Combined Immune Deficiency Research Articles

Hematopoietic stem cell transplantation from unrelated donors in children with DOCK8 deficiency

DIDS is a unique form of combined immune deficiency characterized by an unusual susceptibility to cutaneous viral infections, severe allergies with eosinophilia and elevated immunoglobulin E titers, autoimmunity, and cancer. HSCT is considered the standard of care for this deadly disease. We have retrospectively analyzed the outcome of allogeneic HSCT from unrelated donors in patients with DIDS. Data from four patients, with five transplants, are presented. All patients received transplants from unrelated donors' BM, except for one patient who received a cord blood transplant. The conditioning regimens were based on myeloablative protocols for BM derived transplants; a NM regimen was pursued for the patient who received a cord blood transplant, which resulted in graft rejection. Although recurrent pneumonia and skin infections resolved immediately after transplantation, all patients subsequently developed human herpesvirus infection, including cutaneous herpetic lesions, cytomegalovirus reactivation, and zona zoster, which could be attributed to the use of ATG. Despite the presence of serious morbidities prior to transplantation, all patients recovered successfully. DIDS can be successfully treated with allogeneic HSCT from unrelated donors following a myeloablative conditioning regimen, with a reasonable safety profile.

Improving Gene Therapy Efficiency through the Enrichment of Human Hematopoietic Stem Cells

Lentiviral vector (LV)-based hematopoietic stem cell (HSC) gene therapy is becoming a promising clinical strategy for the treatment of genetic blood diseases. However, the current approach of modifying 1× 108 to 1× 109 CD34+ cells per patient requires large amounts of LV, which is expensive and technically challenging to produce at clinical scale. Modification of bulk CD34+ cells uses LV inefficiently, because the majority of CD34+ cells are short-term progenitors with a limited post-transplant lifespan. Here, we utilized a clinically relevant, immunomagnetic bead (IB)-based method to purify CD34+CD38- cells from human bone marrow (BM) and mobilized peripheral blood (mPB). IB purification of CD34+CD38- cells enriched severe combined immune deficiency (SCID) repopulating cell (SRC) frequency an additional 12-fold beyond standard CD34+ purification and did not affect gene marking of long-term HSCs. Transplant of purified CD34+CD38- cells led to delayed myeloid reconstitution, which could be rescued by the addition of non-transduced CD38+ cells. Importantly, LV modification and transplantation of IB-purified CD34+CD38- cells/non-modified CD38+ cells into immune-deficient mice achieved long-term gene-marked engraftment comparable with modification of bulk CD34+ cells, while utilizing ∼7-fold less LV. Thus, we demonstrate a translatable method to improve the clinical and commercial viability of gene therapy for genetic blood cell diseases.

Adult venous endothelium is a niche for highly proliferative and vasculogenic endothelial colony-forming cells

ObjectivePostnatal resident endothelium of blood vessels has been proposed to represent terminally differentiated tissue that does not replicate. We previously isolated endothelial colony-forming cells (ECFCs) from human umbilical cord blood (CB) and term placenta by using colony-forming assays and immunocytochemistry. We showed that ECFCs are highly proliferative and form functioning vessels in vivo, the defining characteristics of a true endothelial progenitor cell. This exploratory investigation was conducted to determine whether the endothelium of healthy adult blood vessels contained resident ECFCs. MethodsThe endothelium of great saphenous vein (GSV) obtained from vein stripping procedures was collected with mechanical scraping, and ECFCs were isolated according to established protocols. ResultsGSV ECFCs incorporated acetylated low-density lipoprotein, formed tubules in Matrigel (BD Biosciences, San Jose, Calif) at 24 hours, and expressed endothelial antigens cluster of differentiation (CD) 144, CD31, CD105, and kinase insert domain receptor but not hematopoietic antigen CD45. Using cumulative population doublings and single-cell assays, we demonstrated that GSV ECFCs exhibited comparable proliferative capacities compared with CB ECFCs, including similar numbers of highly proliferative cells. When injected in collagen/fibronectin gels implanted in nonobese diabetic/severe combined immune deficiency mice, GSV ECFCs formed blood vessels with circulating murine red blood cells, demonstrating their vasculogenic potential. ConclusionsThe ECFCs of the GSV contain a hierarchy of progenitor cells with a comparable number of highly proliferative clones as ECFCs of CB. The results of this investigation demonstrate that the adult endothelium contains resident progenitor cells that may have a critical role in vascular homeostasis and repair and could potentially be used as a source of autologous cells for cell therapies focusing on vasculogenesis.

