RESUMO
Retinitis pigmentosa (RP) is a group of progressive retinal degenerations of mostly monogenic inheritance, which cause blindness in about 1:3,500 individuals worldwide. Heterozygous variants in the rhodopsin (RHO) gene are the most common cause of autosomal dominant RP (adRP). Among these, missense variants at C-terminal proline 347, such as p.Pro347Ser, cause severe adRP recurrently in European affected individuals. Here, for the first time, we use CRISPR/Cas9 to selectively target the p.Pro347Ser variant while preserving the wild-type RHO allele in vitro and in a mouse model of adRP. Detailed in vitro, genomic, and biochemical characterization of the rhodopsin C-terminal editing demonstrates a safe downregulation of p.Pro347Ser expression leading to partial recovery of photoreceptor function in a transgenic mouse model treated with adeno-associated viral vectors. This study supports the safety and efficacy of CRISPR/Cas9-mediated allele-specific editing and paves the way for a permanent and precise correction of heterozygous variants in dominantly inherited retinal diseases.
Assuntos
Edição de Genes , Retinose Pigmentar/genética , Retinose Pigmentar/terapia , Rodopsina/genética , Alelos , Animais , Sistemas CRISPR-Cas , Linhagem Celular , Dependovirus/genética , Modelos Animais de Doenças , Eletrorretinografia , Terapia Genética , Humanos , Mutação INDEL , Camundongos , Camundongos Transgênicos , Mutação de Sentido Incorreto , Células Fotorreceptoras de Vertebrados/metabolismo , Retina/metabolismo , Retina/fisiopatologia , Rodopsina/metabolismoRESUMO
Inherited retinal disorders (IRDs) affect millions of people worldwide and are a major cause of irreversible blindness. Therapies based on drugs, gene augmentation or transplantation approaches have been widely investigated and proposed. Among gene therapies for retinal degenerative diseases, the fast-evolving genome-editing CRISPR/Cas technology has emerged as a new potential treatment. The CRISPR/Cas system has been developed as a powerful genome-editing tool in ophthalmic studies and has been applied not only to gain proof of principle for gene therapies in vivo, but has also been extensively used in basic research to model diseases-in-a-dish. Indeed, the CRISPR/Cas technology has been exploited to genetically modify human induced pluripotent stem cells (iPSCs) to model retinal disorders in vitro, to test in vitro drugs and therapies and to provide a cell source for autologous transplantation. In this review, we will focus on the technological advances in iPSC-based cellular reprogramming and gene editing technologies to create human in vitro models that accurately recapitulate IRD mechanisms towards the development of treatments for retinal degenerative diseases.
Assuntos
Células-Tronco Pluripotentes Induzidas , Degeneração Retiniana , Humanos , Edição de Genes , Sistemas CRISPR-Cas/genética , Degeneração Retiniana/genética , Degeneração Retiniana/terapia , Terapia GenéticaRESUMO
Sleeping Beauty (SB) is the first DNA transposon employed for efficient transposition in vertebrate cells, opening new applications for genetic engineering and gene therapies. A transposon-based gene delivery system holds the favourable features of non-viral vectors and an attractive safety profile. Here, we employed SB to engineer HEK293 cells for optimizing the production of a chimpanzee Adenovector (chAd) belonging to the Human Mastadenovirus C species. To date, chAd vectors are employed in several clinical settings for infectious diseases, last but not least COVID-19. A robust, efficient and quick viral vector production could advance the clinical application of chAd vectors. To this aim, we firstly swapped the hAd5 E1 with chAd-C E1 gene by using the CRISPR/Cas9 system. We demonstrated that in the absence of human Ad5 E1, chimp Ad-C E1 gene did not support HEK293 survival. To improve chAd-C vector production, we engineered HEK293 cells to stably express the chAd-C precursor terminal protein (ch.pTP), which plays a crucial role in chimpanzee Adenoviral DNA replication. The results indicate that exogenous ch.pTP expression significantly ameliorate the packaging and amplification of recombinant chAd-C vectors thus, the engineered HEK293ch.pTP cells could represent a superior packaging cell line for the production of these vectors.
