID patients who lack a suitable bone marrow donor. We designed lentiviral vectors with different internal promoters driving codon-optimized RAG1 to ensure optimal expression. We used Rag1−/− mice as a preclinical model for RAG1-SCID to assess the efficacy of the various vectors. We observed that B and T cell reconstitution directly correlated with RAG1 expression. Mice with low RAG1 expression showed poor immune reconstitution; however, higher expression resulted in phenotypic and functional lymphocyte reconstitution comparable to mice receiving wild-type stem cells. No signs of genotoxicity were found. Additionally, RAG1-SCID patient CD34+ cells transduced with our clinical RAG1 vector and transplanted into NSG mice led to improved human B and T cell development. Considering this efficacy outcome, together with favorable safety data, these results substantiate the need for a clinical trial for RAG1-SCID.

Evaluation of antitumor effects of folate-conjugated methyl-β-cyclodextrin in melanoma.

Melanoma is a life-threatening disorder and its incidence is increasing gradually. Despite the numerous treatment approaches, conventional systemic chemotherapy has not reduced the mortality rate among melanoma patients, probably due to the induction of toxicity to normal tissues. Recently, we have developed folate-conjugated methyl-β-cyclodextrin (FA-M-β-CyD) and clarified its potential as a new antitumor agent involved in autophagic cell death. However, it remains uncertain whether FA-M-β-CyD exerts anticancer effects against melanomas. Therefore, in this study, we investigated the effects of FA-M-β-CyD on the folate receptor-α (FR-α)-expressing melanoma cell-selective cytotoxic effect. FA-M-β-CyD showed cytotoxic effects in Ihara cells, a human melanoma cell line expressing FR-α. In sharp contrast to methyl-β-cyclodextrin, FA-M-β-CyD entered Ihara cells [FR-α(+)] through FR-α-mediated endocytosis. Additionally, FA-M-β-CyD elicited the formation of autophagosomes in Ihara cells. Notably, FA-M-β-CyD suppressed melanoma growth in BALB/c nude recombinase-activating gene-2 (Rag-2)/Janus kinase 3 (Jak3) double deficient mice bearing Ihara cells. Therefore, these results suggest that FA-M-β-CyD could be utilized as a potent anticancer agent for melanoma chemotherapy by regulating autophagy.

Loss of cholinergic phenotype in septohippocampal projection neurons: Relation to brain versus peripheral IL-2 deficiency

In the peripheral immune system, IL-2 is essential for immune homeostasis, normal T regulatory cell function, and self-tolerance. IL-2 knockout (IL-2KO) mice develop spontaneous autoimmunity characterized by increased T cell trafficking to multiple organs. The IL-2 gene is also expressed in the brain, and in vitro studies have shown that IL-2 is a potent modulator of acetylcholine release from septohippocampal neurons and exerts trophic effects on septal neurons in culture. We previously described the apparent loss of cholinergic cell bodies in the medial septum of IL-2KO mice. Here we investigated if loss of brain-derived IL-2, or autoimmunity stemming from loss of peripheral IL-2, is responsible for the alteration in choline acetyltransferase (ChAT) expression in the medial septum of IL-2KO mice. To accomplish this objective, we compared ChAT-positive neurons between wild-type (WT) mice, IL-2KO mice, and congenic mice with a double gene deletion for the IL-2 gene and the recombinase activating gene-2 (RAG-2) which are referred to as IL-2KO/RAG-2KO mice (congenic mice which lack mature T and B cells as well as peripheral and brain-derived IL-2). We found that the loss of ChAT staining did not coincide with an overall loss of cells in the medial septum, suggesting that loss of brain IL-2 results in a change in cholinergic phenotype unrelated to cell death. No differences were noted in the endogenous expression of cytokines and chemokines tested in the medial septum. Evaluation of BDNF and NGF levels between WT and IL-2KO mice in medial septal homogenates revealed that IL-2KO mice have markedly higher levels of NGF in the medial septum compared to WT mice. Our findings suggest that brain-derived IL-2 plays an essential role in the maintainance of septohippocampal projection neurons in vivo.

