Recombinase-activating Gene Research Articles

Lack of activity of HIV-1 integrase strand-transfer inhibitors on recombinase activating gene (RAG) activity at clinically relevant concentrations.

Human immunodeficiency virus 1 (HIV-1) infection remains a global health concern, with nearly 30 million people on antiretroviral (ARV) treatment. Integrase strand-transfer inhibitors (INSTIs) that block HIV-1 integrase are crucial components of first-line combination ARV therapies recommended in most international guidelines and have significantly improved HIV-1 treatment due to their efficacy and safety. This study evaluates potential off-target effects of INSTIs on recombinase activating genes (RAG1 and RAG2), which are essential for adaptive immune system function. We performed a comprehensive assessment of the off-target effects of clinically approved INSTIs on RAG activity, utilizing both biochemical and cellular assays. We purified the first catalytically active recombinant human core RAG1-RAG2 complex and tested it in the presence of the co-factor human HMGB1 protein for the gel-based biochemical RAG DNA cleavage assay. Additionally, we optimized an extrachromosomal V(D)J recombination cellular assay using murine mCherry-core RAG1, full-length murine mCherry-RAG2, and a plasmid substrate green fluorescent protein (GFP) reporter system, transfecting them into cells in the absence or presence of inhibitors. This setup enabled high-throughput analysis of V(D)J recombination for multiple compounds in a dose-response manner via flow cytometry. Physiologically relevant concentrations of INSTIs were examined for their potential impact on RAG activity and V(D)J recombination, with approved INSTIs showing minimal to no effects on recombinase activity. Consequently, the findings support the continued use of INSTIs in HIV-1 treatment without substantial concern for adverse effects on V(D)J recombination and immune system function.IMPORTANCEINSTIs are a crucial component of antiretroviral treatments for HIV-1 infection. This study provides a careful and thorough analysis of the impact of approved INSTIs on recombinase activating gene (RAG1 and RAG2) activity, which plays a pivotal role in the adaptive immune system. The concentrations tested were derived from several clinical studies and accounted for the maximum free fraction of the drug available in patients. This approach ensures that our findings are directly applicable to clinical scenarios by providing meaningful insights into the potential drug side effects in patients. We developed biochemical and cellular assays to measure the impact of INSTIs on RAG activity. All tested INSTIs did not inhibit RAG at supratherapeutic concentrations in the RAG1/RAG2 biochemical cleavage and cellular V(D)J recombination assays. Our assessment supports the continued use of INSTIs in HIV-1 treatments without concern for adverse effects.

Transplantation after CD45-ADC corrects Rag1 immunodeficiency in congenic and haploidentical settings

Mutations in the recombinase-activating genes 1 and 2 (RAG1, RAG2) cause a spectrum of phenotypes, ranging from severe combined immune deficiency to combined immune deficiency with immune dysregulation (CID-ID). Hematopoietic cell transplantation is a curative option. Use of conditioning facilitates robust and durable stem cell engraftment and immune reconstitution but may cause toxicity. Transplantation from haploidentical donors is associated with poor outcome in patients with CID-ID. We sought to evaluate multilineage engraftment and immune reconstitution after conditioning with CD45-antibody drug conjugate (CD45-ADC) as a single agent in hypomorphic mice with Rag1 mutation treated with congenic and haploidentical hematopoietic cell transplantation. Rag1-F971L mice, a model of CID-ID, were conditioned with various doses of CD45-ADC, total body irradiation, or isotype-ADC, and then given transplants of total bone marrow cells from congenic or haploidentical donors. Flow cytometry was used to assess chimerism and immune reconstitution. Histology was used to document reconstitution of thymic architecture. Conditioning with CD45-ADC as a single agent allowed robust engraftment and immune reconstitution, with restoration of thymus, bone marrow, and peripheral compartments. The optimal doses of CD45-ADC were 1.5 mg/kg and 5 mg/kg for congenic and haploidentical transplantation, respectively. No graft-versus-host disease was observed. Conditioning with CD45-ADC alone allows full donor chimerism and immune reconstitution in Rag1 hypomorphic mice even following haploidentical transplantation, opening the way for the implementation of similar approaches in humans.

