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To the editor Wiskott–Aldrich syndrome (WAS) is a rare X-linked disorder caused by mutations in the WAS gene, and it is characterized by the triad of thrombocytopenia, eczema, and susceptibility to infection1. WAS exhibits extensive clinical variability, and the genotypes of WAS patients are also highly variable1. Mutations impairing but not abolishing WAS protein expression can cause X-linked thrombocytopenia (XLT), an attenuated form of WAS with minimal or no immunodeficiency1. Autoimmune complications are exceedingly common in WAS/XLT and affect 40–70% of patients according to retrospective cohort studies1. Glomerulonephritis is a frequent complication of WAS/XLT. It is found in 3.5–19% of cases and can progress to chronic renal failure requiring renal transplantation2. Immunoglobulin A (IgA) nephropathy (IgAN) has been diagnosed in a majority of WAS/XLT patients2, 3. Previous reports revealed that aberrantly O-glycosylated IgA which exhibits galactose deficiency in the O-linked glycans in the hinge region of the heavy chain may play a pivotal role in the pathogenesis of these IgAN4. We have previously reported that Was-knockout mice develop proliferative glomerulonephritis reminiscent of human IgAN5. We measured serum levels of aberrantly glycosylated IgA in Was- knockout mice using lectin-binding assays with elderberry (Sambucus nigra) bark and ricinus communis agglutinin I, which recognize terminal sialic acid and galactose residues6. These results indicated aberrant IgA production may be critically involved in the pathogenesis of glomerulonephritis in these mice5. Furthermore, aberrant O-glycosylation of serum IgA with characteristics similar to that observed in IgAN was detected in a WAS-carrier patient who presented with Henoch-Schonlein purpura (HSP)6. In this report, we evaluated the role of aberrant IgA production in the development of autoimmunity and glomerulonephritis in WAS/XLT patients. Serum samples were obtained from 26 patients with WAS or XLT and 20 age matched control. The clinical characteristics of the patients with WAS/XLT are shown in Table e1. Eleven patients presented with autoimmune complications including IgAN, vasculitis, arthritis, colitis, autoimmune hemolytic anemia, and autoimmune thrombocytopenia. To examine the terminal galactosylation of IgA molecules, we used lectin-binding assays with Helix aspersa (HAA), which recognizes terminal N-acetylgalactosamine (GalNAc) residues7 (for more information, see the Methods section in this article's Online Repository.) Table e1 The serum levels of galactose-deficient IgA in WAS/XLT patients were significantly higher than those in the control group (p

Mutations affecting the expression of the Janus family kinase JAK3 were recently shown to be responsible for autosomal recessive severe combined immunodeficiency (SCID). JAK3-deficient patients present with a clinical phenotype virtually indistinguishable from boys affected by X-linked SCID, a disease caused by genetic defects of the common gamma chain (gamma c) that is a shared component of the receptors for IL-2, IL-4, IL-7, IL-9, and IL-15. The specific interaction of JAK3 and gamma c represents the biochemical basis for the similarities between these two immunodeficiencies. Both forms of SCID are characterized by recurrent, severe infections leading to death in infancy unless successfully treated by allogeneic bone marrow transplantation. Because of the potentially lethal complications associated with allogeneic bone marrow transplantation and the frequent lack of suitable marrow donors, the development of alternative forms of therapy is highly desirable. To this end, we investigated a retroviral-mediated gene correction approach for JAK3-deficiency. A vector carrying a copy of JAK3 cDNA was constructed and used to transduce B cell lines derived from patients with JAK3-deficient SCID. We demonstrate restoration of JAK3 expression and phosphorylation upon IL-2 and IL-4 stimulation. Furthermore, patients' cells transduced with JAK3 acquired the ability to proliferate normally in response to IL-2. These data indicate that the biological defects of JAK3-deficient cells can be efficiently corrected in vitro by retroviral-mediated gene transfer, thus providing the basis for future investigation of gene therapy as treatment for JAK3-deficient SCID.

Abstract 2049 Gene transfer into hematopoietic stem cells has been used successfully to treat a variety of human genetic diseases. Although protocols have shown positive clinical outcomes, the successes of clinical trials have been tempered by adverse events in which the integration of the viral vectors increased transcription of cancer-related genes and thereby contributed to development of leukemias. The use of gamma-retroviral vectors containing full-length, long terminal repeats (LTRs) with strong promoter and enhancer activity has been well documented to have the potential of resulting in activation of expression of genes neighboring the vector insertion site. Assessing safety of integrating viral vectors for future clinical use is therefore of paramount importance. In preparation for gene therapy approaches for the Wiskott-Aldrich syndrome (WAS), we used an in vitro assay of murine bone marrow (BM) cell immortalization to compare the consequences of hematopoietic stem cell transduction by three different kinds of viral vectors, including Moloney murine leukemia virus (MMLV), lentivirus (LV), and foamy virus (FV) constructs. To evaluate critical elements for cell immortalization by MMLV vectors, we also tested five different MMLV LTR forms: unmodified (full-MMLV), deleted of most of the two 75-bp repeats associated with the viral enhancer (delE1), deleted of all the two 75-bp repeats and negative control region (NCR) (delE2), deleted of the viral promoter sequences (delP), and with full deletion of enhancer and promoter sequences (delEP). All vectors carried an internal expression cassette including the eGFP gene under the control of a UCOE (ubiquitously acting chromatin opening element) or the WAS endogenous promoter (WASp). In this assay, BM cells are harvested from C57BL6 mice, exposed to retroviral supernatants and cultured long-term. Derived lines are considered immortalized based on their ability to continue to grow in vitro for more than six weeks in the presence of interleukin-3 and stem cell factor. Real-time PCR was performed to verify comparable transduction efficiency of bone marrow cells by different vectors. To date, full-MMLV and delE1 transduction of 123 and 132 cultures, respectively, has given rise to 48 and 43 immortalized lines (39.0% and 32.5% immortalization rate, respectively). The difference in immortalization rate between full-MMLV and delE1 was not statistically significant. In contrast, transduction of 114 and 62 cultures with LV and FV vectors, respectively, resulted in no immortalized lines. In our analysis of MMLV LTR mutants, full-MMLV and delE1 transduction of 56 and 72 cultures, respectively, has given rise to 24 and 26 immortalized lines (43% and 36% immortalization rate). Again, the difference in immortalization rate between full-MMLV and delE1 was not statistically significant. In contrast, delE2, delP and delEP transduction of 24 cultures each has given rise to 2, 5 and 3 immortalized lines (8.3%, 21% and 13% immortalized ratio, respectively). The difference between the immortalization caused by delE1 and delE2 vectors was statistically significant (p These preliminary results confirm that gamma-retroviral vectors are prone to causing immortalization of hematopoietic cells and indicate that deletion of viral enhancer and/or promoter sequences may not be adequate to eliminate the insertional oncogenesis risk. Importantly, our data point to the NCR as a crucial element for immortalization and justify additional studies to evaluate its specific role in MMLV-mediated insertional oncogenesis. Finally, our results suggest that vectors based on LV and FV backbones are safer alternatives for clinical gene transfer into hematopoietic stem cells. Disclosures: No relevant conflicts of interest to declare.

