Human Severe Combined Immunodeficiency Research Articles

Zebrafish Model of Severe Combined Immunodeficiency (SCID) Due to JAK3 Mutation.

JAK3 is principally activated by members of the interleukin-2 receptor family and plays an essential role in lymphoid development, with inactivating JAK3 mutations causing autosomal-recessive severe combined immunodeficiency (SCID). This study aimed to generate an equivalent zebrafish model of SCID and to characterize the model across the life-course. Genome editing of zebrafish jak3 created mutants similar to those observed in human SCID. Homozygous jak3 mutants showed reduced embryonic T lymphopoiesis that continued through the larval stage and into adulthood, with B cell maturation and adult NK cells also reduced and neutrophils impacted. Mutant fish were susceptible to lymphoid leukemia. This model has many of the hallmarks of human SCID resulting from inactivating JAK3 mutations and will be useful for a variety of pre-clinical applications.

Lentivector cryptic splicing mediates increase in CD34+ clones expressing truncated HMGA2 in human X-linked severe combined immunodeficiency

X-linked Severe Combined Immunodeficiency (SCID-X1) due to IL2RG mutations is potentially fatal in infancy where ‘emergency’ life-saving stem cell transplant may only achieve incomplete immune reconstitution following transplant. Salvage therapy SCID-X1 patients over 2 years old (NCT01306019) is a non-randomized, open-label, phase I/II clinical trial for administration of lentiviral-transduced autologous hematopoietic stem cells following busulfan (6 mg/kg total) conditioning. The primary and secondary objectives assess efficacy in restoring immunity and safety by vector insertion site analysis (VISA). In this ongoing study (19 patients treated), we report VISA in blood lineages from first eight treated patients with longer follow up found a > 60-fold increase in frequency of forward-orientated VIS within intron 3 of the High Mobility Group AT-hook 2 gene. All eight patients demonstrated emergence of dominant HMGA2 VIS clones in progenitor and myeloid lineages, but without disturbance of hematopoiesis. Our molecular analysis demonstrated a cryptic splice site within the chicken β-globin hypersensitivity 4 insulator element in the vector generating truncated mRNA transcripts from many transcriptionally active gene containing forward-oriented intronic vector insert. A two base-pair change at the splice site within the lentiviral vector eliminated splicing activity while retaining vector functional capability. This highlights the importance of functional analysis of lentivectors for cryptic splicing for preclinical safety assessment and a redesign of clinical vectors to improve safety.

Transplantation of human cells into Interleukin-2 receptor gamma gene knockout pigs under several conditions.

IntroductionPreviously, we performed gene knockout (KO) of interleukin-2 receptor gamma (IL2RG) in porcine fetal fibroblasts using zinc finger nuclease-encoding mRNAs, subsequently generating IL2RG KO pigs using these cells through somatic cell nuclear transfer. The IL2RG KO pigs lacked a thymus and were deficient in T lymphocytes and natural killer cells, similar to human X-linked severe combined immunodeficiency (SCID) patients. The present study aimed to evaluate whether pigs can support the growth of xenografted human cells and have the potential to be an effective animal model.MethodsThe IL2RG XKOY pigs used in this study were obtained by mating IL2RG XKOX females with wild-type boars. This permitted the routine production of IL2RG KO pigs via natural breeding without complicated somatic cell cloning procedures; therefore, a sufficient number of pigs could be prepared. We transplanted human HeLa S3 cells expressing the tandem dimer tomato into the ears and pancreas of IL2RG KO pigs. Additionally, a newly developed method for the aseptic rearing of SCID pigs was used in case of necessity.ResultsTumors from the transplanted cells quickly developed in all pigs and were verified by histology and immunohistochemistry. We also transplanted these cells into the pancreas of designated pathogen-free pigs housed in novel biocontainment facilities, and large tumors were confirmed.ConclusionsIL2RG KO pigs have the potential to become useful animal models in a variety of translational biology fields.

CD3ε+ Cells in Pigs With Severe Combined Immunodeficiency Due to Defects in ARTEMIS.

