RAG1 Deficiency Research Articles

Hypomorphic RAG2 Deficiency Promotes Selection of Self-Reactive B Cells

Reduced function or hypomorphic variants in recombination-activating genes (RAG) 1 or 2 result in a broad clinical phenotype including common variable immunodeficiency (CVID) and even adult-onset disease. Milder RAG variants are less characterized. Here we describe the longitudinal course of a milder combined RAG deficiency in 3 of 7 siblings sharing the same RAG2 mutations over a 50-year study. Whole-genome and repertoire sequencing, bacteriophage immunizations, and deep immunophenotyping were used to compare affected and unaffected family members. The clinical phenotype of three affected siblings with hypomorphic RAG deficiency ranged from combined immunodeficiency and early mortality to a late-onset CID with hyper-IgM phenotype. T cells were remarkably similar across affected siblings, yet CDR3 skewing and regulatory T cell defects were not observed. B cell analysis showed elevated unswitched CD27+ and CD21low cells as well as features of an autoreactive antibody repertoire and presence of secreted autoantibodies, yet no clinical autoimmunity was present. Most striking was an expanded polyclonal marginal zone-like B cell population (IgM+IgD+CD27+) utilizing the self-reactive unmutated VH4-34 receptor demonstrating that hypomorphic RAG deficiency can promote expansion of self-reactive B cells. This process, however, was not sufficient to trigger clinical autoimmunity. Utilizing multiple approaches, we functionally measured the specific RAG2 variant effects and assessed how selection and secondary triggers altered the BCR repertoire and immunophenotype overtime. Overall, we demonstrate a broad disease spectrum in siblings with identical hypomorphic RAG deficiency, highlighting that phenotypic divergence can result from expansion of IgM + memory B cells.

Assessment of non-myelotoxic agents as a preparatory regimen for hematopoietic stem cell gene therapy.

RAG2 deficiency is characterized by a lack of B and T lymphocytes, causing severe lethal infections. Currently, RAG2 deficiency is treated with a Hematopoietic Stem Cell transplantation (HSCT). Most conditioning regimens used before HSCT consist of alkylating myelotoxic agents with or without irradiation and affect growth and development of pediatric patients. Here, we developed a non-myelotoxic regimen using G-CSF, VLA-4I or AMD3100. These agents are known HSC mobilizers or affect bone marrow (BM) permeability and may support the homing of HSCs to the BM, without inducing major side effects. Female Rag2-/- mice were pre-treated with Busulfan (BU), G-CSF, VLA-4I or AMD3100 and transplanted with male BM cells transduced with a lentiviral vector carrying codon optimized human RAG2 (RAG2co). Peripheral blood cell counts increased significantly after G-CSF, VLA-4I and AMD3100 treatment, but not after BU. Reconstitution of PB lymphocytes was comparable for all groups with full immune reconstitution at 6months post transplantation, despite different methods of conditioning. Survival of mice pre-treated with non-myelotoxic agents was significantly higher than after BU treatment. Here, we show that the non-myelotoxic agents G-CSF, VLA-4I, and AMD3100 are highly effective as conditioning regimen before HSC gene therapy and can be used as an alternative to BU.

112 A Pediatric Case of RAG1 Deficiency Caused by Novel Variants: From Diagnosis to Bone Marrow Transplant and Immune Reconstitution

112 A Pediatric Case of RAG1 Deficiency Caused by Novel Variants: From Diagnosis to Bone Marrow Transplant and Immune Reconstitution

Abstract LB365: Generation of a novel severely immunodeficient Sprague Dawley rat model for xenograft studies