Polyomavirus-associated Prostatitis in Wistar Han Rats Following Immunosuppression in a Chronic Toxicity Study.

Chronic prostatitis characterized on light microscopic examination by moderate, multifocal, predominantly lymphocytic inflammation associated with epithelial atypia and intranuclear and cytoplasmic inclusion-like material was identified in the prostate gland of 2 Wistar Han rats administered an immunomodulatory test article in a 6-month chronic toxicity study. Transmission electron microscopy of the prostate glands identified 45-nm, nonenveloped, icosahedral virions arranged in paracrystalline array within the cell nuclei in 1 of the 2 rats. The size, shape, location, and array pattern were most consistent with a polyomavirus. The light and electron microscopic findings after immunosuppression in our case have a resemblance to a polyomavirus recently reported to affect prostate gland epithelium in a colony of immunocompromised X-linked severe combined immune deficiency rats. To the best of our knowledge, this is the first report of light and electronic microscopic lesions in the reproductive tract associated with polyomavirus following chronic immunosuppression in a widely used, wild-type Wistar Han rat.

Gene Therapy 2017: Progress and Future Directions.

Gene therapy has changed dramatically in the 28 years since the first human gene transfer experiment in 1989. Alipogene tiparvovec, GlyberaR®, a recombinant adeno‐associated virus (rAAV) product for lipoprotein lipase deficiency, and Strimvelis®, a lentivirus vector for severe combined immune deficiency are approved in Europe. An rAAV2 product for a congenital form of blindness is currently under review in the United States, likely to be followed by numerous other gene therapies. Nonviral gene transfer The success of gene therapy has largely been driven by improvements in nonviral and viral gene transfer vectors. An array of physical and chemical nonviral methods have been used to transfer DNA and mRNA to mammalian cells and a substantial number of these have been developed as clinical stage technologies for gene therapy, both ex vivo and in vivo. Cationic liposome technology is based on the ability of amphipathic lipids, possessing a positively charged head group and a hydrophobic lipid tail, to bind to negatively charged DNA or RNA and form particles that generally enter cells by endocytosis. Some cationic liposomes also contain a neutral co‐lipid, thought to enhance liposome uptake by mammalian cells.4, 5, 6, 7 Similarly, other polycations, such as poly‐l‐lysine and polyethylene‐imine, complex with nucleic acids via charge interaction and aid in the condensation of DNA or RNA into nanoparticles, which are then substrates for endosome‐mediated uptake.8 Several of these cationic‐nucleic acid complex technologies have been developed as potential clinical products, including complexes with plasmid DNA (pDNA), oligodeoxynucleotides, and various forms of synthetic RNA.9, 10, 11 Modified (and unmodified or “naked”) DNA, RNA, and oligonucleotides have also been shown to mediate successful gene transfer in a number of circumstances. These include the use of pDNA by direct intramuscular injection for DNA vaccines, the use of intratumoral injection of pDNA to deliver cytokine and/or suicide genes, systemic (s.c. or i.v.) injection of antisense nucleotides to induce RNAse H1 or exon‐skipping.12, 13, 14 The most recent of these developed for induction of RNAi are discussed in a later section. Ex vivo introduction of pDNA and/or other nucleotides using physical methods has been well developed for certain cell types, including T lymphocytes.15 Electroporation techniques have become the standard with T cells for the introduction of a variety of molecular cargoes, including ribonucleoproteins composed of Cas9 and short‐guide RNAs for genome editing (see section below) and transposons for long‐term integration of transgenes.

Podoplanin promotes progression of malignant pleural mesothelioma by regulating motility and focus formation.