Severe combined immunodeficiency (SCID) is described as the lack of functional T and B cells. In some cases, mutant genes encoding proteins involved in the process of VDJ recombination retain partial activity and are classified as hypomorphs. Hypomorphic activity in the products from these genes can function in the development of T and B cells and is referred to as a leaky phenotype in patients and animals diagnosed with SCID. We previously described two natural, single nucleotide variants in ARTEMIS (DCLR1EC) in a line of Yorkshire pigs that resulted in SCID. One allele contains a splice site mutation within intron 8 of the ARTEMIS gene (ART16), while the other mutation is within exon 10 that results in a premature stop codon (ART12). While initially characterized as SCID and lacking normal levels of circulating lymphoid cells, low levels of CD3ε+ cells can be detected in most SCID animals. Upon further assessment, we found that ART16/16, and ART12/12 SCID pigs had abnormally small populations of CD3ε+ cells, but not CD79α+ cells, in circulation and lymph nodes. Newborn pigs (0 days of age) had CD3ε+ cells within lymph nodes prior to any environmental exposure. CD3ε+ cells in SCID pigs appeared to have a skewed CD4α+CD8α+CD8β− T helper memory phenotype. Additionally, in some pigs, rearranged VDJ joints were detected in lymph node cells as probed by PCR amplification of TCRδ V5 and J1 genomic loci, as well as TCRβ V20 and J1.1, providing molecular evidence of residual Artemis activity. We additionally confirmed that TCRα and TCRδ constant region transcripts were expressed in the thymic and lymph node tissues of SCID pigs; although the expression pattern was abnormal compared to carrier animals. The leaky phenotype is important to characterize, as SCID pigs are an important tool for biomedical research and this additional phenotype may need to be considered. The pig model also provides a relevant model for hypomorphic human SCID patients.

Novel Engraftment and T Cell Differentiation of Human Hematopoietic Cells in ART-/-IL2RG-/Y SCID Pigs.

Pigs with severe combined immunodeficiency (SCID) are an emerging biomedical animal model. Swine are anatomically and physiologically more similar to humans than mice, making them an invaluable tool for preclinical regenerative medicine and cancer research. One essential step in further developing this model is the immunological humanization of SCID pigs. In this work we have generated T− B− NK− SCID pigs through site directed CRISPR/Cas9 mutagenesis of IL2RG within a naturally occurring DCLRE1C (ARTEMIS)−/− genetic background. We confirmed ART−/− IL2RG−/Y pigs lacked T, B, and NK cells in both peripheral blood and lymphoid tissues. Additionally, we successfully performed a bone marrow transplant on one ART−/− IL2RG−/Y male SCID pig with bone marrow from a complete swine leukocyte antigen (SLA) matched donor without conditioning to reconstitute porcine T and NK cells. Next, we performed in utero injections of cultured human CD34+ selected cord blood cells into the fetal ART−/− IL2RG−/Y SCID pigs. At birth, human CD45+ CD3ε+ cells were detected in cord and peripheral blood of in utero injected SCID piglets. Human leukocytes were also detected within the bone marrow, spleen, liver, thymus, and mesenteric lymph nodes of these animals. Taken together, we describe critical steps forwards the development of an immunologically humanized SCID pig model.

Severe combined immunodeficiency pig as an emerging animal model for human diseases and regenerative medicines

Severe combined immunodeficiency (SCID) is a group of inherited disorders characterized by compromised T lymphocyte differentiation related to abnormal development of other lymphocytes [i.e., B and/or natural killer (NK) cells], leading to death early in life unless treated immediately with hematopoietic stem cell transplant. Functional NK cells may impact engraftment success of life-saving procedures such as bone marrow transplantation in human SCID patients. Therefore, in animal models, a T cell−/B cell−/NK cell+ environment provides a valuable tool for understanding the function of the innate immune system and for developing targeted NK therapies against human immune diseases. In this review, we focus on underlying mechanisms of human SCID, recent progress in the development of SCID animal models, and utilization of SCID pig model in biomedical sciences. Numerous physiologies in pig are comparable to those in human such as immune system, X-linked heritability, typical T−B+NK− cellular phenotype, and anatomy. Due to analogous features of pig to those of human, studies have found that immunodeficient pig is the most appropriate model for human SCID.