Brief Report: T-Cell Receptor α Repertoire Diversity at Birth After in utero Exposure to HIV Integrase Strand-Transfer Inhibitors.

Effectiveness of anti-HIV in the prevention of perinatal transmission has been established. Assessing the tolerance of drug exposure during pregnancy is of the utmost importance given the number of children exposed. HIV integrase and the recombinase-activating gene enzyme involved in the establishment of the T-lymphocyte repertoire show structural similarity. The inhibition of recombinase-activating (RAG) gene by anti-integrases is observed in vitro, in a variable way according to the molecules. Here, we show that in utero exposure to raltegravir did not alter the T-lymphocyte repertoire of 12 newborns. These reassuring data merit verification for other anti-integrases. ( ClinicalTrial.org NCT04024150).

Diverse mutational landscapes in human lymphocytes

The lymphocyte genome is prone to many threats, including programmed mutation during differentiation1, antigen-driven proliferation and residency in diverse microenvironments. Here, after developing protocols for expansion of single-cell lymphocyte cultures, we sequenced whole genomes from 717 normal naive and memory B and T cells and haematopoietic stem cells. All lymphocyte subsets carried more point mutations and structural variants than haematopoietic stem cells, with higher burdens in memory cells than in naive cells, and with T cells accumulating mutations at a higher rate throughout life. Off-target effects of immunological diversification accounted for approximately half of the additional differentiation-associated mutations in lymphocytes. Memory B cells acquired, on average, 18 off-target mutations genome-wide for every on-target IGHV mutation during the germinal centre reaction. Structural variation was 16-fold higher in lymphocytes than in stem cells, with around 15% of deletions being attributable to off-target recombinase-activating gene activity. DNA damage from ultraviolet light exposure and other sporadic mutational processes generated hundreds to thousands of mutations in some memory cells. The mutation burden and signatures of normal B cells were broadly similar to those seen in many B-cell cancers, suggesting that malignant transformation of lymphocytes arises from the same mutational processes that are active across normal ontogeny. The mutational landscape of normal lymphocytes chronicles the off-target effects of programmed genome engineering during immunological diversification and the consequences of differentiation, proliferation and residency in diverse microenvironments.

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.

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).

Efficacy and safety of anti-CD45–saporin as conditioning agent for RAG deficiency

Background:Mutations in the recombinase-activating genes cause severe immunodeficiency, with a spectrum of phenotypes ranging from severe combined immunodeficiency to immune dysregulation. Hematopoietic stem cell transplantation is the only curative option, but a high risk of graft failure and poor immune reconstitution have been observed in the absence of myeloablation.Objectives:Our aim was to improve multilineage engraftment; we tested nongenotoxic conditioning with anti-CD45 mAbs conjugated with saporin CD45 (CD45-SAP).Methods:Rag1-KO and Rag1-F971L mice, which represent models of severe combined immune deficiency and combined immune deficiency with immune dysregulation, respectively, were conditioned with CD45-SAP, CD45-SAP plus 2 Gy of total body irradiation (TBI), 2 Gy of TBI, 8 Gy of TBI, or no conditioning and treated by using transplantation with lineage-negative bone marrow cells from wild-type mice. Flow cytometry and immunohistochemistry were used to assess engraftment and immune reconstitution. Antibody responses to 2,4,6-trinitrophenyl–conjugated keyhole limpet hemocyanin were measured by ELISA, and presence of autoantibody was detected by microarray.Results:Conditioning with CD45-SAP enabled high levels of multilineage engraftment in both Rag1 mutant models, allowed overcoming of B- and T-cell differentiation blocks and thymic epithelial cell defects, and induced robust cellular and humoral immunity in the periphery.Conclusions:Conditioning with CD45-SAP allows multilineage engraftment and robust immune reconstitution in mice with either null or hypomorphic Rag mutations while preserving thymic epithelial cell homeostasis.

