Combined Immunodeficiency Patients Research Articles

Integration of retroviral vectors induces minor changes in the transcriptional activity of T cells from ADA-SCID patients treated with gene therapy

AbstractGene transfer into hematopoietic stem cells by γ-retroviral vectors (RVs) is an effective treatment for inherited blood disorders, although potentially limited by the risk of insertional mutagenesis. We evaluated the genomic impact of RV integration in T lymphocytes from adenosine deaminase-deficient severe combined immunodeficiency (ADA-SCID) patients 10 to 30 months after infusion of autologous, genetically corrected CD34+ cells. Expression profiling on ex vivo T-cell bulk population revealed no difference with respect to healthy controls. To assess the effect of vector integration on gene expression at the single-cell level, primary T-cell clones were isolated from 2 patients. T-cell clones harbored either 1 (89.8%) or 2 (10.2%) vector copies per cell and displayed partial to full correction of ADA expression, purine metabolism, and T-cell receptor-driven functions. Analysis of RV integration sites indicated a high diversity in T-cell origin, consistently with the polyclonal T-cell receptor-Vβ repertoire. Quantitative transcript analysis of 120 genes within a 200-kb window around RV integration sites showed modest (2.8- to 5.2-fold) dysregulation of 5.8% genes in 18.6% of the T-cell clones compared with controls. Nonetheless, affected clones maintained a stable phenotype and normal in vitro functions. These results confirm that RV-mediated gene transfer for ADA-SCID is safe, and provide crucial information for the development of future gene therapy protocols. The trials described herein have been registered at http://www.clinicaltrials.gov as #NCT00598481 and #NCT00599781.

ADA-deficient SCID is associated with a specific microenvironment and bone phenotype characterized by RANKL/OPG imbalance and osteoblast insufficiency

AbstractAdenosine deaminase (ADA) deficiency is a disorder of the purine metabolism leading to combined immunodeficiency and systemic alterations, including skeletal abnormalities. We report that ADA deficiency in mice causes a specific bone phenotype characterized by alterations of structural properties and impaired mechanical competence. These alterations are the combined result of an imbalanced receptor activator of nuclear factor-κB ligand (RANKL)/osteoprotegerin axis, causing decreased osteoclastogenesis and an intrinsic defect of osteoblast function with subsequent low bone formation. In vitro, osteoblasts lacking ADA displayed an altered transcriptional profile and growth reduction. Furthermore, the bone marrow microenvironment of ADA-deficient mice showed a reduced capacity to support in vitro and in vivo hematopoiesis. Treatment of ADA-deficient neonatal mice with enzyme replacement therapy, bone marrow transplantation, or gene therapy resulted in full recovery of the altered bone parameters. Remarkably, untreated ADA–severe combined immunodeficiency patients showed a similar imbalance in RANKL/osteoprotegerin levels alongside severe growth retardation. Gene therapy with ADA-transduced hematopoietic stem cells increased serum RANKL levels and children's growth. Our results indicate that the ADA metabolism represents a crucial modulatory factor of bone cell activities and remodeling. The trials were registered at www.clinicaltrials.gov as #NCT00598481 and #NCT00599781.

Identification of a high incidence region for retroviral vector integration near exon 1 of the LMO2locus

Therapeutic retroviral vector integration near the oncogene LMO2 is thought to be a cause of leukemia in X-SCID gene therapy trials. However, no published studies have evaluated the frequency of vector integrations near exon 1 of the LMO2 locus. We identified a high incidence region (HIR) of vector integration using PCR techniques in the upstream region close to the LMO2 transcription start site in the TPA-Mat T cell line. The integration frequency of the HIR was one per 4.46 × 104 cells. This HIR was also found in Jurkat T cells but was absent from HeLa cells. Furthermore, using human cord blood-derived CD34+ cells we identified a HIR in a similar region as the TPA-Mat T cell line. One of the X-linked severe combined immunodeficiency (X-SCID) patients that developed leukemia after gene therapy had a vector integration site in this HIR. Therefore, the descriptions of the location and the integration frequency of the HIR presented here may help us to better understand vector-induced leukemogenesis.