Malignant pleural mesothelioma (MPM) is characterized by dissemination and aggressive growth in the thoracic cavity. Podoplanin (PDPN) is an established diagnostic marker for MPM, but the function of PDPN in MPM is not fully understood. The purpose of this study was to determine the pathogenetic function of PDPN in MPM. Forty‐seven of 52 tumors (90%) from Japanese patients with MPM and 3/6 (50%) MPM cell lines tested positive for PDPN. Knocking down PDPN in PDPN‐high expressing MPM cells resulted in decreased cell motility. In contrast, overexpression of PDPN in PDPN‐low expressing MPM cells enhanced cell motility. PDPN stimulated motility was mediated by activation of the RhoA/ROCK pathway. Moreover, knocking down PDPN with short hairpin (sh) RNA in PDPN‐high expressing MPM cells resulted in decreased development of a thoracic tumor in mice with severe combined immune deficiency (SCID). In sharp contrast, transfection of PDPN in PDPN‐low expressing MPM cells resulted in an increase in the number of Ki‐67‐positive proliferating tumor cells and it promoted progression of a thoracic tumor in SCID mice. Interestingly, PDPN promoted focus formation in vitro, and a low level of E‐cadherin expression and YAP1 activation was observed in PDPN‐high MPM tumors. These findings indicate that PDPN is a diagnostic marker as well as a pathogenetic regulator that promotes MPM progression by increasing cell motility and inducing focus formation. Therefore, PDPN might be a pathogenetic determinant of MPM dissemination and aggressive growth and may thus be an ideal therapeutic target.

Homozygous deletion of RAG1, RAG2 and 5' region TRAF6 causes severe immune suppression and atypical osteopetrosis.

Mutations of several genes have been implicated in autosomal recessive osteopetrosis (OP), a disease caused by impaired function and differentiation of osteoclasts. Severe combined immune deficiencies (SCID) can likewise result from different genetic mutations. We report two siblings with SCID and an atypical phenotype of OP. A biallelic microdeletion encompassing the 5' region of TRAF6, RAG1 and RAG2 genes was identified. TRAF6, a tumor necrosis factor receptor-associated family member, plays an important role in T cell signaling and in RANKL-dependent osteoclast differentiation and activation but its role in human OP has not been previously reported. The RAG proteins are essential for recombination of B and T cell receptors, and for the survival and differentiation of these cells. This is the first study to report a homozygous deletion of TRAF6 as a cause of human disease.

Noninvasive Imaging of Human Immune Responses in a Human Xenograft Model of Graft-Versus-Host Disease.

The immune system plays a crucial role in many diseases. Activation or suppression of immunity is often related to clinical outcome. Methods to explore the dynamics of immune responses are important to elucidate their role in conditions characterized by inflammation, such as infectious disease, cancer, or autoimmunity. Immuno-PET is a noninvasive method by which disease and immune cell infiltration can be explored simultaneously. Using radiolabeled antibodies or fragments derived from them, it is possible to image disease-specific antigens and immune cell subsets. Methods: We developed a method to noninvasively image human immune responses in a relevant humanized mouse model. We generated a camelid-derived single-domain antibody specific for human class II major histocompatibility complex products and used it to noninvasively image human immune cell reconstitution in nonobese diabetic severe combined immune deficiency γ-/- mice reconstituted with human fetal thymus, liver, and liver-derived hematopoietic stem cells (BLT mice). Results: We showed imaging of infiltrating immunocytes in BLT mice that spontaneously developed a graft-versus-host-like condition, characterized by alopecia and blepharitis. In diseased animals, we showed an increased PET signal in the liver, attributable to infiltration of activated class II major histocompatibility complex+ T cells. Conclusion: Noninvasive imaging of immune infiltration and activation could thus be of importance for diagnosis and evaluation of treatment of graft-versus-host disease and holds promise for other diseases characterized by inflammation.

Abstract P1-02-10: Reduced serum B-cell maturation antigen levels predict poor outcome in metastatic breast cancer patients in a phase 3 randomized 2nd-line hormone therapy trial