Finding the Role of Helicobacter pylori in Colorectal Cancer

Colorectal cancer (CRC) is the third most common cancer and the fourth leading cause of cancer-related death globally; nonetheless, its etiology is still not established. CRC has a strong association with certain hereditary gene mutations, but only 3-5% of cases are due to these known mutations alone. Epidemiological data has established an association between CRC development and various environmental factors, such as high-calorie diet and obesity; however, these are not high-risk factors and there is apparently contradictory evidence. Besides host genetic and environmental factors, chronic inflammation, induced by bacteria, viruses or parasites, can also cause gastrointestinal tract (GIT) cancers. The human intestine is rich in nutrients and is a habitat for over 500 different bacterial species and viruses, with the highest concentration of bacteria found in the colon. The long list of infectious microbes, potentially implicated in the pathogenesis of CRC, includes Streptococcus bovis, Helicobacter pylori (H. pylori), Escherichia coli, Bacteroides, polyoma viruses (JC and SV40), human papillomavirus, Epstein Barr virus and cytomegalovirus. Among these microorganisms, H. pylori-induced carcinogenesis involves inflammation, as well as deregulation of the cell cycle via CagA, a bacterial oncoprotein that binds and activates SHP2 (a human oncogenic phosphatase), resulting in cell proliferation and motility. Due to the strong association between H. pylori and gastric cancer, the bacterium is classified as a class I carcinogen. Given that H. pylori is strongly associated with carcinogenesis in the human stomach, it may also be relevant to the pathogenesis of other GIT cancers, including CRC. However, the association between H. pylori infection and the development of CRC had been highly contentious. Zumkeller et al. (2006), who conducted a systematic review and meta-analysis of 11 published studies (between 1991 and 2002) involving 899 CRC patients and 1,476 controls, concluded that there is a possible small increase in the risk of CRC because of H. pylori. Similarly, in a recent large-scale retrospective population-based case-control study, conducted in the Rhine-Neckar region southwest of Germany involving 1,712 histologically confirmed CRC cases and 1,669 controls, H. pylori seropositivity was also reported to be associated with small but relevant increased risk confined to left-sided CRC. While these epidemiological studies showed a correlative relation, the association between H. pylori and CRC remained weak. Moving beyond epidemiological data, another recent study using the Helicobacter hepaticus-infected mice lacking the recombinase-activating gene-2 (rag2) – emulating many aspects of human inflammatory bowel disease (including the development of colitis and CRC) – supported the hypothesis of Helicobacterrelated inflammation-mediated carcinogenesis by demonstrating that Helicobacter induced molecular damage and altered gene expression during disease progression in vivo. In this issue of Immuno-Gastroenterology, Kapetanakis et al. went further to demonstrate that H. pylori may cause chronic inflammatory damage in the colonic mucosa, stimulating cancer stem cells and/or recruiting bone marrow-derived stem cells, thereby affecting oncogenesis and immune surveillance. The authors suggest that this may have an impact on the sequential transformation of colon epithelium to adenoma, leading to moderate-severe dysplasia and eventually CRC development and progression. Here, Kapetanakis et al. detected the presence of H. pylori in histological colonic tissue samples using cresyl violet staining further confirmed by imunohistochemistry (IHC) using anti-H. pylori. Previously, Jones et al. (2007) has also visualized H. pylori in human colonic tissues using IHC. Can H. pylori truly colonize the human colon? Serological detection of antibody against H. pylori indicates only past or current infection with no information on the site of colonization. Polymerase chain reaction and immunohistochemical detection of H. pylori in colonic tissues are sensitive, specific and correlate to present infection but are still limited by their inability to distinguish transient H. pylori infection from a true colonization of the colon or dead from viable bacteria. We need further direct and indirect evidence of extra-gastric colonization of H. pylori before passing a verdict. Despite having a clearer understanding of the potential role of H. pylori in the progression of pathological events leading up to CRC, many questions still remained to be answered. The development and progression of CRC is likely to be a multi-factorial process, involving a delicate interplay between factors such as host genetics, infectious agents (including but not limited to H. pylori or a combination of multiple agents), gut microbiome and environmental factors. The microbial pathogens-cancers relationship is indeed a complex one. Clarification of the role of infectious agents in carcinogenesis will strongly influence the clinical management of various cancers in the future.