Expression of the leukemia oncogene Lmo2 is controlled by an array of tissue-specific elements dispersed over 100 kb and bound by Tal1/Lmo2, Ets, and Gata factors

AbstractThe Lmo2 gene encodes a transcriptional cofactor critical for the development of hematopoietic stem cells. Ectopic LMO2 expression causes leukemia in T-cell acute lymphoblastic leukemia (T-ALL) patients and severe combined immunodeficiency patients undergoing retroviral gene therapy. Tightly controlled Lmo2 expression is therefore essential, yet no comprehensive analysis of Lmo2 regulation has been published so far. By comparative genomics, we identified 17 highly conserved noncoding elements, 9 of which revealed specific acetylation marks in chromatin-immunoprecipitation and microarray (ChIP-chip) assays performed across 250 kb of the Lmo2 locus in 11 cell types covering different stages of hematopoietic differentiation. All candidate regulatory regions were tested in transgenic mice. An extended LMO2 proximal promoter fragment displayed strong endothelial activity, while the distal promoter showed weak forebrain activity. Eight of the 15 distal candidate elements functioned as enhancers, which together recapitulated the full expression pattern of Lmo2, directing expression to endothelium, hematopoietic cells, tail, and forebrain. Interestingly, distinct combinations of specific distal regulatory elements were required to extend endothelial activity of the LMO2 promoter to yolk sac or fetal liver hematopoietic cells. Finally, Sfpi1/Pu.1, Fli1, Gata2, Tal1/Scl, and Lmo2 were shown to bind to and transactivate Lmo2 hematopoietic enhancers, thus identifying key upstream regulators and positioning Lmo2 within hematopoietic regulatory networks.

Murine Leukemias with Retroviral Insertions at Lmo2 Are Predictive of the Leukemias Induced in SCID-X1 Patients Following Retroviral Gene Therapy

Five X-linked severe combined immunodeficiency patients (SCID-X1) successfully treated with autologous bone marrow stem cells infected ex vivo with an IL2RG-containing retrovirus subsequently developed T-cell leukemia and four contained insertional mutations at LMO2. Genetic evidence also suggests a role for IL2RG in tumor formation, although this remains controversial. Here, we show that the genes and signaling pathways deregulated in murine leukemias with retroviral insertions at Lmo2 are similar to those deregulated in human leukemias with high LMO2 expression and are highly predictive of the leukemias induced in SCID-X1 patients. We also provide additional evidence supporting the notion that IL2RG and LMO2 cooperate in leukemia induction but are not sufficient and require additional cooperating mutations. The highly concordant nature of the genetic events giving rise to mouse and human leukemias with mutations at Lmo2 are an encouraging sign to those wanting to use mice to model human cancer and may help in designing safer methods for retroviral gene therapy.

Outcomes of 48 Severe Combined Immunodeficiency Patients Treated with Hematopoietic Stem Cell Transplantation at a Single Center Between 1981 and 2007

Outcomes of 48 Severe Combined Immunodeficiency Patients Treated with Hematopoietic Stem Cell Transplantation at a Single Center Between 1981 and 2007

The actin regulator coronin 1A is mutant in a thymic egress-deficient mouse strain and in a patient with severe combined immunodeficiency.

Mice carrying the recessive peripheral T cell deficiency (Ptcd) locus have a block in thymic egress but the mechanism responsible is undefined. Here we found that Ptcd T cells have an intrinsic migration defect, impaired lymphoid tissue trafficking and irregularly shaped protrusions. Characterization of the Ptcd locus revealed an E26K point mutation within the actin regulator coronin-1A (Coro1a) that enhanced its inhibition of the actin regulator Arp2/3 and resulted in its mislocalization from the leading edge of migrating T cells. Discovery of another Coro1a mutant during an N-ethyl-N-nitrosourea (ENU) mutagenesis screen for T cell lymphopenic mice prompted us to evaluate a T cell-deficient, B cell- and NK cell-sufficient (T−B+NK+) severe combined immunodeficiency (SCID) patient, whom we found had mutations in both CORO1A alleles. These findings establish a role for coronin-1A in T cell egress, identify a surface of coronin involved in Arp2/3 regulation, and reveal actin regulation as a biological process defective in human and mouse SCID.

