Publications by authors named "Anna Villa"

162 Publications

NKG2A expression identifies a subset of human Vδ2 T cells exerting the highest antitumor effector functions.

Cell Rep 2021 Oct;37(3):109871

Laboratory of Clinical and Experimental Immunology, IRCCS Humanitas Research Hospital, 20089 Rozzano, Milan, Italy; Department of Medical Biotechnologies and Translational Medicine, University of Milan, Milan, Italy. Electronic address:

Human Vδ2 cells are innate-like γδ T effectors performing potent immune surveillance against tumors. The constitutive expression of NKG2A identifies a subset of Vδ2 T cells licensed with an intrinsic hyper-responsiveness against cancer. Indeed, the transcriptomic profiles of NKG2A and NKG2A cells characterize two distinct "intralineages" of Vδ2 T lymphocytes that appear early during development, keep their phenotypes, and show self-renewal capabilities in adult life. The hyper-responsiveness of NKG2A Vδ2 T cells is counterbalanced by the inhibitory signaling delivered by human leukocyte antigen E (HLA-E) expressed on malignant cells as a tumor-escape mechanism. However, either masking or knocking out NKG2A restores the capacity of Vδ2 T cells to exert the highest effector functions even against HLA-E tumors. This is highly relevant in the clinic, as the different degrees of engagement of the NKG2A-HLA-E checkpoint in hepatocellular carcinoma, glioblastoma, and non-small cell lung cancer directly impact patients' overall survival. These findings open avenues for developing combined cellular and immunologic anticancer therapies.
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http://dx.doi.org/10.1016/j.celrep.2021.109871DOI Listing
October 2021

Insights into Cadmium-Induced Carcinogenesis through an In Vitro Study Using C3H10T1/2Cl8 Cells: The Multifaceted Role of Mitochondria.

Int J Mol Sci 2021 Oct 7;22(19). Epub 2021 Oct 7.

Department of Earth and Environmental Sciences, University of Milan Bicocca, Piazza della Scienza 1, 20126 Milan, Italy.

In this paper, we report the metabolic characterization of two , F1 and F3, obtained at the end of Cell Transformation Assay (CTA), performed by treating C3H10T1/2Cl8 mouse embryo fibroblasts with 1 μM CdCl for 24 h. The elucidation of the cadmium action mechanism can be useful both to improve the in vitro CTA and to yield insights into carcinogenesis. The metabolism of the two was investigated through Seahorse and enzyme activity assays; mitochondria were studied in confocal microscopy and reactive oxygen species were detected by flow cytometry. The results showed that F1 has higher glycolytic and TCA fluxes compared to F3 , and a more negative mitochondrial membrane potential, so that most ATP synthesis is performed through oxidative phosphorylation. Confocal microscopy showed mitochondria crowded in the perinuclear region. On the other hand, F3 showed lower metabolic rates, with ATP mainly produced by glycolysis and damaged mitochondria. Overall, our results showed that cadmium treatment induced lasting metabolic alterations in both . Triggered by the loss of the Pasteur effect in F1 and by mitochondrial impairment in F3 , these alterations lead to a loss of coordination among glycolysis, TCA and oxidative phosphorylation, which leads to malignant transformation.
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http://dx.doi.org/10.3390/ijms221910837DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8509127PMC
October 2021

Ablation of Collagen VI leads to the release of platelets with altered function.

Blood Adv 2021 Sep 21. Epub 2021 Sep 21.

University of Pavia, Pavia, Italy.

Hemostatic abnormalities and impaired platelet function have been described in patients affected by connective tissue disorders. We observed a moderate bleeding tendency in patients affected by Collagen VI-related disorders and investigated the defects in platelet functionality whose mechanisms are unknown. We demonstrated that megakaryocytes express Collagen VI that is involved in the regulation of functional platelet production. By exploiting a Collagen VI null mouse model (Col6a1-/-), we found that Collagen VI null platelets display significantly increased susceptibility to activation and intracellular calcium signaling. Col6a1-/- megakaryocytes and platelets showed increased expression of STIM1 and ORAI1, the components of Store-Operated Calcium Entry (SOCE), and activation of the mTOR signaling pathway. In vivo mTOR inhibition by rapamycin reduced both STIM1 and ORAI1 expression and calcium flows, resulting in a normalization of platelet susceptibility to activation. These defects were cell-autonomous, as transplantation of lineage-negative bone marrow cells from Col6a1-/- mice into lethally irradiated wild-type animals showed the same alteration in SOCE and platelet activation as compared to Col6a1-/- mice. Peripheral blood platelets of patients affected by Collagen VI-related diseases, Bethlem myopathy and Ullrich congenital muscular dystrophy, displayed increased expression of STIM1 and ORAI1 and were more prone to activation. Altogether, these data demonstrate the importance of Collagen VI in the production of functional platelets by megakaryocytes in mouse models and in Collagen VI-related diseases.
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http://dx.doi.org/10.1182/bloodadvances.2020002671DOI Listing
September 2021

A novel intronic variant in PIGB in Acrofrontofacionasal dysostosis type 1 patients expands the spectrum of phenotypes associated with GPI biosynthesis defects.

Bone 2021 12 14;153:116152. Epub 2021 Aug 14.

