Molecular form of adenosine deaminase in severe combined immunodeficiency

The specific activity of adenosine deaminase was reduced to approximately 0.5% of normal in splenic tissue obtained from a patient with severe combined immunodeficiency. Sedimentation analysis of splenic homogenate from this patient revealed a major peak of adenosine deaminase activity which corresponded with respect to the sedimentation coefficient of one of three molecular species observed in control spleens but had markedly reduced activity. These findings suggest that the molecular heterogeneity of human adenosine deaminase is under the control of a single genetic locus and that the deficiency of adenosine deaminase activity in severe combined immunodeficiency is not due to a genetic deletion.     

Adenosine deaminase deficiency and severe combined immunodeficiency disease.

The nature of the association of adenosine deaminase deficiency and severe combined immunodeficiency disease is reviewed. The basis for the molecular heterogeneity exhibited by adenosine deaminase in human tissue and the mechanisms whereby a deficiency of this activity results in the extreme perturbation of the immune system as observed in severe combined immunodeficiency are critically discussed.     

Defective thymocyte maturation in horses with severe combined immunodeficiency

Six monoclonal antibodies, designated EqT2, EqT3, EqT6, EqT7, EqT12, and EqT13, which identify T lymphocyte antigens present at different stages of T cell maturation were used to examine T lymphocyte development in foals with severe combined immunodeficiency (SCID). Flow microfluorimetry demonstrated the presence of EqT12+ and EqT13+ prothymocytes and a few phenotypically mature EqT2+ and EqT3+ thymocytes within the thymic remnants of SCID foals. However, very few EqT6+ and EqT7+ resident cortical thymocytes were detected. The near absence of EqT6+ and EqT7+ cortical thymocytes was confirmed by immunofluorescence analysis of thymic tissue from SCID foals. Those cells present were larger than normal cortical thymocytes. Furthermore, their activities of adenosine deaminase, adenosine monophosphate-deaminase, and 5' nucleotidase differed from those of normal cortical thymocytes. The combined evidence of monoclonal antibody analysis, size parameters, and purine enzyme activities demonstrate the near absence of cortical thymocytes in horses with this genetically defined immunodeficiency disorder.     

Metabolic defects in severe combined immunodeficiency in man and animals

Severe combined immunodeficiency (SCID) was originally thought to be one disease. Accumulating evidence indicates that SCID is a heterogeneous group of diseases that are clinically similar but are caused by quite different biochemical abnormalities. The best-studied form of SCID is that associated with an autosomal recessive inheritance pattern of adenosine deaminase (ADA) deficiency. Several biochemical mechanisms have been postulated to explain how a deficiency of ADA causes immune dysfunction. In forms of SCID not associated with ADA deficiency, other biochemical abnormalities have been detected. These abnormalities include deficiency in biotin-dependent carboxylases, alteration in lymphocyte surface membranes and irregularities in cytokine production. Two animal models for SCID now exist. Neither of these models is associated with ADA deficiency. Evidence for a possible defect in purine metabolism in one model has been demonstrated.     

Persistent rotavirus infection in mice with severe combined immunodeficiency.

Rotaviruses are important pathogens of human infants and the infants of many animal species. The disease produced by these viruses can be described as an acute, self-limiting diarrheal disease, with virus replication localized to the differentiated epithelial enterocytes of the small intestine. Immunologically normal infants shed virus for approximately 5 to 12 days after the onset of infection. Recently, it has been shown that rotavirus can produce a chronic infection in severely immunocompromised children, with virus shedding and intermittent diarrhea lasting from 6 weeks to 2 years (G. A. Losonsky, J. P. Johnson, J. A. Winkelstein, and R. H. Yolken, J. Clin. Invest. 76:2362-2367, 1985; F. T. Saulsbury, J. A. Winkelstein, and R. H. Yolken, J. Pediatr. 97:61-65, 1980). These findings point to an important role for the immune system in recovery from the disease. The study described here examined the outcome of murine rotavirus infection in mice with severe combined B- and T-cell immunodeficiency (SCID) and in immunologically normal seronegative BALB/c mice. Persistent rotavirus infection was established in all mice with SCID which had been inoculated orally as pups. Low levels of virus replication and constant fecal virus shedding characterized the chronic infection. This is the first report of a persistent rotavirus infection in an animal model.     