Abstract Background: B-cell maturation antigen (BCMA) is a member of the tumor necrosis factor receptor family and has two ligands, B-cell activating factor (BAFF) and a proliferation inducing ligand (APRIL). These ligands activate cell proliferation and inhibit apoptosis of normal and malignant B-cells including in multiple myeloma (MM) cell lines. Berenson et al have recently reported that circulating BCMA levels are elevated in B-cell malignancies and can be used to monitor disease and predict PFS and OS for patients with MM, Waldenstroms's macroglobulinemia and chronic lymphocytic leukemia (CLL). On the other hand, recent studies have shown that serum BCMA levels are very low among patients with MM in complete remission with low antibody levels and those with primary immune deficiencies specifically those with combined variable immune deficiency and X-linked agammaglobulinemia. Studies of the potential role of serum BCMA for patients with solid tumors has not been evaluated to date. Methods: The pretreatment serum from 139 patients with hormone receptor-positive metastatic breast cancer who were enrolled in a phase 3 randomized clinical trial of second-line hormone therapy was evaluated using an ELISA for BCMA. The BCMA ELISA was from R&D Systems (Minneapolis, MN). Serum BCMA was correlated with TTP using categorical serum BCMA cutpoints. Results: Pretreatment serum BCMA levels had a median of 55.61 ng/ml, an interquartile range of 34.20 and 78.79 ng/ml, and full range from 3.99 to 1193.26 ng/ml. In univariate analysis for TTP, reduced serum BCMA correlated with shorter TTP at the following dichotomous cutpoints: 15 ng/ml [HR=2.60, p=0.064, n=6 (4.3%) of patients below cutpoint]; 20 ng/ml [HR=2.88, p=0.005, n=10 (7.2%) of patients below cutpoint]; 25 ng/ml [HR=2.16, p=0.023, n=13 (9.4%) of patients below cutpoint]; and 30 ng/ml [HR=1.77, p=0.016, n=27 (19.4%) of patients below the cutpoint]. Conclusions: In a phase 3 randomized clinical trial of second-line hormone therapy among patients with hormone receptor-positive metastatic breast cancer, reduced pretreatment serum BCMA was associated with shorter TTP. This may be due to the association of reduced serum BCMA with immune deficiency; and, thus, lead to shorter TTP among patients with metastatic breast cancer. Evaluation of serum BCMA as a new biomarker to predict outcomes for breast cancer and other solid tumor patients deserves further study. Citation Format: Ali SM, Leitzel K, Li M, Udd K, Wang J, Sanchez E, Chen H, Berenson J, Lipton A. Reduced serum B-cell maturation antigen levels predict poor outcome in metastatic breast cancer patients in a phase 3 randomized 2nd-line hormone therapy trial [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P1-02-10.

How We Manage Adenosine Deaminase-Deficient Severe Combined Immune Deficiency (ADA SCID).

Adenosine deaminase-deficient severe combined immune deficiency (ADA SCID) accounts for 10-15% of cases of human SCID. From what was once a uniformly fatal disease, the prognosis for infants with ADA SCID has improved greatly based on the development of multiple therapeutic options, coupled with more frequent early diagnosis due to implementation of newborn screening for SCID. We review the various treatment approaches for ADA SCID including allogeneic hematopoietic stem cell transplantation (HSCT) from a human leukocyte antigen-matched sibling or family member or from a matched unrelated donor or a haplo-identical donor, autologous HSCT with gene correction of the hematopoietic stem cells (gene therapy-GT), and enzyme replacement therapy (ERT) with polyethylene glycol-conjugated adenosine deaminase. Based on growing evidence of safety and efficacy from GT, we propose a treatment algorithm for patients with ADA SCID that recommends HSCT from a matched family donor, when available, as a first choice, followed by GT as the next option, with allogeneic HSCT from an unrelated or haplo-identical donor or long-term ERT as other options.

Granulomatous Lymphocytic Interstitial Lung Disease (GLILD) in 22q11.2 Deletion Syndrome with Combined Immune Deficiency (CID)

Immune dysregulation and autoimmuity are well-known complications of 22q11.2 deletion syndrome (22q11.2DS). GLILD has classically been assoicated with common variable immune deficiency (CVID) and has not generally been reported in other immunodeficiencies. We describe the clinicopathological and radiological features of GLILD based on positron emission tomography (PET), computed tomography (CT), and lung biopsy in an adolescent male with 22q11.2DS and CID. The patient had 22q11.2DS and CID as defined by low naive T-cell numbers, decreased lymphoproliferative responses to mitogens and antigens, hypogammaglobulinemia, and suboptimal vaccine responses. He received monthly intravenous immunoglobulin and inhaled pentamidine. Routine cardiac imaging incidentally discovered bulky mediastinal lymphadenopathy, splenomegaly, and pulmonary nodules as confirmed by PET and CT. Scattered groundglass nodular opacities and central bronchiectasis were also seen. Lung biopsy histopathology and immunophenotyping showed patchy unencapsulated lymphohistiocytic nodules containing poorly formed non-necrotizing granulomata with a predominance of T-cells and follicular bronchiolitis. Following exclusion of infectious and neoplastic processes, the patient received rituximab with marked improvement. His GLILD progressed 5 months later, incidentally with repopulation of B-cells. He was re-treated with rituximab followed by mercaptopurine with good response. We believe this is only the second reported case of GLILD in a patient with 22q11.2DS. GLILD may not be unique to CVID and should be considered as a potential cause of interstitial lung disease in other immunodeficiencies. GLILD is associated with poorer outcomes in CVID. Recognition of GLILD as a rare phenotype of 22q11.2DS and other immunodeficiencies may therefore have important therapeutic and prognostic implications.

