SPATA2 restricts OTULIN-dependent LUBAC activity independently of CYLD.

SPATA2 mediates the recruitment of CYLD to immune receptor complexes by bridging the interaction of CYLD with the linear ubiquitylation assembly complex (LUBAC) component HOIP. Whether SPATA2 exhibits functions independently of CYLD is unclear. Here, we show that, while Cyld-/- and Spata2-/- mice are viable, double mutants exhibit highly penetrant perinatal lethality, indicating independent functions of SPATA2 and CYLD. Cyld-/-Spata2-/- fibroblasts show increased M1-linked TNFR1-SC ubiquitylation and, similar to Cyld-/-Spata2-/- macrophages and intestinal epithelial cells, elevated pro-inflammatory gene expression compared with Cyld-/- or Spata2-/- cells. We show that SPATA2 competes with OTULIN for binding to HOIP via its PUB-interacting motif (PIM) and its zinc finger domain, thereby promoting autoubiquitylation of LUBAC. Consistently, increased pro-inflammatory signaling in Cyld-/-Spata2-/- cells depends on the presence of OTULIN. Our data therefore indicate that SPATA2 counteracts, independently of CYLD, the deubiquitylation of LUBAC by OTULIN and thereby attenuates LUBAC activity and pro-inflammatory signaling.

CD4+ T cells require Ikaros to inhibit their differentiation toward a pathogenic cell fate.

The production of proinflammatory cytokines, particularly granulocyte-macrophage colony-stimulating factor (GM-CSF), by pathogenic CD4+ T cells is central for mediating tissue injury in inflammatory and autoimmune diseases. However, the factors regulating the T cell pathogenic gene expression program remain unclear. Here, we investigated how the Ikaros transcription factor regulates the global gene expression and chromatin accessibility changes in murine T cells during Th17 polarization and after activation via the T cell receptor (TCR) and CD28. We found that, in both conditions, Ikaros represses the expression of genes from the pathogenic signature, particularly Csf2, which encodes GM-CSF. We show that, in TCR/CD28-activated T cells, Ikaros binds a critical enhancer downstream of Csf2 and is required to regulate chromatin accessibility at multiple regions across this locus. Genome-wide Ikaros binding is associated with more compact chromatin, notably at multiple sites containing NFκB or STAT5 target motifs, and STAT5 or NFκB inhibition prevents GM-CSF production in Ikaros-deficient cells. Importantly, Ikaros also limits GM-CSF production in TCR/CD28-activated human T cells. Our data therefore highlight a critical conserved transcriptional mechanism that antagonizes GM-CSF expression in T cells.

Requirement of GSK-3 for PUMA induction upon loss of pro-survival PI3K signaling.

Growth factor withdrawal induces rapid apoptosis via mitochondrial outer membrane permeabilization. We had previously observed that cell death of IL-3-dependent Ba/F3 cells, induced by removal of the growth factor, required the activity of the kinase GSK-3. Employing CRISPR/Cas9-mediated gene knockout, we aimed to identify pro-apoptotic GSK-3 regulated factors in this process. Knockout of either Puma or Bim demonstrated that the induction of Puma, but not Bim, was crucial for apoptosis induced by IL-3 deprivation. Thus, we aimed at identifying the GSK-3-dependent PUMA regulator. Loss of FOXO3A reduced the induction of Puma, while additional loss of p53 completely repressed induction upon growth factor withdrawal. A constitutively active mutant of FOXO3A, which cannot be controlled by AKT directly, still required active GSK-3 for the full transcriptional induction of Puma and cell death upon IL-3 withdrawal. Thus, the suppression of GSK-3 is the key function of PI3K signaling in order to prevent the induction of Puma by FOXO3A and p53 and thereby apoptosis upon growth factor withdrawal.

CDK9-mediated phosphorylation controls the interaction of TIP60 with the transcriptional machinery.

