Chronic granulomatous disease (CGD) is an inherited disorder of phagocytes in which NADPH oxidase is defective in generating reactive oxygen species. In this study, we reprogrammed three normal unrelated patient's fibroblasts (p47(phox) and gp91(phox)) to pluripotency by lentiviral transduction with defined pluripotency factors. These induced pluripotent stem cells (iPSC) share the morphological features of human embryonic stem cells, express the key pluripotency factors, and possess high telomerase activity. Furthermore, all the iPSC lines formed embryoid bodies in vitro containing cells originating from all three germ layers and were capable of teratoma formation in vivo. They were isogenic with the original patient fibroblasts, exhibited normal karyotype, and retained the p47(phox) or gp91(pho)(x) mutations found in the patient fibroblasts. We further demonstrated that these iPSC could be differentiated into monocytes and macrophages with a similar cytokine profile to blood-derived macrophages under resting conditions. Most importantly, CGD-patient-specific iPSC-derived macrophages showed normal phagocytic properties but lacked reactive oxygen species production, which correlates with clinical diagnosis of CGD in the patients. Together these results suggest that CGD-patient-specific iPSC lines represent an important tool for modeling CGD disease phenotypes, screening candidate drugs, and the development of gene therapy.

BACKGROUND Aspergillus spp infection is a potentially lethal disease in patients with neutropenia or impaired neutrophil function. We showed previously that Aspergillus hyphae, too large for neutrophil phagocytosis, are inhibited by reactive oxygen species-dependent neutrophil extracellular trap (NET) formation. This process is defective in chronic granulomatous disease (CGD) because of impaired phagocyte nicotinamide adenine dinucleotide phosphate (NADPH) oxidase function. OBJECTIVE To determine the antifungal agent and mechanism responsible for reconstitution of Aspergillus growth inhibition within NETs after complementation of NADPH oxidase function by gene therapy (GT) for CGD. METHODS Antifungal activity of free and NET-released calprotectin was assessed by incubation of Aspergillus nidulans with purified calprotectin, induced NETs from human controls, and CGD neutrophils after GT in the presence or absence of Zn(2+) or α-S100A9 antibody, and with induced NETs from wild-type or S100A9(-/-) mouse neutrophils. RESULTS We identified the host Zn(2+) chelator calprotectin as a neutrophil-associated antifungal agent expressed within NETs, reversibly preventing A nidulans growth at low concentrations, and leading to irreversible fungal starvation at higher concentrations. Specific antibody-blocking and Zn(2+) addition abolished calprotectin-mediated inhibition of A nidulans proliferation in vitro. The role of calprotectin in anti-Aspergillus defense was confirmed in calprotectin knockout mice. CONCLUSION Reconstituted NET formation by GT for human CGD was associated with rapid cure of pre-existing therapy-refractory invasive pulmonary aspergillosis in vivo, underlining the role of functional NADPH oxidase in NET formation and calprotectin release for antifungal activity. These results demonstrate the critical role of calprotectin in human innate immune defense against Aspergillus infection.

Chronic granulomatous disease is a primary immunodeficiency, comprising five molecular defects, characterized by an impaired respiratory burst activity of myeloid cells. We are currently developing a gene therapy vector for the p47phox-deficient form of chronic granulomatous disease. Classic intracellular immunostaining of the cytoplasmic p47phox transgene product, however, interferes with respiratory burst activity. In this study we report a new system for measuring p47phox expression: A single open reading frame encoding the surface marker protein ΔLNGFR (truncated low-affinity nerve growth factor receptor) linked to the p47phox transgene by the 2A oligopeptide coexpression technology. Translation generates two discrete products: p47phox localizing to the cytoplasm and 'ΔLNGFR-2A' localizing to the cell surface. Six weeks after transplantation of transduced autologous hematopoietic stem cells into p47-/- mice, the intracellular p47phox fluorescence-activated cell sorting (FACS) signal intensities corresponded to surface ΔLNGFR staining in monocytes, B cells, T cells and Sca I+ bone marrow cells in vivo. The p47phox cleavage product restored nicotinamide adenine dinucleotide phosphate-oxidase activity in granulocytes differentiated from transduced p47phox-/- murine hematopoietic stem cells ex vivo, in murine granulocytes/monocytes in vivo, and in transduced human monocyte derived macrophages from p47phox-deficient chronic granulomatous disease patients. In conclusion, this new marker system allows highly efficient, indirect detection of cytoplasmic transgene products by FACS surface staining.

Background. Chronic granulomatous disease (CGD) is a rare inherited disease of the phagocyte NADPH oxidase system that causes defective production of toxic oxygen metabolites, impaired bacterial and fungal killing, and recurrent life-threatening infections, mostly by catalase-producing organisms. We report for the first time, to our knowledge, chronic infections with Actinomyces species in 10 patients with CGD. Actinomycosis is a chronic granulomatous condition that commonly manifests as cervicofacial, pulmonary, or abdominal disease, caused by slowly progressive infection with oral and gastrointestinal commensal Actinomyces species. Treatment of actinomycosis is usually simple in immunocompetent individuals, requiring long-term, high-dose intravenous penicillin, but is more complicated in those with CGD because of delayed diagnosis and an increased risk of chronic invasive or debilitating disease. Methods. Actinomyces was identified by culture, staining, 16S ribosomal DNA polymerase chain reaction, and/or a complement fixation test in 10 patients with CGD. Results. All 10 patients presented with a history of fever and elevated inflammatory signs without evident focus. Diagnosis was delayed and clinical course severe and protracted despite high-dose intravenous antibiotic therapy and/or surgery. These results suggest an unrecognized and unanticipated susceptibility to weakly pathogenic Actinomyces species in patients with CGD because these are catalase-negative organisms previously thought to be nonpathogenic in CGD. Conclusions. Actinomycosis should be vigorously sought and promptly treated in patients with CGD presenting with uncommon and prolonged clinical signs of infection. Actinomycosis is a catalase-negative infection important to consider in CGD.