CNR-IRGB, Milan Unit, Milan, Italy; IRCCS Humanitas Research Hospital, Rozzano (MI), Italy. Electronic address:

Acrofrontofacionasal dysostosis type 1 (AFFND1) is an extremely rare disorder characterized by several dysmorphic features, skeletal abnormalities and intellectual disability, and described only in seven patients in the literature. A biallelic variant in the Neuroblastoma Amplified Sequence (NBAS) gene was recently identified in two Indian patients with AFFND1. Here we report genetic investigation of AFFND1 in the originally described Brazilian families and the identification of an extremely rare, recessively-inherited, intronic variant in the Phosphatidylinositol Glycan class B (PIGB) gene NC_000015.10 (NM_004855.4): c.795-19T > G) in the affected individuals. The PIGB gene encodes an enzyme involved in the biosynthesis of the glycosylphosphatidylinositol (GPI) anchor, which is required for the post-translational modification of a large variety of proteins, enabling their correct cellular localization and function. Recessive variants in PIGB have previously been reported in individuals with a neurodevelopmental syndrome having partial overlap with AFFND1. In vitro assays demonstrated that the intronic variant leads to exon skipping, suggesting the Brazilian AFFND1 patients may be null for PIGB, in agreement with their severe clinical phenotype. These data increase the number of pathogenic variants in the PIGB gene, place AFFND1 among GPI deficiencies and extend the spectrum of phenotypes associated with GPI biosynthesis defects.
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http://dx.doi.org/10.1016/j.bone.2021.116152DOI Listing
December 2021

Thymic Epithelial Cell Alterations and Defective Thymopoiesis Lead to Central and Peripheral Tolerance Perturbation in MHCII Deficiency.

Front Immunol 2021 15;12:669943. Epub 2021 Jun 15.

San Raffaele Telethon Institute for Gene Therapy (SR-Tiget), IRCCS San Raffaele Scientific Institute, Milan, Italy.

Major Histocompatibility Complex (MHC) class II (MHCII) deficiency (MHCII-D), also known as Bare Lymphocyte Syndrome (BLS), is a rare combined immunodeficiency due to mutations in genes regulating expression of MHCII molecules. MHCII deficiency results in impaired cellular and humoral immune responses, leading to severe infections and autoimmunity. Abnormal cross-talk with developing T cells due to the absence of MHCII expression likely leads to defects in thymic epithelial cells (TEC). However, the contribution of TEC alterations to the pathogenesis of this primary immunodeficiency has not been well characterized to date, in particular in regard to immune dysregulation. To this aim, we have performed an in-depth cellular and molecular characterization of TEC in this disease. We observed an overall perturbation of thymic structure and function in both MHCII mice and patients. Transcriptomic and proteomic profiling of murine TEC revealed several alterations. In particular, we demonstrated that impairment of lymphostromal cross-talk in the thymus of MHCII mice affects mTEC maturation and promiscuous gene expression and causes defects of central tolerance. Furthermore, we observed peripheral tolerance impairment, likely due to defective Treg cell generation and/or function and B cell tolerance breakdown. Overall, our findings reveal disease-specific TEC defects resulting in perturbation of central tolerance and limiting the potential benefits of hematopoietic stem cell transplantation in MHCII deficiency.
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http://dx.doi.org/10.3389/fimmu.2021.669943DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8239840PMC
October 2021

Premature Senescence and Increased Oxidative Stress in the Thymus of Down Syndrome Patients.

Front Immunol 2021 1;12:669893. Epub 2021 Jun 1.

San Raffaele Telethon Institute for Gene Therapy (SR-TIGET), IRCCS San Raffaele Scientific Institute, Milan, Italy.

Down syndrome (DS) patients prematurely show clinical manifestations usually associated with aging. Their immune system declines earlier than healthy individuals, leading to increased susceptibility to infections and higher incidence of autoimmune phenomena. Clinical features of accelerated aging indicate that trisomy 21 increases the biological age of tissues. Based on previous studies suggesting immune senescence in DS, we hypothesized that induction of cellular senescence may contribute to early thymic involution and immune dysregulation. Immunohistochemical analysis of thymic tissue showed signs of accelerated thymic aging in DS patients, normally seen in older healthy subjects. Moreover, our whole transcriptomic analysis on human Epcam-enriched thymic epithelial cells (hTEC), isolated from three DS children, which revealed disease-specific transcriptomic alterations. Gene set enrichment analysis (GSEA) of DS TEC revealed an enrichment in genes involved in cellular response to stress, epigenetic histone DNA modifications and senescence. Analysis of senescent markers and oxidative stress in hTEC and thymocytes confirmed these findings. We detected senescence features in DS TEC, thymocytes and peripheral T cells, such as increased β-galactosidase activity, increased levels of the cell cycle inhibitor p16, telomere length and integrity markers and increased levels of reactive oxygen species (ROS), all factors contributing to cellular damage. In conclusion, our findings support the key role of cellular senescence in the pathogenesis of immune defect in DS while adding new players, such as epigenetic regulation and increased oxidative stress, to the pathogenesis of immune dysregulation.
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http://dx.doi.org/10.3389/fimmu.2021.669893DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8204718PMC
October 2021

Autosomal recessive osteopetrosis: mechanisms and treatments.

Dis Model Mech 2021 May 10;14(5). Epub 2021 May 10.

San Raffaele Telethon Institute for Gene Therapy (SR-Tiget), IRCCS San Raffaele Scientific Institute, Milan 20132, Italy.

Autosomal recessive osteopetrosis (ARO) is a severe inherited bone disease characterized by defective osteoclast resorption or differentiation. Clinical manifestations include dense and brittle bones, anemia and progressive nerve compression, which hamper the quality of patients' lives and cause death in the first 10 years of age. This Review describes the pathogenesis of ARO and highlights the strengths and weaknesses of the current standard of care, namely hematopoietic stem cell transplantation (HSCT). Despite an improvement in the overall survival and outcomes of HSCT, transplant-related morbidity and the pre-existence of neurological symptoms significantly limit the success of HSCT, while the availability of human leukocyte antigen (HLA)-matched donors still remains an open issue. Novel therapeutic approaches are needed for ARO patients, especially for those that cannot benefit from HSCT. Here, we review preclinical and proof-of-concept studies, such as gene therapy, systematic administration of deficient protein, in utero HSCT and gene editing.
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http://dx.doi.org/10.1242/dmm.048940DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8188884PMC
May 2021

Gut Microbiota-Host Interactions in Inborn Errors of Immunity.