Recognition of Epstein—Barr virus (EBV)-infected cells by T cell colonies from a human chemera: restriction by allogeneic determinants

SUMMARYThe anti-EBV T cell response was studied in a severe combined immunodeficiency patient (PS) who received two transplants of fetal liver cells. His peripheral blood mononuclear cells (PBMC) were incubated with EBV and cultured during 15 days. Eleven colonies were derived from the T lymphocytes causing the regression of the infected cell foci: nine were constituted with CD3+CD4+CD8− lymphocytes and two with CD3+CD4−CD8+ cells. HLA typing of six colonies showed that two of them derived from the first transplant and four from the second one. The colonies killed the cells of the lymphoblastoid line (LCL) derived from the recipient (PS-LCL), but failed to kill the LCL matched with the transplants. With only one exception, they all lysed also the LCL derived from the mother or from the father, but they were ineffective on the EBV-negative lymphoblasts. Two colonies recognized determinants which did not appear to be HLA antigens, although they were shared by PS and by one of his parents, two (CD4−CD8+) reacted against the LCL which shared HLA-A3 or -A33 with PS-LCL, and four (CD4+CD8−) lysed the LCL sharing HLA-A3, -A33 or -DR5 with PS-LCL, among which only one was demonstrated to interact directly with host HLA-class I determinants. These data indicate that T lymphocytes differentiating in contact with histo-incompatible determinants may express the capability to recognize viral antigens and to lyse virus-infected cells in the context of allogeneic MHC or non-MHC molecules.

Value of bronchoalveolar lavage before haematopoietic stem cell transplantation for primary immunodeficiency or autoimmune diseases

Pulmonary infection, often insidious, is frequent in primary immunodeficiency (PID) and acquired immunodeficiency. Pulmonary complications are serious obstacles to success of haematopoietic SCT (HSCT) for these conditions. Bronchoalveolar lavage (BAL) permits identification of lower respiratory tract pathogens that may direct specific treatment and influence prognosis. There are no reports about the utility of pre-HSCT BAL for immunodeficient patients. We prospectively studied the value of 'routine' BAL before commencing transplantation in patients undergoing HSCT for severe immunological disease. Routine non-bronchoscopic BAL was performed under general anaesthetic, a few days before commencing pre-HSCT cytoreductive chemotherapy. Patients were categorized as symptomatic or asymptomatic with respect to pulmonary disease or infection. Samples were sent for microbiological processing. Complications arising from the procedure, pathogens isolated and treatments instituted were recorded. Results were available from 69/75 patients transplanted during the study period; 26 (38%) had pathogens identified (six asymptomatic patients), 10 (14.5%) developed complications post-procedure (two asymptomatic patients)-all recovered, 21 had management changes. There was no statistically significant difference in the number of positive isolates from severe combined or other immunodeficient patients, or of symptomatic or asymptomatic patients. Routine non-bronchoscopic BAL is safe in immunodeficient patients about to undergo HSCT, and leads to management changes.

Anti-CD25 Targeted Killing of Bicistronically Transduced Cells: A Novel Safety Mechanism Against Retroviral Genotoxicity