Chronic granulomatous disease (CGD) patients have impaired NADPH oxidase function, resulting in poor antimicrobial activity of neutrophils including the inability to generate Neutrophil Extracellular Traps (NETs). Invasive aspergillosis is the leading cause of death in patients with CGD; it is unclear how neutrophils control Aspergillus species in healthy individuals. The aim of this study was to determine whether gene therapy (GT) restores NET formation in CGD by complementation of NADPH oxidase function, and whether NETs have antimicrobial activity against Aspergillus nidulans. Here we show that reconstitution of NET formation by GT in a patient with CGD restores neutrophil elimination of Aspergillus nidulans conidia and hyphae and is associated with rapid cure of pre-existing therapy refractory invasive pulmonary aspergillosis, underlining the role of functional NADPH oxidase in NET formation and antifungal activity.

Allogeneic hematopoietic stem cell transplantation from an human leukocyte antigen (HLA)-identical donor is currently the only proven curative treatment for chronic granulomatous disease. Hematopoietic stem cell transplantation with alternative donors is associated with higher morbidity and mortality. Therefore, we performed in vitro fertilization and preimplantation HLA matching combined with female sexing for hematopoietic stem cell transplantation in chronic granulomatous disease. Ethical and psychological issues were considered carefully. We used in vitro fertilization with X-enriched spermatozoa followed by preimplantation genetic diagnosis to identify female HLA-genoidentical embryos in a family in need of a suitable donor for their boy affected with severe X-linked chronic granulomatous disease. Two preimplantation genetic diagnosis cycles were performed in the family. In the second cycle, 2 HLA-genoidentical female embryos were transferred and a singleton pregnancy was obtained, resulting in the birth of an unaffected girl at term. Because of insufficient cell numbers in the cord-blood source, conventional hematopoietic stem cell transplantation had to be performed at 12 months of age of the donor and 5 years of age of the recipient and resulted in complete stable donor chimerism and immunologic reconstitution up to 25 months post-hematopoietic stem cell transplantation. Hematopoietic stem cell transplantation after in vitro fertilization and combined female sexing and HLA matching offers a new and relatively rapid therapeutic option for patients with X-linked primary immunodeficiency such as chronic granulomatous disease who need hematopoietic stem cell transplantation but lack an HLA-genoidentical donor.

CD31(+)CD45RA(+)RO(-) lymphocytes contain high numbers of T cell receptor circle (TREC)-bearing T cells; however, the correlation between CD31(+)CD4(+) lymphocytes and TREC during aging and under lymphopenic conditions has not yet been sufficiently investigated. We analyzed TREC, telomere length and telomerase activity within sorted CD31(+) and CD31(-) CD4(+) lymphocytes in healthy individuals from birth to old age. Sorted CD31(+)CD45RA(+)RO(-) naive CD4(+) lymphocytes contained high TREC numbers, whereas CD31(+)CD45RA(-)RO(+) cells (comprising </=5% of CD4(+) cells during aging) did not contain TREC. CD31(+) overall CD4(+) cells remained TREC rich despite an age-related tenfold reduction from neonatal (100 : 1000) to old age (10 : 1000). Besides a high TREC content, CD31(+)CD45RA(+)RO(-)CD4(+) cells exhibited significantly longer telomeres and higher telomerase activity than CD31(-)CD45RA(+)RO(-)CD4(+) cells, suggesting that CD31(+)CD45RA(+)RO(-)CD4(+) cells represent a distinct population of naive T cells with particularly low replicative history. To analyze the value of CD31 in lymphopenic conditions, we investigated six children after allogeneic hematopoietic stem cell transplantation (HSCT). Reemerging overall CD4(+) as well as naive CD45RA(+)RO(-)CD4(+) cells predominantly expressed CD31 and correlated well with the recurrence of TREC 5-12 months after HSCT. Irrespective of limitations in the elderly, CD31 is an appropriate marker to monitor TREC-rich lymphocytes essentially in lymphopenic children after HSCT.

Gene transfer into hematopoietic stem cells has been successfully used to correct immunodeficiencies affecting the lymphoid compartment. However, similar results have not been reported for diseases affecting myeloid cells, mainly due to low engraftment levels of gene-modified cells observed in unconditioned patients. Here we review the developments leading to a gene therapy approach for the treatment of Chronic Granulomatous Disease (CGD), a primary life threatening immunodeficiency caused by a defect in the oxidative antimicrobial activity of phagocytes. Although the disease can be cured by bone marrow transplantation, this treatment is only available to patients with HLA-identical sibling or matched unrelated donors. One therapeutic option for patients without suitable donor is the genetic modification of autologous hematopoietic stem cells. Although early attempts to correct CGD by gene therapy were unsuccessful, these studies demonstrated the safety and limitations of gene transfer into hematopoietic stem cells (HSC) of CGD patients using retroviral vectors. The recent development of advanced gene transduction protocols together with improved retroviral vectors, combined with low intensity chemotherapy conditioning, allowed partial correction of the granulocytic function with a significant clinical benefit in treated patients. These results may have important implications for future applications of gene therapy in myeloid disorders and inherited diseases using hematopoietic stem cells.