B-cell reconstitution after lentiviral vector-mediated gene therapy in patients with Wiskott-Aldrich syndrome

Background Wiskott-Aldrich syndrome (WAS) is a severe X-linked immunodeficiency characterized by microthrombocytopenia, eczema, recurrent infections, and susceptibility to autoimmunity and lymphomas. Hematopoietic stem cell transplantation is the treatment of choice; however, administration of WAS gene-corrected autologous hematopoietic stem cells has been demonstrated as a feasible alternative therapeutic approach. Objective Because B-cell homeostasis is perturbed in patients with WAS and restoration of immune competence is one of the main therapeutic goals, we have evaluated reconstitution of the B-cell compartment in 4 patients who received autologous hematopoietic stem cells transduced with lentiviral vector after a reduced-intensity conditioning regimen combined with anti-CD20 administration. Methods We evaluated B-cell counts, B-cell subset distribution, B cell-activating factor and immunoglobulin levels, and autoantibody production before and after gene therapy (GT). WAS gene transfer in B cells was assessed by measuring vector copy numbers and expression of Wiskott-Aldrich syndrome protein. Results After lentiviral vector-mediated GT, the number of transduced B cells progressively increased in the peripheral blood of all patients. Lentiviral vector-transduced progenitor cells were able to repopulate the B-cell compartment with a normal distribution of B-cell subsets both in bone marrow and the periphery, showing a WAS protein expression profile similar to that of healthy donors. In addition, after GT, we observed a normalized frequency of autoimmune-associated CD19+CD21-CD35- and CD21low

Lentiviral gene therapy corrects platelet phenotype and function in patients with Wiskott-Aldrich syndrome

BACKGROUND: Thrombocytopenia is a serious issue for all patients with classical Wiskott-Aldrich syndrome (WAS) and X-linked thrombocytopenia (XLT) because it causes severe and life-threatening bleeding. Lentiviral gene therapy (GT) for WAS has shown promising results in terms of immune reconstitution. However, despite the reduced severity and frequency of bleeding events, platelet counts remain low in GT-treated patients. OBJECTIVE: We carefully investigated platelet defects in terms of phenotype and function in untreated patients with WAS and assessed the effect of GT treatment on platelet dysfunction. METHODS: We analyzed a cohort of 20 patients with WAS/XLT, 15 of them receiving GT. Platelet phenotype and function were analyzed by using electron microscopy, flow cytometry, and an aggregation assay. Platelet protein composition was assessed before and after GT by means of proteomic profile analysis. RESULTS: We show that platelets from untreated patients with WAS have reduced size, abnormal ultrastructure, and a hyperactivated phenotype at steady state, whereas activation and aggregation responses to agonists are decreased. GT restores platelet size and function early after treatment and reduces the hyperactivated phenotype proportionally to WAS protein expression and length of follow-up. CONCLUSIONS: Our study highlights the coexistence of morphologic and multiple functional defects in platelets lacking WAS protein and demonstrates that GT normalizes the platelet proteomic profile with consequent restoration of platelet ultrastructure and phenotype, which might explain the observed reduction of bleeding episodes after GT. These results are instrumental also from the perspective of a future clinical trial in patients with XLT only presenting with microthrombocytopenia

Lentiviral Hematopoietic Stem Cell Gene Therapy in Patients with Wiskott-Aldrich Syndrome.

iskott-Aldrich syndrome (WAS) is an inherited immunodeficiency caused by mutations in the gene encoding WASP, a protein regulating the cytoskeleton. Hematopoietic stem/progenitor cell (HSPC) transplants can be curative, but, when matched donors are unavailable, infusion of autologous HSPCs modified ex vivo by gene therapy is an alternative approach. We used a lentiviral vector encoding functional WASP to genetically correct HSPCs from three WAS patients and reinfused the cells after a reduced-intensity conditioning regimen. All three patients showed stable engraftment of WASP-expressing cells and improvements in platelet counts, immune functions, and clinical scores. Vector integration analyses revealed highly polyclonal and multilineage haematopoiesis resulting from the gene-corrected HSPCs. Lentiviral gene therapy did not induce selection of integrations near oncogenes, and no aberrant clonal expansion was observed after 20 to 32 months. Although extended clinical observation is required to establish long-term safety, lentiviral gene therapy represents a promising treatment for WAS

NKp46-expressing human gut-resident intraepithelial V\u3b41 T cell subpopulation exhibits high anti-tumor activity against colorectal cancer

\u3b3\u3b4 T cells account for a large fraction of human intestinal intraepithelial lymphocytes (IELs) endowed with potent anti-tumor activities. However, little is known about their origin, phenotype and clinical relevance in colorectal cancer (CRC). To determine \u3b3\u3b4 IEL gut-specificity, homing and functions, \u3b3\u3b4 T cells were purified from human healthy blood, lymph nodes, liver, skin, intestine either disease-free or affected by CRC or generated from thymic precursors. The constitutive expression of NKp46 specifically identifies a new subset of cytotoxic V\u3b41 T cells representing the largest fraction of gut-resident IELs. The ontogeny and gut-tropism of NKp46pos/V\u3b41 IELs depends both on distinctive features of V\u3b41 thymic precursors and gut-environmental factors. Either the constitutive presence of NKp46 on tissue-resident V\u3b41 intestinal IELs or its induced-expression on IL-2/IL-15 activated V\u3b41 thymocytes are associated with anti-tumor functions. Higher frequencies of NKp46pos/V\u3b41 IELs in tumor-free specimens from CRC patients correlate with a lower risk of developing metastatic III/IV disease stages. Additionally, our in vitro settings reproducing CRC tumor-microenvironment inhibited the expansion of NKp46pos/V\u3b41 cells from activated thymic precursors. These results parallel the very low frequencies of NKp46pos/V\u3b41 IELs able to infiltrate CRC, thus providing new insights to either follow-up cancer progression or develop novel adoptive cellular therapies

EXTL3 mutations cause skeletal dysplasia, immune deficiency, and developmental delay.