Cholesterol-Containing Nuclease-Resistant siRNA Accumulates in Tumors in a Carrier-free Mode and Silences MDR1 Gene.

Chemical modifications are an effective way to improve the therapeutic properties of small interfering RNAs (siRNAs), making them more resistant to degradation in serum and ensuring their delivery to target cells and tissues. Here, we studied the carrier-free biodistribution and biological activity of a nuclease-resistant anti-MDR1 cholesterol-siRNA conjugate in healthy and tumor-bearing severe combined immune deficiency (SCID) mice. The attachment of cholesterol to siRNA provided its efficient accumulation in the liver and in tumors, and reduced its retention in the kidneys after intravenous and intraperitoneal injection. The major part of cholesterol-siRNA after intramuscular and subcutaneous injections remained in the injection place. Confocal microscopy data demonstrated that cholesterol-siRNA spread deep in the tissue and was present in the cytoplasm of almost all the liver and tumor cells. The reduction of P-glycoprotein level in human KB-8-5 xenograft overexpressing the MDR1 gene by 60% was observed at days 5–6 after injection. Then, its initial level recovered by the eighth day. The data showed that, regardless of the mode of administration (intravenous, intraperitoneal, or peritumoral), cholesterol-siMDR efficiently reduced the P-glycoprotein level in tumors. The designed anti-MDR1 conjugate has potential as an adjuvant therapeutic for the reversal of multiple drug resistance of cancer cells.

Establishing a colorectal cancer liver metastasis patient-derived tumor xenograft model for the evaluation of personalized chemotherapy.

PurposeIn order to suggest optimal anticancer drugs for patient-tailored chemotherapy, we developed a colorectal cancer (CRC)-liver metastasis patient-derived tumor xenograft (PDTX) model.MethodsTissue obtained from a patient with CRC-liver metastasis (F0) was transplanted in a nonobese female mouse with diabetic/severe combined immune deficiency (F1) and the tumor tissue was retransplanted into nude mice (F2). When tumor volumes reached ~500 mm3, the F2 mice were randomly divided into 4 groups (n = 4/group) of doxorubicin, cisplatin, docetaxel, and nontreated control groups. The tumor tissues were investigated using H&E staining, terminal deoxynucleotidyl transferase dUTP nick end labeling assays, and immunohistochemistry. To determine where the mutant allele frequencies varied across the different passages, we isolated genomic DNA from the primary tumor, liver metastasis, and PDTX models (F1/F2).ResultsThe physiological properties of the tumor were in accord with those of the patient's tumors. Anticancer drugs delayed tumor growth, inhibited proliferation, and caused apoptosis. Histological assessments revealed no observable heterogeneity among the intragenerational PDTX models. Target exon sequencing analysis without high-quality filter conditions revealed some genetic variations in the 83 cancer-related genes across the generations. However, when de novo mutations were defined as a total count of zero in F0 and ≥5 in F2, exactly prognostic impact of clone cancer profiling (EGFR, KRAS, BRAF, PIK3CA, NRAS, APC and TP53) were detected in the paired.ConclusionA CRC liver metastasis PDTX model was established for the evaluation of chemotherapeutic efficacy. This model retained the physiological characters of the patient tumors and potentially provides a powerful means of assessing chemotherapeutic efficacy.

Neonatal Screening for Severe Combined Immune Deficiency in Russia: Glorious Future or Tomorrow’s Reality?

