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DANIELA BENATI

Ricercatore t.d. art. 24 c. 3 lett. A
Dipartimento di Scienze della Vita sede Centro di Medicina Rigenerativa

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Pubblicazioni

2022 - CRISPR-mediated T cell engineering against Non-Small Cell Lung Cancer [Abstract in Atti di Convegno]
Benati, Daniela; Ferrari, Tommaso; Masciale, Valentina; Grisendi, Giulia; Aramini, Beatrice; Dominici, Massimo; Recchia, Alessandra
abstract

2022 - CRISPR/Cas9 Allele-Specific Design To Inactivate A Dominant-Negative Mutation In COL6A1 Causing Ullrich Muscular Dystrophy [Abstract in Atti di Convegno]
Benati, Daniela; Patrizi, Clarissa; Cattin, Eleonora; Ferrari, Tommaso; Pedrazzoli, Eleonora; Marchionni, Matteo; Rossi, Rachele; D’Amico, Adele; Merlini, Luciano; Sabatelli, Patrizia; Ferlini, Alessandra; Gualandi, Francesca; Recchia, Alessandra
abstract

2022 - CRISPR/Cas9 allele-specifc design to inactivate a dominant-negative mutation in COL6A1 causing Ullrich muscular dystrophy. [Abstract in Atti di Convegno]
Benati, Daniela; Patrizi, Clarissa; Cattin, Eleonora; Ferrari, Tommaso; Pedrazzoli, Eleonora; Marchionni, Matteo; Rossi, Rachele; D'Amico, Adele; Merlini, Luciano; Sabatelli, Patrizia; Ferlini, Alessandra; Gualandi, Francesca; Recchia, Alessandra.
abstract

2022 - Engineered Sleeping Beauty Transposon as Efficient System to Optimize Chimp Adenoviral Production [Articolo su rivista]
Baldassarri, S.; Benati, D.; D'Alessio, F.; Patrizi, C.; Cattin, E.; Gentile, M.; Raggioli, A.; Recchia, A.
abstract

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.

2022 - Induced Pluripotent Stem Cells and Genome-Editing Tools in Determining Gene Function and Therapy for Inherited Retinal Disorders [Articolo su rivista]
Benati, Daniela; Leung, Amy; Perdigao, Pedro; Toulis, Vasileios; van der Spuy, Jacqueline; Recchia, Alessandra
abstract

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.

2021 - Allele-specific editing ameliorates dominant retinitis pigmentosa in a transgenic mouse model [Articolo su rivista]
Patrizi, C.; Llado, M.; Benati, D.; Iodice, C.; Marrocco, E.; Guarascio, R.; Surace, E. M.; Cheetham, M. E.; Auricchio, A.; Recchia, A.
abstract

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.

2021 - Alternative splicing of NF-YA promotes prostate cancer aggressiveness and represents a new molecular marker for clinical stratification of patients [Articolo su rivista]
Belluti, Silvia; Semeghini, Valentina; Rigillo, Giovanna; Ronzio, Mirko; Benati, Daniela; Torricelli, Federica; Reggiani Bonetti, Luca; Carnevale, Gianluca; Grisendi, Giulia; Ciarrocchi, Alessia; Dominici, Massimo; Recchia, Alessandra; Dolfini, Diletta; Imbriano, Carol
abstract

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.

2021 - Alternative splicing of NF-YA promotes prostate cancer aggressiveness and represents a new molecular marker for clinical stratification of patients [Poster]
Belluti, Silvia; Semeghini, Valentina; Rigillo, Giovanna; Ronzio, Mirko; Benati, Daniela; Torricelli, Federica; REGGIANI BONETTI, Luca; Carnevale, Gianluca; Grisendi, Giulia; Ciarrocchi, Alessia; Dominici, Massimo; Recchia, Alessandra; Dolfini, Diletta; Imbriano, Carol
abstract

2021 - CRISPR-Mediated Genome Editing to Redirect T Cells against Non-Small Cell Lung Cancer [Abstract in Atti di Convegno]
Benati, Daniela; Masciale, Valentina; Grisendi, Giulia; Marchionni, Matteo; Ferrari, Tommaso; Aramini, Beatrice; Dominici, Massimo; Recchia, Alessandra
abstract