RAG gene defects at the verge of immunodeficiency and immune dysregulation.

Mutations of the recombinase activating genes (RAG) in humans underlie a broad spectrum of clinical and immunological phenotypes that reflect different degrees of impairment of T- and B-cell development and alterations of mechanisms of central and peripheral tolerance. Recent studies have shown that this phenotypic heterogeneity correlates, albeit imperfectly, with different levels of recombination activity of the mutant RAG proteins. Furthermore, studies in patients and in newly developed animal models carrying hypomorphic RAG mutations have disclosed various mechanisms underlying immune dysregulation in this condition. Careful annotation of clinical outcome and immune reconstitution in RAG-deficient patients who have received hematopoietic stem cell transplantation has shown that progress has been made in the treatment of this disease, but new approaches remain to be tested to improve stem cell engraftment and durable immune reconstitution. Finally, initial attempts have been made to treat RAG deficiency with gene therapy.

Abstract 2157: Chemotherapeutic tolerability and estrogen dose response in the B6;129-Rag2 tm1Fwa IL2rg tm1Rsky/DwlHsd (R2G2) mouse model

Abstract The B6;129-Rag2tm1FwaIL2rgtm1Rsky/DwlHsd (R2G2) knockout mouse was developed by backcrossing an IL2rg (common gamma) knockout model to a RAG2 (recombinase activating gene) knockout model. The resulting mouse lacks various cytokines including IL-2, IL-4, IL-7, IL-9 and IL-15. In addition, this model lacks B cells, T cells, NK cells and has a deficit in lymphocyte development. This model was developed to provide another immunodeficient option for the oncology and immunology fields. The literature supports better tolerability of DNA-damaging oncology treatments for models that do not carry the SCID mutation. We have already reported in a white paper that the R2G2 mouse model is more tolerant of whole-body radiation than a model with the SCID mutation. Herein we describe a study examining chemotherapeutic tolerability of common DNA-damaging oncology drugs including 5-fluorouracil (5-FU), doxorubicin (Doxo), and cyclophosphamide (CTX) (n = 10 per group). 5-FU was given at 30, 60 or 100 mg/kg, intraperitoneally, twice weekly for five weeks. Doxo was given at 2 or 5 mg/kg, intraperitoneally, once weekly for three weeks. CTX was given at 100 or 140 mg/kg intraperitoneally, once weekly for three weeks. The low dose was chosen based on the average dose used in immunodeficient mouse models and the high dose was chosen based on the average dose used in immunocompetent mouse models. This was done to determine if the R2G2 mouse model can tolerate higher doses of these chemotherapeutic agents compared to other immunodeficient mouse models. Results show that the R2G2 mouse model tolerates higher doses of these chemotherapeutic drugs than doses found in the literature for SCID models. Exogenous estrogen tolerance is another common concern in oncology research as some immunodeficient mouse models cannot tolerate the subcutaneous estrogen pellets, developing negative secondary effects resulting in removal from study. We performed an estrogen pellet dose-response study using four doses of 60-day release 17-β estradiol pellets at 0.18, 0.36, 0.72, and 1.7 mg/pellet (n = 10 per group). R2G2 mice show dose-dependent effects of estrogen on morbidity. These data will allow researchers to determine the optimal dose for use in the R2G2 model. In conclusion, these data support that the R2G2 mouse model may be a good alternative to SCID models when administering DNA-damaging chemotherapies or when estrogen supplementation is required for xenograft growth. Citation Format: Jamie L. Naden, Michele Melton, Athena Bast, Jermaine Gardner. Chemotherapeutic tolerability and estrogen dose response in the B6;129-Rag2tm1FwaIL2rgtm1Rsky/DwlHsd (R2G2) mouse model [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 2157.