Abstract 1208 Introduction: The adenylate kinase (AK) gene family consists of 7 different members that contribute to energy cell metabolism by converting ATP + AMP to 2 molecules of ADP. AKs are critical players in ensuring cellular energy homeostasis in all tissues and are generally involved in a broad range of cellular functions. Among AKs, AK2 has unique features such as its location in the mitochondrial intermembrane space and critical role in human lymphopoiesis and granulopoiesis. Indeed, mutations of the AK2 gene cause reticular dysgenesis (RD), an autosomal recessive form of severe combined immunodeficiency (SCID) characterized by an early differentiation arrest in the granulocyte lineage and impaired lymphoid maturation. The mechanisms underlying the pathophysiology of RD remain unclear. The phenotype of AK2 deficient animals has not been reported in the literature, but murine lines carrying homozygous inactivating retroviral insertions are embryonically lethal (our personal observations). Objectives: To study the role of AK2 in hematopoietic system development and define the effects of AK2 deficiency, we set out to generate a zebrafish model of RD. Methods: We injected zebrafish embryos with morpholino oligomers specific for the two zebrafish AK2 isoforms and analyzed the serial expression pattern of several hematopoietic markers in developing AK2 morphants. To confirm our observations in AK2 knockdown embryos, we screened a zebrafish DNA library of ENU-induced mutations and recovered a mutant fish line carrying a T371C/L124P missense mutation within the exon 4 of AK2 gene that is predicted to be deleterious for protein stability and function. Results: The downregulation of zebrafish AK2 expression phenocopied the human disease and resulted in strong reduction of developing lymphocytes. In addition, in situ hybridization for GATA1, alpha-globin 1, L-plastin and Odianisidine staining indicated that erythroid development was affected in AK2 morphants during primitive hematopoiesis, while myeloid development was conserved. Furthermore, in situ hybridization studies of the expression of markers of zebrafish definitive hematopoiesis showed abnormalities distributed among all hematopoietic lineages suggesting a broad role of AK2 in zebrafish hematopoiesis. Importantly, the ENU-induced Ak2 mutant recapitulated all the primitive and definitive hematopoietic phenotypes observed in AK2 morphants. Finally, preliminary data suggest that AK2 deficiency (both in morphant and mutant embryos) induces an increased level of reactive oxygen species (ROS) triggering oxidative stress and consequent apoptosis in hematopoietic progenitor cells. Conclusions: Our data provide new insights into the AK2 function and indicate that zebrafish represents a good model for studying the molecular mechanisms involved in RD. To date, our mutant line represents the first example of animal model of this rare and unique human disease. Disclosures: No relevant conflicts of interest to declare.

Background Dermatofibrosarcoma protuberans (DFSP) is a rare malignant skin tumor associated with a characteristic chromosomal translocation (t[17;22][q22;q13]) resulting in the COL1A1 -platelet-derived growth factor β (PDGFB) fusion gene. This malignancy is rarely diagnosed in childhood. Objective We observed an unexpected high incidence of this DFSP in children affected with adenosine deaminase–deficient severe combined immunodeficiency (ADA-SCID) and set out to evaluate the association of these 2 clinical entities. Methods Twelve patients with ADA-SCID were evaluated with a complete dermatologic examination and skin biopsy when indicated. Conventional cytogenetic and molecular analyses (fluorescence in situ hybridization, RT-PCR, or both) were performed when possible. Results Eight patients were found to have DFSP. Six patients had multicentric involvement (4-15 lesions), primarily of the trunk and extremities. Most lesions presented as 2- to 15-mm, round atrophic plaques. Nodular lesions were present in 3 patients. In all cases CD34 expression was diffusely positive, and diagnosis was confirmed either by means of cytogenetic analysis, molecular testing, or both. The characteristic DFSP-associated translocation, t(17;22)(q22;q13), was identified in 6 patients; results of fluorescence in situ hybridization were positive for fusion of the COL1A1 and PDGFB loci in 7 patients; and RT-PCR showed the COL1A1 - PDGFB fusion transcript in 6 patients. Conclusions We describe a previously unrecognized association between ADA-SCID and DFSP with unique features, such as multicentricity and occurrence in early age. We hypothesize that the t(17;22)(q22;q13) translocation that results in dermal overexpression of PDGFB and favors the development of fibrotic tumors might arise because of the known DNA repair defect in patients with ADA-SCID. Although the natural course of DFSP in the setting of ADA-SCID is unknown, this observation should prompt regular screening for DFSP in patients with ADA-SCID.

Abstract 2222 The thrombocytopenia associated with the Wiskott-Aldrich Syndrome (WAS) is thought to be due to the combined effects of impaired platelet production and accelerated platelet consumption. In a murine model of WAS, we have previously demonstrated that platelet consumption is accelerated. Also, we have shown that antibody opsonization accelerates both in vivo consumption and ex vivo phagocytosis of murine WASP(-) platelets in comparison to opsonized WT platelets. Based on these findings, we tested the susceptibility of platelets from WAS patients to ex vivo phagocytosis by activated THP-1 cells. We used a lipophilic fluorescent marker (DIO) to label platelets, and distinguished between platelet uptake and adsorption with a fluorescent anti-CD61 antibody. In comparison to previous methods using CMFDA-labeled platelets, use of DIO resulted in a tenfold reduction in the number of platelets needed per assay. This in turn allowed us to perform ex vivo phagocytosis studies with the very small number of platelets available for study in peripheral blood specimens from thrombocytopenic WAS patients. We report that untreated platelets from WAS patients are taken up more rapidly than control platelets by activated THP-1 cells. Specifically, the fraction of macrophages demonstrating DIO uptake, and showing no adsorbed platelets is consistently increased in comparison to cells showing adsorbed platelets (figure 1). Using a numerical analysis method, we distinguish the effect of WASP deficiency on the probability of phagocytosis per adsorbed platelet (p) from effects on the fluorescence intensity imparted to the macrophage per internalized platelet (alpha) and from variation in the platelet/macrophage ratio (m). This was done by predicting the type of results shown in figure 1 for approximately 32,000 possible combinations of p, alpha, and m, and finding among the possible combinations those for which the predicted results best fit our observations. We validated the sensitivity of the method to changes in p (via the use of opsonized vs. non-opsonized platelets), m (via changing the platelet/macrophage ratio) and alpha (via changing the mean fluorescence intensity of the platelets). Applied to the data in figure 1, the numerical analysis method demonstrates an increased p value for each of the WAS patients studied, and a reduced alpha value (as might be expected for smaller platelets) in 5 of 6 studies (figure 2). Opsonization with murine anti-CD61 antibody did not accelerate uptake of WAS platelets in comparison to controls. However, we observed significantly increased levels of surface IgM, and possibly IgG, on platelets from WAS patients. In addition to inhibiting opsonization ex vivo, this level of surface antibody could contribute to accelerated phagocytosis of platelets in clinical WAS in the same way that ex vivo opsonization augments the in vivo clearance and ex vivo phagocytosis of murine WASP(-) platelets. Our results provide additional support for the role of accelerated platelet consumption in generating the thrombocytopenia of WAS. Disclosures: No relevant conflicts of interest to declare.