Int J Mol Sci 2021 Jan 31;22(3). Epub 2021 Jan 31.

Laboratory of Clinical Immunology and Microbiology, Division of Intramural Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20814, USA.

Inborn errors of immunity (IEI) are a group of disorders that are mostly caused by genetic mutations affecting immune host defense and immune regulation. Although IEI present with a wide spectrum of clinical features, in about one third of them various degrees of gastrointestinal (GI) involvement have been described and for some IEI the GI manifestations represent the main and peculiar clinical feature. The microbiome plays critical roles in the education and function of the host's innate and adaptive immune system, and imbalances in microbiota-immunity interactions can contribute to intestinal pathogenesis. Microbial dysbiosis combined to the impairment of immunosurveillance and immune dysfunction in IEI, may favor mucosal permeability and lead to inflammation. Here we review how immune homeostasis between commensals and the host is established in the gut, and how these mechanisms can be disrupted in the context of primary immunodeficiencies. Additionally, we highlight key aspects of the first studies on gut microbiome in patients affected by IEI and discuss how gut microbiome could be harnessed as a therapeutic approach in these diseases.
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http://dx.doi.org/10.3390/ijms22031416DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7866830PMC
January 2021

Modeling, optimization, and comparable efficacy of T cell and hematopoietic stem cell gene editing for treating hyper-IgM syndrome.

EMBO Mol Med 2021 03 21;13(3):e13545. Epub 2021 Jan 21.

San Raffaele Telethon Institute for Gene Therapy, IRCCS San Raffaele Scientific Institute, Milan, Italy.

Precise correction of the CD40LG gene in T cells and hematopoietic stem/progenitor cells (HSPC) holds promise for treating X-linked hyper-IgM Syndrome (HIGM1), but its actual therapeutic potential remains elusive. Here, we developed a one-size-fits-all editing strategy for effective T-cell correction, selection, and depletion and investigated the therapeutic potential of T-cell and HSPC therapies in the HIGM1 mouse model. Edited patients' derived CD4 T cells restored physiologically regulated CD40L expression and contact-dependent B-cell helper function. Adoptive transfer of wild-type T cells into conditioned HIGM1 mice rescued antigen-specific IgG responses and protected mice from a disease-relevant pathogen. We then obtained ~ 25% CD40LG editing in long-term repopulating human HSPC. Transplanting such proportion of wild-type HSPC in HIGM1 mice rescued immune functions similarly to T-cell therapy. Overall, our findings suggest that autologous edited T cells can provide immediate and substantial benefits to HIGM1 patients and position T-cell ahead of HSPC gene therapy because of easier translation, lower safety concerns and potentially comparable clinical benefits.
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http://dx.doi.org/10.15252/emmm.202013545DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7933961PMC
March 2021

Innovative Cell-Based Therapies and Conditioning to Cure RAG Deficiency.

Front Immunol 2020 19;11:607926. Epub 2020 Nov 19.

San Raffaele Telethon Institute for Gene Therapy (SR-Tiget), IRCCS San Raffaele Scientific Institute, Milan, Italy.

Genetic defects in recombination activating genes (RAG) 1 and 2 cause a broad spectrum of severe immune defects ranging from early severe and repeated infections to inflammation and autoimmune manifestations. A correlation between recombination activity and immune phenotype has been described. Hematopoietic cell transplantation is the treatment of care; however, the availability of next generation sequencing and whole genome sequencing has allowed the identification of novel genetic RAG variants in immunodeficient patients at various ages, raising therapeutic questions. This review addresses the recent advances of novel therapeutic approaches for RAG deficiency. As conventional myeloablative conditioning regimens are associated with acute toxicities and transplanted-related mortality, innovative minimal conditioning regimens based on the use of monoclonal antibodies are now emerging and show promising results. To overcome shortage of compatible donors, gene therapy has been developed in various RAG preclinical models. Overall, the transplantation of autologous gene corrected hematopoietic precursors and the use of non-genotoxic conditioning will open a new era, offering a cure to an increasing number of RAG patients regardless of donor availability and severity of clinical conditions.
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http://dx.doi.org/10.3389/fimmu.2020.607926DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7711106PMC
June 2021

Pathobiologic Mechanisms of Neurodegeneration in Osteopetrosis Derived From Structural and Functional Analysis of 14 ClC-7 Mutants.

J Bone Miner Res 2021 03 29;36(3):531-545. Epub 2020 Nov 29.

Consiglio Nazionale delle Ricerche, Istituto di Biofisica (CNR-IBF), Dulbecco Telethon Laboratory, Genoa, Italy.

ClC-7 is a chloride-proton antiporter of the CLC protein family. In complex with its accessory protein Ostm-1, ClC-7 localizes to lysosomes and to the osteoclasts' ruffled border, where it plays a critical role in acidifying the resorption lacuna during bone resorption. Gene inactivation in mice causes severe osteopetrosis, neurodegeneration, and lysosomal storage disease. Mutations in the human CLCN7 gene are associated with diverse forms of osteopetrosis. The functional evaluation of ClC-7 variants might be informative with respect to their pathogenicity, but the cellular localization of the protein hampers this analysis. Here we investigated the functional effects of 13 CLCN7 mutations identified in 13 new patients with severe or mild osteopetrosis and a known ADO2 mutation. We mapped the mutated amino acid residues in the homology model of ClC-7 protein, assessed the lysosomal colocalization of ClC-7 mutants and Ostm1 through confocal microscopy, and performed patch-clamp recordings on plasma-membrane-targeted mutant ClC-7. Finally, we analyzed these results together with the patients' clinical features and suggested a correlation between the lack of ClC-7/Ostm1 in lysosomes and severe neurodegeneration. © 2020 American Society for Bone and Mineral Research (ASBMR).
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http://dx.doi.org/10.1002/jbmr.4200DOI Listing
March 2021

Genome-first approach for the characterization of a complex phenotype with combined NBAS and CUL4B deficiency.