PO-217 Loss of Kmt2c methyltransferase activity contributes to prostate tumorigenesis in a mouse model of prostate cancer

IntroductionThe B6;129-Rag2tm1FwaIL2rgtm1Rsky/DwlHsd (R2G2) knockout mouse was developed by backcrossing an IL2rg (common gamma) knockout model to a RAG2 (recombinase activating gene) knockout model. The resulting mouse lacks various cytokines including IL-2, IL-4, IL-7, IL-9 and IL-15. In addition, this model lacks B cells, T cells, NK cells and has a deficit in lymphocyte development. This model was developed to provide another immunodeficient option for the oncology and immunology fields. The literature supports better tolerability of DNA damaging oncology treatments for models that do not carry the SCID mutation. We have already reported in a white paper that the R2G2 mouse model is more tolerant of whole body radiation than a model with the SCID mutation. Herein we describe a study examining chemotherapeutic tolerability of common DNA damaging oncology drugs including 5-fluorouracil (5-FU), doxorubicin (Doxo), and cyclophosphamide (CTX) (n=10 per group).Material and methods5-FU was given at 30, 60 or 100 mg/kg, intraperitoneally, twice weekly for five weeks. Doxo was given at 2 or 5 mg/kg, intraperitoneally, once weekly for three weeks. CTX was given at 100 or 140 mg/kg intraperitoneally, once weekly for three weeks. The low dose was chosen based on the average dose used in immunodeficient mouse models and the high dose was chosen based on the average dose used in immunocompetent mouse models. This was done to determine if the R2G2 mouse model can tolerate higher doses of these chemotherapeutic agents compared to other immunodeficient mouse models.Results and discussionsResults show that the R2G2 mouse model tolerates higher doses of these chemotherapeutic drugs than doses found in the literature for SCID models. Exogenous oestrogen tolerance is another common concern in oncology research as some immunodeficient mouse models cannot tolerate the subcutaneous oestrogen pellets, developing negative secondary effects resulting in removal from study. We performed an oestrogen pellet dose response study using four doses of 60 day release 17-β estradiol pellets at 0.18, 0.36, 0.72, and 1.7 mg/pellet (n=10 per group). R2G2 mice show dose dependent effects of oestrogen on morbidity. These data will allow researchers to determine the optimal dose for use in the R2G2 model.ConclusionIn conclusion, these data support that the R2G2 mouse model may be a good alternative to SCID models when administering DNA damaging chemotherapies or when oestrogen supplementation is required for xenograft growth.

PO-216 Chemotherapeutic tolerability and oestrogen dose response in the B6;129-Rag2tm1FwaIL2rgtm1Rsky/DwlHsd (R2G2) mouse model

Introduction The B6;129- Rag2 tm1Fwa IL2rg tm1Rsky /DwlHsd (R2G2) knockout mouse was developed by backcrossing an IL2rg (common gamma) knockout model to a RAG2 (recombinase activating gene) knockout model. The resulting mouse lacks various cytokines including IL-2, IL-4, IL-7, IL-9 and IL-15. In addition, this model lacks B cells, T cells, NK cells and has a deficit in lymphocyte development. This model was developed to provide another immunodeficient option for the oncology and immunology fields. The literature supports better tolerability of DNA damaging oncology treatments for models that do not carry the SCID mutation. We have already reported in a white paper that the R2G2 mouse model is more tolerant of whole body radiation than a model with the SCID mutation. Herein we describe a study examining chemotherapeutic tolerability of common DNA damaging oncology drugs including 5-fluorouracil (5-FU), doxorubicin (Doxo), and cyclophosphamide (CTX) (n=10 per group). Material and methods 5-FU was given at 30, 60 or 100 mg/kg, intraperitoneally, twice weekly for five weeks. Doxo was given at 2 or 5 mg/kg, intraperitoneally, once weekly for three weeks. CTX was given at 100 or 140 mg/kg intraperitoneally, once weekly for three weeks. The low dose was chosen based on the average dose used in immunodeficient mouse models and the high dose was chosen based on the average dose used in immunocompetent mouse models. This was done to determine if the R2G2 mouse model can tolerate higher doses of these chemotherapeutic agents compared to other immunodeficient mouse models. Results and discussions Results show that the R2G2 mouse model tolerates higher doses of these chemotherapeutic drugs than doses found in the literature for SCID models. Exogenous oestrogen tolerance is another common concern in oncology research as some immunodeficient mouse models cannot tolerate the subcutaneous oestrogen pellets, developing negative secondary effects resulting in removal from study. We performed an oestrogen pellet dose response study using four doses of 60 day release 17-β estradiol pellets at 0.18, 0.36, 0.72, and 1.7 mg/pellet (n=10 per group). R2G2 mice show dose dependent effects of oestrogen on morbidity. These data will allow researchers to determine the optimal dose for use in the R2G2 model. Conclusion In conclusion, these data support that the R2G2 mouse model may be a good alternative to SCID models when administering DNA damaging chemotherapies or when oestrogen supplementation is required for xenograft growth.