Optimal titer of anti-HBs in blood components derived from donors with anti-HBc.

The ultimate strategy to cope with transfusion-transmitted hepatitis B virus (HBV) infection caused by transfusion of blood from donors with historical HBV infection is to reject all donors having anti-HBV core antigen (anti-HBc). However, this strategy would result in a huge loss of blood donors and subsequent blood inventory collapse. On the other hand, anti-HBc-positive blood is reportedly not infectious when containing more than 100 mIU/mL of anti-HBV surface antigen (anti-HBs). In Japan, anti-HBc-positive blood has been used for transfusion if it contained 200 mIU/mL or more of anti-HBs. First, to verify the screening policy, clinical outcomes for transfusion of such blood were analyzed for the 2008-2012 period. Second, human hepatocyte-repopulated severe combined immunodeficiency mice were inoculated with HBV preincubated with varying doses of anti-HBs, then viremic status was followed. The effects of anti-HBs across different HBV genotypes were also investigated. Twenty-three transfusion-transmitted HBV infections related to anti-HBc-positive blood components were identified. None of the blood responsible for these cases contained 200 mIU/mL or more of anti-HBs. When 100 μL of plasma containing 104 copies of HBV and 20 mIU of anti-HBs was injected into severe combined immunodeficiency mice, no viremia was detected within 13 weeks. Genotype C anti-HBs was capable of total inhibition of genotype A HBV replication, whereas genotype A anti-HBs inhibited genotype C HBV to a lesser extent. Anti-HBc-positive blood containing 200 mIU/mL or more of anti-HBs appears safe as a transfusion component. HBV vaccination seems effective between HBV genotypes A and C.

The R229Q mutation of Rag2 does not characterize severe immunodeficiency in mice

RAG1 or RAG2 mutations are associated with defects in V(D)J recombination activity, causing severe immunodeficiency with a wide spectrum of clinical phenotypes. A R229Q mutation of RAG2 was identified in patients with severe combined immunodeficiency (SCID) or Omenn syndrome (OS). Although some factors determining the clinical features between SCID and OS were not clear, the molecular mechanism of OS was studied in a mouse model in which an EGFP tag is fused to Rag2 with the R229Q mutation. To design the human disease model mimicking severe immunodeficiency, we generated Rag2-R229Q knock-in mice without an epitope tag. Mutant mice showed impaired T and B cell differentiation with reduced V(D)J recombination activity; however, the extent to which the R229Q mutation affects severe immunodeficiency was not severe. While Rag2-R229Q mutation under some conditions may cause severe immunological and clinical phenotypes similar to human SCID or OS, R229Q mutation per se did not cause severe immunodeficiency in mice, suggesting that additional factors other than R229Q mutation are required to induce severe immunodeficiency. Thus, our report implies that the effects of genetic background and/or a tagged protein sequence may alter the mouse immune system, revealing the mechanism of phenotypic heterogeneity arising from an identical mutation.

Bacterial and Pneumocystis Infections in the Lungs of Gene-Knockout Rabbits with Severe Combined Immunodeficiency.

Using the CRISPR/Cas9 gene-editing technology, we recently produced a number of rabbits with mutations in immune function genes, including FOXN1, PRKDC, RAG1, RAG2, and IL2RG. Seven founder knockout rabbits (F0) and three male IL2RG null (−/y) F1 animals demonstrated severe combined immunodeficiency (SCID), characterized by absence or pronounced hypoplasia of the thymus and splenic white pulp, and absence of immature and mature T and B-lymphocytes in peripheral blood. Complete blood count analysis showed severe leukopenia and lymphocytopenia accompanied by severe neutrophilia. Without prophylactic antibiotics, the SCID rabbits universally succumbed to lung infections following weaning. Pathology examination revealed severe heterophilic bronchopneumonia caused by Bordetella bronchiseptica in several animals, but a consistent feature of lung lesions in all animals was a severe interstitial pneumonia caused by Pneumocystis oryctolagi, as confirmed by histological examination and PCR analysis of Pneumocystis genes. The results of this study suggest that these SCID rabbits could serve as a useful model for human SCID to investigate the disease pathogenesis and the development of gene and drug therapies.