Human Taenia in severe combined immunodeficiency (SCID) mice

A rodent model for the development of the larval stages of human taeniid tapeworms would help advance immunodiagnosis in human and domestic animals and vaccine development for cysticercosis cellulosae or bovis in domestic animals. Here, Akira Ito and Mamoru Ito review recent results demonstrating the potential of the severe combined immunodeficiency (SCID) mouse for supporting development of the larval stages of Taenia saginata asiatica, T. saginata and T. solium.     

Retroviral producer cells cause sarcoma in severe combined immunodeficiency mice

Direct in situ introduction of retroviral producer cells might provide a form of treatment for localized tumors. A possible undesirable consequence of this treatment could be uncontrolled proliferation of the injected producer cells. To test this possibility, severe combined immunodeficiency (SCID) mice were reconstituted with human peripheral blood lymphocytes which were marked with a retroviral vector using a coculture method. Although specific measures were taken to remove the possible contaminating producer cells, a high percentage of mice developed fibrosarcoma 2–6 weeks after reconstitution. We hypothesized that tumors arose from a small number of contaminating producer cells in the inoculum. Tumor cells were consistently DNA tetraploid, a characteristic of the producer cell line. DNA extracted from tumor tissue was found to contain the gene (neomycin phosphotransferase) used to mark the producer cell line. Furthermore, SCID mice injected with 1 × 10⁴ producer cells developed tumors with analogous characteristics. This report indicates that the retroviral producer cell line is tumorigenic in immune-deficient animals.     

The function of antigen-presenting cells in mice with severe combined immunodeficiency

We have examined the antigen-presenting function of spleen cells in the C.B-17 scid mouse, a mutation that severely impairs the development of T and B lymphocytes. We show that antigen-presenting cells (APC) of SCID mice function normally in antigen-specific proliferative responses of primed T cells and in the antigen-specific activation of IL 2-producing T cell hybridomas. In both quantitative and qualitative terms, APC of SCID mice are equivalent to those of normal mice. These results indicate that the development and differentiation of APC function in vivo is independent of signals from mature, functional T or B lymphocytes.     

B-cell-negative severe combined immunodeficiency associated with a common γ chain mutation

Severe combined immunodeficiency (SCID) is caused by a variety of underlying defects. Approximately 40% of cases are thought to be of the X-linked type (SCIDX1), which is phenotypically characterised by the absence, or very low numbers, of T cells, but normal or even high B cell numbers. The gene responsible for SCIDX1 is that coding for the common γ chain (γ_c), a component of multiple cytokine receptors. Mutations in this gene have been demonstrated in a large number of boys affected by typical SCIDX1. We describe a sporadic case of a boy who had SCID with absent B cells and absent T cells, but in whom a mutation in the γ_c gene has been demonstrated. In the absence of a typical X-linked pedigree, the phenotype in this boy suggested an autosomal recessive form of SCID and the family would usually have been counselled accordingly. This family raises the question of the true frequency of SCIDX1 amongst sporadic male cases of SCID and highlights the need to screen these boys for γ chain mutations. Received: 4 December 1996     

A germline-competent embryonic stem cell line from NOD.Cg-Prkdc scid Il2rg tm1Wjl /SzJ (NSG) mice

The NOD.Cg-Prkdc ^scid Il2rg ^tm1Wjl /SzJ mouse strain, commonly known as NSG (for NOD SCID Gamma) is severely immunodeficient and thus is an excellent recipient for xenografts, and in particular for engrafting human tumor cells and human hematopoietic stem cells. In the latter case, these cells give rise to many human hematopoetic lineages in their NSG hosts, resulting in recapitulation of many of the features of a human immune system. However, the immune system of these ??humanized mice?? (huMice) is not completely functional, in part because of a lack of expression of necessary human cytokines and HLA molecules by NSG host tissues. In order to facilitate the genetic modification of this strain in order to improve the huMouse model, we have created germline competent ES cells of this strain in which such modifications can be carried out.     