Bone 2020 11 6;140:115571. Epub 2020 Aug 6.

Division of Biology and Genetics, Department of Molecular and Translational Medicine, University of Brescia, 25123 Brescia, Italy. Electronic address:

Biallelic variants in neuroblastoma-amplified sequence (NBAS) cause an extremely broad spectrum of phenotypes. Clinical features range from isolated recurrent episodes of liver failure to multisystemic syndrome including short stature, skeletal osteopenia and dysplasia, optic atrophy, and a variable immunological, cutaneous, muscular, and neurological abnormalities. Hemizygous variants in CUL4B cause syndromic X-linked intellectual disability characterized by limitations in intellectual functions, developmental delays in gait, cognitive, and speech functioning, and other features including short stature, dysmorphism, and cerebral malformations. In this study, we report on a 4.5-month-old preterm infant with a complex phenotype mainly characterized by placental-related severe intrauterine growth restriction, post-natal growth failure with spontaneous bone fractures, which led to a suspicion of osteogenesis imperfecta, and lethal bronchopulmonary dysplasia with pulmonary hypertension. Whole exome sequencing identified compound heterozygosity for a known frameshift and a novel missense variant in NBAS and hemizygosity for a known CUL4B nonsense mutation. In vitro functional studies on the novel NBAS missense substitution demonstrated altered Golgi-to-endoplasmic reticulum retrograde vesicular trafficking and reduced collagen secretion, likely explaining part of the patient's phenotype. We also provided a comprehensive overview of the phenotypic features of NBAS and CUL4B deficiency, thus updating the recently emerging NBAS genotype-phenotype correlations. Our findings highlight the power of a genome-first approach for an early diagnosis of complex phenotypes.
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http://dx.doi.org/10.1016/j.bone.2020.115571DOI Listing
November 2020

Artificial thymic organoids represent a reliable tool to study T-cell differentiation in patients with severe T-cell lymphopenia.

Blood Adv 2020 06;4(12):2611-2616

Laboratory of Clinical Immunology and Microbiology, Division of Intramural Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health (NIH), Bethesda, MD.

The study of early T-cell development in humans is challenging because of limited availability of thymic samples and the limitations of in vitro T-cell differentiation assays. We used an artificial thymic organoid (ATO) platform generated by aggregating a DLL4-expressing stromal cell line (MS5-hDLL4) with CD34+ cells isolated from bone marrow or mobilized peripheral blood to study T-cell development from CD34+ cells of patients carrying hematopoietic intrinsic or thymic defects that cause T-cell lymphopenia. We found that AK2 deficiency is associated with decreased cell viability and an early block in T-cell development. We observed a similar defect in a patient carrying a null IL2RG mutation. In contrast, CD34+ cells from a patient carrying a missense IL2RG mutation reached full T-cell maturation, although cell numbers were significantly lower than in controls. CD34+ cells from patients carrying RAG mutations were able to differentiate to CD4+CD8+ cells, but not to CD3+TCRαβ+ cells. Finally, normal T-cell differentiation was observed in a patient with complete DiGeorge syndrome, consistent with the extra-hematopoietic nature of the defect. The ATO system may help determine whether T-cell deficiency reflects hematopoietic or thymic intrinsic abnormalities and define the exact stage at which T-cell differentiation is blocked.
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http://dx.doi.org/10.1182/bloodadvances.2020001730DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7322962PMC
June 2020

Efficacy and safety of anti-CD45-saporin as conditioning agent for RAG deficiency.

J Allergy Clin Immunol 2021 01 6;147(1):309-320.e6. Epub 2020 May 6.

Laboratory of Clinical Immunology and Microbiology, Division of Intramural Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Md. Electronic address:

Background: Mutations in the recombinase-activating genes cause severe immunodeficiency, with a spectrum of phenotypes ranging from severe combined immunodeficiency to immune dysregulation. Hematopoietic stem cell transplantation is the only curative option, but a high risk of graft failure and poor immune reconstitution have been observed in the absence of myeloablation.

Objectives: Our aim was to improve multilineage engraftment; we tested nongenotoxic conditioning with anti-CD45 mAbs conjugated with saporin CD45 (CD45-SAP).

Methods: Rag1-KO and Rag1-F971L mice, which represent models of severe combined immune deficiency and combined immune deficiency with immune dysregulation, respectively, were conditioned with CD45-SAP, CD45-SAP plus 2 Gy of total body irradiation (TBI), 2 Gy of TBI, 8 Gy of TBI, or no conditioning and treated by using transplantation with lineage-negative bone marrow cells from wild-type mice. Flow cytometry and immunohistochemistry were used to assess engraftment and immune reconstitution. Antibody responses to 2,4,6-trinitrophenyl-conjugated keyhole limpet hemocyanin were measured by ELISA, and presence of autoantibody was detected by microarray.

Results: Conditioning with CD45-SAP enabled high levels of multilineage engraftment in both Rag1 mutant models, allowed overcoming of B- and T-cell differentiation blocks and thymic epithelial cell defects, and induced robust cellular and humoral immunity in the periphery.