2021 - CRISPR-mediated genome editing to redirect T cells against Non-Small Cell Lung Cancer [Abstract in Atti di Convegno]
Benati, Daniela; Masciale, Valentina; Grisendi, Giulia; Ferrari, Tommaso; Cattin, Eleonora; Aramini, Beatrice; Dominici, Massimo; Recchia, Alessandra
abstract

2021 - CRISPR-mediated genome editing to redirect T cells against Non-Small Cell Lung Cancer [Abstract in Atti di Convegno]
Benati, Daniela; Masciale, Valentina; Grisendi, Giulia; Ferrari, Tommaso; Cattin, Eleonora; Marchionni, Matteo; Aramini, Beatrice; Dominici, Massimo; Recchia, Alessandra
abstract

2020 - Gene editing prospects for treating inherited retinal diseases [Articolo su rivista]
Benati, D.; Patrizi, C.; Recchia, A.
abstract

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.

2020 - Specific Knock-down of P347S Dominant Mutation in Rhodopsin Gene by CRISPR/Cas9 System [Abstract in Atti di Convegno]
Patrizi, Clarissa; Llado, Manel; Benati, Daniela; Guarascio, Rosellina; Cheetham, Mike; Auricchio, Alberto; Recchia, Alessandra
abstract

2019 - CRISPR/Cas9 gene editing in vitro and in retinal cells in vivo [Capitolo/Saggio]
Benati, D.; Marigo, V.; Recchia, A.
abstract

CRISPR/Cas9 is an efficient tool to knock down specific genes in various organisms. In this chapter, we describe how to assess knock-down 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.

2018 - A High Frequency of HIV-Specific Circulating Follicular Helper T Cells Is Associated with Preserved Memory B Cell Responses in HIV Controllers [Articolo su rivista]
Claireaux, M; Galperin, M; Benati, D; Nouël, A; Mukhopadhyay, M; Klingler, J; de Truchis, P; Zucman, D; Hendou, S; Boufassa, F; Moog, C; Lambotte, O; Chakrabarti, L A
abstract

Follicular helper T cells (Tfh) play an essential role in the affinity maturation of the antibody response by providing help to B cells. To determine whether this CD4+ T cell subset may contribute to the spontaneous control of HIV infection, we analyzed the phenotype and function of circulating Tfh (cTfh) in patients from the ANRS CO21 CODEX cohort who naturally controlled HIV-1 replication to undetectable levels and compared them to treated patients with similarly low viral loads. HIV-specific cTfh (Tet+), detected by Gag-major histocompatibility complex class II (MHC-II) tetramer labeling in the CD45RA- CXCR5+ CD4+ T cell population, proved more frequent in the controller group (P = 0.002). The frequency of PD-1 expression in Tet+ cTfh was increased in both groups (median, >75%) compared to total cTfh (<30%), but the intensity of PD-1 expression per cell remained higher in the treated patient group (P = 0.02), pointing to the persistence of abnormal immune activation in treated patients. The function of cTfh, analyzed by the capacity to promote IgG secretion in cocultures with autologous memory B cells, did not show major differences between groups in terms of total IgG production but proved significantly more efficient in the controller group when measuring HIV-specific IgG production. The frequency of Tet+ cTfh correlated with HIV-specific IgG production (R = 0.71 for Gag-specific and R = 0.79 for Env-specific IgG, respectively). Taken together, our findings indicate that key cTfh-B cell interactions are preserved in controlled HIV infection, resulting in potent memory B cell responses that may play an underappreciated role in HIV control.IMPORTANCE The rare patients who spontaneously control HIV replication in the absence of therapy provide a unique model to identify determinants of an effective anti-HIV immune response. HIV controllers show signs of particularly efficient antiviral T cell responses, while their humoral response was until recently considered to play only a minor role in viral control. However, emerging evidence suggests that HIV controllers maintain a significant but "silent" antiviral memory B cell population that can be reactivated upon antigenic stimulation. We report that cTfh help likely contributes to the persistence of controller memory B cell responses, as the frequency of HIV-specific cTfh correlated with the induction of HIV-specific antibodies in functional assays. These findings suggest that T follicular help may contribute to HIV control and highlight the need for inducing such help in HIV vaccine strategies that aim at eliciting persistent B cell responses.