Adult-onset manifestation of idiopathic T-cell lymphopenia due to a heterozygous RAG1 mutation

Adult-onset manifestation of idiopathic T-cell lymphopenia due to a heterozygous RAG1 mutation

Relationship among circulating leukocytes, platelets, and microvascular responses during induction of chronic colitis

The mechanisms by which microvascular alterations contribute to the pathogenesis of the inflammatory bowel diseases (IBDs; Crohn's disease, ulcerative colitis) have not been clearly delineated. The purpose of the current study was to characterize the inflammatory events, microvascular alterations, and blood cell changes that occur in a mouse model of IBD. In this model, CD4 + T-lymphocytes obtained from interleukin-10-deficient mice were injected intraperitoneally into lymphopenic, recombinase-activating gene-1 deficient (RAG −/−) mice. Two groups of control mice were also included: RAG −/− mice and C57BL/6 mice that were injected with phosphate-buffered saline but did not receive the T-cells. Four weeks later, the RAG −/− mice that had received the T-cell transfer showed significant signs of colonic inflammation, but without significant decreases in either body weight or mean arterial blood pressure. T-cell transfer increased the volume % of circulating platelets, while decreasing the number of circulating red blood cells. Additionally, the T-cell transfer tended to increase the circulating numbers of both lymphocytes and neutrophils when compared to unmanipulated RAG −/− mice. First-order colonic arterioles and venules tended to dilate in the colitic mice; however, the dilation was considerably more substantial with higher numbers of circulating leukocytes. The possibility that circulating inflammatory cells initiate the microvascular alterations in colitis warrants further investigation.

Why Newborn Screening for Severe Combined Immunodeficiency Is Essential: A Case Report

Physicians caring for infants in the first months of life need to know the normal ranges for absolute lymphocyte counts (ALCs) during that age. Any ALC <2500/microL is potentially pathogenic in early infancy and should be evaluated. We report the case of a 4-month-old white girl with a 2-month history of an oral ulcer, intermittent fever, recurrent otitis, decreased appetite, weight loss, and a new respiratory illness with hypoxemia. She had been in an in-home day care since birth. The patient's primary care physician had seen her frequently and obtained blood counts, but her persistent lymphopenia had not been appreciated. The infant was ultimately diagnosed with T(-)B(-)NK(+) (lacking both B and T lymphocytes and having primarily natural killer [NK] cells), recombinase-activating gene 2 (RAG2)-deficient severe combined immunodeficiency (SCID). However, because she had already developed 2 difficult-to-treat viral infections (parainfluenza 3 and adenovirus), she did not survive long enough to receive a bone marrow transplant. Newborn screening would not only have made the diagnosis at birth but would have led to measures to protect her from becoming infected before she could receive a transplant. Newborn screening would also reveal the true incidence of SCID and define the range of conditions characterized by severely impaired T-cell development. Until screening for SCID and other T-cell defects becomes available for all neonates (either by quantifying T-cell receptor excision circles in Guthrie spots or using other tests that quantify T cells), all pediatricians should know the normal range for ALCs according to age. Recognition of the characteristic lymphopenia of SCID can facilitate early diagnosis.