Conclusions: Conditioning with CD45-SAP allows multilineage engraftment and robust immune reconstitution in mice with either null or hypomorphic Rag mutations while preserving thymic epithelial cell homeostasis.
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http://dx.doi.org/10.1016/j.jaci.2020.04.033DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8322962PMC
January 2021

Successful Preclinical Development of Gene Therapy for Recombinase-Activating Gene-1-Deficient SCID.

Mol Ther Methods Clin Dev 2020 Jun 31;17:666-682. Epub 2020 Mar 31.

Department of Immunohematology and Blood Transfusion, Leiden University Medical Center, 2333ZA Leiden, the Netherlands.

Recombinase-activating gene-1 (RAG1)-deficient severe combined immunodeficiency (SCID) patients lack B and T lymphocytes due to the inability to rearrange immunoglobulin and T cell receptor genes. Gene therapy is an alternative for those RAG1-SCID patients who lack a suitable bone marrow donor. We designed lentiviral vectors with different internal promoters driving codon-optimized to ensure optimal expression. We used mice as a preclinical model for RAG1-SCID to assess the efficacy of the various vectors. We observed that B and T cell reconstitution directly correlated with expression. Mice with low expression showed poor immune reconstitution; however, higher expression resulted in phenotypic and functional lymphocyte reconstitution comparable to mice receiving wild-type stem cells. No signs of genotoxicity were found. Additionally, RAG1-SCID patient CD34 cells transduced with our clinical RAG1 vector and transplanted into NSG mice led to improved human B and T cell development. Considering this efficacy outcome, together with favorable safety data, these results substantiate the need for a clinical trial for RAG1-SCID.
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http://dx.doi.org/10.1016/j.omtm.2020.03.016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7163047PMC
June 2020

Cutaneous barrier leakage and gut inflammation drive skin disease in Omenn syndrome.

J Allergy Clin Immunol 2020 11 18;146(5):1165-1179.e11. Epub 2020 Apr 18.

Laboratory of Pathology, Center for Cancer Research, National Cancer Institute, NIH, Bethesda, Md.

Background: Severe early-onset erythroderma and gut inflammation, with massive tissue infiltration of oligoclonal activated T cells are the hallmark of Omenn syndrome (OS).

Objective: The impact of altered gut homeostasis in the cutaneous manifestations of OS remains to be clarified.

Methods: We analyzed a cohort of 15 patients with OS and the 129Sv/C57BL/6 knock-in Rag2 (Rag2) mouse model. Homing phenotypes of circulating lymphocytes were analyzed by flow cytometry. Inflammatory cytokines and chemokines were examined in the sera by ELISA and in skin biopsies by immunohistochemistry and in situ RNA hybridization. Experimental colitis was induced in mice by dextran sulfate sodium salt.

Results: We show that memory/activated T cells from patients with OS and from the Rag2 mouse model of OS abundantly express the skin homing receptors cutaneous lymphocyte associated antigen and CCR4 (Ccr4), associated with high levels of chemokine C-C motif ligands 17 and 22. Serum levels of LPS are also elevated. A broad T1/T2/T17 inflammatory signature is detected in the periphery and in the skin. Increased Tlr4 expression in the skin of Rag2 mice is associated with enhanced cutaneous inflammation on local and systemic administration of LPS. Likewise, boosting colitis in Rag2 mice results in increased frequency of Ccr4 splenic T cells and worsening of skin inflammation, as indicated by epidermal thickening, enhanced epithelial cell activation, and dermal infiltration by T1 effector T cells.

Conclusions: These results support the existence of an interplay between gut and skin that can sustain skin inflammation in OS.
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http://dx.doi.org/10.1016/j.jaci.2020.04.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7649331PMC
November 2020

Chromosome Transplantation: A Possible Approach to Treat Human X-linked Disorders.

Mol Ther Methods Clin Dev 2020 Jun 21;17:369-377. Epub 2020 Jan 21.

National Research Council (CNR)-IRGB/UOS, Milan, Italy.

Many human genetic diseases are associated with gross mutations such as aneuploidies, deletions, duplications, or inversions. For these "structural" disorders, conventional gene therapy, based on viral vectors and/or on programmable nuclease-mediated homologous recombination, is still unsatisfactory. To correct such disorders, chromosome transplantation (CT), defined as the perfect substitution of an endogenous defective chromosome with an exogenous normal one, could be applied. CT re-establishes a normal diploid cell, leaving no marker of the procedure, as we have recently shown in mouse pluripotent stem cells. To prove the feasibility of the CT approach in human cells, we used human induced pluripotent stem cells (hiPSCs) reprogrammed from Lesch-Nyhan (LN) disease patients, taking advantage of their mutation in the X-linked gene, making the LN cells selectable and distinguishable from the resistant corrected normal cells. In this study, we demonstrate, for the first time, that CT is feasible in hiPSCs: the normal exogenous X chromosome was first transferred using an improved chromosome transfer system, and the extra sex chromosome was spontaneously lost. These CT cells were functionally corrected and maintained their pluripotency and differentiation capability. By inactivation of the autologous gene, CT paves the way to the correction of hiPSCs from several X-linked disorders.
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http://dx.doi.org/10.1016/j.omtm.2020.01.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7029378PMC
June 2020

Expanded circulating hematopoietic stem/progenitor cells as novel cell source for the treatment of TCIRG1 osteopetrosis.

Haematologica 2021 01 1;106(1):74-86. Epub 2021 Jan 1.

San Raffaele Telethon Institute for Gene Therapy (SR-Tiget), IRCCS San Raffaele Scientific Institute.