Abstract 2185 Objective: The Adenylate Kinase (AK) gene family consists of 7 different members (AK1-7) that contribute to energy metabolism of the cells by converting ATP (or GTP) and free AMP to ADP (or GDP) and free ADP. AKs are critical players in ensuring cellular energy homeostasis in all tissues and are generally involved in a broad range of cellular functions. Among AKs, AK2 is uniquely located in the mitochondrial intermembrane space and has been implicated in Caspase 10-mediated apoptosis, although the published data remain controversial. More recently, it was demonstrated that mutations of the AK2 gene cause reticular dysgenesis, an autosomal recessive form of severe combined immunodeficiency (SCID). Reticular dysgenesis is characterized by an early differentiation arrest in the granulocyte lineage and impaired lymphoid maturation resulting in overwhelming infections and high lethality in affected patients. Moreover, patients commonly present with bilateral sensorineural deafness. The mechanisms underlying the biological consequences of AK2-defieincy remain unclear and the generation and characterization study of model systems is expected to provide useful insigths. Ak2 gene-targeted mice have not been reported in the literature, but lines carrying homozygous inactivating retroviral insertions have been shown to be embryonically lethal (our unpublished observations). Because of the known advantages of zebrafish as model system for developmental studies and the similarities of hematopoiesis in zebrafish and higher vertebrates, we set out to investigate the function of the zebrafish ak2 gene in development, with particular emphasis on hematopoiesis. Results: Similar to humans, we found that two different alternatively spliced isoforms of the ak2 gene (Isoform A and Isoform B) are expressed in zebrafish. By Real-Time PCR and In situ Hybridization (ISH) we analyzed the expression of both ak2 isoforms during embryo development. Preliminary data indicate that Isoform A is more abundantly represented than Isoform B during embryo development. ISH analysis showed that the two isoforms have different spatial expression patterns. These data suggest different functionalities for ak2 isoforms during embryo development. To explore such hypothesis, we injected two different morpholinos (MOs) targeting the ak2 isoforms. Downregulation of both ak2 isoforms phenocopied the human disease and resulted in a strong reduction of developing lymphocytes. Moreover we observed a hypochromic phenotype that also suggested impairment of the erythroid lineage. ISH experiments are underway to better define the affected hematopoietic lineages. Interestingly, ak2 MOs-injected embryos showed also developmental defects beyond the hematopoietic system, such as abnormal jaw development. Future studies will focus on the characterization of the specific function of the alternatively spliced ak2 isoforms. Conclusions: We show that the transcription features of the AK2 gene are conserved in zebrafish. The observed differential expression patterns of the zebrafish ak2 isoforms may provide new insights into the function of AK2 in the development of the hematopoietic system, as well as other organs and offers prospects for the understanding of the molecular mechanisms involved in reticular dysgenesis. Disclosures: No relevant conflicts of interest to declare.

Abstract 3116 Therapeutic gene transfer has been used successfully to treat a variety of human genetic diseases. Although protocols have shown positive clinical outcomes, the success of clinical trials were tempered by adverse events, in which integration of the viral vectors increased transcription of cancer-related genes and thereby contributed to development of leukemia. In all documented cases of insertional mutagenesis, the viral vectors used contained full-length, gamma-retrovirus long terminal repeats (LTRs) that are able to exert strong promoter and enhancer activity driving not only the expression of the transgene carried by the vector, but also that of genes neighboring the insertion site. Assessing safety of integrating viral vectors for future clinical use is therefore of paramount importance. In preparation for gene therapy approaches for the Wiskott-Aldrich syndrome (WAS), we used an in vitro assay of murine bone marrow (BM) cell immortalization to compare the consequences of transduction by four different kinds of viral vectors, including a full-length LTR Moloney leukemia virus (MLV), a self-inactivating MLV carrying a 3' LTR deleted of the viral enhancer region (SIN), a lentivirus (LV), and a foamy virus (FV) construct. All vectors carried EGFP under the control of a ubiquitously acting chromatin opening element (UCOE) or the WAS endogenous promoter (WASp). In this assay, BM cells are harvested from C57BL6 mice, exposed to retroviral supernatants and cultured long-term. Derived lines are considered immortalized based on their ability to continue to grow in vitro for more than 6 weeks in the presence of interleukin-3 and stem cell factor. To date, MLV and SIN transduction of 123 and 132 cultures, respectively, gave rise to 48 and 43 immortalized lines (39.0% and 32.6% immortalization rate). The difference in immortalization rate between MLV and SIN vectors was not statistically significant (Chi square: p=0.30). As expected, immortalized cells were negative/low for IgER, cKit and Sca1 expression, and positive to different degrees for expression of the myeloid markers CD11b/Mac1 and Ly6g/Gr1. Transduction of 114 and 62 cultures with LV and FV vectors, respectively, resulted in no immortalized lines. Real-time PCR was performed to evaluate transduction efficiency of bone marrow cells and immortalized lines. Integrated vector sequences in bone marrow cells transduced by LV and FV were detected in significantly higher quantities than in cells transduced with MLV and SIN vectors. However, expression of the EGFP transgene was markedly reduced in LV- and FV-transduced cells compared to cells exposed to MLV vectors (MFI: 14.0, 1.88, 93.2, respectively). These preliminary results confirm that gamma-retroviral gene transfer vectors are prone to causing immortalization of hematopoietic cells and suggest the vectors based on LV and FV backbones may be safer alternatives for WAS and other genetic disorders, provided that effective gene expression levels can be achieved in the biological model system under study.Table.Summary of immortalization results using MLV, SIN, LV and FV vectorsVirusMOITransduction efficiencyTransduction experiments% ImmortalizationMLV/UCOE/EGFP2067%5643MLV/WASp/EGFP2065%6736SIN/UCOE/EGFP532%7236SIN/WASp/EGFP537%6028LV/UCOE/EGFP1040%570LV/WASp/EGFP1049%570FV/UCOE/EGFP1027%280FV/WASp/EGFP1022%340negativeN.A.N.A.720 Disclosures: No relevant conflicts of interest to declare.