Gene therapy for Fabry disease, a deficiency in alpha-galactosidase A (alpha-gal A) activity, has the potential to provide a cure for the disorder with a single treatment. Despite modifications to existing vectors, concerns have arisen regarding the risk of genotoxicity associated with the use of retroviruses. To address safety concerns, we propose that expression of a cell surface protein, human CD25 (huCD25) in a bicistronic format, with any therapeutic gene such as alpha-gal A can provide a target that can be used to kill transduced cells selectively should transformative events occur. We show that an anti-CD25 antibody and immunotoxin can specifically target and eliminate transduced leukemia cells expressing CD25. In a murine leukemia model, antibody treatment reduced tumor burden 32-fold and increased survival compared with untreated mice. Furthermore, after a bone marrow transplant of therapeutically transduced cells into Fabry mice, antibody treatment reduced the number of retrovirally transduced huCD25-expressing cells in the peripheral blood. A systemic loss of transduced cells with functional consequences was also evident in the liver and spleen. This proof-of-principle study demonstrates that a targeted antibody can reduce tumor burden and selectively clear bicistronically transduced hematopoietic cells that express a target antigen, thus acting as a built-in safety mechanism.

Transplantation of mesenchymal stem cells to enhance engraftment of hematopoietic stem cells

Seven patients underwent treatment with mesenchymal stem cells (MSCs), together with allogeneic hematopoietic stem cell transplantation (HSCT). MSCs were given to three patients for graft failure and four patients were included in a pilot study. HSCT donors were three human leukocyte antigen (HLA)-identical siblings, three unrelated donors and one cord blood unit. The conditioning was myeloablative in four patients and reduced in three patients. MSC donors were HLA-identical siblings in three cases and haploidentical in four cases. Neutrophil counts >0.5 x 10(9)/l was reached at a median of 12 (range 10-28) days. Platelet counts >30 x 10(9)/l was achieved at a median of 12 (8-36) days. Acute graft-versus-host disease (GVHD) grade 0-I was seen in five patients. Two patients developed grade II, which in one patient evolved into chronic GVHD. One severe combined immunodeficiency (SCID) patient died of aspergillosis, the others are alive and well. One patient, diagnosed with aplastic anemia had graft failure after her first transplantation and severe Henoch-Schönlein Purpura (HSP). After retransplantation of MSCs and HSCs, she recovered from both the HSP and aplasia. Thus, co-transplantation of MSC resulted in fast engraftment of absolute neutrophil count (ANC) and platelets and 100% donor chimerism, even in three patients regrafted for graft failure/rejection.

Anti-CD25 Targeted Killing of Bicistronically Transduced Cells: A Novel Safety Mechanism Against Retroviral Genotoxicity

Anti-CD25 Targeted Killing of Bicistronically Transduced Cells: A Novel Safety Mechanism Against Retroviral Genotoxicity

Receptor specific downregulation of cytokine signaling by autophosphorylation in the FERM domain of Jak2

The tyrosine kinase, Janus kinase-2 (Jak2), plays a pivotal role in signal transduction through a variety of cytokine receptors, including the receptor for erythropoietin (Epo). Although the physiological relevance of Jak2 has been definitively established, less is known about its regulation. In studies assessing the roles of sites of tyrosine phosphorylation, we identified Y(119) in the FERM (band 4.1, Ezrin, radixin and moesin) domain as a phosphorylation site. In these studies, we demonstrate that the phosphorylation of Y(119) in response to Epo downregulates Jak2 kinase activity. Using a phosphorylation mimic mutation (Y(119)E), downregulation is shown to involve dissociation of Jak2 from the receptor complex. Conversely, a Y(119)F mutant is more stably associated with the receptor complex. Thus, in cytokine responses, ligand binding induces activation of receptor associated Jak2, autophosphorylation of Y(119) in the FERM domain and the subsequent dissociation of the activated Jak2 from the receptor and degradation. This regulation occurs with the receptors for Epo, thrombopoietin and growth hormone but not with the receptor for interferon-gamma.