Correlation between recombinase activating gene 1 ubiquitin ligase activity and V(D)J recombination

The really interesting new gene (RING) finger ubiquitin ligase domain of the recombinase activating gene 1 (RAG1) V(D)J recombinase protein adopts a standard cross-brace architecture but co-ordinates three zinc ions as opposed to the canonical two. We demonstrated previously that disruption of the conserved zinc co-ordination sites resulted in loss of structural integrity and ubiquitin ligase (E3) activity and interfered with the ability of full-length RAG1 to support recombination. Here we present evidence that amino acids surrounding the third, non-canonical site also contribute to functional interaction with the ubiquitin conjugating (E2) enzyme CDC34, while certain residues on the RING domain's surface important for interaction between other E2-E3 pairs are less critical to the functional RAG1-CDC34 interaction in this assay. Partial reduction of ubiquitin ligase activity was significantly correlated with reduction in the ability of RAG1 to support recombination of extra-chromosomal substrates (r = 0.805, P = 0.009). While poly-ubiquitin chains could be generated, RAG1 did not promote rapid chain extension following mono-ubiquitylation of substrate, regardless of the E2 enzyme used. No single ubiquitin lysine mutant disrupted the ability of CDC34 to form ubiquitin chains on RAG1, and mass spectrometric analysis of the poly-ubiquitylated products indicated ubiquitin chain linkages through lysines 48 and 11. These data suggest that RAG1 promotes a mono-ubiquitylation reaction that is required for optimal levels of V(D)J recombination.

Recombinase‐activating gene 1 immunodeficiency: different immunological phenotypes in three siblings

We report different immunological phenotypes in three siblings from consanguineous family with recombinase-activating gene 1 (RAG1) gene mutations. Null mutations of RAG genes result in severe combined immunodeficiency (SCID) with absent T and B cells. Hypomorphic mutations with retained activity of RAG genes may lead to a 'leaky' SCID with some features of Omenn syndrome (OS) or typical OS. In our three patients, homozygous, hypomorphic RAG1 gene mutation (g.368-369delAA) was detected. Two patients presented with T-B-SCID phenotype while the youngest patient developed T+B+NK+SCID phenotype with expansion of autologous T-cell receptor (TCR) gammadelta-positive T cells, increased immunoglobulin levels and retained ability for antibody production. Similar to originally reported patients with this newly recognized immune phenotype, our patient developed disseminated cytomegalovirus (CMV) infection and autoimmune cytopenia. In infants with disseminated cytomegalovirus infection and autoimmune cytopenia, even if basic immunologic investigation appears normal, RAG1 immunodeficiency should be considered.

Noncoding transcription controls downstream promoters to regulate T-cell receptor α recombination

The T early alpha (TEA) promoter in the murine Tcra locus generates noncoding transcripts that extend across the 65 kb Jalpha array. Here, we have analyzed the significance of TEA transcription for Tcra locus regulation through the targeted introduction of a transcription terminator downstream of the TEA promoter. We demonstrate that noncoding transcription driven by this single promoter can instruct both positively and negatively the activity of downstream Jalpha promoters, and can similarly instruct alterations in Jalpha chromatin structure and Jalpha recombination. TEA transcription activates promoters associated with relatively proximal Jalpha segments and stimulates histone acetylation, histone methylation and chromatin accessibility in this region. In contrast, at more distal locations, TEA transcription inhibits promoter activity through transcriptional interference and suppresses chromatin accessibility. In combination, these effects target initial Valpha-to-Jalpha recombination to TEA-proximal Jalpha segments and promote the ordered usage of the Jalpha array. The ability of TEA transcription to coordinate the activity of multiple downstream promoters maximizes the biological potential of the Jalpha array and diversifies the Tcra repertoire.

T cell-associated CD18 but not CD62L, ICAM-1, or PSGL-1 is required for the induction of chronic colitis