Assuntos
COVID-19 , Pan troglodytes , Adenoviridae/genética , Animais , Elementos de DNA Transponíveis/genética , Técnicas de Transferência de Genes , Vetores Genéticos/genética , Células HEK293 , Humanos , Pan troglodytes/genéticaRESUMO
Retinal diseases (RD) include inherited retinal dystrophy (IRD), for example, retinitis pigmentosa and Leber's congenital amaurosis, or multifactorial forms, for example, age-related macular degeneration (AMD). IRDs are clinically and genetically heterogeneous in nature. To date, more than 200 genes are known to cause IRDs, which perturb the development, function and survival of rod and cone photoreceptors or retinal pigment epithelial cells. Conversely, AMD, the most common cause of blindness in the developed world, is an acquired disease of the macula characterised by progressive visual impairment. To date, available therapeutic approaches for RD include nutritional supplements, neurotrophic factors, antiangiogenic drugs for wet AMD and gene augmentation/interference strategy for IRDs. However, these therapies do not aim at correcting the genetic defect and result in inefficient and expensive treatments. The genome editing technology based on clustered regularly interspaced short palindromic repeat (CRISPR)-associated protein (Cas) and an RNA that guides the Cas protein to a predetermined region of the genome, represents an attractive strategy to tackle IRDs without available cure. Indeed, CRISPR/Cas system can permanently and precisely replace or remove genetic mutations causative of a disease, representing a molecular tool to cure a genetic disorder. In this review, we will introduce the mechanism of CRISPR/Cas system, presenting an updated panel of Cas variants and delivery systems, then we will focus on applications of CRISPR/Cas genome editing in the retina, and, as emerging treatment options, in patient-derived induced pluripotent stem cells followed by transplantation of retinal progenitor cells into the eye.
Assuntos
Sistemas CRISPR-Cas/genética , Técnicas de Transferência de Genes/tendências , Degeneração Macular/terapia , Doenças Retinianas/terapia , Edição de Genes/métodos , Humanos , Células-Tronco Pluripotentes Induzidas/transplante , Degeneração Macular/genética , Atrofia Óptica Hereditária de Leber/genética , Atrofia Óptica Hereditária de Leber/terapia , Retina/patologia , Retina/transplante , Doenças Retinianas/genética , Retinose Pigmentar/genética , Retinose Pigmentar/terapiaRESUMO
Rare patients who spontaneously control HIV replication provide a useful model to inform HIV vaccine development. HIV controllers develop particularly efficient antiviral CD4+ T cell responses mediated by shared high-affinity TCRs. To determine whether the candidate DNA vaccine ADVAX could induce similar responses, we analyzed Gag-specific primary CD4+ T cells from healthy volunteers who received ADVAX DNA by electroporation. Vaccinated volunteers had an immunodominant response to the Gag293 epitope with a functional avidity intermediate between that of controllers and treated patients. The TCR repertoire of Gag293-specific CD4+ T cells proved highly biased, with a predominant usage of the TCRß variable gene 2 (TRBV2) in vaccinees as well as controllers. TCRα variable gene (TRAV) gene usage was more diverse, with the dominance of TRAV29 over TRAV24 genes in vaccinees, whereas TRAV24 predominated in controllers. Sequence analysis revealed an unexpected degree of overlap between the specific repertoires of vaccinees and controllers, with the sharing of TRAV24 and TRBV2 public motifs (>30%) and of public clonotypes characteristic of high-affinity TCRs. MHC class II tetramer binding revealed a broad HLA-DR cross-restriction, explaining how Gag293-specific public clonotypes could be selected in individuals with diverse genetic backgrounds. TRAV29 clonotypes also proved cross-restricted, but conferred responses of lower functional avidity upon TCR transfer. In conclusion, DNA vaccination by electroporation primed for TCR clonotypes that were associated with HIV control, highlighting the potential of this vaccine delivery method. To our knowledge, this study provides the first proof-of-concept that clonotypic analysis may be used as a tool to monitor the quality of vaccine-induced responses and modulate these toward "controller-like" responses.