The Wiskott-Aldrich syndrome (WAS) is a severe X-linked, recessive disorder, with a high mortality rate at early age due to hemorrhages, infections, and lymphoid malignancies. The molecular pathogenesis of the disease is unknown. Carrier females of WAS are clinically and immunologically normal, thus precluding carrier detection by simple laboratory tests. Major advances in molecular genetics have allowed mapping of the WAS gene to the pericentromeric short arm of the X chromosome, and have made carrier detection and prenatal diagnosis feasible by segregation analysis with closely linked polymorphic DNA markers. Furthermore, the observation that carriers of WAS exhibit a unilateral inactivation of the X chromosome in hematopoietic cells has provided a new tool for carrier detection. However, critical interpretation of molecular analysis data is essential to provide accurate genetic counseling to WAS families.

Natural Tregs from both WAS patients and WASp-deficient (WKO) mice were previously reported as unable to suppress the proliferation of conventional T-cells in vitro. Because WAS T-cells have a known defect of IL-2 production and this cytokine is important for the generation of Treg cells induced from naïve T-cells, we assessed the efficiency with which WKO naïve T-cells can differentiate in Tregs and observed a significant defect compared to WT. Such defect was not corrected by increasing concentration of anti-CD3 and TGF-β. Preliminary experiments indicated a role for endogenous IL-2 production in the generation of iTregs and pointed again to the IL-2 pathway as a major determinant of the Treg cells defects in WAS. Furthermore, we previously observed that the impaired suppression of T-cell proliferation could be partially rescued by pre-activation of nTregs. Because activated nTregs are known to be able to induce B-cell apoptosis, we set out to evaluate the function of WKO nTregs on B-cells. We found that pre-activated WKO Treg cells fail to effectively suppress B-cell proliferation. The reduced granzyme-B secretion by WKO nTregs compared to WT, suggest that the defective suppressive activity on B-cells may be due to a degranulation defect of WKO cells. In summary, our data suggest that both natural and induced arms of T regulatory cells are defective in WAS and that defective peripheral Treg induction may be involved in the pathogenesis of autoimmunity in this disease.

Abstract 1321 Poster Board I-343 Wiskott-Aldrich syndrome is characterized by immune deficiency and thrombocytopenia. There is also a qualitative platelet defect in Wiskott-Aldrich syndrome, typically presenting on aggregometry as a failure of the second wave of aggregation. The pathophysiology of this defect has not been studied in detail. To investigate this phenomenon, we enumerated dense bodies and measured serotonin content in the platelets of 7 patients with Wiskott-Aldrich syndrome. Patients ranged in age from 14 to 45 years old. Five of 7 patients had had splenectomy. In these patients, the platelet count ranged from 120,000/mcL to 521,000/mcL. Platelet counts were 20,000/mcL and 46,000/mcL in the 2 patients who had not had splenectomy. The latter 2 patients had occasional petechiae, as did 2 of the splenectomized patients. Three patients had no clinical evidence of platelet dysfunction. One of these 3 had tolerated abdominal surgery without untoward bleeding. Blood specimens were taken in the course of routine follow-up, when patients were otherwise well. Platelets were prepaed for electron microscopy by the whole mount technique. For serotonin content, platelets were counted and lysed in distilled water. Serotonin in the lysate was chemically acetylated and then measured by ELISA. Serotonin levels were normalized to ng of serotonin per 10 9 platelets. The average ± standard deviation dense body count in our patient group was 1.1 ± 0.4 dense bodies per platelet (normal count 6-8 dense bodies/platelet). Serotonin content was measured in three patients, who showed 273 ± 103 ng serotonin per 10 9 platelets (normal value 621-1064 ng/10 9 platelets). Our results are the first quantitative analysis of platelet dense body counts and serotonin level in patients with Wiskott-Aldrich syndrome. In addition to further elucidating the pathophysiology of this disease, these data have implications for understanding the role of the Wiskott-Aldrich syndrome protein in the genesis of dense bodies. Disclosures No relevant conflicts of interest to declare.

10570 Background: DFSP is a rare malignant skin tumor associated with a characteristic chromosomal translocation (t(17;22)(q22;q13)), resulting in the COL1A1-PDGFB fusion gene. We originally diagnosed DFSP in two patients affected with a rare form of severe combined immunodeficiency due to adenosine deaminase deficiency (ADA-SCID). The association of these two rare conditions has been described in two other cases, which prompted us to screen for DFSP systematically in patients with ADA-SCID. Methods: Eight ADA-SCID patients were evaluated with complete dermatological exam and skin biopsy. Molecular analysis (FISH and/or RT-PCR) and karyotype were performed when possible. Results: Six patients (age 2, 2, 5, 9, 12 and 22 years) were found to have DFSP. Five patients had between 4 and 12 multicentric lesions over the trunk and extremities. One patient had a single lesion. Most lesions appeared as 2–15 mm tan atrophic plaques. Nodular lesions were present in 3 patients. All lesions showed a spindle cell proliferation of the dermis, extending into the subcutaneous fat. A storiform pattern was only noticed in one adult patient. In all cases, CD34 expression was diffusely positive and FXIIIa was negative. Karyotype showed t(17;22)(q22;q13) in the 2 patients in whom it was performed. FISH was positive for COL1A1-PDGFB in 2 of 4 patients studied. RT-PCR showed the COL1A1-PDGFB fusion transcript in one case in which FISH was inconclusive.FISH and RT-PCR analyses are being conducted in 2 and 5 remaining cases, respectively. Conclusions: We describe a previously unrecognized association between multicentric DFSP and ADA-SCID. Multicentricity of DFSP to this extent has not previously been reported . We hypothesize that t(17;22)(q22;q13) may arise due to the known DNA repair defect in ADA-SCID and that the known dermal overexpression of PDGFB in this condition may favor the development of fibrotic tumors, as opposed to other skin cancers. Our observations can provide further insight into the pathogenesis of DFSP and should facilitate early diagnosis of DFSP in ADA-SCID. No significant financial relationships to disclose.

Dermatofibrosarcoma protuberans (DFSP) is a rare malignant skin tumor with characteristic cytogenetic findings (t(17;22)(q22;q13)) resulting in a fusion COL1A-PDGFB gene. Here we report an association between DFSP and Adenosine Deaminase Deficient Severe Combined Immunodeficiency (ADA-SCID), a rare congenital immunodeficiency syndrome. We observed DFSP in 3 ADA-SCID patients. The first patient is a 3.6-year-old boy who developed a 1.5 cm soft tissue mass on the right medial thigh. The second patient is a 2.4-year-old girl with 12 plaques (0.4– 0.8 cm) distributed on the trunk, thigh and lower extremities. The third patient is a 6-year-old boy with 5 depressed plaques ranging from 1–2 cm. The diagnosis in the first two patients was confirmed by immunohistochemistry (CD34 positive/Factor XIIIa negative) and demonstration of the COL1A1-PDGFB fusion by Fluorescent In Situ Hybridization (FISH). In the third patient, lesional tissue was positive for CD34 and negative for Factor XIIIa. FISH was inconclusive and further molecular analysis (RT-PCR) is ongoing. We came across two additional ADA-SCID patients in the literature who developed DFSP and we conclude therefore that this unique and unreported association between two rare disease entities merits further study. Importantly, our observations describe the first 2 cases presenting with multiple multicentric DFSPs. DFSP may be overlooked particularly in children who may present with an indolent plaque-like lesion mistaken as birthmark or benign vascular proliferation. DFSP in ADA-SCID may be caused by an unknown virus in the setting of defective immune surveillance. The search for a viral etiology is ongoing. Alternatively, the increased DNA breakage characteristic of ADA-SCID may play a role in the onset of this malignancy.