Preclinical modeling highlights the therapeutic potential of hematopoietic stem cell gene editing for correction of SCID-X1.

Targeted genome editing in hematopoietic stem/progenitor cells (HSPCs) is an attractive strategy for treating immunohematological diseases. However, the limited efficiency of homology-directed editing in primitive HSPCs constrains the yield of corrected cells and might affect the feasibility and safety of clinical translation. These concerns need to be addressed in stringent preclinical models and overcome by developing more efficient editing methods. We generated a humanized X-linked severe combined immunodeficiency (SCID-X1) mouse model and evaluated the efficacy and safety of hematopoietic reconstitution from limited input of functional HSPCs, establishing thresholds for full correction upon different types of conditioning. Unexpectedly, conditioning before HSPC infusion was required to protect the mice from lymphoma developing when transplanting small numbers of progenitors. We then designed a one-size-fits-all IL2RG (interleukin-2 receptor common γ-chain) gene correction strategy and, using the same reagents suitable for correction of human HSPC, validated the edited human gene in the disease model in vivo, providing evidence of targeted gene editing in mouse HSPCs and demonstrating the functionality of the IL2RG-edited lymphoid progeny. Finally, we optimized editing reagents and protocol for human HSPCs and attained the threshold of IL2RG editing in long-term repopulating cells predicted to safely rescue the disease, using clinically relevant HSPC sources and highly specific zinc finger nucleases or CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR-associated protein 9). Overall, our work establishes the rationale and guiding principles for clinical translation of SCID-X1 gene editing and provides a framework for developing gene correction for other diseases.

T Cell Lymphoma and Leukemia in Severe Combined Immunodeficiency Pigs following Bone Marrow Transplantation: A Case Report.

After the discovery of naturally occurring severe combined immunodeficiency (SCID) within a selection line of pigs at Iowa State University, we found two causative mutations in the Artemis gene: haplotype 12 (ART12) and haplotype 16 (ART16). Bone marrow transplants (BMTs) were performed to create genetically SCID and phenotypically immunocompetent breeding animals to establish a SCID colony for further characterization and research utilization. Of nine original BMT transfer recipients, only four achieved successful engraftment. At approximately 11 months of age, both animals homozygous for the ART16 mutation were diagnosed with T cell lymphoma. One of these ART16/ART16 recipients was a male who received a transplant from a female sibling; the tumors in this recipient consist primarily of Y chromosome-positive cells. The other ART16/ART16 animal also presented with leukemia in addition to T cell lymphoma, while one of the ART12/ART16 compound heterozygote recipients presented with a nephroblastoma at a similar age. Human Artemis SCID patients have reported cases of lymphoma associated with a “leaky” Artemis phenotype. The naturally occurring Artemis SCID pig offers a large animal model more similar to human SCID patients and may offer a naturally occurring cancer model and provides a valuable platform for therapy development.

SCID pigs: An emerging large animal NK model

Severe Combined ImmunoDeficiency (SCID) is defined as the lack or impairment of an adaptive immune system. Although SCID phenotypes are characteristically absent of T and B cells, many such SCID cellular profiles include the presence of NK cells. In human SCID patients, functional NK cells may impact the engraftment success of life saving procedures such as bone marrow transplantation. However, in animal models, a T cell-, B cell-, NK cell+ environment provides a valuable tool for asking specific questions about the extent of the innate immune system function as well as emerging NK targeted therapies against cancer. Physiologically and immunologically the pig is more similar to the human than common rodent research animals. This review discusses why the T- B- NK+ SCID pig may offer a more relevant model for development of human SCID patient therapies as well as provide an opportunity for systematic exploration of the role of NK cells in artiodactyl immunity.

The development of T cells from stem cells in mice and humans.