DNA-PK autophosphorylation facilitates Artemis endonuclease activity

The Artemis nuclease is defective in radiosensitive severe combined immunodeficiency patients and is required for the repair of a subset of ionising radiation induced DNA double-strand breaks (DSBs) in an ATM and DNA-PK dependent process. Here, we show that Artemis phosphorylation by ATM and DNA-PK in vitro is primarily attributable to S503, S516 and S645 and demonstrate ATM dependent phosphorylation at serine 645 in vivo. However, analysis of multisite phosphorylation mutants of Artemis demonstrates that Artemis phosphorylation is dispensable for endonuclease activity in vitro and for DSB repair and V(D)J recombination in vivo. Importantly, DNA-dependent protein kinase catalytic subunit (DNA-PKcs) autophosphorylation at the T2609-T2647 cluster, in the presence of Ku and target DNA, is required for Artemis-mediated endonuclease activity. Moreover, autophosphorylated DNA-PKcs stably associates with Ku-bound DNA with large single-stranded overhangs until overhang cleavage by Artemis. We propose that autophosphorylation triggers conformational changes in DNA-PK that enhance Artemis cleavage at single-strand to double-strand DNA junctions. These findings demonstrate that DNA-PK autophosphorylation regulates Artemis access to DNA ends, providing insight into the mechanism of Artemis mediated DNA end processing.

Re-Examining the Role of IL-7 in Human B Lymphopoiesis.

The role of IL-7 in human B-cell development is still not well understood in contrast to its role in murine B-cell development. Adult mice with targeted disruptions in the IL-7 receptor (R) alpha chain or IL-7 genes completely lack B-lineage cells, while human severe combined immunodeficiency (SCID) patients with mutations in the IL-7R alpha chain have normal numbers of circulating B-lymphocytes. The goal of this study was to re-examine the functional consequences of IL-7R signaling in human B-cell precursors. This was accomplished using a xenogeneic culture system consisting of human CD34+ cord blood hematopoietic stem cells (HSC) plated on the MS-5 murine stromal cell line supplemented with G-CSF and SCF. Following the robust development of monocyte-like precursors and cells encompassing the later stages of granulocyte differentiation by 2 weeks, cells that emerged at 2.5–4 weeks were phenotypically pro-B cells (CD19+/CD10+/CD20lo/CD22+/CD24+/CD34-/pre-BCR-). The pro-B cells harbored functional heavy chain rearrangements, since re-plating them on MS-5 stromal cells supplemented with anti-CD40 and IL-4 led to the development of pre-BCR+ cells. CD19+ cells purified from HSC/MS-5 cultures underwent rapid cell death within 24 hours, and IL-7 had minimal effect on survival. Purified CD19+ cells re-plated in direct contact with MS-5 stromal cells survived for 7–10 days, but did not proliferate. However, addition of human (h) IL-7 from 0.01 to 10 ng/mL promoted dose-dependent proliferation of CD19+ cells re-plated on MS-5. Approximately one-third of the CD19+ cells that emerged at 2.5–4 weeks expressed the IL-7R. FACS-purified IL-7R+/CD19+ cells were larger and proliferated on MS-5 in response to hIL-7, while IL-7R-/CD19+ cells were smaller and did not expand on MS-5 after stimulation with hIL-7. These results suggested that the IL-7R+/CD19+ cells that emerged in the HSC/MS-5 culture might be responding to murine (m) IL-7 produced by MS-5. In order to determine if endogenous IL-7 could be contributing to B-lymphopoiesis, murine and human-specific ELISAs were used to screen HSC/MS-5 culture supernatants. Supernatants from 3 and 4 week HSC/MS-5 cultures contained 20–30 pg/mL mIL-7 and 0.1–2.0 pg/mL hIL-7. Analysis of the kinetics of mIL-7 accumulation in steady-state cultures of confluent MS-5 (no HSC present) showed an increase from 8.0 pg/mL at 2 days, to nearly 150 pg/mL after 2.5 weeks. Flow cytometric analysis of STAT5 phosphorylation in IL-7R+/CD19+ cells demonstrated that hIL-7 and mIL-7 were capable of transducing a signal through the human IL-7R resulting in STAT5 phosphorylation. Furthermore, a monoclonal antibody to the human IL-7R alpha chain blocked STAT5 phosphorylation by both hIL-7 and mIL-7. Inclusion of goat anti-mouse IL-7 neutralizing antibody in HSC/MS-5 cultures effectively reduced the concentration of bioactively available mIL-7 to less than 6 pg/mL. This resulted in a 33% and 67% reduction in the number of CD19+ cells by 3 and 4 weeks, respectively. These collective results are the first to reveal a crucial role for stromal cell-derived IL-7 in promoting the proliferative expansion of human CD19+ B-lineage cells, and support a model wherein IL-7 can transduce a proliferative/replicative signal that cooperates with a stromal cell-derived co-stimulus to regulate development of human B-lineage cells.