Abstract The recombinant-activating gene 2 (Rag2) plays a critical role in the recombination of the immunoglobulin and T cell receptor genes during the maturation of T and B cells. Loss of Rag2 protein leads to a lack of functional T and B cells, which eliminates the adaptive immune response. However, Rag2 deficiency alone may not guarantee the success of xenograft implantation, due to NK cells and other innate cells remaining. Il2rg is a key component of several cytokine receptors involved in differentiation and/or function of numerous adaptive and innate immune cells, including NK cells, mast cells, basophils, and dendritic cells. To generate a more severely immunodeficient rat model, both the Rag2 and Il2rg genes were knocked out in Sprague Dawley (SD) rats; this model was termed B-SDG rats. The immune profile and organ weights of thymus and spleen were investigated in B-SDG rats in comparison with Rag2-/- and wild-type SD rats. The spleen weights of both Rag2-/- and B-SDG rats were similar, but significantly reduced compared to wild-type SD rats. Notably, the thymus weights of B-SDG male rats were significantly smaller than those of Rag2-/- males. The immune cell profiles in thymus, spleen and blood were next evaluated by flow cytometry. A complete lack of B and T cells was observed in both Rag2-/- and B-SDG rats. B-SDG rats lacked CD161+ NK cells in the spleen and blood, which were maintained in Rag2-/- rats. Dendritic cells, monocytes, macrophages, and neutrophils were detected in all strains. In vivo xenograft implantation with human lung, bladder, breast, pancreas, acute monocytic and myelomonocytic leukemia tumor cell lines was next evaluated in B-SDG rats, which demonstrated successful engraftment. Furthermore, several efficacy studies were performed in B-SDG rats, including cisplatin chemotherapy on human breast invasive ductal carcinoma cell (HCC1954) and human large cell lung cancer cell (NCI-H460) xenografts, and a HER2-targeting ADC therapy was tested in B-SDG rats harboring human non-small cell lung cancer tumors (NCI-H1975). Taken together, these data confirm that B-SDG rats provide a powerful immunodeficient model for preclinical in vivo evaluation of novel drug candidates. Citation Format: Yanhui Nie, Meiqi Zhang, Wanling Zhong, Jia Feng, Bahetiyaer Huwatibieke, Zhaoxue Yu. Generation of a novel severely immunodeficient Sprague Dawley rat model for xenograft studies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB365.

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

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

Exonic knockout and knockin gene editing in hematopoietic stem and progenitor cells rescues RAG1 immunodeficiency.

Recombination activating genes (RAGs) are tightly regulated during lymphoid differentiation, and their mutations cause a spectrum of severe immunological disorders. Hematopoietic stem and progenitor cell (HSPC) transplantation is the treatment of choice but is limited by donor availability and toxicity. To overcome these issues, we developed gene editing strategies targeting a corrective sequence into the human RAG1 gene by homology-directed repair (HDR) and validated them by tailored two-dimensional, three-dimensional, and in vivo xenotransplant platforms to assess rescue of expression and function. Whereas integration into intron 1 of RAG1 achieved suboptimal correction, in-frame insertion into exon 2 drove physiologic human RAG1 expression and activity, allowing disruption of the dominant-negative effects of unrepaired hypomorphic alleles. Enhanced HDR-mediated gene editing enabled the correction of human RAG1 in HSPCs from patients with hypomorphic RAG1 mutations to overcome T and B cell differentiation blocks. Gene correction efficiency exceeded the minimal proportion of functional HSPCs required to rescue immunodeficiency in Rag1-/- mice, supporting the clinical translation of HSPC gene editing for the treatment of RAG1 deficiency.

Genetically corrected RAG2-SCID human hematopoietic stem cells restore V(D)J-recombinase and rescue lymphoid deficiency

AbstractRecombination-activating genes (RAG1 and RAG2) are critical for lymphoid cell development and function by initiating the variable (V), diversity (D), and joining (J) (V(D)J)-recombination process to generate polyclonal lymphocytes with broad antigen specificity. The clinical manifestations of defective RAG1/2 genes range from immune dysregulation to severe combined immunodeficiencies (SCIDs), causing life-threatening infections and death early in life without hematopoietic cell transplantation (HCT). Despite improvements, haploidentical HCT without myeloablative conditioning carries a high risk of graft failure and incomplete immune reconstitution. The RAG complex is only expressed during the G0-G1 phase of the cell cycle in the early stages of T- and B-cell development, underscoring that a direct gene correction might capture the precise temporal expression of the endogenous gene. Here, we report a feasibility study using the CRISPR/Cas9-based “universal gene-correction” approach for the RAG2 locus in human hematopoietic stem/progenitor cells (HSPCs) from healthy donors and RAG2-SCID patient. V(D)J-recombinase activity was restored after gene correction of RAG2-SCID-derived HSPCs, resulting in the development of T-cell receptor (TCR) αβ and γδ CD3+ cells and single-positive CD4+ and CD8+ lymphocytes. TCR repertoire analysis indicated a normal distribution of CDR3 length and preserved usage of the distal TRAV genes. We confirmed the in vivo rescue of B-cell development with normal immunoglobulin M surface expression and a significant decrease in CD56bright natural killer cells. Together, we provide specificity, toxicity, and efficacy data supporting the development of a gene-correction therapy to benefit RAG2-deficient patients.