Assuntos
Vacinas contra a AIDS/imunologia , Linfócitos T CD4-Positivos/imunologia , Infecções por HIV/imunologia , HIV/fisiologia , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Produtos do Gene gag do Vírus da Imunodeficiência Humana/imunologia , Células Clonais , Reações Cruzadas , Eletroporação , ELISPOT , Antígenos HLA-DR/metabolismo , Humanos , Ativação Linfocitária , Ligação Proteica , Vacinas de DNA , Replicação ViralRESUMO
Deficiency of basement membrane heterotrimeric laminin 332 component, coded by LAMA3, LAMB3, and LAMC2 genes, causes junctional epidermolysis bullosa (JEB), a severe skin adhesion defect. Herein, we report the first application of CRISPR/Cas9-mediated homology direct repair (HDR) to in situ restore LAMB3 expression in JEB keratinocytes in vitro and in immunodeficient mice transplanted with genetically corrected skin equivalents. We packaged an adenovector carrying Cas9/guide RNA (gRNA) tailored to the intron 2 of LAMB3 gene and an integration defective lentiviral vector bearing a promoterless quasi-complete LAMB3 cDNA downstream a splice acceptor site and flanked by homology arms. Upon genuine HDR, we exploited the in vitro adhesion advantage of laminin 332 production to positively select LAMB3-expressing keratinocytes. HDR and restored laminin 332 expression were evaluated at single-cell level. Notably, monoallelic-targeted integration of LAMB3 cDNA was sufficient to in vitro recapitulate the adhesive property, the colony formation typical of normal keratinocytes, as well as their cell growth. Grafting of genetically corrected skin equivalents onto immunodeficient mice showed a completely restored dermal-epidermal junction. This study provides evidence for efficient CRISPR/Cas9-mediated in situ restoration of LAMB3 expression, paving the way for ex vivo clinical application of this strategy to laminin 332 deficiency.
Assuntos
Sistemas CRISPR-Cas/genética , Moléculas de Adesão Celular/genética , Epidermólise Bolhosa Juncional/terapia , Terapia Genética , Animais , Membrana Basal/patologia , Moléculas de Adesão Celular/antagonistas & inibidores , Moléculas de Adesão Celular/deficiência , Reparo do DNA/genética , DNA Complementar/genética , Epidermólise Bolhosa Juncional/genética , Epidermólise Bolhosa Juncional/patologia , Regulação da Expressão Gênica , Humanos , Íntrons/genética , Queratinócitos/metabolismo , Queratinócitos/patologia , Laminina/genética , Lentivirus/genética , Camundongos , Mutação , Edição de RNA/genética , CalininaRESUMO
HIV controllers are rare individuals who spontaneously control HIV replication in the absence of antiretroviral therapy. To identify parameters of the CD4 response that may contribute to viral control rather than merely reflect a persistently low viremia, we compared the T helper profiles in two groups of patients with more than 10 years of viral suppression: HIV controllers from the Agence Nationale de Recherche sur le SIDA et les Hépatites Virales (ANRS) CO18 cohort (n = 26) and efficiently treated patients (n = 16). Cells specific for immunodominant Gag and cytomegalovirus (CMV) peptides were evaluated for the production of 10 cytokines and cytotoxicity markers and were also directly quantified ex vivo by major histocompatibility complex (MHC) class II tetramer staining. HIV controller CD4(+) T cells were characterized by a higher frequency of gamma interferon (IFN-γ) production, perforin(+)/CD107a(+) expression, and polyfunctionality in response to Gag peptides. While interleukin 4 (IL-4), IL-17, and IL-21 production did not differ between groups, the cells of treated patients produced more IL-10 in response to Gag and CMV peptides, pointing to persistent negative immunoregulation after long-term antiretroviral therapy. Gag293 tetramer-positive cells were detected at a high frequency (0.12%) and correlated positively with IFN-γ-producing CD4(+) T cells in the controller group (R = 0.73; P = 0.003). Tetramer-positive cells were fewer in the highly active antiretroviral therapy (HAART) group (0.04%) and did not correlate with IFN-γ production, supporting the notion of a persistent immune dysfunction in HIV-specific CD4(+) T cells of treated patients. In conclusion, HIV controllers maintained a population of highly efficient Th1 effectors directed against Gag in spite of a persistently low antigenemia, while patients treated in the long term showed a loss of CD4 effector functions.