Eradication of established hepatic human neuroblastoma metastases in mice with severe combined immunodeficiency by antibody-targeted interleukin-2

 A major problem in the treatment of solid tumors is the eradication of established, disseminated metastases. Here we describe an effective treatment for established experimental hepatic metastases of human neuroblastoma in C. B.-17 scid/scid mice. This was accomplished with an antibody-cytokine fusion protein, combining the unique targeting ability of antibodies with the multifunctional activity of cytokines. An anti-(ganglioside GD2) antibody (ch14.18) fusion protein with interleukin-2 (ch14.18-IL2), constructed by fusion of a synthetic sequence coding for human interleukin-2 (IL-2) to the carboxyl end of the Cγ1 gene of ch14.18, was tested for its therapeutic efficacy against xenografted human neuroblastoma in vivo. The ch14.18-IL2 fusion protein markedly inhibited growth of established hepatic metastases in SCID (severe combined immunodeficiency) mice previously reconstituted with human lymphokine-activated killer cells. Animals treated with ch14.18-IL2 showed an absence of macroscopic liver metastasis. In contrast, treatment with combinations of ch14.18 and recombinant IL2 at dose levels equivalent to the fusion protein only reduced the tumor load. Survival times of SCID mice treated with the fusion protein were more than double that of control animals. These results demonstrate that an immunotherapeutic approach using a cytokine targeted by an antibody to tumor sites is highly effective in eradicating the growth of established tumor metastases. Received: 7 November 1995 / Accepted: 15 December 1995     

Two mutational hotspots in the interleukin-2 receptor gamma chain gene causing human X-linked severe combined immunodeficiency.

Human severe combined immunodeficiency (SCID), a syndrome of profoundly impaired cellular and humoral immunity, is most commonly caused by mutations in the X-linked gene for interleukin-2 (IL-2) receptor gamma chain (IL2RG). For mutational analysis of IL2RG in males with SCID, SSCP screening was followed by DNA sequencing. Of 40 IL2RG mutations found in unrelated SCID patients, 6 were point mutations at the CpG dinucleotide at cDNA 690-691, encoding amino acid R226. This residue lies in the extracellular domain of the protein in a region not previously recognized to be significantly conserved in the cytokine receptor gene family, 11 amino acids upstream from the highly conserved WSXWS motif. Three additional instances of mutation at another CpG dinucleotide at cDNA 879 produced a premature termination signal in the intracellular domain of IL2RG, resulting in loss of the SH2-homologous intracellular domain known to be essential for signaling from the IL-2 receptor complex. Mutations at these two hotspots constitute > 20% of the X-linked SCID mutations found by our group and a similar proportion of all reported IL2RG mutations.     

X-linked severe combined immunodeficiency: from molecular cause to gene therapy within seven years

X-linked severe combined immunodeficiency (XSCID) is the most common form of SCID. The discovery of the genetic defect in this disease, namely mutations in the gene encoding the common cytokine receptor gamma chain, gammac, was reported just over seven years ago. In the subsequent period, a tremendous amount of knowledge about the biology and function of this protein has been generated. Moreover, gammac-knockout mice have been generated and their immune systems successfully reconstituted by gene therapy. Furthermore, initial attempts at using gene therapy to treat patients with XSCID have been successful for more than ten months, making this disease perhaps the most promising to date for treatment with such a strategy.     

Human peripheral blood lymphocyte severe combined immunodeficiency (hu-PBL SCID) models of toxoplasmosis