Chemotherapeutic tolerability and Estrogen dose response in the B6;129‐Rag2tm1FwaIL2rgtm1Rsky/DwlHsd (R2G2) mouse model

The B6;129‐Rag2tm1FwaIL2rgtm1Rsky/DwlHsd (R2G2) knockout mouse was developed by backcrossing an IL2rg (common gamma) knockout model to a RAG2 (recombinase activating gene) knockout model. The resulting mouse lacks various cytokines including IL‐2, IL‐4, IL‐7, IL‐9 and IL‐15. In addition, this model lacks B cells, T cells, NK cells and has a deficit in lymphocyte development. This model was developed to provide another immunodeficient option for the oncology and immunology fields. The literature supports better tolerability of DNA damaging oncology treatments for models that do not carry the SCID mutation. We have already reported in a white paper that the R2G2 mouse model is more tolerant of whole body radiation than a model with the SCID mutation. Herein we describe a study examining chemotherapeutic tolerability of common DNA damaging oncology drugs including 5‐fluorouracil (5‐FU), doxorubicin (Doxo), and cyclophosphamide (CTX) (n = 10 per group). 5‐FU was given at 30, 60 or 100 mg/kg, intraperitoneally, twice weekly for five weeks. Doxo was given at 2 or 5 mg/kg, intraperitoneally, once weekly for three weeks. CTX was given at 100 or 140 mg/kg intraperitoneally, once weekly for three weeks. The low dose was chosen based on the average dose used in immunodeficient mouse models and the high dose was chosen based on the average dose used in immunocompetent mouse models. This was done to determine if the R2G2 mouse model can tolerate higher doses of these chemotherapeutic agents compared to other immunodeficient mouse models. Results show that the R2G2 mouse model tolerates higher doses of these chemotherapeutic drugs than doses found in the literature for SCID models. Exogenous estrogen tolerance is another common concern in oncology research as some immunodeficient mouse models cannot tolerate the subcutaneous estrogen pellets, developing negative secondary effects resulting in removal from study. We performed an estrogen pellet dose response study using four doses of 60‐day release 17‐β estradiol pellets at 0.18, 0.36, 0.72, and 1.7 mg/pellet (n = 10 per group). R2G2 mice show dose dependent effects of estrogen on morbidity. These data will allow researchers to determine the optimal dose for use in the R2G2 model. In conclusion, these data support that the R2G2 mouse model may be a good alternative to SCID models when administering DNA damaging chemotherapies or when estrogen supplementation is required for xenograft growth.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Natural Killer Cells from Patients with Recombinase-Activating Gene and Non-Homologous End Joining Gene Defects Comprise a Higher Frequency of CD56bright NKG2A+++ Cells, and Yet Display Increased Degranulation and Higher Perforin Content.