The acetyltransferase TIP60 is regulated by phosphorylation, and we have previously shown that phosphorylation of TIP60 on S86 by GSK-3 promotes p53-mediated induction of the BCL-2 protein PUMA. TIP60 phosphorylation by GSK-3 requires a priming phosphorylation on S90, and here, we identify CDK9 as a TIP60S90 kinase. We demonstrate that a phosphorylation-deficient mutant, TIP60S90A, exhibits reduced interaction with chromatin, histone 3 and RNA Pol II, while its association with the TIP60 complex subunit EPC1 is not affected. Consistently, we find a diminished association of TIP60S90A with the MYC gene. We show that cells expressing TIP60S90A, but also TIP60S86A, which retains S90 phosphorylation, exhibit reduced histone 4 acetylation and proliferation. Thus, our data indicate that, during transcription, phosphorylation of TIP60 at two sites has different regulatory effects on TIP60, whereby S90 phosphorylation controls association with the transcription machinery, and S86 phosphorylation is regulating TIP60 HAT activity.

SPATA2 promotes CYLD activity and regulates TNF-induced NF-κB signaling and cell death.

K63- and Met1-linked ubiquitylation are crucial posttranslational modifications for TNF receptor signaling. These non-degradative ubiquitylations are counteracted by deubiquitinases (DUBs), such as the enzyme CYLD, resulting in an appropriate signal strength, but the regulation of this process remains incompletely understood. Here, we describe an interaction partner of CYLD, SPATA2, which we identified by a mass spectrometry screen. We find that SPATA2 interacts via its PUB domain with CYLD, while a PUB interaction motif (PIM) of SPATA2 interacts with the PUB domain of the LUBAC component HOIP SPATA2 is required for the recruitment of CYLD to the TNF receptor signaling complex upon TNFR stimulation. Moreover, SPATA2 acts as an allosteric activator for the K63- and M1-deubiquitinase activity of CYLD In consequence, SPATA2 substantially attenuates TNF-induced NF-κB and MAPK signaling. Conversely, SPATA2 is required for TNF-induced complex II formation, caspase activation, and apoptosis. Thus, this study identifies SPATA2 as an important factor in the TNF signaling pathway with a substantial role for the effects mediated by the cytokine.

Phosphorylation of Tip60 by GSK-3 determines the induction of PUMA and apoptosis by p53.

Activation of p53 by DNA damage results in either cell-cycle arrest, allowing DNA repair and cell survival, or induction of apoptosis. As these opposite outcomes are both mediated by p53 stabilization, additional mechanisms to determine this decision must exist. Here, we show that glycogen synthase kinase-3 (GSK-3) is required for the p53-mediated induction of the proapoptotic BH3 only-protein PUMA, an essential mediator of p53-induced apoptosis. Inhibition of GSK-3 protected from cell death induced by DNA damage and promoted increased long-term cell survival. We demonstrate that GSK-3 phosphorylates serine 86 of the p53-acetyltransferase Tip60. A Tip60(S86A) mutant was less active to induce p53 K120 acetylation, histone 4 acetylation, and expression of PUMA. Our data suggest that GSK-3 mediated Tip60S86 phosphorylation provides a link between PI3K signaling and the choice for or against apoptosis induction by p53.

Vector integration is nonrandom and clustered and influences the fate of lymphopoiesis in SCID-X1 gene therapy.

Recent reports have challenged the notion that retroviruses and retroviral vectors integrate randomly into the host genome. These reports pointed to a strong bias toward integration in and near gene coding regions and, for gammaretroviral vectors, around transcription start sites. Here, we report the results obtained from a large-scale mapping of 572 retroviral integration sites (RISs) isolated from cells of 9 patients with X-linked SCID (SCID-X1) treated with a retrovirus-based gene therapy protocol. Our data showed that two-thirds of insertions occurred in or very near to genes, of which more than half were highly expressed in CD34(+) progenitor cells. Strikingly, one-fourth of all integrations were clustered as common integration sites (CISs). The highly significant incidence of CISs in circulating T cells and the nature of their locations indicate that insertion in many gene loci has an influence on cell engraftment, survival, and proliferation. Beyond the observed cases of insertional mutagenesis in 3 patients, these data help to elucidate the relationship between vector insertion and long-term in vivo selection of transduced cells in human patients with SCID-X1.

Gammaretrovirus-mediated correction of SCID-X1 is associated with skewed vector integration site distribution in vivo.