T cells develop from hematopoietic stem cells in the specialized microenvironment of the thymus. The main transcriptional players of T-cell differentiation such as Notch, Tcf-1, Gata3 and Bcl11b have been identified, but their role and regulation are not yet completely understood. In humans, functional experiments on T-cell development have traditionally been rather difficult to perform, but novel in vitro culture systems and in vivo xenograft models have allowed detailed studies on human T-cell development. Recent work has allowed the use of human severe combined immunodeficiency stem cells to unravel developmental checkpoints for human thymocyte development.

The functional relationship between hematopoietic stem cells and developing T lymphocytes

In contrast to all other blood and immune cells, T lymphocytes do not develop in the bone marrow (BM), but in the specialized microenvironment provided by the thymus. Similar to the other lineages, however, all T cells arise from multipotent hematopoietic stem cells (HSCs) that reside in the BM. Not all HSCs give rise to T cells; but how many and what kind of developmental checkpoints are located along this intricate differentiation path is the subject of intense research. Traditionally, this process has been studied almost exclusively using mouse cells, but recent advances in immunodeficient mouse models, high-speed cell sorting, lentiviral transduction protocols, and deep sequencing techniques have allowed these questions to be addressed using human cells. Here we review the process of thymic seeding by BM-derived cells and T cell commitment in humans, discussing recent insights into the clonal composition of the thymus and the definition of developmental checkpoints, on the basis of insights from human severe combined immunodeficiency patients.

Lymphopoiesis in transgenic mice over-expressing Artemis.

Artemis is a factor of the non-homologous end joining pathway involved in DNA double-strand break repair that has a critical role in V(D)J recombination. Mutations in DCLRE1C/ARTEMIS gene result in radiosensitive severe combined immunodeficiency in humans owing to a lack of mature T and B cells. Given the known drawbacks of allogeneic hematopoietic stem cell transplantation (HSCT), gene therapy appears as a promising alternative for these patients. However, the safety of an unregulated expression of Artemis has to be established. We developed a transgenic mouse model expressing human Artemis under the control of the strong CMV early enhancer/chicken beta actin promoter through knock-in at the ROSA26 locus to analyze this issue. Transgenic mice present a normal development, maturation and function of T and B cells with no signs of lymphopoietic malignancies for up to 15 months. These results suggest that the over-expression of Artemis in mice (up to 40 times) has no deleterious effects in early and mature lymphoid cells and support the safety of gene therapy as a possible curative treatment for Artemis-deficient patients.

AK2 deficiency compromises the mitochondrial energy metabolism required for differentiation of human neutrophil and lymphoid lineages.

Reticular dysgenesis is a human severe combined immunodeficiency that is primarily characterized by profound neutropenia and lymphopenia. The condition is caused by mutations in the adenylate kinase 2 (AK2) gene, resulting in the loss of mitochondrial AK2 protein expression. AK2 regulates the homeostasis of mitochondrial adenine nucleotides (ADP, ATP and AMP) by catalyzing the transfer of high-energy phosphate. Our present results demonstrate that AK2-knocked-down progenitor cells have poor proliferative and survival capacities and are blocked in their differentiation toward lymphoid and granulocyte lineages. We also observed that AK2 deficiency impaired mitochondrial function in general and oxidative phosphorylation in particular – showing that AK2 is critical in the control of energy metabolism. Loss of AK2 disrupts this regulation and leads to a profound block in lymphoid and myeloid cell differentiation.

Relieving oxidative stress in immune cells.

![Figure][1] Insight from Kamil Kranc (left) and Elaine Dzierzak (right) Reticular dysgenesis (RD) is the most severe form of human severe combined immunodeficiency (SCID). It is associated with mutations in adenylate kinase 2 (AK2), the mitochondrial intermembrane space enzyme that

Association between hepatitis B virus and MHC class I polypeptide-related chain A in human hepatocytes derived from human-mouse chimeric mouse liver