The induction and perpetuation of chronic colitis are thought to involve a complex set of adhesive interactions between T cells and endothelial cells located on the vasculature within secondary lymphoid tissue and the intestine. The objective of this study was to assess the roles of T cell-associated CD18, CD62L (L-selectin), ICAM-1, and P-selectin glycoprotein ligand-1 (PSGL-1) in the induction of chronic colitis in mice. CD4(+)CD25(-) T cells derived from either wild-type (WT), CD18-deficient [CD18 knockout (KO)], CD62L KO, ICAM-1 KO, or PSGL-1 KO mice were adoptively transferred into recombinase activating gene-1 (RAG-1)-deficient mice (RAG KO mice) to assess the potential of these T cells to induce chronic colitis. At 8-10 wk following T cell transfer, we observed moderate to severe colitis as assessed by increases in colon weight-to-length ratios and by blinded histopathological analysis. In contrast, we found that transfer of CD18 KO T cells into RAG KO recipients resulted in the significant attenuation of colonic inflammation in these mice. Furthermore, we observed fewer infiltrating CD4(+) T cells in the colonic lamina propria in the CD18 KO-->RAG KO group compared with the WT-->RAG KO group. Finally, message levels of colonic TNF-alpha, IL-1beta, and IFN-gamma were significantly reduced in CD18 KO-->RAG KO mice compared with colitic control animals. We conclude that T cell-associated CD18, but not CD62L, ICAM-1, or PSGL-1, is required for the development of chronic colitis.

IL9 leads to airway inflammation by inducing IL13 expression in airway epithelial cells

Constitutive expression of IL9 in the lungs of transgenic (Tg) mice resulted in an asthma-like phenotype consisting of lymphocytic and eosinophilic lung inflammation, mucus hypersecretion and mast cell hyperplasia. Several T(h)2 cytokines including IL4, IL5 and IL13 were expressed in the lung in response to Tg IL9. IL13 was absolutely necessary for the development of lung pathology. To understand how IL9 induces IL13-dependent lung inflammation and mucus production, we sought the IL13-producing cells. Surprisingly, we found that the absence of T cells and B cells in recombinase-activating gene 1 (RAG1)-deficient IL9 Tg mice enhanced lung inflammation and dramatically enhanced IL13 production. In addition, the lack of mast cells or eosinophils in IL9 Tg mice did not affect IL13 levels in the lung. In situ hybridization for IL13 on lung sections from RAG1-/- IL9 Tg mice revealed that airway epithelial cells were the major IL13-producing cell type. Our results implicate the lung epithelium as a potentially important source of inflammatory cytokines in asthma.

B cell clones that sustain long-term plasmablast growth in T-independent extrafollicular antibody responses

Some antigens induce Ab responses without T lymphocyte help. Among these, many polysaccharide-based antigens cause marginal zone B cells to proliferate and differentiate into plasma cells. B1 cells also respond to some of these antigens. In this article, we report that antigen-specific B1b cells, in response to the T-independent antigen (4-hydroxy-3-nitrophenyl)-acetyl (NP)-Ficoll, develop into clones that sustain Ab production for months with continued production of plasma cells and the accumulation of antigen-specific B cells in follicles. The persistence of this T-independent plasmablast response contrasts with the short-term plasmablast growth associated with T-dependent extrafollicular responses. The nature of the cells responding to NP-Ficoll was probed by using chimeras that have B1 cells but lack primary B lymphopoietic capacity and have very few B2 cells or T cells. The chimeras were constructed by transferring 10(5) IgM(+) IgD(-) peritoneal exudate cells into mice unable to produce their own T and B cells because of deficiency in recombinase-activating gene 1 (RAG-1). The chimeras mounted sustained IgM and IgG3 anti-NP Ab responses to NP-Ficoll. This finding was associated with continued NP-specific extrafollicular plasmablast growth and the accumulation of NP-specific B cells in follicles. B cells were not found in the marginal zones of chimeras, and they also lacked recirculating IgD(+) cells and CD3(+) cells. The absence of B2 and T cells confirms that hemopoietic cell chimerism leading to primary lymphopoiesis had not been established.

Regulation of T-cell receptor beta-chain gene assembly by recombination signals: the beyond 12/23 restriction.