Adenosine deaminase (ADA) deficiency is a form of severe combined immunodeficiency (SCID) that has long been considered a good candidate for gene therapy (GTx). In 2001–2002, we treated 4 ADA-SCID patients in a clinical trial evaluating the efficacy of 2 different retroviral vectors while continuing enzyme replacement with pegylated bovine ADA (PEG-ADA). No chemotherapy was used. All patients have been monitored for 6 years. No treatment-related serious adverse events occurred. A mild transient elevation in absolute lymphocyte count (ALC) was seen in 2 patients early post-treatment, however, no durable immunologic changes were observed. Low levels (0.1–0.7%) of vector-marked peripheral blood mononuclear cells (PBMCs) persist in 2 patients treated at the age of 4–5 years. All patients remain on PEG-ADA, prophylactic antibiotics and intravenous immunoglobulins. In 2004, we revised the protocol in order to facilitate engraftment and selective advantage of gene-corrected cells by withdrawing PEG-ADA and giving busulfan (75 mg/m^2) before GTx. In November 2005, a first patient was treated who developed unexpected prolonged bone marrow (BM) aplasia. Cytogenetics revealed trisomy 8 aberrations that were found to be present on a BM specimen obtained pre-GTx (Blood2007; 109:503). Our second patient enrolled in January 2007. He received 5x10^6 CD34+ cells/kg that showed 40–200 units (U) of ADA activity (normal range 58–128). Over 6 months, this patient showed a slow increase in ALC (up to 750/mcL) and lymphocyte function. PBMC ADA activity has been up to 50U. The deoxyadenosine metabolite (dAXP) level has decreased to 500/mcL were 36 and 45 days, respectively. In >200 combined days of observation, there has been only one temperature above 38 °C, which resolved with acetaminophen. These data are consistent with the positive results of GTx for ADA-SCID obtained in Milan and London and show that PEG-ADA withdrawal and reduced conditioning improve the outcome of GTx for this disease. Longer follow-up should allow us to study engraftment of cells containing vector-specific sequences and conclude if either vector contributes more to recovery of lymphoid immunity.

Recent studies have demonstrated that cell populations intended for therapeutic purposes that are cultured in heterologous animal products can acquire xenoantigens, potentially limiting their utility. In investigations of the immune response to murine ES cells, we found that a strong antibody response was generated after the second infusion. Both polyclonal and monoclonal antibody responses, derived from immunized mice, were found to be specific for bovine apolipoprotein B-100 which binds to abundant low density lipoprotein receptor on the cell surface and is internalized. Critically, we have shown that in the majority of patients administered three different types of cell-based therapies utilizing cells grown in FCS containing media, an antibody response to bovine apoB-100 develops following the second infusion and is the dominant specificity. The low level of engraftment of genetically modified autologous cells in an ADA-SCID patient with a strong bovine apoB response suggests that such antibodies may influence cell engraftment.

In the previous meeting, we reported the initial follow-up of the two patients with adenosine deaminase (ADA)-deficiency whom we had treated in a clinical trial of retroviral-mediated stem cell gene therapy (SCGT). Our protocol is unique in that patients do not receive any kinds of marrow-preparative conditioning prior to infusion of gene-corrected autologous bone marrow (BM) cells. Since the successful cases of ADA gene therapy reported from Italy used low-dose busulfan to enhance the engraftment of gene-transduced BM cells, it is intriguing to see how the lack of conditioning affects the hematological and immunological reconstitution in our patients.

Integrating gene transfer vectors based on murine oncoretroviruses have been widely used in clinical gene therapy trials to attempt correction of genetic diseases and provide novel therapeutic approaches for HIV-1 infection and cancer. However, the recent occurrence of uncontrolled lymphoproliferation in patients treated with retroviral gene transfer for X-linked severe combined immune deficiency has substantiated the risk of insertional oncogenesis associated with the clinical use of such vectors. Data on safety of retroviral T-cell-directed gene transfer is lacking, therefore we set out to analyze the pattern of retroviral integrations in a patient affected with adenosine deaminase (ADA) deficiency who received eleven infusions retroviral vector-transduced cells between 1990 and 1992. No adverse events have been observed in this patient who still carries |[sim]|15|[ndash]|20% of gene-corrected peripheral blood lymphocytes. Genomic DNA was prepared from stored lymphocyte samples dating 1991, 1992, 1995, 1998, 2000, and 2003. By inverse PCR and ligation-mediated PCR, we have identified |[sim]|760 bona fide insertion sites. Search for homology within the human genome using BLAT returned |[sim]|275 unique hits that involved a variety of genes, including transcription factors and oncogenes (e.g. RUNX1, STAT5, FYN). We found high prevalence of integrations within 10 kb upstream and downstream of genes that was significantly different (p

Patients carrying genetic defects in the IL-12 signaling pathway (IL-12 p40 or IL-12Rb1) show defective immunity against intracellular pathogens, resulting in increased vulnerability to weakly pathogenic strains of Mycobacteria and Salmonella. Despite early diagnosis and treatment with antibiotics and IFN-g, IL12Rb1-deficient patients can still succumb to complications of serious infections. Reversion of patients' susceptibility by corrective gene transfer could be beneficial by preventing the infectious episodes. We showed that gene transfer of IL-12Rb1 chain in PHA-activated T cells blasts of IL-12Rb1 deficient patients through retroviral-mediated transduction restored the expression of the receptor chain and reconstituted IL-12 signaling, as demonstrated by STAT4 phosphorylation and IFN-g production._In addition, to test the feasibility and safety of retroviral-mediated gene correction in vivo, we established a murine model of gene therapy in IL-12Rb1-deficient mice. Treated mice did not show adverse effects upon gene therapy. Transplantation of gene-corrected hematopoietic stem cells resulted in IL-12Rb1 expression in PBMCs, thymocytes and SP cells of IL-12Rb1 KO mice. In addition, splenocytes isolated from gene-corrected IL-12Rb1 KO mice acquired the ability to respond to IL-12, as demonstrated by the production of IFN-g, which is completely impaired in IL-12Rb1 KO mice. However, restoration of IL-12Rb1 membrane expression as well as the production of IFN-g in response to IL-12 where significantly lower than those detected in wild type control mice. Interestingly, we couldn't detect in the periphery of gene corrected mice the presence of cells expressing high levels of IL-12Rb1 chain, comparable to those detected in their thymuses. These data indicate the possibility of a selective elimination of potentially dangerous IL-12Rb1 high expressing cells in the periphery that prevented the achievement of a complete correction of gene therapy treated mice. In addition, production of IFN-g upon challenge with LPS from Salmonella enteriditis provided in vivo evidence of partial correction of IL-12 signaling pathway in IL-12Rb1 KO mice after gene therapy. We are performing experiments of infections with intracellular pathogens, such as Mycobacterium tuberculosis, to establish if the degree of gene correction reached in our experimental model is sufficient to overcome their immunodeficiency

WAS is an immunodeficiency characterized by recurrent infection, thrombocytopenia, eczema, and increased susceptibility to autoimmunity and malignancy. WAS results from deficient function of the WAS protein (WASp) which functions in receptor-mediated actin cytoskeletal rearrangement. Gene therapy represents an attractive potential therapeutic approach for WAS patients.