Due to the lack of efficient hepatitis B virus (HBV) infection systems, progress in understanding the role of innate immunity in HBV infection has remained challenging. Here we used human hepatocytes from a humanized severe combined immunodeficiency albumin promoter/enhancer driven-urokinase-type plasminogen activator mouse model for HBV infection. HBV DNA levels in culture medium from these human hepatocytes were 4.8–5.7 log IU/mL between day 16 and day 66 post-infection by HBV genotype C inoculum. HBV surface antigen (HBsAg) was also detected by chemiluminescent immunoassay from day 7 to day 66 post-infection. Western blot analysis revealed that major histocompatibility complex class I-related chain A (MICA), which plays a role in the innate immune system, was induced in HBV-infected human hepatocytes 27 days after infection compared with the uninfected control. MICA was reduced at day 62 and undetectable at day 90. Of interest, MICA expression by human hepatocytes increased after HBV infection and decreased before HBsAg loss. Human hepatocytes derived from chimeric mice with hepatocyte-humanized liver could support HBV genome replication. Further studies of the association between HBV replication and MICA induction should be conducted.

Establishment of the human acute promyelocytic leukemia pathological model in the SCID beige mice by using NB4 cell line

This study was to establish a stable, effective and reproducible human acute promyelocytic leukemia model in severe combined immunodeficient (SCID) mice by using NB4 cell line, and to investigate the disease course character and biological behaviors. Three-five-week-old SCID beige mice were divided randomly into two groups: experimental and control group. SCID mice of experimental group were transplanted by tail vein (iv) injection of 5×10(6) NB4 cells. The WBC cell count and the positive rate of promyelocytes in peripheral blood were dynamically monitored by using smears. Morphological examination and histopathological assay were employed to confirm NB4 cell infiltration in organs (liver, spleen, lung, kidney and brain). The expression level of PML-RARα fusion protein was detected by Western blot. Within two weeks there was no significant difference in peripheral blood WBC count between two groups (P > 0.05), meanwhile, NB4 cells were not found. At the day 21 and 28 after inoculation, the peripheral blood white blood cell count of experimental group reached to (4.79 ± 1.13)×10(9)/L and (7.62 ± 2.24)×10(9)/L respectively, which were significantly higher than that in control group (P < 0.05); simultaneously, the positive rates of promyelocytes on smears were (2.14 ± 0.63)% and (6.6 ± 2.76)%, respectively. Morphological observation showed single or multiple tumor lumps at day 21 after inoculation; HE staining of tissue biopsies demonstrated a large number of promyelocyte in the liver, spleen, lung, kidney and brain tissue. Cell immunofluorescence results showed that the CD33 expression of bone marrow cells in mice of experimental group was strongly positive (P < 0.05). Western blot confirmed that the PML-RARα fusion protein was expressed variously in liver, kidney and brain tissue. The human acute promyelocytic leukemia SCID mouse model is succesfully established by tail vein injection of NB4 cells. This model can mimic the characters of involved bone marrow and diffuse growth of cells. This model is a useful tool to explore the pathogenic mechanism and experimental treatment of human leukemia.

The expanding spectrum of human coronin 1A deficiency.

Since the first discovery of coronin in the amoeba Dictyostelium discoideum, remarkable insights have been gained regarding the structure and function of coronins, highly conserved from yeast to humans. It has been speculated that coronins have evolved from actin-binding molecules in lower eukaryotes to regulators of various cellular processes in mammals. Indeed, coronins are not only involved in cytokinesis, cell motility, and other actin-related processes but they are also implicated in immune homeostasis and calcium-calcineurin signaling. Most strikingly, coronin 1 deficiencies give rise to immune deficiencies in mice and humans that are characterized by severe T lymphocytopenia. Whereas complete absence of coronin 1A is associated with severe combined immunodeficiency in humans, hypomorphic mutations lead to a profound defect in naïve T cells, expansion of oligoclonal memory T cells, and exquisite susceptibility to EBV-associated B cell lymphoproliferation. Recent publications show that coronin 1A also plays a role in natural killer cell cytotoxic function and in neurobehavioral processes. It can be expected that future identification of coronin 1A-deficient patients will further extend the phenotypic spectrum thereby increasing our knowledge of this fascinating molecule.