Mass screening of newborns in Russia for five hereditary diseases does not meet the requirements of the world community for the neonatal screening program. Success in the development of laboratory diagnostic technologies and active introduction of achievements in genetics and molecular biology into medical practice allow for the revision of the list of nosologies included in the national neonatal screening program by replacing the disease or adding new nosologies. The article discusses the possibility of including genetic testing for severe combined immune deficiency in the Newborn Screening Program in Russia. The results from a retrospective study of markers of naive T- and B-lymphocytes (TREC and KREC) in the group of children with immuno-dependent pathology developed in the first year of life are discussed.

In Vivo Study of Human Endothelial-Pericyte Interaction Using the Matrix Gel Plug Assay in Mouse.

Angiogenesis is the process by which new blood vessels are formed from existing vessels. New vessel growth requires coordinated endothelial cell proliferation, migration, and alignment to form tubular structures followed by recruitment of pericytes to provide mural support and facilitate vessel maturation. Current in vitro cell culture approaches cannot fully reproduce the complex biological environment where endothelial cells and pericytes interact to produce functional vessels. We present a novel application of the in vivo matrix gel plug assay to study endothelial-pericyte interactions and formation of functional blood vessels using severe combined immune deficiency mutation (SCID) mice. Briefly, matrix gel is mixed with a solution containing endothelial cells with or without pericytes followed by injection into the back of anesthetized SCID mice. After 14 days, the matrix gel plugs are removed, fixed and sectioned for histological analysis. The length, number, size and extent of pericyte coverage of mature vessels (defined by the presence of red blood cells in the lumen) can be quantified and compared between experimental groups using commercial statistical platforms. Beyond its use as an angiogenesis assay, this matrix gel plug assay can be used to conduct genetic studies and as a platform for drug discovery. In conclusion, this protocol will allow researchers to complement available in vitro assays for the study of endothelial-pericyte interactions and their relevance to either systemic or pulmonary angiogenesis.

Foamy Virus Backbone Has Insulator Properties Which Remarkably Reduce Its Genotoxicity Potential

Strong viral enhancers in γ-retrovirus vectors (GV) have caused cellular proto-oncogene activation and leukemia in gene therapy trials, necessitating the use of cellular promoters in enhancer-less integrating vectors. However, data is now emerging that inadequate transgene expression from cellular promoters may limit successful correction. Vectors derived from foamy virus (FV), a nonpathogenic retrovirus, have a higher preference for non-genic integrations than GV/lentiviral vectors (LV), and may be less genotoxic. We constructed GV, LV and FV driven either by the spleen focus forming virus (SFFV) or the murine stem cell virus (MSCV) enhancer/promoters, all driving eGFP expression, and compared their relative genotoxicity using an in vitro immortalization assay on primary hematopoietic stem/progenitor cells (HSPC). In this assay, integration near a protooncogene/gene promoting cell proliferation results in quantifiable HSPC immortalization. Strong viral enhancer/promoters from SFFV or MSCV in FV caused a remarkably low immortalization of HSPC compared to analogous LV or GV: compared to the immortalization frequency of HSPC with the SFFV-GV in this assay, SFFV-LV and MSCV-LV had 12- and 14-fold lower immortalization frequency, while the SFFV-FV and MSCV-FV showed a 155- and 414-fold lower immortalization frequency, respectively. Immortalized clones had multiple (3-10) integrated copies. Integration site analysis of FV immortalized clones revealed a majority of integrants in non-gene regions; those in genic regions targeted cell proliferation or proto-oncogenes, as previously reported. FV has been previously reported to have 2-fold higher insertions in non-genic regions and higher, but nearly half the propensity to target promoters compared to GV. However, this remarkably reduced genotoxicity with FV was not explained by the integration pattern. We therefore hypothesized that FV backbone may contain sequences that have an enhancer blocking/insulator effect. Studies on chromatin insulators have shown that enhancer-blocking property of insulators is mediated via binding of CTCF to its consensus sequences within the insulator. Indeed, an in silico analysis for CCCTC-binding factor (CTCF) binding sites in the vector backbone sequences showed more predicted CTCF binding sites in the FV than in GV or LV (26, 8, and 6, respectively). To functionally validate the enhancer-blocking effect of the FV backbone and ensure that only effects of the vector backbone would be measured, without the confounding influence of integration site or the enhancer/promoter/transgene, we inserted SFFV-GV, SFFV-LV and SFFV-FV into a clinically relevant proto-oncogene, LMO2, using CRISPR/Cas9, and assessed LMO2 expression. LMO2 upregulation has previously resulted in leukemias in the X-linked severe combined immune-deficiency and Wiscott-Aldrich syndrome (WAS) GV-mediated gene transfer trials; notably SFFV-GV was used in the WAS trial and caused leukemias in 8 of 10 patients from insertional oncogenesis. HeLa cells were transfected with the proviral donor plasmids and the guide-RNA/spCas9 plasmids and GFP+ cells sorted and cloned. Nearly all clones derived had one intact LMO2 allele, while the other alleles had GV/LV/FV proviral insertions. We next assessed LMO2 mRNA and protein expression in these clones. We found a minimal increase in LMO2 mRNA expression with SFFV-FV, in sharp contrast to significantly increased LMO2 expression with SFFV-GV and SFFV-LV by qRT-PCR (Figure 1A). Overall, the SFFV enhancer in GV demonstrated the greatest fold-increase in LMO2 expression (median increase of 280+/-23-fold over unmodified HeLa cells), followed by the SFFV enhancer in LV (median 200+/-27-fold increase). However, the same SFFV enhancer in FV only showed a 45+/-7-fold median increase in expression. Western blot analysis for LMO2 protein expression in three clones for each vector showed no detectable LMO2 expression in SFFV-FV clones, which was similar to baseline in mock (non-targeted) HeLa cells (Figure 1B). However, significantly higher LMO2 protein was detectable in GV and LV clones. Hence, the remarkably low genotoxic potential of FV, even those carrying strong viral enhancers appears to be, in large part, from an insulator property of FV sequences, making FV ideal for situations where high transgene expression, necessitating strong enhancers is required for a therapeutic effect. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