Gene transfer into peripheral blood lymphocytes has several potential applications including the correction of genetic diseases and therapeutic approaches for HIV-1 infection and cancer. Integrating gene transfer system based on murine oncoretroviruses are a convenient tool for such strategies. However, the recent occurrence of uncontrolled clonal T cell expansions in two patients treated with retroviral gene transfer for X-linked severe combined immune deficiency has raised the concern of the risk of insertional oncogenesis associated with the clinical use of integrating viral systems. In vitro studies have indicated that murine viral vectors tend to integrate in the vicinity of transcription start regions of the genome, thus providing a possible mechanism for oncogene activation, however, data from clinical gene transfer trials is lacking. We are following patients affected with adenosine deaminase (ADA) deficiency who have received T-lymphocyte-directed, retroviral-mediated gene transfer starting in 1990. The first treated patient received the last infusion of gene-corrected cells 12 years ago, has never shown any sign of lymphoproliferation and still carries ~20% of gene-corrected peripheral blood lymphocytes. We set out to study the integration sites in the cells of this patient with the aim of mapping the regions involved by retroviral integrations, determining their localization with respect to known genes, and assessing whether a preferred pattern could be defined. Genomic DNA was prepared from stored lymphocyte samples dating 1991, 1992, 1995, 1998, 2000, and 2003. By inverse PCR and ligation-mediated PCR, we have identified ~860 bona fide insertion sites. Search for homology within the human genome using BLAT returned ~330 unique hits that involved a variety of genes, including transcription factors and oncogenes (e.g. RUNX1, STAT5, FYN). To evaluate the distribution pattern of these integration sites, 2000 randomly generated data sets of genomic coordinates were assembled and their distribution relative to annotations of the human genome was analyzed. A preliminary comparison of the random distribution to our experimental samples showed that retroviral integrations in cells obtained from the patient were significantly skewed toward regions within 2 kb of genes (p These results suggest that, similar to what observed in murine fibroblast and human cancer cell lines, transcriptionally active regions of the genome may be preferred targets of retroviral vectors in human primary T lymphocytes. At the same time, our observations show that the resulting integration events are compatible with long-term, event-free in vivo survival of gene-modified cells in clinical settings.

For ADA-deficiency, a form of severe combined immunodeficiency, retroviral-mediated gene correction of peripheral T cells or hematopoietic stem/progenitor cells (HSCs) is a treatment of choice alternative to bone marrow (BM) transplantation. Recently, two new such clinical trials aiming for the correction of patients' HSCs were conducted in US and Italy, resulting in remarkably different outcomes: Italian patients showed immune reconstitution whereas the US trial led to no clinical improvement. There are two major differences between their protocols, either or both of which may explain these consequences. 1) In Italy, patients were mildly myeloablated, while not in the US trial. 2) US patients were on the concomitant enzyme replacement therapy (PEG-ADA) that may lessen in vivo selective advantages for gene-corrected cells and thus the efficacy of gene therapy. Italian group has not given the enzyme throughout the studies. We thus have proposed a trial that differs from these two to see If only the absence of PEG-ADA is enough to benefit patients, or if any kind of myeloablation is absolutely necessary. Two ADA-deficiency patients (#1, a 4 yr-old girl; #2, a 13 yr-old boy) were treated under the approval by the Japanese authorities. First, PEG-ADA was discontinued prior to the treatment. Rapid decrease in peripheral counts of lymphocytes and of neutrophils, liver enzyme abnormalities, and mild anorexia became evident, suggesting that the generalized toxicity due to ADA-deficiency had deteriorated. Nevertheless, both patients well tolerated the first several weeks with no serious problems. Good purity CD34+ BM cells (>80%) were transduced with the retroviral vector GCsapM-ADA/PG13. The use of serum-free medium, a cocktail of cytokines (SCF, TPO, flt3-L, IL-6, and soluble IL-6 receptor) and the fibronectin fragments resulted in ~50–70% transduction efficiencies and normal levels of ADA activities in the infused cell populations. No immediate adverse reactions were noted upon cell infusion (1.3 and 0.9 × 106 CD34+ cells/kg for #1 and #2, respectively). Patients did not receive any myeloablative conditionings. Although still preliminary in the limited time frames after treatment (6 weeks for #1 and several days for #2), we have observed normalization of liver enzyme abnormalities in #1 with gradual recovering from anorexia. Moreover, her neutrophil counts, not lymphocytes' though, have also returned to normal by week 6, suggesting that gene-corrected HSCs have engrafted and started expanding at least towards the myeloid lineage, while providing the patient with significant amount of ADA enough to detoxify the affected organs. In light of the serious adverse events in the French gene therapy trial for XSCID, careful follow-up of the patients is mandatory and thus underway, including monitoring a clonal expansion of any type of hematopoietic cells. More detailed outcomes will be presented.

Genetic deficiency of human IL-12 and IL-23 receptor β1 chain (IL-12Rβ1) results in increased vulnerability to weakly pathogenic strains of Mycobacteria and Salmonella. The main pathogenic mechanism in IL-12Rβ1 deficiency is the insufficient production of interferon gamma (IFN-γ) by T lymphocytes in response to IL-12 and IL-23, which prevents the efficient elimination of pathogens by effector cells. Despite early diagnosis and treatment with antibiotics and IFN-γ, IL12Rβ1-deficient patients can still succumb to complications of serious infections. Reversion of patients' susceptibility by corrective gene transfer could prevent the infectious episodes, thus providing a beneficial alternative. Because T cell development is intact in IL12Rβ1-deficiency, T lymphocyte-directed IL-12Rβ1 gene transfer correction could theoretically restore IL-12/IL-23 responsiveness and provide a polyclonal pool of gene-corrected T cells able to produce IFN-γ in response to exposure to pathogens. We therefore set out to investigate the feasibility of retroviral-mediated gene correction of T lymphocytes from IL-12Rβ1-deficient patients. Transduction of the human IL-12Rβ1 cDNA into immortalized and primary T cells from three IL-12Rβ1-deficient patients restored the expression of IL-12Rβ1 and reconstituted IL-12 signaling, as demonstrated by STAT4 phosphorylation and IFN-γ indicated that the biological defects of T cells from IL-12Rβ1 deficiency could be corrected by gene transfer. To evaluate the feasibility of T lymphocyte-directed gene therapy of IL-12Rβ1 deficiency in vivo, we used the IL-12Rβ1 knockout mouse model that closely mimics the human phenotype. These mice present with normal development of T cells that, however, fail to produce IFN-γ and proliferate in response to IL-12 and show impaired ability to produce IFN-γ in response to endotoxin (LPS) administration in vivo. Retroviral-mediated transduction of the murine IL-12Rβ1 cDNA into splenocytes from IL-12Rβ1 knockout mice activated with Concavalin A plus IL-2 was followed by FACS sorting of IL-12Rβ1-positive cells to obtain 100% gene-corrected cells. Sorted cells were stimulated with IL-12 and showed restored production of IFN-γ at levels comparable to normal control cells. In a preliminary experiment, five million IL-12Rβ1-positive cells were then injected intraperitoneally into two IL-12Rβ1 knockout mice and expression of IL-12Rβ1 was assessed in PBMCs from the recipient animals 2 weeks after transplantation. No clear evidence of gene corrected cells was demonstrated. Additional experiments are being performed to assess the optimal numbers of corrected T cells to be infused and to follow their survival in recipient animals.

