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In the setting of hematopoietic stem cell transplantation (HSCT), Rituximab (RTX) is used for the treatment and prevention of EBV-associated post-transplantation lymphoproliferative disease or autoimmune phenomena such as autoimmune hemolytic anemia (AIHA). Persistent hypogammaglobulinemia and immunoglobulin substitution dependence has been observed in several patients after RTX treatment despite the normalization of total B cell numbers. We aimed to study whether this is a B cell intrinsic phenomenon. We analyzed four patients with different primary diseases who were treated with myeloablative conditioning and matched unrelated donor HSCT who developed persistent hypogammaglobulinemia after receiving RTX treatment. They all received RTX early after HSCT to treat EBV infection or AIHA post-HSCT. All patients showed normalized total B cell numbers but absent to very low IgG positive memory B cells, and three lacked IgA positive memory B cells. All of the patients had full donor chimerism, and none had encountered graft-versus-host disease. Sorted peripheral blood naïve B cells from these patients, when stimulated with CD40L, IL21, IL10 and anti-IgM, demonstrated intact B cell differentiation including the formation of class-switched memory B cells and IgA and IgG production. Peripheral blood T cell numbers including CD4 follicular T-helper (Tfh) cells were all within the normal reference range. In conclusion, in these four HSCT patients, the persistent hypogammaglobulinemia observed after RTX cannot be attributed to an acquired intrinsic B cell problem nor to a reduction in Tfh cell numbers.
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Agammaglobulinemia , Trasplante de Células Madre Hematopoyéticas , Humanos , Rituximab/uso terapéutico , Agammaglobulinemia/tratamiento farmacológico , Agammaglobulinemia/etiología , Trasplante de Células Madre Hematopoyéticas/efectos adversos , Inmunoglobulina G , Inmunoglobulina ARESUMEN
Primary immunodeficiency diseases comprise a group of heterogeneous genetic defects that affect immune system development and/or function. Here we use in vitro differentiation of human induced pluripotent stem cells (iPSCs) generated from patients with different recombination-activating gene 1 (RAG1) mutations to assess T-cell development and T-cell receptor (TCR) V(D)J recombination. RAG1-mutants from severe combined immunodeficient (SCID) patient cells showed a failure to sustain progression beyond the CD3(--)CD4(-)CD8(-)CD7(+)CD5(+)CD38(-)CD31(-/lo)CD45RA(+) stage of T-cell development to reach the CD3(-/+)CD4(+)CD8(+)CD7(+)CD5(+)CD38(+)CD31(+)CD45RA(-) stage. Despite residual mutant RAG1 recombination activity from an Omenn syndrome (OS) patient, similar impaired T-cell differentiation was observed, due to increased single-strand DNA breaks that likely occur due to heterodimers consisting of both an N-terminal truncated and a catalytically dead RAG1. Furthermore, deep-sequencing analysis of TCR-ß (TRB) and TCR-α (TRA) rearrangements of CD3(-)CD4(+)CD8(-) immature single-positive and CD3(+)CD4(+)CD8(+) double-positive cells showed severe restriction of repertoire diversity with preferential usage of few Variable, Diversity, and Joining genes, and skewed length distribution of the TRB and TRA complementary determining region 3 sequences from SCID and OS iPSC-derived cells, whereas control iPSCs yielded T-cell progenitors with a broadly diversified repertoire. Finally, no TRA/δ excision circles (TRECs), a marker of TRA/δ locus rearrangements, were detected in SCID and OS-derived T-lineage cells, consistent with a pre-TCR block in T-cell development. This study compares human T-cell development of SCID vs OS patients, and elucidates important differences that help to explain the wide range of immunologic phenotypes that result from different mutations within the same gene of various patients.