Assuntos
Infecções por HIV/sangue , Infecções por HIV/virologia , Células Th1/virologia , ADP-Ribosil Ciclase 1/biossíntese , Adulto , Idoso , Antirretrovirais/farmacologia , Linfócitos T CD4-Positivos/citologia , Estudos de Coortes , Citocinas/metabolismo , Citometria de Fluxo/métodos , Produtos do Gene gag/metabolismo , Humanos , Interferon gama/metabolismo , Interleucina-10/metabolismo , Interleucina-17/metabolismo , Interleucina-4/metabolismo , Interleucinas/metabolismo , Leucócitos Mononucleares/citologia , Proteína 1 de Membrana Associada ao Lisossomo/biossíntese , Pessoa de Meia-Idade , FenótipoRESUMO
BACKGROUND: Approaches based on expression signatures of prostate cancer (PCa) have been proposed to predict patient outcomes and response to treatments. The transcription factor NF-Y participates to the progression from benign epithelium to both localized and metastatic PCa and is associated with aggressive transcriptional profile. The gene encoding for NF-YA, the DNA-binding subunit of NF-Y, produces two alternatively spliced transcripts, NF-YAs and NF-YAl. Bioinformatic analyses pointed at NF-YA splicing as a key transcriptional signature to discriminate between different tumor molecular subtypes. In this study, we aimed to determine the pathophysiological role of NF-YA splice variants in PCa and their association with aggressive subtypes. METHODS: Data on the expression of NF-YA isoforms were extracted from the TCGA (The Cancer Genome Atlas) database of tumor prostate tissues and validated in prostate cell lines. Lentiviral transduction and CRISPR-Cas9 technology allowed the modulation of the expression of NF-YA splice variants in PCa cells. We characterized 3D cell cultures through in vitro assays and RNA-seq profilings. We used the rank-rank hypergeometric overlap approach to identify concordant/discordant gene expression signatures of NF-YAs/NF-YAl-overexpressing cells and human PCa patients. We performed in vivo studies in SHO-SCID mice to determine pathological and molecular phenotypes of NF-YAs/NF-YAl xenograft tumors. RESULTS: NF-YA depletion affects the tumorigenic potential of PCa cells in vitro and in vivo. Elevated NF-YAs levels are associated to aggressive PCa specimens, defined by Gleason Score and TNM classification. NF-YAl overexpression increases cell motility, while NF-YAs enhances cell proliferation in PCa 3D spheroids and xenograft tumors. The transcriptome of NF-YAs-spheroids has an extensive overlap with localized and metastatic human PCa signatures. According to PCa PAM50 classification, NF-YAs transcript levels are higher in LumB, characterized by poor prognosis compared to LumA and basal subtypes. A significant decrease in NF-YAs/NF-YAl ratio distinguishes PCa circulating tumor cells from cancer cells in metastatic sites, consistently with pro-migratory function of NF-YAl. Stratification of patients based on NF-YAs expression is predictive of clinical outcome. CONCLUSIONS: Altogether, our results indicate that the modulation of NF-YA isoforms affects prostate pathophysiological processes and contributes to cancer-relevant phenotype, in vitro and in vivo. Evaluation of NF-YA splicing may represent a new molecular strategy for risk assessment of PCa patients.
Assuntos
Processamento Alternativo/genética , Fator de Ligação a CCAAT/metabolismo , Edição de Genes/métodos , Neoplasias da Próstata/genética , Animais , Humanos , Masculino , Camundongos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
CRISPR/Cas9 is an efficient tool to knock down specific genes in various organisms. In this chapter, we describe how to assess knockdown of human rhodopsin (RHO) gene carrying the P23H mutation in vitro, in engineered HeLa cells, and in vivo, in P23H RHO transgenic mice. To this aim, we report two molecular assays: site-specific PCR on P23H RHO cells treated with CRISPR/Cas9 and Western blotting analysis on retinal cells prepared from P23H RHO transgenic mice electroporated with CRISPR/Cas9 and GFP plasmids.
Assuntos
Sistemas CRISPR-Cas , Edição de Genes , Retina/citologia , Retina/metabolismo , Animais , Clonagem Molecular , Biologia Computacional/métodos , Imunofluorescência , Expressão Gênica , Técnicas de Silenciamento de Genes , Marcação de Genes , Células HeLa , Humanos , Camundongos , Reação em Cadeia da Polimerase , RNA Guia de Cinetoplastídeos , Rodopsina/genéticaRESUMO
The Src family consists of eight non-receptor protein tyrosine kinases characterised by a common structure. Based on their amino acid sequence, Src family kinases are grouped into two subfamilies, which are also characterised by different tissue specificity. Src kinases are involved in signal transduction pathways triggered by a wide variety of surface receptors, including receptor tyrosine kinases, integrins, G-protein-coupled receptors and antigen receptors. Several pieces of evidence implicate Src family kinases in cancer development, as a consequence of changes in protein expression and/or kinase activity, and have prompted the design of potent specific inhibitors, the most common of which are adenine mimetics, as tools of relevant clinical interest for the treatment of both solid tumours and leukaemias. In addition, the finding that some Src kinases expressed in haematopoietic cells play pivotal roles in lymphocyte maturation and activation has fostered the development of safe and effective inhibitors selective for specific Src family members, which are currently being tested in clinical trials as immunosuppressants for the treatment of immunological disorders. Here we shall review the recent literature on the involvement of Src family kinases in human neoplasias and immunological disorders and the goals reached in the search for selective pharmacological inhibitors.