Allogeneic hematopoietic stem cell transplantation is the treatment of choice for autosomal recessive osteopetrosis caused by defects in the TCIRG1 gene. Despite recent progress in conditioning, a relevant number of patients are not eligible for allogeneic stem cell transplantation because of the severity of the disease and significant transplant-related morbidity. We exploited peripheral CD34+ cells, known to circulate at high frequency in the peripheral blood of TCIRG1-deficient patients, as a novel cell source for autologous transplantation of gene corrected cells. Detailed phenotypical analysis showed that circulating CD34+ cells have a cellular composition that resembles bone marrow, supporting their use in gene therapy protocols. Transcriptomic profile revealed enrichment in genes expressed by hematopoietic stem and progenitor cells (HSPCs). To overcome the limit of bone marrow harvest/ HSPC mobilization and serial blood drawings in TCIRG1 patients, we applied UM171-based ex-vivo expansion of HSPCs coupled with lentiviral gene transfer. Circulating CD34+ cells from TCIRG1-defective patients were transduced with a clinically-optimized lentiviral vector (LV) expressing TCIRG1 under the control of phosphoglycerate promoter and expanded ex vivo. Expanded cells maintained long-term engraftment capacity and multi-lineage repopulating potential when transplanted in vivo both in primary and secondary NSG recipients. Moreover, when CD34+ cells were differentiated in vitro, genetically corrected osteoclasts resorbed the bone efficiently. Overall, we provide evidence that expansion of circulating HSPCs coupled to gene therapy can overcome the limit of stem cell harvest in osteopetrotic patients, thus opening the way to future gene-based treatment of skeletal diseases caused by bone marrow fibrosis.
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http://dx.doi.org/10.3324/haematol.2019.238261DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7776247PMC
January 2021

Immune dysregulation in patients with RAG deficiency and other forms of combined immune deficiency.

Blood 2020 02;135(9):610-619

Laboratory of Clinical Immunology and Microbiology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD.

Traditionally, primary immune deficiencies have been defined based on increased susceptibility to recurrent and/or severe infections. However, immune dysregulation, manifesting with autoimmunity or hyperinflammatory disease, has emerged as a common feature. This is especially true in patients affected by combined immune deficiency (CID), a group of disorders caused by genetic defects that impair, but do not completely abolish, T-cell function. Hypomorphic mutations in the recombination activating genes RAG1 and RAG2 represent the prototype of the broad spectrum of clinical and immunological phenotypes associated with CID. The study of patients with RAG deficiency and with other forms of CID has revealed distinct abnormalities in central and peripheral T- and B-cell tolerance as the key mechanisms involved in immune dysregulation. Understanding the pathophysiology of autoimmunity and hyperinflammation in these disorders may also permit more targeted therapeutic interventions.
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http://dx.doi.org/10.1182/blood.2019000923DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7046604PMC
February 2020

Generation of an immunodeficient mouse model of tcirg1-deficient autosomal recessive osteopetrosis.

Bone Rep 2020 Jun 7;12:100242. Epub 2020 Jan 7.

CNR-IRGB, Milan Unit, via Fantoli 16/15, 20138 Milan, Italy.

Background: Autosomal recessive osteopetrosis is a rare skeletal disorder with increased bone density due to a failure in osteoclast bone resorption. In most cases, the defect is cell-autonomous, and >50% of patients bear mutations in the gene, encoding for a subunit of the vacuolar proton pump essential for osteoclast resorptive activity. The only cure is hematopoietic stem cell transplantation, which corrects the bone pathology by allowing the formation of donor-derived functional osteoclasts. Therapeutic approaches using patient-derived cells corrected through viral transduction or gene editing can be considered, but to date functional rescue cannot be demonstrated because a relevant animal model for xenotransplant is missing.

Methods: We generated a new mouse model, which we named NSG oc/oc, presenting severe autosomal recessive osteopetrosis owing to the mutation, and profound immunodeficiency caused by the NSG background. We performed neonatal murine bone marrow transplantation and xenotransplantation with human CD34 cells.

Results: We demonstrated that neonatal murine bone marrow transplantation rescued NSG oc/oc mice, in line with previous findings in the oc/oc parental strain and with evidence from clinical practice in humans. Importantly, we also demonstrated human cell chimerism in the bone marrow of NSG oc/oc mice transplanted with human CD34 cells. The severity and rapid progression of the disease in the mouse model prevented amelioration of the bone pathology; nevertheless, we cannot completely exclude that minor early modifications of the bone tissue might have occurred.

Conclusion: Our work paves the way to generating an improved xenograft model for evaluation of functional rescue of patient-derived corrected cells. Further refinement of the newly generated mouse model will allow capitalizing on it for an optimized exploitation in the path to novel cell therapies.
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http://dx.doi.org/10.1016/j.bonr.2020.100242DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6953598PMC
June 2020

Generation of 3 clones of induced pluripotent stem cells (iPSCs) from a patient affected by Autosomal Recessive Osteopetrosis due to mutations in TCIRG1 gene.

Stem Cell Res 2020 01 20;42:101660. Epub 2019 Nov 20.

A. Nocivelli Institute for Molecular Medicine, Department of Molecular and Translational Medicine, University of Brescia, 25123 Brescia, Italy.

Autosomal recessive osteopetrosis (ARO) is a rare inherited disorder leading to increased bone density with impairment in bone resorption. Among the genes responsible for ARO, the TCIRG1 gene, coding for the a3 subunit of the osteoclast proton pump, is mutated in more than 50% of the cases, increasing the importance of TCIRG1-iPSCs as disease model. We generated 3 iPSC clones derived from Peripheral Blood Mononuclear Cells (PBMCs) of a patient carrying the heterozygous mutations p.Y512X and c.2236 + 1G > A. A Sendai virus-based vector was used and the iPSCs were characterized for genetic identity to parental cells, genomic integrity, pluripotency, and differentiation ability.
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http://dx.doi.org/10.1016/j.scr.2019.101660DOI Listing
January 2020

NKp46-expressing human gut-resident intraepithelial Vδ1 T cell subpopulation exhibits high antitumor activity against colorectal cancer.