Mutations of the recombinase-activating genes 1 and 2 (RAG1 and RAG2) in humans are associated with a broad range of phenotypes. For patients with severe clinical presentation, hematopoietic stem cell transplantation (HSCT) represents the only curative treatment; however, high rates of graft failure and incomplete immune reconstitution have been observed, especially after unconditioned haploidentical transplantation. Studies in mice have shown that Rag−/− natural killer (NK) cells have a mature phenotype, reduced fitness, and increased cytotoxicity. We aimed to analyze NK cell phenotype and function in patients with mutations in RAG and in non-homologous end joining (NHEJ) genes. Here, we provide evidence that NK cells from these patients have an immature phenotype, with significant expansion of CD56bright CD16−/int CD57− cells, yet increased degranulation and high perforin content. Correlation was observed between in vitro recombinase activity of the mutant proteins, NK cell abnormalities, and in vivo clinical phenotype. Addition of serotherapy in the conditioning regimen, with the aim of depleting the autologous NK cell compartment, may be important to facilitate engraftment and immune reconstitution in patients with RAG and NHEJ defects treated by HSCT.

Abstract 2812: Development and characterization of the ultra immunodeficient B6;129-Rag2 tm1Fwa IL2rg tm1Rsky/DwlHsd (R2G2) mouse model

Abstract The “R2G2” (B6;129-Rag2tm1FwaIL2rgtm1Rsky/DwlHsd) knockout mouse is the latest advancement to provide an alternative option in the highly immunodeficient mouse model category for the oncology, immunology and other biomedical research communities. This model was generated by backcrossing an IL2rg (common gamma) knockout model to a RAG2 (recombinase activating gene) knockout model. The resulting mouse lacks various cytokines including IL-2, IL-4, IL-7, IL-9 and IL-15. In addition, this model lacks B cells, T cells, NK cells and has a deficit in lymphocyte development. The R2G2 model is not only ultra immunodeficient, but provides a model that is less leaky and more tolerant to gamma radiation than traditional SCID models. This model is particularly useful for both tumor transplantation and humanization studies. Herein we describe the development and characterization of the R2G2 model, which includes breeding history, growth curve, CBC/Chemistry, flow cytometry and both huHSC and huPMBC humanization studies. Citation Format: Sheryl J. Wildt, Jamie McClellan, Mandy Horn. Development and characterization of the ultra immunodeficient B6;129-Rag2tm1FwaIL2rgtm1Rsky/DwlHsd (R2G2) mouse model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2812. doi:10.1158/1538-7445.AM2017-2812

Corrigendum: Natural Killer Cells from Patients with Recombinase-Activating Gene and Non-Homologous End Joining Gene Defects Comprise a Higher Frequency of CD56bright NKG2A+++ Cells, and Yet Display Increased Degranulation and Higher Perforin Content

[This corrects the article on p. 798 in vol. 8, PMID: 28769923.].

Innate Lymphoid Cells Are the Predominant Source of IL-17A during the Early Pathogenesis of Acute Respiratory Distress Syndrome.

IL-17A is purported to help drive early pathogenesis in acute respiratory distress syndrome (ARDS) by enhancing neutrophil recruitment. Although IL-17A is the archetypal cytokine of T-helper 17 cells, it is produced by a number of lymphocytes, the source during ARDS being unknown. To identify the cellular source and the role of IL-17A in the early phase of lung injury. Lung injury was induced in wild-type (C57BL/6) and IL-17 knockout (KO) mice with aerosolized LPS (100 μg) or Pseudomonas aeruginosa infection. Detailed phenotyping of the cells expressing RORγt, the transcriptional regulator of IL-17 production, in the mouse lung at 24 hours was performed by flow cytometry. A 100-fold reduction in neutrophil infiltration was observed in the lungs of the IL-17A KO compared with wild-type mice. The majority of RORγt(+) cells in the mouse lung were the recently identified group 3 innate lymphoid cells (ILC3s). Detailed characterization revealed these pulmonary ILC3s (pILC3s) to be discrete from those described in the gut. The critical role of these cells was verified by inducing injury in recombinase-activating gene 2 KO mice, which lack T cells but retain innate lymphoid cells. No amelioration of pathology was observed in the recombinase-activating gene 2 KO mice. IL-17 is rapidly produced during lung injury and significantly contributes to early immunopathogenesis. This is orchestrated largely by a distinct population of pILC3s. Modulation of the activity of pILC3s may potentiate early control of the inflammatory dysregulation seen in ARDS, opening up new therapeutic targets.