We studied three patients with severe skeletal dysplasia, T cell immunodeficiency, and developmental delay. Whole-exome sequencing revealed homozygous missense mutations affecting exostosin-like 3 (EXTL3), a glycosyltransferase involved in heparan sulfate (HS) biosynthesis. Patient-derived fibroblasts showed abnormal HS composition and altered fibroblast growth factor 2 signaling, which was rescued by overexpression of wild-type EXTL3 cDNA. Interleukin-2-mediated STAT5 phosphorylation in patients' lymphocytes was markedly reduced. Interbreeding of the extl3-mutant zebrafish (box) with Tg(rag2:green fluorescent protein) transgenic zebrafish revealed defective thymopoiesis, which was rescued by injection of wild-type human EXTL3 RNA. Targeted differentiation of patient-derived induced pluripotent stem cells showed a reduced expansion of lymphohematopoietic progenitor cells and defects of thymic epithelial progenitor cell differentiation. These data identify EXTL3 mutations as a novel cause of severe immune deficiency with skeletal dysplasia and developmental delay and underline a crucial role of HS in thymopoiesis and skeletal and brain development

Tractography passes the test: Results from the diffusion-simulated connectivity (disco) challenge.

Estimating structural connectivity from diffusion-weighted magnetic resonance imaging is a challenging task, partly due to the presence of false-positive connections and the misestimation of connection weights. Building on previous efforts, the MICCAI-CDMRI Diffusion-Simulated Connectivity (DiSCo) challenge was carried out to evaluate state-of-the-art connectivity methods using novel large-scale numerical phantoms. The diffusion signal for the phantoms was obtained from Monte Carlo simulations. The results of the challenge suggest that methods selected by the 14 teams participating in the challenge can provide high correlations between estimated and ground-truth connectivity weights, in complex numerical environments. Additionally, the methods used by the participating teams were able to accurately identify the binary connectivity of the numerical dataset. However, specific false positive and false negative connections were consistently estimated across all methods. Although the challenge dataset doesn't capture the complexity of a real brain, it provided unique data with known macrostructure and microstructure ground-truth properties to facilitate the development of connectivity estimation methods

Heterozygous FOXN1 Variants Cause Low TRECs and Severe T Cell Lymphopenia, Revealing a Crucial Role of FOXN1 in Supporting Early Thymopoiesis

FOXN1 is the master regulatory gene of thymic epithelium development. FOXN1 deficiency leads to thymic aplasia, alopecia, and nail dystrophy, accounting for the nude/severe combined immunodeficiency (nu/SCID) phenotype in humans and mice. We identified several newborns with low levels of T cell receptor excision circles (TRECs) and T cell lymphopenia at birth, who carried heterozygous loss-of-function FOXN1 variants. Longitudinal analysis showed persistent T cell lymphopenia during infancy, often associated with nail dystrophy. Adult individuals with heterozygous FOXN1 variants had in most cases normal CD4+ but lower than normal CD8+ cell counts. We hypothesized a FOXN1 gene dosage effect on the function of thymic epithelial cells (TECs) and thymopoiesis and postulated that these effects would be more prominent early in life. To test this hypothesis, we analyzed TEC subset frequency and phenotype, early thymic progenitor (ETP) cell count, and expression of FOXN1 target genes (Ccl25, Cxcl12, Dll4, Scf, Psmb11, Prss16, and Cd83) in Foxn1nu/+ (nu/+) mice and age-matched wild-type (+/+) littermate controls. Both the frequency and the absolute count of ETP were significantly reduced in nu/+ mice up to 3 weeks of age. Analysis of the TEC compartment showed reduced expression of FOXN1 target genes and delayed maturation of the medullary TEC compartment in nu/+ mice. These observations establish a FOXN1 gene dosage effect on thymic function and identify FOXN1 haploinsufficiency as an important genetic determinant of T cell lymphopenia at birth

Arp2/3 Complex Regulates Asymmetric Division and Cytokinesis in Mouse Oocytes

Mammalian oocyte meiotic maturation involves oocyte polarization and a unique asymmetric division, but until now, the underlying mechanisms have been poorly understood. Arp2/3 complex has been shown to regulate actin nucleation and is widely involved in a diverse range of processes such as cell locomotion, phagocytosis and the establishment of cell polarity. Whether Arp2/3 complex participates in oocyte polarization and asymmetric division is unknown. The present study investigated the expression and functions of Arp2/3 complex during mouse oocyte meiotic maturation. Immunofluorescent staining showed that the Arp2/3 complex was restricted to the cortex, with a thickened cap above the meiotic apparatus, and that this localization pattern was depended on actin. Disruption of Arp2/3 complex by a newly-found specific inhibitor CK666, as well as by Arpc2 and Arpc3 RNAi, resulted in a range of effects. These included the failure of asymmetric division, spindle migration, and the formation and completion of oocyte cytokinesis. The formation of the actin cap and cortical granule-free domain (CGFD) was also disrupted, which further confirmed the disruption of spindle migration. Our data suggest that the Arp2/3 complex probably regulates oocyte polarization through its effect on spindle migration, asymmetric division and cytokinesis during mouse oocyte meiotic maturation

DOCK8 deficiency impairs CD8 T cell survival and function in humans and mice

As shown by analysis of mice and humans bearing DOCK8-inactivating mutations, DOCK8 plays a cell-autonomous role in survival of naive CD8 T cells, LFA-1 polarization toward the immune synapse, and CD8 T cell memory and recall responses following viral infection