JCI Insight 2019 12 19;4(24). Epub 2019 Dec 19.

Unit of Clinical and Experimental Immunology, Humanitas Clinical and Research Center, Rozzano, Milan, Italy.

γδ T cells account for a large fraction of human intestinal intraepithelial lymphocytes (IELs) endowed with potent antitumor activities. However, little is known about their origin, phenotype, and clinical relevance in colorectal cancer (CRC). To determine γδ IEL gut specificity, homing, and functions, γδ T cells were purified from human healthy blood, lymph nodes, liver, skin, and intestine, either disease-free, affected by CRC, or generated from thymic precursors. The constitutive expression of NKp46 specifically identifies a subset of cytotoxic Vδ1 T cells representing the largest fraction of gut-resident IELs. The ontogeny and gut-tropism of NKp46+/Vδ1 IELs depends both on distinctive features of Vδ1 thymic precursors and gut-environmental factors. Either the constitutive presence of NKp46 on tissue-resident Vδ1 intestinal IELs or its induced expression on IL-2/IL-15-activated Vδ1 thymocytes are associated with antitumor functions. Higher frequencies of NKp46+/Vδ1 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 NKp46+/Vδ1 cells from activated thymic precursors. These results parallel the very low frequencies of NKp46+/Vδ1 IELs able to infiltrate CRC, thus providing insights to either follow-up cancer progression or to develop adoptive cellular therapies.
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http://dx.doi.org/10.1172/jci.insight.125884DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6975269PMC
December 2019

Anomalous Intrinsic Fluorescence of HCl and NaOH Aqueous Solutions.

J Phys Chem Lett 2019 Nov 11;10(22):7230-7236. Epub 2019 Nov 11.

Department of Biotechnology and Biosciences , University of Milano-Bicocca , Piazza della Scienza 2 , 20126 Milan , Italy.

The unique properties of liquid water mainly arise from its hydrogen bond network. The geometry and dynamics of this network play a key role in shaping the characteristics of soft matter, from simple solutions to biosystems. Here we report an anomalous intrinsic fluorescence of HCl and NaOH aqueous solutions at room temperature that shows important differences in the excitation and emission bands between the two solutes. From ab initio time-dependent density functional theory modeling we propose that fluorescence emission could originate from hydrated ion species contained in transient cavities of the bulk solvent. These cavities, which are characterized by a stiff surface, could provide an environment that, upon trapping the excited state, suppresses the fast nonradiative decay and allows the slower radiative channel to become a possible decay pathway.
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http://dx.doi.org/10.1021/acs.jpclett.9b02163DOI Listing
November 2019

International collaboration generates high quality clusters of pharmaceutical patents.

Pharm Pat Anal 2019 Sep;8(5):193-202

Swiss Federal Institute of Intellectual Property, Stauffacherstrasse 65, CH-3003 Bern, Switzerland.

The worldwide active patent portfolio has nearly doubled in numbers and strength since 2000. The number of active pharmaceutical patent families has tripled in the same time period. The quantitative growth results mostly from a surge of patents from China, half of them classified in A61K36 ('medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants'). High-quality patents exhibit a slower growth curve, and cluster within the three areas biologicals; heterocyclic compounds, and cancer drugs. However, the highest concentration of high-quality patents was found when selecting patents listing inventors from at least two out of the five most important countries of origin for pharmaceutical patents: China, EP countries, Japan, South Korea and the USA.
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http://dx.doi.org/10.4155/ppa-2019-0017DOI Listing
September 2019

Absence of Dipeptidyl Peptidase 3 Increases Oxidative Stress and Causes Bone Loss.

J Bone Miner Res 2019 11 9;34(11):2133-2148. Epub 2019 Sep 9.

Consiglio Nazionale delle Ricerche-Istituto di Ricerca Genetica e Biomedica (CNR-IRGB), Milan Unit, Milan, Italy.

Controlling oxidative stress through the activation of antioxidant pathways is crucial in bone homeostasis, and impairments of the cellular defense systems involved contribute to the pathogenesis of common skeletal diseases. In this work we focused on the dipeptidyl peptidase 3 (DPP3), a poorly investigated ubiquitous zinc-dependent exopeptidase activating the Keap1-Nrf2 antioxidant pathway. We showed Dpp3 expression in bone and, to understand its role in this compartment, we generated a Dpp3 knockout (KO) mouse model and specifically investigated the skeletal phenotype. Adult Dpp3 KO mice showed a mild growth defect, a significant increase in bone marrow cellularity, and bone loss mainly caused by increased osteoclast activity. Overall, in the mouse model, lack of DPP3 resulted in sustained oxidative stress and in alterations of bone microenvironment favoring the osteoclast compared to the osteoblast lineage. Accordingly, in vitro studies revealed that Dpp3 KO osteoclasts had an inherent increased resorptive activity and ROS production, which on the other hand made them prone to apoptosis. Moreover, absence of DPP3 augmented bone loss after estrogen withdrawal in female mice, further supporting its relevance in the framework of bone pathophysiology. Overall, we show a nonredundant role for DPP3 in the maintenance of bone homeostasis and propose that DPP3 might represent a possible new osteoimmunological player and a marker of human bone loss pathology. © 2019 American Society for Bone and Mineral Research.
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http://dx.doi.org/10.1002/jbmr.3829DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7203631PMC
November 2019

Gene Modification and Three-Dimensional Scaffolds as Novel Tools to Allow the Use of Postnatal Thymic Epithelial Cells for Thymus Regeneration Approaches.