Separating the Wheat From the Chaff in Asthma and Bronchiectasis: The Saga Trajectory of a Patient With Adult-Onset RAG1 Deficiency.

Separating the Wheat From the Chaff in Asthma and Bronchiectasis: The Saga Trajectory of a Patient With Adult-Onset RAG1 Deficiency.

AT 1 inhibition mediated neuroprotection after experimental traumatic brain injury is dependent on neutrophils in male mice

After traumatic brain injury (TBI) cerebral inflammation with invasion of neutrophils and lymphocytes is a crucial factor in the process of secondary brain damage. In TBI the intrinsic renin-angiotensin system is an important mediator of cerebral inflammation, as inhibition of the angiotensin II receptor type 1 (AT1) reduces secondary brain damage and the invasion of neutrophil granulocytes into injured cerebral tissue. The current study explored the involvement of immune cells in neuroprotection mediated by AT1 inhibition following experimental TBI. Four different cohorts of male mice were examined, investigating the effects of neutropenia (anti-Ly6G antibody mediated neutrophil depletion; C57BL/6), lymphopenia (RAG1 deficiency, RAG1−/−), and their combination with candesartan-mediated AT1 inhibition. The present results showed that reduction of neutrophils and lymphocytes, as well as AT1 inhibition in wild type and RAG1−/− mice, reduced brain damage and neuroinflammation after TBI. However, in neutropenic mice, candesartan did not have an effect. Interestingly, AT1 inhibition was found to be neuroprotective in RAG1−/− mice but not in neutropenic mice. The findings suggest that AT1 inhibition may exert neuroprotection by reducing the inflammation caused by neutrophils, ultimately leading to a decrease in their invasion into cerebral tissue.

Diffuse large B-cell lymphoma in 1-year male with founder Slavic RAG1 mutation: case report

Background and AimsLymphomas in RAG1 deficiency are extremely rare, especially in early childhood. Only a few cases of lymphoma in RAG1 deficiency have been previously described in the literature. MethodsSanger sequencing of Slavic founders mutation (RAG1-c.256_257del, p.Lys86ValfsTer33, IL7Rα - c.132C>G, p.Ser44Arg, NBN1 -c.657_661del5, p.Lys219AsnfsTer16 genes) in DNA of died children with malignancy in early age in Belarus. ResultsWe report a case of 14 month-old boy affected by diffuse large B-cell lymphoma diagnosed with RAG1 deficiency. A male patient of Belarus origin was born full-term from non-consanguineous parents. He had a history of recurrent pneumonia since 3 months of age. The patient was hospitalized in 14 month-old with subfebrile fever, wet cough, lymphadenopathy and cyanosis of the nasolabial triangle. Chest x-ray showed bilateral polysegmental pneumonia. Microbiological analysis of sputum found Candida. Ultrasound examination showed hepato-(+3 cm)-spleno- (+3 cm)-megaly and lymphadenopathy of multiple groups of lymph nodes (cervical, inguinal and axillary). Lymph-node, liver, and intestines biopsies were suggestive of EBV-positive Diffuse Large B-cell lymphoma, NOS. He had progressive leukopenia (from 3100 cells/µl to 200 cells/µl). Lymphopenia (400 cells/µl), decreased T-cells (300 cells/µl) and elevated activated T-cells (41%) were detected. B-cell percentage was in normal ranges (33%), but absolute number was slightly decreased (160 cells/µl). TRECs – 0 per 1 million leukocytes, KRECs – 0 per 1 million leukocytes. According to the clinical and immunological phenotype, combined immunodeficiency was suspected.The patient died (age 15 m) one month after the start of the chemotherapy cycle due to sepsis and multiple organ failure without genetic diagnosis. His material was studied post-mortem due to the lymphoma at early age and place of birth (Brest, Western Belarus). Targeted Sanger sequencing revealed a homozygous deletion in RAG1 gene (NM_000448.3) c.256_257del (p.Lys86ValfsTer33). ConclusionsWe described a rare case of EBV-positive diffuse large B-cell lymphoma NOS in a patient with homozygous founder Slavic mutation in the RAG1 gene.