T Cell Immune Deficiency in zap70 Mutant Zebrafish.

ZAP70 [zeta-chain (TCR)-associated protein kinase, 70-kDa], is required for T cell activation. ZAP70 deficiencies in humans and null mutations in mice lead to severe combined immune deficiency. Here, we describe a zap70 loss-of-function mutation in zebrafish (zap70 y442 ) that was created using transcription activator-like effector nucleases (TALENs). In contrast to what has been reported for morphant zebrafish, zap70 y442 homozygous mutant zebrafish displayed normal development of blood and lymphatic vasculature. Hematopoietic cell development was also largely unaffected in mutant larvae. However, mutant fish had reduced lck:GFP + thymic T cells by 5 days postfertilization that persisted into adult stages. Morphological analysis, RNA sequencing, and single-cell gene expression profiling of whole kidney marrow cells of adult fish revealed complete loss of mature T cells in zap70 y442 mutant animals. T cell immune deficiency was confirmed through transplantation of unmatched normal and malignant donor cells into zap70 y442 mutant zebrafish, with T cell loss being sufficient for robust allogeneic cell engraftment. zap70 mutant zebrafish show remarkable conservation of immune cell dysfunction as found in mice and humans and will serve as a valuable model to study zap70 immune deficiency.

Anticytokine autoantibodies in infection and inflammation: an update.

Concise overview of the field of anticytokine autoantibodies with a focus on recent developments. Advances in particular in the analysis of autoantibodies to IFNγ, granulocyte-macrophage colony-stimulating factor (GM-CSF) and type I IFN are presented. The target epitope for anti-IFNγ autoantibodies has been found to have high homology to a protein from Aspergillus suggesting molecular mimicry as a mechanism of breaking self-tolerance. A treatment strategy using a recombinant, epitope-depleted version of IFNγ is suggested. Autoantibodies to GM-CSF are associated with disseminated Crytococcus and Nocardia infections thus expanding the spectrum of associated diseases beyond pulmonary alveolar proteinosis. Detailed analysis of anti-GM-CSF autoantibody clones derived from pulmonary alveolar proteinosis patients show evidence of high somatic mutation suggesting T cell-dependent affinity maturation; full GM-CSF neutralization is achieved by synergistic binding of antibodies targeting various distinct noncross-reactive epitopes and leading to antigen sequestration and Fc-mediated clearance. Single mAbs in contrast may lead to higher GM-CSF bioavailability. Anti type I IFN-specific autoantibodies derived from autoimmune polyglandular syndrome type I patients are of extreme high affinity and negatively correlate with the incidence of type I diabetes and may be thus considered to be protective. Hypomorphic severe combined immune deficiency may be associated with complex anticytokine patterns and the emergence of anti type I IFN autoantibodies correlates with severe viral infection histories. Anticytokine autoantibodies may cause susceptibility to infections. In autoimmune/autoinflammatory conditions, anticytokine autoantibodies may be protective or promote disease.