Improved survival after unrelated donor bone marrow transplantation in children with primary immunodeficiency using a reduced-intensity conditioning regimen

AbstractThe optimal approach to stem cell transplantation in children with immunodeficiency who lack a matched family donor is controversial. Unrelated donor stem cell transplantation gives equivalent outcome to mismatched family donor stem cell transplantation in severe combined immunodeficiency, whereas unrelated donors may be preferable in non–severe combined immunodeficiency children. However, unrelated donor stem cell transplantation with conventional conditioning regimens has been associated with significant treatment-related toxicity, particularly in non–severe combined immunodeficiency patients with preexisting organ dysfunction. We report the outcome of a series of 33 consecutive unrelated donor transplantations performed at our center in children with primary immunodeficiency using a reduced-intensity conditioning regimen between 1998 and 2001. We have compared these outcomes with a retrospective control cohort of 19 patients who underwent transplantation with myeloablative conditioning between 1994 and 1998. All children in both groups had primary engraftment. There was no statistical difference in the speed of immune reconstitution or incidence of graft-versus-host disease between the 2 groups. Overall survival was significantly better in the reduced-intensity conditioning group: 31 (94%) of 33 patients survived, compared with 10 (53%) of 19 in the myeloablative conditioning group (P = .014). We conclude that the reduced-intensity conditioning regimen results in improved survival and reduced transplantation-related mortality compared with myeloablative conditioning in high-risk patients undergoing unrelated donor transplantation.

Perspectives of gene therapy for primary immunodeficiencies

Standard therapies for patients with severe primary immunodeficiencies include bone marrow transplantation and, for adenosine deaminase deficiency, enzyme replacement. In the last decade, gene therapy has been developed as an alternative for these conditions. We summarize the recent advances in gene therapy for primary immunodeficiencies and discuss the unexpected occurrence of leukemia in a gene therapy trial for X-linked severe combined immunodeficiency. Eight of 10 infants with X-linked severe combined immunodeficiency who received autologous hematopoietic stem cells transduced with a retroviral vector carrying the IL2RG complementary DNA achieved immune reconstitution. However, the two youngest patients developed leukemic expansions of gene-corrected cells. The first case had proliferation of a gamma delta T cell clone, and the second case had three alpha beta T cell clones derived from a single transduced progenitor. Leukemic cells in both patients aberrantly expressed the LIM domain only-2 transcription factor due to retroviral vector insertions in this locus. After receiving anti-leukemic treatment one patient achieved a lasting remission, but the other relapsed. Four adenosine deaminase deficient severe combined immunodeficiency patients also developed functional immunity after receiving autologous hematopoietic stem cells transduced with the adenosine deaminase gene complementary DNA following submyeloablative chemotherapy. Chronic granulomatous disease, Wiskott-Aldrich syndrome, JAK3 deficiency and RAG2 deficiency are other immunodeficiencies being studied as candidates for gene therapy. Gene therapy is a promising therapeutic option for some primary immunodeficiencies, especially when cells expressing the correct gene have a selective advantage. More clinical trials with closer patient monitoring are under way to define which patients may benefit from this approach, and strategies are being developed to understand and ultimately reduce the risk of leukemia secondary to retroviral vector insertion.