Assuntos
Antineoplásicos/farmacologia , Doenças do Sistema Imunitário/tratamento farmacológico , Neoplasias/tratamento farmacológico , Quinases da Família src/efeitos dos fármacos , Animais , Antineoplásicos/uso terapêutico , Catálise , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/uso terapêutico , Humanos , Doenças do Sistema Imunitário/enzimologia , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/antagonistas & inibidores , Neoplasias/enzimologia , Receptores de Superfície Celular/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Quinases da Família src/antagonistas & inibidores , Quinases da Família src/químicaRESUMO
Inhibition of deregulated protein tyrosine kinases represents an attractive strategy for controlling cancer growth. However, target specificity is an essential aim of this strategy. In this report, pp60(c-Src) kinase and beta-catenin were found physically associated and constitutively activated on tyrosine residues in human colorectal cancer cells. The use of specific small-interfering RNAs (siRNA) validated pp60(c-Src) as the major kinase responsible for beta-catenin tyrosine phosphorylation in colorectal cancer. Src-dependent activation of beta-catenin was prevented by SKI-606, a novel Src family kinase inhibitor, which also abrogated beta-catenin nuclear function by impairing its binding to the TCF4 transcription factor and its trans-activating ability in colorectal cancer cells. These effects were seemingly specific, as cyclin D1, a crucial beta-catenin/TCF4 target gene, was also down-regulated by SKI-606 in a dose-dependent manner accounting, at least in part, for the reduced growth (IC50, 1.5-2.4 micromol/L) and clonogenic potential of colorectal cancer cells. Protein levels of beta-catenin remained substantially unchanged by SKI-606, which promoted instead a cytosolic/membranous retention of beta-catenin as judged by immunoblotting analysis of cytosolic/nuclear extracts and cell immunofluorescence staining. The SKI-606-mediated relocalization of beta-catenin increased its binding affinity to E-cadherin and adhesion of colorectal cancer cells, with ensuing reduced motility in a wound healing assay. Interestingly, the siRNA-driven knockdown of beta-catenin removed the effect of SKI-606 on cell-to-cell adhesion, which was associated with prolonged stability of E-cadherin protein in a pulse-chase experiment. Thus, our results show that SKI-606 operates a switch between the transcriptional and adhesive function of beta-catenin by inhibiting its pp60(c-Src)-dependent tyrosine phosphorylation; this could constitute a new therapeutic target in colorectal cancer.
Assuntos
Compostos de Anilina/farmacologia , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/metabolismo , Nitrilas/farmacologia , Proteínas Proto-Oncogênicas pp60(c-src)/antagonistas & inibidores , Quinolinas/farmacologia , beta Catenina/metabolismo , Caderinas/metabolismo , Processos de Crescimento Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Neoplasias Colorretais/enzimologia , Neoplasias Colorretais/patologia , Humanos , Junções Intercelulares/efeitos dos fármacos , Fosforilação/efeitos dos fármacos , Proteínas Proto-Oncogênicas pp60(c-src)/genética , Proteínas Proto-Oncogênicas pp60(c-src)/metabolismo , Transdução de Sinais/efeitos dos fármacos , Fatores de Transcrição TCF/metabolismo , Proteína 2 Semelhante ao Fator 7 de Transcrição , Ativação Transcricional/efeitos dos fármacos , Tirosina/metabolismoRESUMO
The Sleeping Beauty (SB) transposon is a non-viral integrating system with proven efficacy for gene transfer and functional genomics. To optimize the SB transposon machinery, a transcriptionally regulated hyperactive transposase (SB100X) and T2-based transposon are employed. Typically, the transposase and transposon are provided transiently by plasmid transfection and SB100X expression is driven by a constitutive promoter. Here, we describe an efficient method to deliver the SB components to human cells that are resistant to several physical and chemical transfection methods, to control SB100X expression and stably integrate a gene of interest (GOI) through a "cut and paste" SB mechanism. The expression of hyperactive transposase is tightly controlled by the Tet-ON system, widely used to control gene expression since 1992. The gene of interest is flanked by inverted repeats (IR) of the T2 transposon. Both SB components are packaged in integration defective lentiviral vectors transiently produced in HEK293T cells. Human cells, either cell lines or primary cells from human tissue, are in vitro transiently transduced with viral vectors. Upon addition of doxycycline (dox, tetracycline analog) into the culture medium, a fine-tuning of transposase expression is measured and results in a long-lasting integration of the gene of interest in the genome of the treated cells. This method is efficient and applicable to the cell line (e.g., HeLa cells) and primary cells (e.g., human primary keratinocytes), and thus represents a valuable tool for genetic engineering and therapeutic gene transfer.