The Wiskott-Aldrich Syndrome (WAS) is an X-linked disorder characterized by opportunistic, viral and bacterial infections, thrombocytopenia, eczema, and an increased incidence of autoimmune disorders and malignancies. While transplantation using HLA-matched bone marrow can be curative for young patients, the success rate for matched unrelated donor (MUD) transplantation falls precipitously with increasing age. Introduction of the WASp gene into autologous hematopoietic stem cells (HSC) by gene therapy would provide an alternative approach for correcting the deficiency, and may lead to long-term immunologic reconstitution. Several observations document the strong selective pressure for hematopoietic stem cells and their progeny expressing normal WASp. We have generated a series of WASp lentiviral vectors using an optimized SIN vector (“SMPU”) and a variety of internal promoters including the modified MMLV-based, MND LTR. Viral supernatants were used to transduce EBV- or HVS-transformed, wild-type and WASp-deficient B-cell and T cell lines, respectively. WASp expression was monitored using flow cytometry-based intracellular staining and Western blot analysis. Viral integration was quantified using Q-PCR based analysis. Using the vectors, SMPU-MND-WASp-GFP and SMPU-MND-WASp, we consistently achieved endogenous levels of WASp expression with an average of one viral integration/ transduced B or T cell. Strikingly, in WASp-vector transduced, WASp(−/−) T cells lines, we observed a progressive increase in the level of GFP/WASp+ cells over time. In one representative experiment we observed an increase in GFP/WASp expressing cells from ~2% at 14 days to 40% at 42 days post-transduction. In contrast, WASp(−/−) T cells concurrently transduced with the control SMPU-MND-GFP vector remained unchanged, exhibiting ~2–4 % GFP+ cells at Days 14 to 42 post-transduction. These data strongly suggest that transduced, WASp expressing T cells exhibit a progressive selective advantage in culture. We observed no similar selective advantage in the WASp (−/−) B cell lines transduced with the identical viral vector, SMPU-MND-WASp-GFP vector. The proliferative response of WASp reconstituted WASp(−/−) T-cells was also evaluated by [3H]thymidine incorporation. FACS purified WASp gene-corrected T-cells proliferated similarly to wild-type T-cells when exposed to anti-CD3, demonstrating correction of the T-cell proliferative defect after gene transfer. As an approach to potentially limit viral enhancer mediated insertional mutagenesis, and further stabilize transgene expression, we added defined insulator/boundary elements into these lentiviral-WASp gene constructs. A 243bp core element of the cHS4 insulator was inserted into the 3'-LTR (+39bp) in SMPU-MND-WASp-GFP. Experiments are underway to evaluate the capacity of these vectors to rescue WASp dependent function in WASp(−/−) B, and T-cell lines.

Hematopoietic stem cells (HSCs) differentiate in the thymus in to T cells along precisely defined steps. In this process the interaction between the thymic stroma, the epithelium and the hematopoietic precursors is indispensable. Previously it has been shown that human CD34 + cells from fetal liver, umbilical cord blood and adult bone marrow could differentiate meaningful numbers of T cells when seeded either into fetal thymic organ cultures (FTOCs) or human and Rhesus fetal thymic stromal monolayers. In severe combined immunodeficient (SCID) mouse models, development of T cells from transplanted human CD34 + cells requires previous engraftment of a human fetal thymus. All the mentioned assays require fetal tissues and are technically cumbersome. We set out to explore the possibility of using murine thymic stromal microenvironment to support differentiation of lymphocytes from human HSCs. Here we report that highly purified CD34 + or AC133 + CD34 + human umbilical cord blood cells generate T cells and natural killer (NK) cells when cultured on irradiated murine thymic stromal monolayers in the presence of recombinant human IL-12 and recombinant murine SCF. In this system, the human stem cells differentiate sequentially into CD4 + CD8 − CD3 − , CD4 + CD8 + CD3 − , CD4 + CD8 + CD3 + , and finally, CD4 + CD8 − CD3 + and CD4 − CD8 + CD3 + cells expressing T cell receptor α/β. CD56 + CD3 − NK cells were also differentiated and all cells strongly expressed human leucocyte-common antigen CD45. The kinetics of differentiation from HSCs to mature T cells were completed in about 6 weeks. Mature T-cells appeared to be functional as assessed by their normal response to mitogenic stimuli. This novel chimeric human-mouse thymic stromal culture offers a tool to study human T-cell ontogeny in vitro and is a rapid assay for T-cell precursor activity of genetically modified human stem cells. It also provides a preclinical model for analyzing the stability of transgene expression in differentiating lymphocytes.

Combined immune deficiencies comprise a spectrum of genetic disorders characterized by developmental or functional defects of both T and B lymphocytes. Recent progress in cell biology and molecular genetics has unraveled the pathophysiology of most of these defects. In particular, the most common form of severe combined immune deficiency in humans, with lack of circulating T cells, a normal or increased number of B lymphocytes, and an X-linked pattern of inheritance (SCIDXI) has been shown to be due to defects of the IL2RG gene, encoding for the common gamma chain (gammac), shared by several cytokine receptors. Furthermore, defects of the JAK3 gene, encoding for an intracellular tyrosine kinase required for signal transduction through gammac-containing cytokine receptors, have been identified in patients with autosomal recessive T-B+ SCID. Characterization of the functional properties of cytokines that signal through the gammac-JAK3 signaling pathway has been favored by the detailed analysis of SCID patients. Specifically, the key role of IL-7 in promoting T cell development has been substantiated by the identification of rare patients with T-B+ SCID who have a defect in the alpha subunit of the IL-7 receptor (IL7Ralpha). The heterogeneity of genetic defects along the same signaling pathway that may lead to combined immune deficiency is paralleled by the heterogeneity of immunological phenotypes that may associate with defects in the same gene, thus creating a need for detailed immunological and molecular investigations in order to dissect the spectrum of combined immune deficiencies in humans.