High rate of molecular diagnosis among individuals with alopecia with known or suspected inborn errors of immunity

Background and aimsAlopecia is a multifactorial disorder causing hair loss of the scalp, face, and/or body. It affects an estimated 0.1–0.2% of individuals worldwide and can be visually stigmatizing and have significant quality of life consequences. While most cases of alopecia lack an identifiable genetic explanation, this condition is enriched in individuals with Mendelian inborn errors of immunity (IEI). The purpose of this study was to determine Mendelian contributions to alopecia in a mixed cohort of patients with suspected IEI. MethodsThe cohort was comprised of 3,072 unrelated participants with suspected IEI or related inflammatory disorders who underwent exome and/or genome sequencing from 2017–2022. From this pool, we identified 110 (3.58%) probands with alopecia. ResultsProbands were predominantly adult (mean 37.2 years, range 4–76), female (67%), and of European ancestry (64.5%). Alopecia was characterized as universalis (3/110, 2.7%), totalis (4/110, 3.6%), areata (5/110, 4.5%), scarring alopecia of the scalp (2/110, 1.8%), and other or not otherwise specified (96/100, 87.3%). Overall, 33/110 probands (30.0%) were found to have a Mendelian molecular diagnosis. Almost half of the participants with molecular diagnoses (15/33, 45%) had biallelic defects in AIRE, consistent with a molecular diagnosis of autoimmune polyendo-crinopathy-candidiasis-ectodermal dystrophy (APECED). In addition, 4/33 (12.1%) had a molecular diagnosis of STAT3-related hyper-IgE syndrome, 2/33 (6.1%) had STAT1 deficiency, and 2/33 (6.1%) had RAG1 deficiency. The remaining 10/33 (30.3%) included defects in the following genes: CXCR4, FAM111B, FOXN1, GATA2, IL2RG, GJB2, NFKB2, PIK3CD, PLCG2, and PSMB8. Multiple linear regression found that when controlling for sex and ancestry, younger age and greater phenotypic complexity was significantly associated with the likelihood of receiving a molecular diagnosis related to alopecia (OR = 0.93, p-value = 0.004 and OR = 1.27, p-value = 0.034, respectively). ConclusionsIn summary, a high proportion of individuals with alopecia in an IEI cohort received a molecular diagnosis via WES/WGS. Further, there was a small but significant association between younger age and higher phenotypic complexity and likelihood of receiving a molecular diagnosis. Most molecular diagnoses for alopecia in this cohort were disorders of the immune system, while two (hereditary fibrosing poikiloderma and keratitis-ichthyosis deafness syndrome) involved primarily non-immune etiologies.

Rare immune diseases paving the road for genome editing-based precision medicine.

Clustered regularly interspaced short palindromic repeats (CRISPR) genome editing platform heralds a new era of gene therapy. Innovative treatments for life-threatening monogenic diseases of the blood and immune system are transitioning from semi-random gene addition to precise modification of defective genes. As these therapies enter first-in-human clinical trials, their long-term safety and efficacy will inform the future generation of genome editing-based medicine. Here we discuss the significance of Inborn Errors of Immunity as disease prototypes for establishing and advancing precision medicine. We will review the feasibility of clustered regularly interspaced short palindromic repeats-based genome editing platforms to modify the DNA sequence of primary cells and describe two emerging genome editing approaches to treat RAG2 deficiency, a primary immunodeficiency, and FOXP3 deficiency, a primary immune regulatory disorder.

Cdkn2a inactivation promotes malignant transformation of mouse immature thymocytes before the β-selection checkpoint

CDKN2A deletion is the most frequent genetic alteration in T-cell acute lymphoblastic leukemia (T-ALL), occurring across all molecular and immunophenotypic subtypes. CDKN2A encodes two functionally unrelated tumor suppressor proteins, ARF and INK4a, critical regulators of cell cycle and proliferation. Arf has been reported to suppress T-ALL development in post-β-selection thymocytes, but whether CDKN2A acts as a tumor suppressor gene in immature, pre-β-selection thymocytes remains to be elucidated. Resorting to a Rag2-deficient model of T-ALL, driven by the ETV6::JAK2 fusion, we report that Cdkn2a haploinsufficiency at early stages of T-cell development facilitates leukemia development. ETV6::JAK2;Rag2-/-;Cdkn2a+/- T-ALL arose from pre-β-selection thymocytes, since the thymocyte differentiation arrest caused by Rag2 deficiency was maintained in pre-leukemic mice. Furthermore, somatic loss of the Cdkn2a wild-type allele was frequently observed in ETV6::JAK2;Rag2-/-;Cdkn2a+/- T-ALL cells, suggesting a selective advantage for total inactivation of Cdkn2a. Both Cdkn2a-sufficient and Cdkn2a-defective T-ALL presented additional genetic alterations, such as Notch1 mutations and gains of chromosomes 13 and 15. These data indicate that Cdkn2a acts as a gatekeeper for leukemogenesis from the most immature stages of thymocyte development.