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Proteínas de Homeodominio/genética , Células Madre Pluripotentes Inducidas/patología , Inmunodeficiencia Combinada Grave/genética , Inmunodeficiencia Combinada Grave/patología , Linfocitos T/patología , Células Cultivadas , Roturas del ADN , Genes RAG-1 , Humanos , Lactante , Mutación , Receptores de Antígenos de Linfocitos T alfa-beta/genética , Recombinación V(D)JAsunto(s)
Proteínas de Unión al ADN/genética , Proteínas de Homeodominio/genética , Síndromes de Inmunodeficiencia/genética , Síndromes de Inmunodeficiencia/inmunología , Proteínas Nucleares/genética , Receptores de Antígenos de Linfocitos T/inmunología , Linfocitos T/inmunología , Autoinmunidad , Niño , Preescolar , Humanos , Lactante , Recién NacidoRESUMEN
PURPOSE OF REVIEW: Provide an overview of the landmark accomplishments and state of the art of gene therapy for inborn errors of immunity (IEI). RECENT FINDINGS: Three decades after the first clinical application of gene therapy for IEI, there is one market authorized product available, while for several others efficacy has been demonstrated or is currently being tested in ongoing clinical trials. Gene editing approaches using programmable nucleases are being explored preclinically and could be beneficial for genes requiring tightly regulated expression, gain-of-function mutations and dominant-negative mutations. SUMMARY: Gene therapy by modifying autologous hematopoietic stem cells (HSCs) offers an attractive alternative to allogeneic hematopoietic stem cell transplantation (HSCT), the current standard of care to treat severe IEI. This approach does not require availability of a suitable allogeneic donor and eliminates the risk of graft versus host disease (GvHD). Gene therapy can be attempted by using a viral vector to add a copy of the therapeutic gene (viral gene addition) or by using programmable nucleases (gene editing) to precisely correct mutations, disrupt a gene or introduce an entire copy of a gene at a specific locus. However, gene therapy comes with its own challenges such as safety, therapeutic effectiveness and access. For viral gene addition, a major safety concern is vector-related insertional mutagenesis, although this has been greatly reduced with the introduction of safer vectors. For gene editing, the risk of off-site mutagenesis is a main driver behind the ongoing search for modified nucleases. For both approaches, HSCs have to be manipulated ex vivo, and doing this efficiently without losing stemness remains a challenge, especially for gene editing.
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Enfermedad Injerto contra Huésped , Trasplante de Células Madre Hematopoyéticas , Humanos , Terapia Genética , Edición GénicaRESUMEN
Introduction: Recombination activating genes (RAG) 1 and 2 defects are the most frequent form of severe combined immunodeficiency (SCID). Patients with residual RAG activity have a spectrum of clinical manifestations ranging from Omenn syndrome to delayed-onset combined immunodeficiency, often associated with granulomas and/or autoimmunity (CID-G/AI). Lentiviral vector (LV) gene therapy (GT) has been proposed as an alternative treatment to the standard hematopoietic stem cell transplant and a clinical trial for RAG1 SCID patients recently started. However, GT in patients with hypomorphic RAG mutations poses additional risks, because of the residual endogenous RAG1 expression and the general state of immune dysregulation and associated inflammation. Methods: In this study, we assessed the efficacy of GT in 2 hypomorphic Rag1 murine models (Rag1F971L/F971L and Rag1R972Q/R972Q), exploiting the same LV used in the clinical trial encoding RAG1 under control of the MND promoter. Results and discussion: Starting 6 weeks after transplant, GT-treated mice showed a decrease in proportion of myeloid cells and a concomitant increase of B, T and total white blood cells. However, counts remained lower than in mice transplanted with WT Lin- cells. At euthanasia, we observed a general redistribution of immune subsets in tissues, with the appearance of mature recirculating B cells in the bone marrow. In the thymus, we demonstrated correction of the block at double negative stage, with a modest improvement in the cortical/medullary ratio. Analysis of antigenspecific IgM and IgG serum levels after in vivo challenge showed an amelioration of antibody responses, suggesting that the partial immune correction could confer a clinical benefit. Notably, no overt signs of autoimmunity were detected, with B-cell activating factor decreasing to normal levels and autoantibodies remaining stable after GT. On the other hand, thymic enlargement was frequently observed, although not due to vector integration and insertional mutagenesis. In conclusion, our work shows that GT could partially alleviate the combined immunodeficiency of hypomorphic RAG1 patients and that extensive efficacy and safety studies with alternative models are required before commencing RAG gene therapy in thesehighly complex patients.