We treated 10 children with X-linked SCID (SCID-X1) using gammaretrovirus-mediated gene transfer. Those with sufficient follow-up were found to have recovered substantial immunity in the absence of any serious adverse events up to 5 years after treatment. To determine the influence of vector integration on lymphoid reconstitution, we compared retroviral integration sites (RISs) from peripheral blood CD3(+) T lymphocytes of 5 patients taken between 9 and 30 months after transplantation with transduced CD34(+) progenitor cells derived from 1 further patient and 1 healthy donor. Integration occurred preferentially in gene regions on either side of transcription start sites, was clustered, and correlated with the expression level in CD34(+) progenitors during transduction. In contrast to those in CD34(+) cells, RISs recovered from engrafted CD3(+) T cells were significantly overrepresented within or near genes encoding proteins with kinase or transferase activity or involved in phosphorus metabolism. Although gross patterns of gene expression were unchanged in transduced cells, the divergence of RIS target frequency between transduced progenitor cells and post-thymic T lymphocytes indicates that vector integration influences cell survival, engraftment, or proliferation.

Efficient marking of human cells with rapid but transient repopulating activity in autografted recipients.

Short-term hematopoietic reconstituting cells have been identified in mice, nonhuman primates, and among human cells that engraft xenogeneic hosts. We now present clonal marking data demonstrating a rapid but unsustained contribution of cultured human autografts to the initial phase of hematologic recovery in myeloablated patients. Three patients received transplants of granulocyte colony-stimulating factor-mobilized autologous peripheral blood (PB) cells, of which a portion (8%-25% of the CD34+ cells) had been incubated in vitro with growth factors (5 days) and clinical grade LN retrovirus (3-5 days). More than 9% of the clonogenic and long-term culture-initiating cells harvested were transduced. Semiquantitative and linear amplification-mediated polymerase chain reaction analyses of serial PB samples showed that marked white blood cells appeared in all 3 patients within 11 days and transiently constituted up to 0.1% to 1% of those produced in the first month. However, within another 2 to 9 months, marked cells had permanently decreased to very low levels. Analysis of more than 50 vector insertion sites showed none of the clones detected in the first month were active later. Eighty percent of inserts were located within or near genes, 2 near CXCR4. These findings provide direct evidence of cells with rapid but transient repopulating activity in patients and demonstrate their efficient transduction in vitro.

Clonal evidence for the transduction of CD34+ cells with lymphomyeloid differentiation potential and self-renewal capacity in the SCID-X1 gene therapy trial.

Immune function has been restored in 9 of 10 children with X-linked severe combined immunodeficiency by gamma c gene transfer in CD34+ cells. The distribution of both T-cell receptor (TCR) V beta family usage and TCR V beta complementarity-determining region 3 (CDR3) length revealed a broadly diversified T-cell repertoire. Retroviral integration site analysis in T cells demonstrated a high number of distinct insertion sites, indicating polyclonality of genetically corrected cell clones, in all patients. Detection of gamma c transgene expression on patients' mature myeloid cells has prompted us to investigate the nature of the most immature transduced hematopoietic precursor cells. Insertion sites shared by T and B lymphocytes as well as highly purified granulocytes and monocytes demonstrate the correction of common multipotent progenitor cells. Moreover, our data show that differentiated leukocytes share the same exact insertion sites with CD34+ cells that we obtained 8 months later and that were able to generate long-term culture-initiating cells (LTC-ICs). This finding demonstrates the initial transduction of very primitive multipotent progenitor cells with self-renewal capacity. These results provide a first evidence in the setting of a clinical trial that CD34+ cells maintain both lymphomyeloid potential as well as self-renewal capacity after ex vivo manipulation.

Efficient characterization of retro-, lenti-, and foamyvector-transduced cell populations by high-accuracy insertion site sequencing.

The identification of unknown genomic flanking DNA sequences can be used for the molecular monitoring of retro-, lenti- and foamyviral integration, transgenes in early embryogenesis, insertional mutagenesis, cell fate, and stem cell plasticity. Most existing methods reflect shortcomings in sensitivity and or specificity, thus limiting genomic sequencing of unknown flanking DNA to clonal preparations. The application of linear amplification-mediated PCR (LAM-PCR), a recently developed direct sequencing technique for flanking DNA, should circumvent current limitations in different research fields. This technique combines preamplification of target DNA with a unique succession of enzymatic reactions on solid-phase. Using LAM-PCR, we show the previously unfeasible in vivo retro-, lenti- and foamyvirus integration site analysis in primate peripheral blood hematopoietic cells and human xenograft hematopoiesis. In light of two severe adverse events that occurred in a clinical SCID-X1 gene therapy trial, in vivo monitoring of the reinfused transduced cell pool by integration site analysis will be an important component of each gene transfer and therapy study aimed at clinical use.