Assuntos
Elementos de DNA Transponíveis , Vetores Genéticos/genética , Lentivirus/genética , Transposases/genética , Animais , Células HEK293 , Células HeLa , Humanos , TransfecçãoRESUMO
Rare individuals, termed HIV controllers, spontaneously control HIV infection by mounting efficient T cell responses against the virus. Protective CD4+ T cell responses from HIV controllers involve high-affinity public T cell receptors (TCRs) recognizing an immunodominant capsid epitope (Gag293) presented by a remarkably broad array of human leukocyte antigen (HLA) class II molecules. Here, we determine the structures of a prototypical public TCR bound to HLA-DR1, HLA-DR11, and HLA-DR15 molecules presenting the Gag293 epitope. TCR recognition was driven by contacts with the Gag293 epitope, a feature that underpinned the extensive HLA cross-restriction. These high-affinity TCRs promoted mature immunological synapse formation and cytotoxic capacity in both CD4+ and CD8+ T cells. The public TCRs suppressed HIV replication in multiple genetic backgrounds ex vivo, emphasizing the functional advantage conferred by broad HLA class II cross-restriction.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Infecções por HIV/imunologia , HIV-1/imunologia , Antígenos HLA-D/imunologia , Interações Hospedeiro-Patógeno/imunologia , Animais , Reações Cruzadas/imunologia , Fibroblastos , Células HEK293 , Voluntários Saudáveis , Humanos , Células Jurkat , Leucócitos Mononucleares , Ativação Linfocitária , Camundongos , Cultura Primária de Células , Receptores de Antígenos de Linfócitos T/imunologia , Carga Viral/imunologia , Replicação Viral/imunologiaRESUMO
Major Histocompatibility Complex (MHC) tetramers have been used for two decades to detect, isolate and characterize T cells specific for various pathogens and tumor antigens. In the context of Human Immunodeficiency Virus (HIV) infection, antigen-specific CD8+ T cells have been extensively studied ex vivo, as they can be readily detected by HIV peptide-loaded MHC class I tetramers. In contrast, the detection of HIV-specific CD4+ T cells has proven more challenging, due to the intrinsically lower clonal expansion rates of CD4+ T cells, and to the preferential depletion of HIV-specific CD4+ T cells in the course of HIV infection. In the following protocol, we describe a simple method that facilitates the identification of CD4+ T cells specific for an HIV-1 capsid epitope using peptide-loaded MHC class II tetramers. Tetramer labeled CD4+ T cells can be analyzed for their cell surface phenotype and/or FACS-sorted for further downstream applications. A key point for successful detection of specific CD4+ T cells ex vivo is the choice of a peptide/MHC II combination that results in high-affinity T Cell Receptor (TCR) binding ( Benati et al., 2016 ). A second key point for reliable detection of MHC II tetramer-positive cells is the systematic use of a control tetramer loaded with an irrelevant peptide, with the sample and control tubes being processed in identical conditions.
RESUMO
The bacterial CRISPR/Cas system has proven to be an efficient tool for genetic manipulation in various organisms. Here we show the application of CRISPR-Cas9 technology to edit the human Rhodopsin (RHO) gene in a mouse model for autosomal dominant Retinitis Pigmentosa. We designed single or double sgRNAs to knock-down mutant RHO expression by targeting exon 1 of the RHO gene carrying the P23H dominant mutation. By delivering Cas9 and sgRNAs in a single plasmid we induced an efficient gene editing in vitro, in HeLa cells engineered to constitutively express the P23H mutant RHO allele. Similarly, after subretinal electroporation of the CRISPR/Cas9 plasmid expressing two sgRNAs into P23H RHO transgenic mice, we scored specific gene editing as well as significant reduction of the mutant RHO protein. Successful in vivo application of the CRISPR/Cas9 system confirms its efficacy as a genetic engineering tool in photoreceptor cells.