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Síndromes de Inmunodeficiencia , Inmunodeficiencia Combinada Grave , Humanos , Ratones , Animales , Proteínas de Homeodominio/genética , Síndromes de Inmunodeficiencia/terapia , Linfocitos B , Inmunodeficiencia Combinada Grave/genética , Inmunodeficiencia Combinada Grave/terapia , Terapia Genética , Inmunoproteínas , MutaciónRESUMEN
Primary atopic disorders are a group of inborn errors of immunity that skew the immune system toward severe allergic disease. Defining the biology underlying these extreme monogenic phenotypes reveals shared mechanisms underlying common polygenic allergic disease and identifies potential drug targets. Germline gain-of-function (GOF) variants in JAK1 are a cause of severe atopy and eosinophilia. Modeling the JAK1GOF (p.A634D) variant in both zebrafish and human induced pluripotent stem cells (iPSCs) revealed enhanced myelopoiesis. RNA-Seq of JAK1GOF human whole blood, iPSCs, and transgenic zebrafish revealed a shared core set of dysregulated genes involved in IL-4, IL-13, and IFN signaling. Immunophenotypic and transcriptomic analysis of patients carrying a JAK1GOF variant revealed marked Th cell skewing. Moreover, long-term ruxolitinib treatment of 2 children carrying the JAK1GOF (p.A634D) variant remarkably improved their growth, eosinophilia, and clinical features of allergic inflammation. This work highlights the role of JAK1 signaling in atopic immune dysregulation and the clinical impact of JAK1/2 inhibition in treating eosinophilic and allergic disease.
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Eosinofilia , Hipersensibilidad Inmediata , Hipersensibilidad , Células Madre Pluripotentes Inducidas , Niño , Animales , Humanos , Mutación con Ganancia de Función , Pez Cebra , Hipersensibilidad/genética , Inflamación/genética , Eosinofilia/genética , Janus Quinasa 1/genéticaRESUMEN
Ureaplasma species are increasingly recognized as relevant pathogens in prenatal, perinatal and postnatal morbidity. They are commonly found as commensals on the mucous membranes of the lower urogenital tract of pregnant women, but when ascending, they can cause bacterial vaginosis, chorioamnionitis, premature birth and postnatal morbidities such as bronchopulmonary dysplasia, and early-onset neonatal sepsis and meningitis. The detection of Ureaplasma species is challenging and is not covered by routine diagnostics, and current empiric antibiotic treatment in neonates suspected of infection is not directed against Ureaplasma species. The aim of this review is to discuss the pathophysiology of Ureaplasma infections, the clinical consequences and the current difficulties in diagnosis and treatment by providing an overview of the current literature.
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Enfermedades del Recién Nacido/microbiología , Complicaciones del Embarazo/microbiología , Infecciones por Ureaplasma/microbiología , Ureaplasma , Inmunidad Adaptativa , Corioamnionitis/microbiología , Femenino , Humanos , Inmunidad Innata , Lactante , Recién Nacido/inmunología , Enfermedades del Recién Nacido/diagnóstico , Enfermedades del Recién Nacido/terapia , Embarazo , Complicaciones del Embarazo/diagnóstico , Complicaciones del Embarazo/terapia , Nacimiento Prematuro/microbiología , Infecciones por Ureaplasma/diagnóstico , Infecciones por Ureaplasma/terapiaRESUMEN
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.
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Enfermedades del Desarrollo Óseo/etiología , Discapacidades del Desarrollo/etiología , Síndromes de Inmunodeficiencia/etiología , Mutación , N-Acetilglucosaminiltransferasas/genética , Animales , Preescolar , Femenino , Heparitina Sulfato/fisiología , Humanos , Células Madre Pluripotentes Inducidas/citología , Lactante , Linfocitos/fisiología , Pez CebraRESUMEN
Mutations of the recombinase-activating genes 1 and 2 (RAG1 and RAG2) in humans are associated with a broad range of phenotypes. For patients with severe clinical presentation, hematopoietic stem cell transplantation (HSCT) represents the only curative treatment; however, high rates of graft failure and incomplete immune reconstitution have been observed, especially after unconditioned haploidentical transplantation. Studies in mice have shown that Rag-/- natural killer (NK) cells have a mature phenotype, reduced fitness, and increased cytotoxicity. We aimed to analyze NK cell phenotype and function in patients with mutations in RAG and in non-homologous end joining (NHEJ) genes. Here, we provide evidence that NK cells from these patients have an immature phenotype, with significant expansion of CD56bright CD16-/int CD57- cells, yet increased degranulation and high perforin content. Correlation was observed between in vitro recombinase activity of the mutant proteins, NK cell abnormalities, and in vivo clinical phenotype. Addition of serotherapy in the conditioning regimen, with the aim of depleting the autologous NK cell compartment, may be important to facilitate engraftment and immune reconstitution in patients with RAG and NHEJ defects treated by HSCT.