Comparison of three retroviral vector systems for transduction of nonobese diabetic/severe combined immunodeficiency mice repopulating human CD34+ cord blood cells.

The use of recombinant vectors based on wild-type viruses that are absent in humans and are not associated with any disease in their natural animal hosts or in accidentally infected humans would add an additional level of safety for human somatic gene therapy approaches. These criteria are fulfilled by foamy viruses (FVs), a family of complex retroviruses whose members are widely found among mammals and are apathogenic in all hosts. Here, we show by comparison of identically designed vector constructs that recombinant retroviral vectors based on FVs were as efficient as lentiviral vectors in transducing nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice repopulating human CD34(+) cord blood (CB) cells. The FV vector was able to achieve gene transfer levels up to 84% of engrafted human cells in a short overnight transduction protocol. In contrast, without prestimulation of the target cells, a human immunodeficiency virus type 1 (HIV-1)-based lentiviral vector pseudotyped with gibbon ape leukemia virus envelope (GALV Env) was nearly as inefficient as murine leukemia virus (MLV)-based oncoretroviral vectors in transducing NOD/SCID repopulating cells. The same HIV vector pseudotyped with the vesicular stomatitis virus glycoprotein G (VSV-G) achieved high marking efficiency. Clonality analysis of bone marrow samples showed oligoclonal hematopoiesis with single to multiple insertions per cell, both for FV and HIV vectors. These data demonstrate that vectors based on FVs warrant further investigation and development for medical use.

Clonality analysis after retroviral-mediated gene transfer to CD34+ cells from the cord blood of ADA-deficient SCID neonates.

A clinical trial of retroviral-mediated transfer of the adenosine deaminase (ADA) gene into umbilical cord blood CD34(+) cells was started in 1993. ADA-containing peripheral blood mononuclear cells (PBMCs) have persisted in patients from this trial, with T lymphocytes showing the highest prevalence of gene marking. To gain a greater understanding of the nature and number of the transduced cells that were engrafted, we used linear amplification-mediated PCR (LAM-PCR) to identify clonal vector proviral integrants. In one patient, a single vector integrant was predominant in T lymphocytes at a stable level over most of the eight-year time span analyzed and was also detected in some myeloid samples. T-cell clones with the predominant integrant, isolated after eight years, showed multiple patterns of T-cell receptor (TCR) gene rearrangement, indicating that a single pre-thymic stem or progenitor cell served as the source of the majority of the gene-marked cells over an extended period of time. It is important to distinguish the stable pattern of monoclonal gene marking that we observed here from the progressive increase of a T-cell clone with monoclonal gene marking that results from leukemic transformation, as observed in two subjects in a clinical trial of gene therapy for X-linked severe combined immunodeficiency (SCID).

Polyclonal long-term repopulating stem cell clones in a primate model.

Hematopoietic bone marrow stem cells generate differentiated blood cells and, when transplanted, may contribute to other organs, such as the brain, heart, and liver. An understanding of in vivo clonal behavior of stem cells will have important implications for cellular and gene therapy. For the first time, we have directly demonstrated the derivation of circulating peripheral blood cells from individual stem cell clones. We analyzed the clonal composition of retrovirus-marked peripheral blood leukocyte populations in 2 different primate models by a novel direct genomic sequencing technique allowing the identification of vector insertion sites. More than 80 contributing long-term hematopoietic clones were identified in individual rhesus macaque peripheral blood transplant recipients and more than 25 different clones in a baboon marrow transplant recipient. Up to 5 insertion sequences from each animal were used to trace the long-term contribution of stem cell clones in these primate models. Continuous and mostly pluripotent contributions of peripheral blood leukocytes from each of the traced clones could be detected for the entire follow-up period of 23 to 33 months. Our study provides direct molecular evidence for a polyclonal, multilineage, and sustained contribution of individual stem cells to primate hematopoiesis.