414. Design of an HIV Vector for Site-Specific Gene Insertion

Top of pageAbstract Pluripotent hematopoietic stem cells (HSCs) give rise to all lineages of mature blood cells, and serve as an important gene therapy target. While vectors derived from murine leukemia virus (MLV) are widely used to deliver genes into HSCs in various human gene therapy trials, their transduction efficiency of HSCs is rather low. This could be partly due to the quiescent nature of HSCs. Vectors derived from human immunodeficiency virus (HIV) are developed to circumvent the problem of low transduction efficiency of quiescent cells by the MLV vector. However, since both vectors integrate randomly into the host genome, it can lead to gene disruption or unanticipated gene activation through the enhancer or the promoter element present in the vector. The leukemia incidence induced by MLV vector integration near an oncogene in severe combined immunodeficiency (SCID) patients and recent discoveries of preferential integration by MLV and HIV near or within active genes underscore the importance of designing strategies for site-specific gene insertion. To address this issue, we have incorporated the Cre-loxP system into an HIV vector. We generated a human cell line containing a unique loxP site in the genome to serve as a target for gene insertion. A targeting vector containing the loxP site was produced from 293T cells and used to transduce the loxP-containing cell line. Transduced cells were pooled and the genomic DNA was prepared. PCR analysis of the genomic DNA showed site-specific insertion of the gene carried by the targeting vector into the loxP site in the host genome. From the number of the transduced cells, the efficiency of site-specific gene insertion was determined to be in the range between 0.04% and 0.004%. If we enriched for the site-specific gene insertion event by using an HIV vector deficient in integrase, the efficiency was increased to 0.7%. Our approach demonstrates the first step of designing HIV vectors for therapeutic gene targeting.

Janus kinase 3 (JAK3) deficiency: clinical, immunologic, and molecular analyses of 10 patients and outcomes of stem cell transplantation

AbstractWe found 10 individuals from 7 unrelated families among 170 severe combined immunodeficiency (SCID) patients who exhibited 9 different Janus kinase 3 (JAK3) mutations. These included 3 missense and 2 nonsense mutations, 1 insertion, and 3 deletions. With the exception of 1 individual with persistence of transplacentally transferred maternal lymphocytes, all infants presented with a T–B+NK– phenotype. The patient mutations all resulted in abnormal B-cell Janus kinase 3 (JAK3)–dependent interleukin-2 (IL-2)–induced signal transducer and activator of transcription-5 (STAT5) phosphorylation. Additional analyses of mutations permitting protein expression revealed the N-terminal JH7 (del58A) and JH6 (D169E) domain mutations each inhibited receptor binding and catalytic activity, whereas the G589S JH2 mutation abrogated kinase activity but did not affect γc association. Nine of the 10 patients are currently alive from between 4 years and 18 years following stem cell transplantation, with all exhibiting normal T-cell function. Reconstitution of antibody function was noted in only 3 patients. Natural killer (NK) function was severely depressed at presentation in the 4 patients studied, whereas after transplantation the only individuals with normal NK lytic activity were patients 1 and 5. Hence, bone marrow transplantation is an effective means for reconstitution of T-cell immunity in this defect but is less successful for restoration of B-cell and NK cell functions.

Artemis and p53 cooperate to suppress oncogenic N-myc amplification in progenitor B cells.

The nonhomologous DNA end-joining (NHEJ) pathway contains six known components, including Artemis, a nuclease mutated in a subset of human severe combined immunodeficient patients. Mice doubly deficient for the five previously analyzed NHEJ factors and p53 inevitably develop progenitor B lymphomas harboring der(12)t(12;15) translocations and immunoglobin heavy chain (IgH)/c-myc coamplification mediated by a breakage-fusion-bridge mechanism. In this report, we show that Artemis/p53-deficient mice also succumb reproducibly to progenitor B cell tumors, demonstrating that Artemis is a tumor suppressor in mice. However, the majority of Artemis/p53-deficient tumors lacked der(12)t(12;15) translocations and c-myc amplification and instead coamplified IgH and N-myc through an intra- or interchromosome 12 breakage-fusion-bridge mechanism. We discuss this finding in the context of potential implications for mechanisms that may target IgH locus translocations to particular oncogenes.