RESUMO
The rare patients who are able to spontaneously control HIV replication in the absence of therapy show signs of a particularly efficient cellular immune response. To identify the molecular determinants that underlie this response, we characterized the T cell receptor (TCR) repertoire directed at Gag293, the most immunoprevalent CD4 epitope in the HIV-1 capsid. HIV controllers from the ANRS CODEX cohort showed a highly skewed TCR repertoire that was characterized by a predominance of TRAV24 and TRBV2 variable genes, shared CDR3 motifs, and a high frequency of public clonotypes. The most prevalent public clonotypes generated TCRs with affinities at the higher end of values reported for naturally occurring TCRs. The high-affinity Gag293-specific TCRs were cross-restricted by up to 5 distinct HLA-DR alleles, accounting for the expression of these TCRs in HIV controllers of diverse genetic backgrounds. Transfer of these TCRs to healthy donor CD4+ T cells conferred high antigen sensitivity and polyfunctionality, thus recapitulating key features of the controller CD4 response. Transfer of a high-affinity Gag293-specific TCR also redirected CD8+ T cells to target HIV-1 capsid via nonconventional MHC II restriction. Together, these findings indicate that TCR clonotypes with superior functions are associated with HIV control. Amplification or transfer of such clonotypes may contribute to immunotherapeutic approaches aiming at a functional HIV cure.
Assuntos
Antígenos CD4/imunologia , Infecções por HIV/imunologia , HIV-1 , Receptores de Antígenos de Linfócitos T/imunologia , Transferência Adotiva , Adulto , Animais , Terapia Antirretroviral de Alta Atividade , Linfócitos T CD4-Positivos/imunologia , Genes Codificadores da Cadeia alfa de Receptores de Linfócitos T , Genes Codificadores da Cadeia beta de Receptores de Linfócitos T , Infecções por HIV/tratamento farmacológico , Infecções por HIV/genética , Sobreviventes de Longo Prazo ao HIV , Antígenos HLA-DR/genética , Humanos , Imunidade Celular , Células L , Camundongos , Pessoa de Meia-Idade , Produtos do Gene gag do Vírus da Imunodeficiência Humana/imunologiaRESUMO
Idiopathic CD4 lymphocytopenia (ICL) is a rare immune deficiency characterized by a protracted CD4(+) T cell loss of unknown etiology and by the occurrence of opportunistic infections similar to those seen in AIDS. We investigated whether a defect in responses to cytokines that control CD4(+) T cell homeostasis could play a role in ICL. Immunophenotype and signaling responses to interleukin-7 (IL-7), IL-2, and thymic stromal lymphopoietin (TSLP) were analyzed by flow cytometry in CD4(+) T cells from 15 ICL patients and 15 healthy blood donors. The induction of phospho-STAT5 after IL-7 stimulation was decreased in memory CD4(+) T cells of some ICL patients, which correlated with a decreased expression of the IL-7Rα receptor chain (Râ=â0.74, p<0.005) and with lower CD4(+) T cell counts (Râ=â0.69, p<0.005). IL-2 responses were also impaired, both in the Treg and conventional memory subsets. Decreased IL-2 responses correlated with decreased IL-7 responses (Râ=â0.75, p<0.005), pointing to combined defects that may significantly perturb CD4(+) T cell homeostasis in a subset of ICL patients. Unexpectedly, responses to the IL-7-related cytokine TSLP were increased in ICL patients, while they remained barely detectable in healthy controls. TSLP responses correlated inversely with IL-7 responses (Râ=â-0.41; p<0.05), suggesting a cross-regulation between the two cytokine systems. In conclusion, IL-7 and IL-2 signaling are impaired in ICL, which may account for the loss of CD4(+) T cell homeostasis. Increased TSLP responses point to a compensatory homeostatic mechanism that may mitigate defects in γc cytokine responses.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Citocinas/imunologia , Homeostase/imunologia , Linfopenia/imunologia , Adolescente , Adulto , Idoso , Contagem de Linfócito CD4 , Linfócitos T CD4-Positivos/metabolismo , Citocinas/metabolismo , Citocinas/farmacologia , Feminino , Humanos , Imunofenotipagem , Interleucina-2/imunologia , Interleucina-2/metabolismo , Interleucina-2/farmacologia , Subunidade alfa de Receptor de Interleucina-2/metabolismo , Interleucina-7/imunologia , Interleucina-7/metabolismo , Interleucina-7/farmacologia , Subunidade alfa de Receptor de Interleucina-7/metabolismo , Ativação Linfocitária , Linfopenia/metabolismo , Masculino , Pessoa de Meia-Idade , Fosforilação/efeitos dos fármacos , Fator de Transcrição STAT5/metabolismo , Adulto Jovem , Linfopoietina do Estroma do TimoRESUMO