Glypican-5 suppresses Epithelial-Mesenchymal Transition of the lung adenocarcinoma by competitively binding to Wnt3a.

We previously demonstrated that Glypican-5 (GPC5), one of the members of heparan sulfate proteoglycan, was a novel tumor metastasis suppressor in lung adenocarcinoma (LAC). However, it remains unclear how GPC5 suppresses lung cancer metastasis. Here, we found over-expression GPC5 induced significant Epithelial-Mesenchymal Transition (EMT) process of A549 cells in vitro. Bioinformatic analysis of RNA sequencing data indicated that GPC5 was co-expressed with EMT related markers, E-cadherin and Vimentin. Wnt/β-catenin signaling pathway was also significantly enriched after overexpressing GPC5. Further in vitro experiments demonstrated that overexpressing GPC5 could block the translocation of β-catenin from cytoplasm to nucleus and therefore inactivate the Wnt/β-catenin signaling pathway by competitively binding to Wnt3a. Subsequent rescue experiments demonstrated that GPC5-induced metastatic phenotype and EMT process suppression were significantly reversed when cells cultured in Wnt3a conditioned media. By establishing the metastatic model in severe combined immune deficiency (SCID) mice, we also demonstrated that overexpressing GPC5 suppressed LAC migration and accordingly alerted EMT related markers, which including up-regulated E-cadherin and down-regulated Vimentin in both lung and liver metastasis. Finally, clinical samples of LAC further validated that GPC5 expression was positively correlated with E-cadherin, and negatively correlated with both Twist1 and MMP2. Taken together, these data suggested that GPC5 is able to suppress the LAC metastasis by competitively binding to Wnt3a and inactivating the Wnt/β-catenin signaling pathway. Our findings expanded the role and the molecular mechanism of GPC5 on malignant bionomics of LAC.

Due to interleukin-6 type cytokine redundancy only glycoprotein 130 receptor blockade efficiently inhibits myeloma growth.

Interleukin-6 has an important role in the pathophysiology of multiple myeloma where it supports the growth and survival of the malignant plasma cells in the bone marrow. It belongs to a family of cytokines which use the glycoprotein 130 chain for signal transduction, such as oncostatin M or leukemia inhibitory factor. Targeting interleukin-6 in plasma cell diseases is currently evaluated in clinical trials with monoclonal antibodies. Here, efforts were made to elucidate the contribution of interleukin-6 and glycoprotein 130 signaling in malignant plasma cell growth in vivo In the xenograft severe combined immune deficiency model employing our interleukin-6-dependent plasma cell line INA-6, the lack of human interleukin-6 induced autocrine interleukin-6 production and a proliferative response to other cytokines of the glycoprotein 130 family. Herein, mice were treated with monoclonal antibodies against human interleukin-6 (elsilimomab/B-E8), the interleukin-6 receptor (B-R6), and with an antibody blocking glycoprotein 130 (B-R3). While treatment of mice with interleukin-6 and interleukin-6 receptor antibodies resulted in a modest delay in tumor growth, the development of plasmacytomas was completely prevented with the anti-glycoprotein 130 antibody. Importantly, complete inhibition was also achieved using F(ab')2-fragments of monoclonal antibody B-R3. Tumors harbor activated signal transducer and activator of transcription 3, and in vitro, the antibody inhibited leukemia inhibitory factor stimulated signal transducer and activator of transcription 3 phosphorylation and cell growth, while being less effective against interleukin-6. In conclusion, the growth of INA-6 plasmacytomas in vivo under interleukin-6 withdrawal remains strictly dependent on glycoprotein 130, and other glycoprotein 130 cytokines may substitute for interleukin-6. Antibodies against glycoprotein 130 are able to overcome this redundancy and should be explored for a possible therapeutic window.