2018 - An efficient in vitro transposition method by a transcriptionally regulated sleeping beauty system packaged into an integration defective lentiviral vector [Articolo su rivista]
Benati, Daniela; Cocchiarella, Fabienne; Recchia, Alessandra
abstract

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.

2018 - CD4+ T cell-mediated HLA class II cross-restriction in HIV controllers [Articolo su rivista]
Galperin, Moran; Farenc, Carine; Mukhopadhyay, Madhura; Jayasinghe, Dhilshan; Decroos, Amandine; Benati, Daniela; Tan, Li Lynn; Ciacchi, Lisa; Reid, Hugh H; Rossjohn, Jamie; Chakrabarti, Lisa A; Gras, Stephanie
abstract

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.

2018 - CRISPR/Cas9-Mediated In Situ Correction of LAMB3 Gene in Keratinocytes Derived from a Junctional Epidermolysis Bullosa Patient [Articolo su rivista]
Benati, Daniela; Miselli, Francesca; Cocchiarella, Fabienne; Patrizi, Clarissa; Carretero, Marta; Baldassarri, Samantha; Ammendola, Virginia; Has, Cristina; Colloca, Stefano; Del Rio, Marcela; Larcher, Fernando; Recchia, Alessandra
abstract

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.

2018 - Efficient CRISPR/Cas9-Mediated iIn Situ Correction of LAMB3 Gene in Keratinocytes Derived from Junctional Epidermolysis Bullosa Patient [Abstract in Atti di Convegno]
Benati, Daniela; Miselli, Francesca; Cocchiarella, Fabienne; Patrizi, Clarissa; Latella, Maria Carmela; Baldassarri, Samantha; Pedrazzoli, Eleonora; Ammendola, Virginia; Larcher, Fernando; Recchia, Alessandra
abstract

2018 - Specific cTfh frequency correlates with memory B cell responses in HIV controllers. [Poster]
Claireaux, Mathieu; Galperin, Moran; Benati, Daniela; Nouel, Alexandre; Mukhopadhyay, Madhura; de Truchis, Pierre; Zucman, David; Hendou, Samia; Boufassa, Faroudy; Lambotte, Olivier; La., Chakrabarti
abstract

Background: Follicular helper T cells (Tfh) play an essential role in the affinity maturation of the antibody response by providing help to B cells. To determine whether this CD4+ T cell subset may contribute to the spontaneous control of HIV infection, we analyzed the phenotype and function of circulating Tfh (cTfh) in patients from the ANRS CO21 CODEX cohort who naturally controlled HIV-1 replication to undetectable levels (HIC group), and compared them to treated patients with similarly low viral loads (ART group). Methods: HIV-specific cTfh (Tet+) were detected by Gag MHC-II tetramer labeling in the CD45RA- CXCR5+ CD4+ T cell population. The function of cTfh was analyzed by the capacity to promote IgG secretion in cocultures with autologous memory B cells. Results: HIV-specific cTfh (Tet+) proved more frequent in the controller group than in the treated patient group (P=0.002). The frequency of PD-1 expression in Tet+ cTfh was increased in both groups (median &gt;75%) compared to total cTfh (&lt;30%), but the intensity of PD-1 expression per cell remained higher in the ART group (P=0.02), pointing to the persistence of abnormal immune activation in treated patients. The function of cTfh, analyzed in coculture with memory B cells, did not show major differences between groups in terms of total IgG production, but proved significantly more efficient in the controller group when measuring HIV-specific IgG production. The frequency of Tet+ cTfh correlated with HIV-specific IgG production (R=0.71 for Gag-specific and R=0.79 for Env-specific IgG, respectively). Conclusion: Taken together, these findings indicate that key cTfh/B cell interactions are preserved in controlled HIV infection, resulting in potent memory B cell responses that may play an underappreciated role in HIV control.