Rai/ShcC is a member of the Shc family of protein adaptors expressed with the highest abundance in the central nervous system, where it exerts a protective function by coupling neurotrophic receptors to the PI3K/Akt survival pathway. Rai is also expressed, albeit at lower levels, in other cell types, including T and B lymphocytes. We have previously reported that in these cells Rai attenuates antigen receptor signaling, thereby impairing not only cell proliferation but also, opposite to neurons, cell survival. Here we have addressed the mechanism underlying the inhibitory activity of Rai on TCR signaling. We show that Rai interferes with the TCR signaling cascade one of the earliest steps--recruitment of the initiating kinase ZAP-70 to the phosphorylated subunit of the TCR/CD3 complex, which results in a generalized dampening of the downstream signaling events. The inhibitory activity of Rai is associated to its inducible recruitment to phosphorylated CD3, which occurs in the physiological signaling context of the immune synapse. Rai is moreover found as a pre-assembled complex with ZAP-70 and also constitutively interacts with the regulatory p85 subunit of PI3K, similar to neuronal cells, notwithstanding the opposite biological outcome, i.e. impairment of PI-3K/Akt activation. The data highlight the ability of Rai to establish interactions with the TCR and key signaling mediators which, either directly (e.g. by inhibiting ZAP-70 recruitment to the TCR or sequestering ZAP-70/PI3K in the cytosol) or indirectly (e.g. by promoting the recruitment of effectors responsible for signal extinction) prevent full triggering of the TCR signaling cascade.
Assuntos
Receptores de Antígenos de Linfócitos T/metabolismo , Proteínas Adaptadoras da Sinalização Shc/metabolismo , Transdução de Sinais , Proteína-Tirosina Quinase ZAP-70/antagonistas & inibidores , Animais , Complexo CD3/metabolismo , Ativação Enzimática , Humanos , Sinapses Imunológicas/metabolismo , Células Jurkat , Camundongos , Camundongos Endogâmicos C57BL , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação , Ligação Proteica , Proteínas Adaptadoras da Sinalização Shc/deficiência , Proteína 3 de Transformação que Contém Domínio 2 de Homologia de Src , Proteína-Tirosina Quinase ZAP-70/metabolismoRESUMO
It is unexplained how strepsipteran insects manipulate the physiology of their hosts in order to undergo endoparasitic development without being entrapped by the innate immune defences of the host. Here we present pioneering work that aimed to explore for the first time several components of the cellular and humoral immune response among immature stages of the paper wasp Polistes dominulus, in both unparasitized insects and after infection by the strepsipteran endoparasite Xenos vesparum. We carried out hemocyte counts, phagocytosis assays in vitro and antibacterial response in vivo. On the whole, hemocyte load does not seem to be drastically affected by parasitization: a non-significant increase in hemocyte numbers was observed in parasitized wasps as respect to control, while the two dominant hemocyte types were present with similar proportions in both groups. On the other hand, phagocytosis was significantly reduced in hemocytes from parasitized wasps while the antibacterial response seemed to be less effective in control. These somewhat unexpected results are discussed, along with the implications of a multiple approach in immune response studies.
Assuntos
Interações Hospedeiro-Parasita , Insetos/fisiologia , Vespas/imunologia , Vespas/parasitologia , Animais , Hemócitos/imunologia , Imunocompetência , Larva/crescimento & desenvolvimento , Larva/imunologia , Larva/parasitologia , Fagocitose , Vespas/crescimento & desenvolvimentoRESUMO
Besides lowering circulating cholesterol, statins act as immunomodulators. Although the effects of statins on lymphocyte activation and differentiation have been clearly defined, there is no consensus as to effects of these drugs on phagocytes. We have addressed the outcome of simvastatin treatment on the activation and effector function of human macrophages in the pathophysiologically relevant context of challenge with an opportunistic pathogen. We provide evidence that: simvastatin blocks the biological effects rapidly triggered by IgG-opsonized bacteria (phagocytosis and oxidative burst) while enhancing the delayed effects elicited by FcgammaR stimulation (production of proinflammatory mediators); these opposite effects of simvastatin result from enhancement of the JNK pathway and concomitant impairment of other signaling modules activated by FcgammaR engagement; and these activities are dependent on the capacity of simvastatin to block protein prenylation. The results provide novel mechanistic insight into the activities of statins on phagocytes and are of relevance to the assessment of potential side-effects in patients undergoing long-term hypocholesterolemic therapy.