Recombinase Activating Gene 1 Deficiencies Without Omenn Syndrome May Also Present With Eosinophilia and Bone Marrow Fibrosis.

BackgroundSevere combined immunodeficiency (SCID) syndromes are a heterogenous group of diseases characterized by impairment in both cellular and humoral immunity with a range of genetic disorders. Complete recombinase activating gene (RAG) deficiency is associated with classical T-B-NK+ SCID which is the most common phenotype of Turkish SCID patients. There is a broad spectrum of hypomorfic RAG mutations including Omenn syndrome, leaky or atypical SCID with expansion of γδ T cells, autoimmunity and cytomegalovirus (CMV) infections.MethodsTwenty-one (44%) patients had RAG1 deficiency of all 44 SCID patients followed up by pediatric immunology department. A retrospective analysis was conducted on the medical records of all SCID patients with RAG1 deficiency.ResultsEight patients were classified as T-B-NK+ SCID, five patients as T+B-NK+ SCID (three of these were Omenn phenotype), and eight patients as T+B+NK+ SCID phenotype. Mean age of the whole study group, mean age at onset of symptoms and mean age at diagnosis were 87.7 ± 73.8 (12 - 256), 4.4 ± 8.2 (1 - 36) and 29.1 ± 56.8 (1 - 244) months, respectively. Consanguinity was present in 11 (52%) of 21 patients. Autoimmunity was found in six patients (28%). Ten patients (47%) had CMV infection, four (19%) had Epstein-Barr virus (EBV) infections and three (14%) had Bacillus Calmette-Guerin (BCG) infections. Seven patients who had refractory cytopenia (two pancytopenia and five bicytopenia) underwent bone marrow biopsy, three of whom had bone marrow fibrosis. Future evaluations must be considered about bone marrow fibrosis in RAG1 deficiency patients. Eosinophilia was observed in 10 patients, seven of whom did not have Omenn phenotype.ConclusionNon-Omenn phenotype RAG1 deficiencies can also present with eosinophilia. This report is presented to emphasize that RAG1 mutations may lead to diverse clinical phenotypes.

Anergic B Cells: Precarious On-Call Warriors at the Nexus of Autoimmunity and False-Flagged Pathogens.

A tenet of modern immunology is that the adaptive immune system has evolved so as to prevent, or at least diminish responses targeting self-antigens (1). Self-reactive B cells that arise due to incomplete negative selection in the bone marrow have been shown to be removed or inactivated in the periphery, with the most strongly self-reactive cells subject to clonal deletion. Less self-reactive cells either undergo receptor editing or are rendered anergic (2, 3). Receptor editing entails the reactivation of recombinase activating genes (RAGs) and enables immunoglobulin genes to be rearranged to create new antigen specificities. Anergy is a poorly understood state whereby cells retain the ability to bind to self-antigens but are otherwise rendered insensitive to antigenic stimulation (4). However, anergy is not a perfect solution to control self-reactivity. Many multifactorial autoimmune disorders involve the disruption of B cell anergy as a potential mechanism (5). One of the best known examples is systemic lupus erythematosus (SLE), where the inappropriate activation of anergic B cells and their differentiation into plasma cells that secrete autoreactive antibodies are an important contributing pathogenic mechanism. In particular, a large number of the autoantibodies in SLE is of the 9G4 idiotype (6). It has been shown that 9G4 idiotypic B cells are present and anergic in normal individuals, but actively expand into the plasma and memory cell compartments in SLE patients (7).