2018 - Specific knock-down of C-terminal dominant mutation in Rhodopsin gene by CRISPR/Cas9 system [Abstract in Atti di Convegno]
Benati, Daniela; Patrizi, Clarissa; Marigo, Valeria; Auricchio, Alberto; Recchia, Alessandra
abstract

2017 - CRISPR/Cas9-mediated specific knock-down of dominant mutations in Rhodopsin gene [Poster]
Benati, Daniela; Manel, Llado; Patrizi, Clarissa; Schiroli, Davide; Marigo, Valeria; Auricchio, Alberto; Recchia, Alessandra
abstract

Rhodopsin (RHO) mutations represent a common cause of blindness, accounting for 25% of autosomal dominant Retinitis Pigmentosa (RP) and 8-10% of all RP. Although gene therapy has been successfully applied to retinal degeneration caused by recessive mutations, therapeutic intervention for dominant mutations are still lagging behind. In this study, we explored the efficacy of newly described CRISPR/Cas9 variants with altered PAM specificity and nearly completely reduced off-target effects, to specifically inactivate two highly frequent dominant mutations, P23H and P347S, mapped in the N-terminal and the C-terminal region of the RHO gene, respectively. We designed gRNAs on the mutations to compare allele-specific targeting of the high fidelity SpCas9 (SpCas9-HF1), the respective VQR variant (SpCas9-VQR-HF1) or the SaCas9, and we tested gRNAs in vitro on HeLa clones stably expressing P23H, P347S or wild-type RHO. Analysis of insertions or deletions (Indels) in the genomic DNA specifically in the RHO gene, by Cel-I assay and sequencing, identified the most efficient and mutation-specific system able to induce Indels in the P23H or P347S RHO mutated allele, with almost undetectable editing of the wild-type allele. We are going to package the selected CRISPR/Cas9-gRNA in AAV2/8 particles to test this approach in P23H or P347S RHO transgenic mice, to evaluate retina functionality and vision recovery upon CRISPR/Cas9-mediated editing. Our results will provide clear evidences about the employment of CRISPR/Cas9 system to selectively target dominant mutations and the preclinical application of this strategy for patients affected by RP due to mutations in the RHO gene.

2017 - DNA vaccination by electroporation amplifies broadly cross-restricted public TCR clonotypes shared with HIV Controllers [Articolo su rivista]
Mukhopadhyay, Madhura; Galperin, Moran; Patgaonkar, Mandar; Vasan, Sandhya; Ho, David D.; Nouël, Alexandre; Claireaux, Mathieu; Benati, Daniela; Lambotte, Olivier; Huang, Yaoxing; Chakrabarti, Lisa A.
abstract

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 TCRβ 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 (&gt;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, DNAvaccination 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.

2017 - MHC Class II Tetramer Labeling of Human Primary CD4+ T Cells from HIV Infected Patients [Articolo su rivista]
Galperin, Moran; Benati, Daniela; Claireaux, Mathieu; Chakrabarti, Madhura Mukhopadhyay and Lisa A.
abstract

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.

2016 - Comparison of the antigen sensitivity of Gag-specific CD4+ T cell responses in controlled HIV infection and HIV vaccination [Abstract in Atti di Convegno]
Mukhopadhyay, Madhura; Galperin, Moran; Nouël, Alexandre; Vasan, Sandhya; Ho, David D.; Fast, P.; Lambotte, Olivier; Benati, Daniela; Chakrabarti, and Lisa A.
abstract

Spontaneous control of HIV infection is characterized by a highly efficient cellular immune response. We showed in particular that HIV Controllers from the ANRS CO21 CODEX cohort harbor a population of specific CD4+ T cells that detect the immunodominant CD4 epitope Gag293 with high antigen sensitivity. To determine whether candidate vaccines can induce the high sensitivity responses seen in Controllers, we analyzed Gag293-specific responses in healthy volunteers who received the ADVAX DNA vaccine administered by electroporation. Comparison of Gag293-specific responses in primary CD4+ T cell lines via IFN-γ ELISpot revealed that the median antigen sensitivity in vaccinees was similar to that observed for Controllers (5x10-8 M) but higher than that in treated patients (5x10-7 M). However, antigen sensitivity remained higher in a subset of Controllers compared to vaccinees. TCR repertoire analysis of Gag293-specific CD4+ T cells from vaccinees revealed a preferential amplification of TCRβ family chain TRBV2, which also predominates in Controllers. However, TRAV family gene usage appeared more diverse in vaccinees compared to Controllers. Sequence analysis of the TCR chains amplified in 4 vaccinees revealed a biased TCR repertoire with the presence of public clonotypes (3 TRAV24 and 2 TRBV2) shared with HIV Controllers. In conclusion, DNA vaccination administered by electroporation has the potential to induce Gag-specific CD4+ T cells responses with a high antigen sensitivity and partial TCR repertoire overlap with that of Controllers. Monitoring the amplification of public TCR clonotypes could provide a novel approach to evaluate the quality of HIV vaccine responses.