Assembly of antigen receptor genes is regulated in several important contexts during lymphocyte development. This regulation occurs through modulation of gene segment accessibility to the V(D)J recombinase and/or at the level of the recombination reaction due, in part, to constraints imposed by recombination signal (RS) sequences. RSs are composed of conserved heptamer and nonamer sequences that flank relatively non-conserved spacer sequences of either 12 or 23 base pairs. Recombination occurs only between RSs of dissimilar spacer lengths, a restriction known as the 12/23 rule. Recently, we have shown that RSs can impose significant constraints on antigen receptor gene assembly beyond enforcing the 12/23 rule. This restriction, termed B12/23, was revealed by analysis of T-cell receptor beta (TCRbeta) locus rearrangements, where Dbeta 12RSs and not Jbeta 12RSs are capable of efficiently targeting Vbeta 23RSs' rearrangement. The B12/23 restriction occurs at or prior to the DNA-cleavage step of the V(D)J recombination reaction, relies on features of the Dbeta 12RSs and Vbeta 23RSs, and is not absolutely dependent on lymphoid-specific factors other than the recombinase-activating gene-1 (RAG-1) and RAG-2 proteins. By preserving Dbeta gene segment utilization, the B12/23 restriction is required, at a minimum, for the generation of a diverse repertoire of TCRbeta chains.

Identification of the RAG-1 as a suitable mouse model for mitochondrial DNA disease

Previous studies have shown that transfer of human myoblasts carrying a mitochondrial DNA mutation into muscles of the severe combined immunodeficient mouse may provide an important animal model for mitochondrial myopathy. However, a major drawback of this mouse is its extreme sensitivity to ionising radiation, a pre-treatment which enhances the efficiency of myoblast transfer success. We implanted human myoblasts into the tibialis anterior muscles of another immunodeficient mouse, mutated in the recombinase activating gene-1 (RAG-1), to determine if this mouse could be an alternative to the severe combined immunodeficient for our mitochondrial myoblast transfer model. We also examined several different methods of muscle degeneration prior to myoblast transfer to determine which method resulted in the greatest amount of human tissue in implanted muscles. Our results show that the RAG-1 mouse displayed no sensitivity to the irradiation process compared to the high sensitivity in the severe combined immunodeficient mouse which resulted in early termination of the study. We also show that degeneration of host muscles by the myotoxin barium chloride (BaCl 2) resulted in the greatest amount of regenerating human muscle fibres in both the severe combined immunodeficient and RAG-1 mice. In addition, the maximum amount of human fibres observed in transplanted muscles was similar in each mouse strain. The average number of fibres throughout muscles was significantly greater in severe combined immunodeficient mice injured by BaCl 2, but was similar between all other muscle groups. This study suggests that the RAG-1 mouse is a suitable host for the mitochondrial myoblast transfer model and may also prove valuable for other myoblast transfer models such as muscular dystrophy.

Hepatic dendritic cell subsets in the mouse.

The CD11c(+) cell population in the non-parenchymal cell population of the mouse liver contains dendritic cells (DC), NK cells, B cells and T cells. In the hepatic CD11c(+) DC population from immunocompetent or immunodeficient [recombinase-activating gene-1 (RAG1)(-/-)] C57BL/6 mice (rigorously depleted of T cells, B cells and NK cells), we identified a B220(+) CD11c(int) subset of 'plasmacytoid' DC, and a B220(-) CD11c(+) DC subset. The latter DC population could be subdivided into a major, immature (CD40(lo) CD80(lo) CD86(lo) MHC class II(lo)) CD11c(int) subset, and a minor, mature (CD40(hi) CD80(hi) CD86(hi) MHC class II(hi)) CD11c(hi) subset. Stimulated B220(+) but not B220(-) DC produced type I interferon. NKT cell activation in vivo increased the number of liver B220(-) DC three- to fourfold within 18 h post-injection, and up-regulated their surface expression of activation marker, while it contracted the B220(+) DC population. Early in virus infection, the hepatic B220(+) DC subset expanded, and both, the B220(+) as well as B220(-) DC populations in the liver matured. In vitro, B220(-) but not B220(+) DC primed CD4(+) or CD8(+)T cells. Expression of distinct marker profiles and functions, and distinct early reaction to activation signals hence identify two distinct B220(+) and B220(-) subsets in CD11c(+) DC populations freshly isolated from the mouse liver.