Resting lymphocytes are refractory to gene transfer using Moloney murine leukemia virus (MMLV)-based retroviral vectors because of their quiescent status. Recently, it has been shown that lentiviral vectors are capable of transferring genes into nondividing and terminally differentiated cells. We used human immunodeficiency virus type-1 (HIV-1)–based vectors expressing enhanced green fluorescent protein (EGFP) driven by different promoters (CMV, MPSV, or PGK) and investigated their ability to transduce human T- and B-cell lines, as well as resting or activated primary peripheral and umbilical cord blood lymphocytes. The effects of the presence or the absence of HIV-1 accessory proteins (Vif, Vpr, Vpu, and Nef) in the vector system were also assessed. Flow cytometry analysis showed no differences in the ability of these vectors of transferring the reporter gene into lymphocytic lines and mitogen-stimulated primary lymphocytes in the presence or the absence of HIV-1 accessory proteins (APs). Similarly, viral supernatants generated in the presence of accessory genes could efficiently transduce various subsets of resting lymphocytes and provide long-term expression of the transgene. No significant transduction-induced changes in cell activation or cycling status were observed and Alu-HIV-1 long terminal repeat polymerase chain reaction (LTR PCR) analysis demonstrated integration of the vector sequences at the molecular level. In contrast, in the absence of HIV-1 APs, lentiviral vectors failed to integrate and express the transgene in resting lymphocytes. These results show that transduction of primary resting lymphocytes with HIV-1–based vectors requires the presence of viral accessory proteins.

X-linked severe combined immunodeficiency (XSCID) is a lethal disease caused by a defect in the gene encoding the common gamma chain (gamma- c) of the receptor for interleukin-2 (IL-2), IL-4, IL-7, IL-9, and IL- 15. Allogeneic bone marrow transplantation, the current therapy of choice for this defect, is often complicated by graft-versus-host disease and/or incomplete reconstitution of B-lymphocyte functions. Correction of the gene defect at the level of the autologous lymphohematopoietic progenitors could therefore represent an improvement in the medical management of these patients. To study the feasibility of a gene therapy approach for XSCID, a retroviral vector expressing gamma-c was used to transduce Epstein-Barr virus-transformed B-cell lines derived from patients with XSCID. After transduction, XSCID cells newly expressed gamma-c on the cell surface at levels comparable to those observed on B-cell lines obtained from normal donors. Moreover, the reconstituted gamma-c restored function to the IL- 2 and IL-4 receptors as shown by signal transduction mediated by phosphorylation of the JAK1 and JAK3 members of the Janus family of tyrosine kinases and by restoration of cellular proliferation in response to IL-2.

Defects of the common gamma chain subunit of the cytokine receptors (γc) or of Jak3, a tyrosine kinase required for γc signal transduction, result in T−B+ severe combined immunodeficiency (SCID). However, atypical cases, characterized by progressive development of T lymphocytes, have been also reported. We describe a child with SCID caused by Jak3 gene defects, which strongly but not completely affect Jak3 protein expression and function, who developed a substantial number (>3,000/μL) of autologous CD3+CD4+ T cells. These cells showed a primed/activated phenotype (CD45R0+ Fas+HLA-DR+ CD62Llo), defective secretion of T-helper 1 and T-helper 2 cytokines, reduced proliferation to mitogens, and a high in vitro susceptibility to spontaneous (caused by downregulation of bcl-2 expression) as well as activation-induced cell death. A restricted T-cell receptor repertoire was observed, with oligoclonal expansion within each of the dominant segments. These features resemble those observed in γc-/y and in Jak3−/−mice, in which a population of activated, anergic T cells (predominantly CD4+) also develops with age. These results suggest that residual Jak3 expression and function or other Jak3-independent signals may also permit the generation of CD4+ T cells that undergo in vivo clonal expansion in humans; however, these mechanisms do not allow development of CD8+ T cells, nor do they fully restore the functional properties of CD4+ T lymphocytes.

We previously reported on an X-linked SCID (X-SCID) patient, who also had peripheral growth hormone (GH) hyporesponsiveness and abnormalities of the protein phosphorylation events following GH receptor (GHR) stimulation. In the present study, we examined a potential role of common cytokine receptor γ-chain (γc) in GHR signaling using EBV-transformed lymphocytes from healthy subjects and γc-negative X-SCID patients. We demonstrated that the proliferative response to GH stimulation of the B cell lines of γc-negative patients was impaired despite a comparable cellular expression of GHR molecules to controls. In patients, after GH stimulation, no phosphorylation of STAT5 was observed. In addition, the molecule localization through confocal microscopy revealed that in B cell lines of patients no nuclear translocation of STAT5b following GH stimulation occurred differently from controls. Biochemical analysis of the nuclear extracts of γc-negative cell lines provided further evidence that the amount of STAT5b and its phosphorylated form did not increase following GH stimulation. In patients, cells reconstituted with wild-type γc abnormal biochemical and functional events were restored resulting in nuclear translocation of STAT5. Confocal experiments revealed that GHR and γc were colocalized on the cell membrane. Our study demonstrates the existence of a previously unappreciated relationship between GHR-signaling pathway and γc, which is required for the activation of STAT5b in B cell lines. These data also confirm that growth failure in X-SCID is primarily related to the genetic alteration of the IL2RG gene.

Primary immunodeficiency diseases have been important targets of corrective gene transfer approaches since the very early days of gene therapy. The potential for selective survival advantage of gene-corrected cells over populations carrying the mutated, causative gene translates into the possibility of obtaining clinical meaningful results in patients with primary immunodeficiency diseases even if levels of gene transfer are low. This critical prospect has fueled the interest of researchers since the mid-1980s and has recently determined the success of a clinical trial of gene therapy for X-linked severe combined immunodeficiency.

Measurement of proliferative responses of human lymphocytes is a fundamental technique for the assessment of their biological responses to various stimuli. Most simply, this involves measurement of the number of cells present in a culture before and after the addition of a stimulating agent. This unit contains several different prototype protocols to induce proliferation in lymphocytes following exposure to mitogens, antigens, allogeneic or autologous cells, or soluble factors. Each of these protocols can be used in conjunction with an accompanying protocol, which contains methods to determine cell proliferation by incorporation of [(3)H]thymidine into DNA by nonradioactive methods, e.g., reduction of tetrazolium salts (MTT or WST-1). These protocols provide an estimate of cell proliferation indirectly by measuring DNA synthesis, and cell metabolic activity in an entire cell population, but no data on individual cells is obtained. A protocol for CFSE labeling allows direct detection of single proliferating cells and facilitates the quantification of cell divisions by flow cytometry according to the respective CFSE-dilution, and following costaining with fluorescent labeled antibodies, the characterization of subpopulations in the cell culture.