2016 - Frequent sharing of high-affinity TCRs in naturally controlled HIV infection: implications for vaccination [Poster]
Galperin, Moran; Benati, Daniela; Mukhopadhyay, Madhura; Lambotte, Olivier; Gras, Stephanie; Nouël, Alexandre; Campbell, Kristy-Anne; Claireaux, Mathieu; De Truchis, Pierre; Boufassa, Faroudy; Rossjohn, Jamie; Delfraissy, Jean-Francois; Chakrabarti, and Lisa A.
abstract

The rare patients who spontaneously control HIV replication in the absence of therapy show signs of a particularly efficient cellular immune response. To identify the molecular determinants underlying this response, we characterized the TCR repertoire directed at the most immunodominant CD4 epitope in HIV-1 capsid, Gag293. HIV Controllers from the ANRS CO21 CODEX cohort showed a highly skewed TCR repertoire characterized by a predominance of the TRAV24 and TRBV2 variable gene families. Controllers shared public clonotypes at higher frequencies than treated patients, suggesting the implication of particular TCRs in HIV control. The most prevalent public clonotypes generated TCRs with affinities in the micromolar range, at the higher end of values reported for naturally occurring TCRs. Transfer of the high-affinity Gag293-specific TCRs via lentivector conferred broad HLA II cross-restriction to healthy donor T cells, with up to 5 HLA-DR alleles recognized. In addition, transfer of a high-affinity Gag293-specific TCR was sufficient to confer a series of properties characteristic of HIV Controller CD4+ T cells, including high antigen sensitivity, polyfunctionality, and cytotoxicity. Of note, transduction of a high-affinity Gag293-specific TCR could also redirect CD8+ T cells to target HIV-1 capsid via nonconventional MHC II restriction. These findings indicate that TCR clonotypes with superior functions are associated with HIV control. Due to their broad HLA II cross-restriction, Gag293-specific public clonotypes can be amplified in individuals of different genetic backgrounds. This opens the possibility to test for the induction of these clonotypes in the diverse populations involved in HIV vaccination trials, and rapidly assess the quality of the vaccine responses at the clonotypic level. Analysis of the Gag293-specific repertoire induced by a candidate HIV vaccine reveals significant amplification of public clonotypes found in HIV Controllers, indicating the feasibility and potential of this approach for vaccine evaluation.

2016 - In vitro and in vivo CRISPR/Cas9-mediated genome editing to downregulate dominant mutations in Rhodopsin gene [Poster]
Benati, Daniela; Latella, Maria Carmela; DI SALVO, MARIA TERESA; Cocchiarella, Fabienne; Marigo, Valeria; Recchia, Alessandra
abstract

Many progresses have been made in understanding the genetic basis for Retinitis Pigmentosa (RP), however therapeutic interventions are still lagging behind. Rhodopsin (RHO) mutations represent a common cause of RP, accounting for 25% of autosomal dominant RP and 8-10% of all RP (Hartong et al., 2006) with more than 100 different mutations identified so far. Here we show the application of CRISPR-Cas9 technology to knock out the RHO defective alleles by introducing double strand breaks into the target gene. We designed single or double gRNAs to knock-down mutant RHO expression by targeting exon 1 of the RHO gene carrying the P23H dominant mutation. The two gRNAs were tested singularly or together in vitro in HeLa clones stably expressing P23H RHO. Cel I assay, TIDE and sequencing analyses demonstrated insertions or deletions (indels) in the genomic DNA specifically in the RHO gene, which caused strong reduction of RHO expression up to 90%. The higher effect was obtained with two gRNAs together. The CRISPR/Cas9 plasmid expressing two gRNAs were then in vivo tested in P23H RHO transgenic mice by sub-retinal electroporation, together with EGFP expressing plasmids. Analysis of indels in FACS-sorted EGFP+ cells demonstrated up to 30% of in vivo genome editing of the human P23H RHO gene, without targeting of the murine Rho allele. We also detected reduction of RHO at mRNA and protein levels. Thus, successful in vivo application of the CRISPR/Cas9 system confirms its efficacy as a genetic engineering tool and its potential use in gene therapy.