Intracellular virus sensor MDA5 mutation develops autoimmune myocarditis and nephritis

Mutations in IFIH1 gene encoding viral RNA sensor MDA5 have been reported responsible for many interferonopathies, including Aicardi-Goutières syndrome (AGS) and monogenic lupus, however, the pathological link between IFIH1 mutations and various autoimmune symptoms remains unclear. Here, we generated transgenic mice expressing human MDA5 R779H mutant (R779H Tg), reported in AGS and monogenic lupus patient. Mice spontaneously developed myocarditis and nephritis with upregulation of type I IFNs in the major organs. R779H Tg Mavs−/− and R779H Tg Ifnar−/− showed no phenotypes, indicating direct MDA5-signaling pathway involvement. Rag-2 deficiency and bone marrow cells transfer from wild type to adult mice did not prevent myocarditis development, while mice with cardiomyocyte-specific expression of hMDA5 R779H showed cardiomegaly and high expression of inflammatory cytokines. Taken together, our study clarifies that type I IFNs production and chemokines from cardiomyocytes starts in neonatal period and is critical for the development of myocarditis. Activated lymphocytes and auto-antibodies exacerbate the pathogenesis but are dispensable for the onset.

Rag2 Deficiency Enhances Susceptibility to Systemic Mouse Adenovirus Type 1 Infection

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

Gene Editing Rescues In vitro T Cell Development of RAG2-Deficient Induced Pluripotent Stem Cells in an Artificial Thymic Organoid System.

Severe combined immune deficiency (SCID) caused by RAG1 or RAG2 deficiency is a genetically determined immune deficiency characterized by the virtual absence of T and B lymphocytes. Unless treated with hematopoietic stem cell transplantation (HSCT), patients with RAG deficiency succumb to severe infections early in life. However, HSCT carries the risk of graft-versus-host disease. Moreover, a high rate of graft failure and poor immune reconstitution have been reported after unconditioned HSCT. Expression of the RAG genes is tightly regulated, and preclinical attempts of gene therapy with heterologous promoters have led to controversial results. Using patient-derived induced pluripotent stem cells (iPSCs) and an in vitro artificial thymic organoid system as a model, here we demonstrate that gene editing rescues the progressive T cell differentiation potential of RAG2-deficient cells to normal levels, with generation of a diversified T cell repertoire. These results suggest that targeted gene editing may represent a novel therapeutic option for correction of this immunodeficiency.

Patterns of Immune Dysregulation in Primary Immunodeficiencies: A Systematic Review

Immune dysregulation is as important as susceptibility to infection in defining primary immunodeficiencies (PIDs). Because of the variability and nonspecificity of the symptoms of PIDs, diagnosis can be delayed-especially if a patient presents with immune dysregulation. Diagnosis is then based on certain combinations of symptoms and relies on the clinician's ability to recognize a pattern. So far there is no large report linking patterns of immune dysregulations to the underlying genetic defects. To identify immune dysregulatory patterns associated with PIDs and to help clinicians to detect an underlying PID in certain patients with noninfectious inflammatory diseases. A systematic literature review was performed. We included 186 articles that reported on n= 745 patients. The most common immune dysregulation category was "autoimmunity" (62%, n= 463), followed by "intestinal disease" (38%, n= 283) and "lymphoproliferation" (36%, n=268). Most patients (67%) had 1 or more symptoms of immune dysregulation. Autoimmune hemolytic anemia, the most common autoimmune phenotype, was most frequently reported in patients with LPS responsive beige-like anchor protein deficiency (when combined with hypogammaglobulinemia or gastrointestinal symptoms), activation-induced cytidine deaminase deficiency (when combined with autoimmune hepatitis), or RAG1 deficiency (when it was the only symptom of immune dysregulation). Eczema, allergies, and asthma were reported in 34%, 4%, and 4% of the patients, respectively. Patterns of immune dysregulation may help the physician to recognize specific PIDs. This systematic review provides clinicians with an overview to better assess patients with immune dysregulation.

In Vivo Analysis of Human Immune Responses in Immunodeficient Rats.