Toxoplasmosis is a potentially fatal opportunistic infection of immunocompromised hosts. Improved animal models of toxoplasmosis are needed to more nearly approximate conditions that occur in immunocompromised humans. The development of models of toxoplasmosis using human peripheral blood lymphocytes (hu-PBL) transplanted into severe combined immunodeficiency (SCID) mice is described here. Transplantation of hu-PBL into SCID mice without prior conditioning of the mice resulted in detectable differences in quantitative histological scores of brain inflammation due to Toxoplasma gondii infection, but did not alter mortality when compared to SCID mouse controls. The lack of detectable differences in survival were due to inadequate engraftment of hu-PBL, as assessed by flow cytometry. Unconditioned hu-PBL SCID mice had low titre T. gondii-specific antibody detectable after infection. When pretransplantation conditioning with irradiation and antiasialo GM 1 (n-glucolyl neuraminic acid) antibody was used, prolonged hu-PBL engraftment was observed in SCID mice, which was associated with worsened histopathology and usually impaired survival when compared with SCID mouse controls. When pretransplantation conditioning with irradiation, antiasialo GM antibody and polyethylene glycol-conjugated IL-2 was used, prolonged hu-PBL engraftment was also documented, but this did not affect survival from T. gondii infection when compared with similarly conditioned SCID mouse controls. The latter conditioning protocol resulted in hu-PBL SCID mice producing high titre T. gondii-specific antibody after infection. Conditioned hu-PBL SCID mice had evidence of increased T. gondii-induced inflammatory scores when compared with conditioned SCID mice. These models show promise for the study of the pathogenesis of toxoplasmosis and conditioned hu-PBL SCID mice may have applications for the evaluation of novel therapies for toxoplasmosis in immunocompromised humans.     

Phenotype and function of engrafted maternal T cells in patients with severe combined immunodeficiency

Engrafted maternal T cells from two patients with severe combined immunodeficiency (SCID) and graft-vs-host disease (GVHD) were characterized for surface phenotype, function, and ecto-5'-nucleotidase (ecto-5'-NT) activity. The majority of engrafted T cells from both patients were T6-, T3+, and Ia+; the ratio of T4+:T8+ cells varied from 0.89 to 3.1 for Patient 1 and was 0.17 for Patient 2. The sum of T4+ + T8+ cells was greater than the number of T3+ cells, and approximately one-third of the patients' T cells were T3-. Two-color immunofluorescent staining showed that one-third of the T cells from Patient 1 had a novel cell surface phenotype (T6-, T3-, T4+, T8+) that was not previously described. T cells from Patient 1 failed to proliferate in response to allogeneic cells or specific antigen and provided little help for PWM-driven Ig synthesis in vitro. However, they did suppress Ig synthesis in vitro and proliferate in response to PHA and Con A; thus they appeared to be more mature than the T cells of Patient 2 and of most previously reported patients with SCID and maternal T cell grafts. Both patients lacked detectable lymphocyte ecto-5'-NT activity, suggesting that either the ecto-5'-NT activity of maternal T cells is lost after engraftment or that a specific subset(s) of ecto-5'-NT-negative maternal T cells predominates in infants with SCID and GVHD. Thus, in vitro T cell function and the proportions of T cells bearing T4 and T8 may vary in SCID patients with maternal T cell grafts. However, the presence of the Ia antigen and the absence of ecto-5'-NT activity may be consistent features of activated maternal T cells responsible for GVHD.     

A severe form of human combined immunodeficiency due to mutations in DNA ligase IV

DNA ligase IV (LigIV) deficiency was identified as the molecular basis for a severe form of combined immunodeficiency in two microcephalic siblings with cellular radiosensitivity. In one patient the diagnosis was made directly after birth, allowing analysis of the role of LigIV in the development of specific immune cells. Absolute numbers of B cells were reduced 100-fold and alphabeta T cells 10-fold, whereas gammadelta T cells were normal. Spectratyping of all three cell populations showed a diverse repertoire, but sequencing of IgH V(D)J junctions revealed shorter CDR3 regions due to more extensive nucleotide deletions among D and J elements and fewer N nucleotide insertions. Clonal restriction of IgG-expressing, but not IgM-expressing, B cells and the lack of primary and secondary lymph node follicles indicated impaired class switch recombination. Observations in the older sibling showed that this rudimentary immune system was able to mount specific responses to infection. However, partial Ab responses and extensive amplification of gammadelta T cells could not prevent a life-threatening course of viral and bacterial infections, the development of an EBV-induced lymphoma, and immune dysregulation reflected by severe autoimmune cytopenia. Impaired generation of immune diversity under conditions of limited LigIV activity can cause a human SCID variant with a characteristic immunological phenotype.