The effect of the muscle environment on the regenerative capacity of human skeletal muscle stem cells.

BackgroundMuscle stem cell transplantation is a possible treatment for muscular dystrophy. In addition to the intrinsic properties of the stem cells, the local and systemic environment plays an important role in determining the fate of the grafted cells. We therefore investigated the effect of modulating the host muscle environment in different ways (irradiation or cryoinjury or a combination of irradiation and cryoinjury) in two immunodeficient mouse strains (mdx nude and recombinase-activating gene (Rag)2-/γ chain-/C5-) on the regenerative capacity of two types of human skeletal muscle-derived stem cell (pericytes and CD133+ cells).MethodsHuman skeletal muscle-derived pericytes or CD133+ cells were transplanted into muscles of either mdx nude or recombinase-activating gene (Rag)2-/γ chain-/C5- host mice. Host muscles were modulated prior to donor cell transplantation by either irradiation, or cryoinjury, or a combination of irradiation and cryoinjury. Muscles were analysed four weeks after transplantation, by staining transverse cryostat sections of grafted muscles with antibodies to human lamin A/C, human spectrin, laminin and Pax 7. The number of nuclei and muscle fibres of donor origin and the number of satellite cells of both host and donor origin were quantified.ResultsWithin both host strains transplanted intra-muscularly with both donor cell types, there were significantly more nuclei and muscle fibres of donor origin in host muscles that had been modulated by cryoinjury, or irradiation+cryoinjury, than by irradiation alone. Irradiation has no additive effects in further enhancing the transplantation efficiency than cryodamage. Donor pericytes did not give rise to satellite cells. However, using CD133+ cells as donor cells, there were significantly more nuclei, muscle fibres, as well as satellite cells of donor origin in Rag2-/γ chain-/C5- mice than mdx nude mice, when the muscles were injured by either cryodamage or irradiation+cryodamage.ConclusionsRag2-/γ chain-/C5- mice are a better recipient mouse strain than mdx nude mice for human muscle stem cell transplantation. Cryodamage of host muscle is the most effective method to enhance the transplantation efficiency of human skeletal muscle stem cells. This study highlights the importance of modulating the muscle environment in preclinical studies to optimise the efficacy of transplanted stem cells.Electronic supplementary materialThe online version of this article (doi:10.1186/s13395-015-0036-8) contains supplementary material, which is available to authorized users.

Helicobacter cinaedi induced typhlocolitis in Rag-2-deficient mice.

Helicobacter cinaedi, an enterohepatic helicobacter species (EHS), is an important human pathogen and is associated with a wide range of diseases, especially in immunocompromised patients. It has been convincingly demonstrated that innate immune response to certain pathogenic enteric bacteria is sufficient to initiate colitis and colon carcinogenesis in recombinase-activating gene (Rag)-2-deficient mice model. To better understand the mechanisms of human IBD and its association with development of colon cancer, we investigated whether H.cinaedi could induce pathological changes noted with murine enterohepatic helicobacter infections in the Rag2(-/-) mouse model. Sixty 129SvEv Rag2(-/-) mice mouse were experimentally or sham infected orally with H.cinaedi strain CCUG 18818. Gastrointestinal pathology and immune responses in infected and control mice were analyzed at 3, 6 and 9months postinfection (MPI). H.cinaedi colonized the cecum, colon, and stomach in infected mice. H.cinaedi induced typhlocolitis in Rag2(-/-) mice by 3 MPI and intestinal lesions became more severe by 9 MPI. H.cinaedi was also associated with the elevation of proinflammatory cytokines, interferon-γ, tumor-necrosis factor-α, IL-1β, IL-10; iNOS mRNA levels were also upregulated in the cecum of infected mice. However, changes in IL-4, IL-6, Cox-2, and c-myc mRNA expressions were not detected. Our results indicated that the Rag2(-/-) mouse model will be useful to continue investigating the pathogenicity of H.cinaedi, and to study the association of host immune responses in IBD caused by EHS.