Humanized immune system immunodeficient mice have been extremely useful for the in vivo analyses of immune responses in a variety of models, including organ transplantation and graft versus host disease (GVHD) but they have limitations. Rat models are interesting complementary alternatives presenting advantages over mice, such as their size and their active complement compartment. Immunodeficient rats have been generated but human immune responses have not yet been described. We generated immunodeficient Rat Rag-/- Gamma chain-/- human signal regulatory protein alpha-positive (RRGS) rats combining Rag1 and Il2rg deficiency with the expression of human signal regulatory protein alpha, a negative regulator of macrophage phagocytosis allowing repression of rat macrophages by human CD47-positive cells. We then immune humanized RRGS animals with human peripheral blood mononuclear cells (hPBMCs) to set up a human acute GVHD model. Treatment of GVHD was done with a new porcine antihuman lymphocyte serum active through complement-dependent cytotoxicity. We also established a tumor xenograft rejection model in these hPBMCs immune system RRGS animals by subcutaneous implantation of a human tumor cell line. RRGS animals receiving hPBMCs showed robust and reproducible reconstitution, mainly by T and B cells. A dose-dependent acute GVHD process was observed with progressive weight loss, tissue damage, and death censoring. Antihuman lymphocyte serum (L1S1) antibody completely prevented acute GVHD. In the human tumor xenograft model, detectable tumors were rejected upon hPBMCs injection. hPBMC can be implanted in RRGS animals and elicit acute GVHD or rejection of human tumor cells and these are useful models to test new immunotherapies.

A Large Cohort of RAG1/2-Deficient SCID Patients-Clinical, Immunological, and Prognostic Analysis.

Severe combined immunodeficiency (SCID) is a fatal disorder resulting from various genetic defects. In the Middle East, where consanguineous marriage is prevalent, autosomal recessive mutations in recombination-activating genes (RAG) are a leading cause of SCID. We present a large cohort of SCID patients due to RAG1 or RAG2 mutations. Twenty-six patients with RAG1 or RAG2 deficiency, diagnosed at Sheba Medical Center, were retrospectively investigated. Clinical presentation, immunologic phenotype, genetic analysis, treatment, and outcome were analyzed. Majority of patients were referred from the Palestinian Authority. Most patients were males of Muslim Arab descent, 77% were born to consanguineous parents, and 65% had family history of immunodeficiency. Nearly all patients suffered from various infections before turning 2 months old, eight patients (31%) presented with Omenn and Omenn-like syndrome, and three patients (11%) had maternal engraftment. Notably, seven patients (27%) suffered from vaccine-derived infections, including a rare case of measles encephalitis. Nineteen patients underwent hematopoietic stem cell transplantation (HSCT) at a median age of 6 months, with a successful outcome for 72% of them. Genetic analysis revealed 11 different mutations (7 RAG2, 4 RAG1), two of them novel. Consanguineous marriages account for a genetic "founder effect." SCID is a pediatric emergency that dictates immediate precautions and curative treatment with HSCT. Due to lack of newborn screening for SCID within the Palestinian population, most patients in this cohort were diagnosed upon clinical symptoms, which led to a delayed diagnosis, harmful administration of contra-indicated live vaccines, delay in HSCT, and poor outcome.

RAG-2 deficiency results in fewer phosphorylated histone H2AX foci, but increased retinal ganglion cell death and altered axonal growth

DNA double-strand breaks (DSBs), selectively visualized as γ-H2AX+ foci, occur during the development of the central nervous system, including the retina, although their origin and biological significance are poorly understood. Mutant mice with DSB repair mechanism defects exhibit increased numbers of γ-H2AX+ foci, increased cell death during neural development, and alterations in axonogenesis in the embryonic retina. The aim of this study was to identify putative sources of DSBs. One of the identified DSBs sources is LINE-1 retrotransposition. While we did not detect changes in LINE-1 DNA content during the early period of cell death associated with retinal neurogenesis, retinal development was altered in mice lacking RAG-2, a component of the RAG-1,2-complex responsible for initiating somatic recombination in lymphocytes. Although γ-H2AX+ foci were less abundant in the rag2−/− mouse retina, retinal ganglion cell death was increased and axonal growth and navigation were impaired in the RAG-2 deficient mice, a phenotype shared with mutant mice with defective DNA repair mechanisms. These findings demonstrate that RAG-2 is necessary for proper retinal development, and suggest that both DSB generation and repair are genuine processes intrinsic to neural development.