Stem Cells Transl Med 2019 10 29;8(10):1107-1122. Epub 2019 May 29.

Telethon Institute for Gene Therapy (SR-Tiget), Division of Regenerative Medicine, Stem Cells, and Gene Therapy, IRCCS San Raffaele Scientific Institute, Milan, Italy.

Defective functionality of thymic epithelial cells (TECs), due to genetic mutations or injuring causes, results in altered T-cell development, leading to immunodeficiency or autoimmunity. These defects cannot be corrected by hematopoietic stem cell transplantation (HSCT), and thymus transplantation has not yet been demonstrated to be fully curative. Here, we provide proof of principle of a novel approach toward thymic regeneration, involving the generation of thymic organoids obtained by seeding gene-modified postnatal murine TECs into three-dimensional (3D) collagen type I scaffolds mimicking the thymic ultrastructure. To this end, freshly isolated TECs were transduced with a lentiviral vector system, allowing for doxycycline-induced Oct4 expression. Transient Oct4 expression promoted TECs expansion without drastically changing the cell lineage identity of adult TECs, which retain the expression of important molecules for thymus functionality such as Foxn1, Dll4, Dll1, and AIRE. Oct4-expressing TECs (iOCT4 TEC) were able to grow into 3D collagen type I scaffolds both in vitro and in vivo, demonstrating that the collagen structure reproduced a 3D environment similar to the thymic extracellular matrix, perfectly recognized by TECs. In vivo results showed that thymic organoids transplanted subcutaneously in athymic nude mice were vascularized but failed to support thymopoiesis because of their limited in vivo persistence. These findings provide evidence that gene modification, in combination with the usage of 3D biomimetic scaffolds, may represent a novel approach allowing the use of postnatal TECs for thymic regeneration. Stem Cells Translational Medicine 2019;8:1107-1122.
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http://dx.doi.org/10.1002/sctm.18-0218DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6766605PMC
October 2019

Targeted NGS Platforms for Genetic Screening and Gene Discovery in Primary Immunodeficiencies.

Front Immunol 2019 11;10:316. Epub 2019 Apr 11.

Department of Systems Medicine, University of Rome Tor Vergata, Rome, Italy.

Primary Immunodeficiencies (PIDs) are a heterogeneous group of genetic immune disorders. While some PIDs can manifest with more than one phenotype, signs, and symptoms of various PIDs overlap considerably. Recently, novel defects in immune-related genes and additional variants in previously reported genes responsible for PIDs have been successfully identified by Next Generation Sequencing (NGS), allowing the recognition of a broad spectrum of disorders. To evaluate the strength and weakness of targeted NGS sequencing using custom-made Ion Torrent and Haloplex (Agilent) panels for diagnostics and research purposes. Five different panels including known and candidate genes were used to screen 105 patients with distinct PID features divided in three main PID categories: and . The Ion Torrent sequencing platform was used in 73 patients. Among these, 18 selected patients without a molecular diagnosis and 32 additional patients were analyzed by Haloplex enrichment technology. The complementary use of the two custom-made targeted sequencing approaches allowed the identification of causative variants in 28.6% ( = 30) of patients. Twenty-two out of 73 (34.6%) patients were diagnosed by Ion Torrent. In this group 20 were included in the SCID/CID category. Eight out of 50 (16%) patients were diagnosed by Haloplex workflow. Ion Torrent method was highly successful for those cases with well-defined phenotypes for immunological and clinical presentation. The Haloplex approach was able to diagnose 4 SCID/CID patients and 4 additional patients with complex and extended phenotypes, embracing all three PID categories in which this approach was more efficient. Both technologies showed good gene coverage. NGS technology represents a powerful approach in the complex field of rare disorders but its different application should be weighted. A relatively small NGS target panel can be successfully applied for a robust diagnostic suspicion, while when the spectrum of clinical phenotypes overlaps more than one PID an in-depth NGS analysis is required, including also whole exome/genome sequencing to identify the causative gene.
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http://dx.doi.org/10.3389/fimmu.2019.00316DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6470723PMC
September 2020

The RANKL-RANK Axis: A Bone to Thymus Round Trip.

Front Immunol 2019 29;10:629. Epub 2019 Mar 29.

Milan Unit, Institute for Genetic and Biomedical Research (CNR-IRGB), Milan, Italy.

The identification of Receptor activator of nuclear factor kappa B ligand (RANKL) and its cognate receptor Receptor activator of nuclear factor kappa B (RANK) during a search for novel tumor necrosis factor receptor (TNFR) superfamily members has dramatically changed the scenario of bone biology by providing the functional and biochemical proof that RANKL signaling via RANK is the master factor for osteoclastogenesis. In parallel, two independent studies reported the identification of mouse RANKL on activated T cells and of a ligand for osteoprotegerin on a murine bone marrow-derived stromal cell line. After these seminal findings, accumulating data indicated RANKL and RANK not only as essential players for the development and activation of osteoclasts, but also for the correct differentiation of medullary thymic epithelial cells (mTECs) that act as mediators of the central tolerance process by which self-reactive T cells are eliminated while regulatory T cells are generated. In light of the RANKL-RANK multi-task function, an antibody targeting this pathway, denosumab, is now commonly used in the therapy of bone loss diseases including chronic inflammatory bone disorders and osteolytic bone metastases; furthermore, preclinical data support the therapeutic application of denosumab in the framework of a broader spectrum of tumors. Here, we discuss advances in cellular and molecular mechanisms elicited by RANKL-RANK pathway in the bone and thymus, and the extent to which its inhibition or augmentation can be translated in the clinical arena.
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http://dx.doi.org/10.3389/fimmu.2019.00629DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6450200PMC
June 2020
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