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[This corrects the article on p. 798 in vol. 8, PMID: 28769923.].
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Mutations in the Recombination Activating Gene 1 (RAG1) can cause a wide variety of clinical and immunological phenotypes in humans, ranging from absence of T and B lymphocytes to occurrence of autoimmune manifestations associated with expansion of oligoclonal T cells and production of autoantibodies. Although the mechanisms underlying this phenotypic heterogeneity remain poorly understood, some genotype-phenotype correlations can be made. Currently, mouse models of Rag deficiency are restricted to RAG1-/- mice and to knock-in models carrying severe missense mutations. The Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)/Cas9 system is a novel and powerful gene-editing strategy that permits targeted introduction of DNA double strand breaks with high efficiency through simultaneous delivery of the Cas9 endonuclease and a guide RNA (gRNA). Here, we report on CRISPR-based, single-step generation and characterization of mutant mouse models in which gene editing was attempted around residue 838 of RAG1, a region whose functional role had not been studied previously.
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Sistemas CRISPR-Cas , Edición Génica/métodos , Proteínas de Homeodominio/genética , Ratones Mutantes/genética , Animales , Ratones , Mutagénesis Sitio-Dirigida/métodos , CigotoRESUMEN
Recombination-activating genes 1 and 2 (RAG1 and RAG2) play a critical role in T and B cell development by initiating the recombination process that controls the expression of T cell receptor (TCR) and immunoglobulin genes. Mutations in the RAG1 and RAG2 genes in humans cause a broad spectrum of phenotypes, including severe combined immunodeficiency (SCID) with lack of T and B cells, Omenn syndrome, leaky SCID, and combined immunodeficiency with granulomas or autoimmunity (CID-G/AI). Using next-generation sequencing, we analyzed the TCR and B cell receptor (BCR) repertoire in 12 patients with RAG mutations presenting with Omenn syndrome (n = 5), leaky SCID (n = 3), or CID-G/AI (n = 4). Restriction of repertoire diversity skewed usage of variable (V), diversity (D), and joining (J) segment genes, and abnormalities of CDR3 length distribution were progressively more prominent in patients with a more severe phenotype. Skewed usage of V, D, and J segment genes was present also within unique sequences, indicating a primary restriction of repertoire. Patients with Omenn syndrome had a high proportion of class-switched immunoglobulin heavy chain transcripts and increased somatic hypermutation rate, suggesting in vivo activation of these B cells. These data provide a framework to better understand the phenotypic heterogeneity of RAG deficiency.
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Severe combined immunodeficiency (SCID) and other severe non-SCID primary immunodeficiencies (non-SCID PID) can be treated by allogeneic hematopoietic stem cell (HSC) transplantation, but when histocompatibility leukocyte antigen-matched donors are lacking, this can be a high-risk procedure. Correcting the patient's own HSCs with gene therapy offers an attractive alternative. Gene therapies currently being used in clinical settings insert a functional copy of the entire gene by means of a viral vector. With this treatment, severe complications may result due to integration within oncogenes. A promising alternative is the use of endonucleases such as ZFNs, TALENs, and CRISPR/Cas9 to introduce a double-stranded break in the DNA and thus induce homology-directed repair. With these genome-editing tools a correct copy can be inserted in a precisely targeted "safe harbor." They can also be used to correct pathogenic mutations in situ and to develop cellular or animal models needed to study the pathogenic effects of specific genetic defects found in immunodeficient patients. This review discusses the advantages and disadvantages of these endonucleases in gene correction and modeling with an emphasis on CRISPR/Cas9, which offers the most promise due to its efficacy and versatility.