2016 - In vitro and in vivo genome editing of the RHO gene to downregulate dominant mutations [Abstract in Atti di Convegno]
Latella, Maria C.; Di Salvo, Maria T.; Cocchiarella, Fabienne; Benati, Daniela; Marigo, Valeria; Recchia, Alessandra
abstract

2016 - In vivo editing of the human mutant Rhodopsin gene by electroporation of plasmid-based CRISPR/Cas9 in the mouse retina [Articolo su rivista]
Latella, Maria Carmela; Di Salvo, Maria Teresa; Cocchiarella, Fabienne; Benati, Daniela; Grisendi, Giulia; Comitato, Antonella; Marigo, Valeria; Recchia, Alessandra
abstract

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.

2016 - Public T cell receptors confer high-avidity CD4 responses to HIV controllers [Articolo su rivista]
Benati, Daniela; Galperin, Moran; Lambotte, Olivier; Gras, Stéphanie; Lim, Annick; Mukhopadhyay, Madhura; Nouël, Alexandre; Campbell, Kristy-Anne; Lemercier, Brigitte; Claireaux, Mathieu; Hendou, Samia; Lechat, Pierre; De Truchis, Pierre; Boufassa, Faroudy; Rossjohn, Jamie; Delfraissy, Jean-François; Arenzana-Seisdedos, Fernando; Chakrabarti, Lisa A.
abstract

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.

2016 - Public TCRs confer high-avidity CD4 responses to HIV controllers [Abstract in Atti di Convegno]
Benati, Daniela; Galperin, Moran; Lambotte, Olivier; Gras, Stephanie; Lim, Annick; Mukhopadhyay, Madhura; Nouël, Alexandre; Campbell, Kristy-Anne; Lemercier, Brigitte; Claireaux, Mathieu; Hendou, Samia; Lechat, Pierre; De Truchis, Pierre; Boufassa, Faroudy; Rossjohn, Jamie; Delfraissy, Jean-Francois; Arenzana-Seisdedos, Fernando; Chakrabarti, Lisa
abstract

Rare patients termed HIV Controllers are able to spontaneously control HIV replication to undetectable levels and maintain normal CD4+ T cell counts in the absence of anti-retroviral therapy. Accumulating evidence suggests that control of viral replication in these patients is mediated by a particularly efficient cellular immune response. To identify the molecular determinants underlying this response, we characterized the TCR repertoire directed at the most immunoprevalent CD4 epitope in HIV-1 capsid, Gag293. HIV Controllers from the ANRS CODEX cohort showed a highly skewed TCR repertoire characterized by a predominance of TRAV24 and TRBV2 variable gene families, the presence of conserved motifs in both CDR3 regions, and a high frequency of public clonotypes (n=18 for each TCR chain). The most prevalent public clonotypes generated TCRs with affinities in the micromolar range, at the higher end of values reported for naturally occurring TCRs. These highaffinity Gag293-specific TCRs were cross-restricted by up to 5 distinct HLA-DR alleles, accounting for their expression in HIV Controllers of diverse genetic backgrounds. Transfer of these TCRs to healthy donor CD4+ T cells conferred high antigen sensitivity and polyfunctional cytokine responses, thus recapitulating key features of the Controller CD4 response. Transfer of a high-affinity Gag293-specific TCR could also redirect CD8+ T cells to target HIV-1 capsid via nonconventional MHC II restriction. These findings indicate that TCR clonotypes with superior functions are associated with HIV control. Amplifying or transferring such clonotypes may contribute to immunotherapeutic approaches that aim at a functional HIV cure.

2016 - T cell receptors from the HIV-specific repertoire, means for their production and therapeutic uses thereof [Brevetto]
Amita Chakrabarti, Lisa; Benati, Daniela; Galperin, Moran
abstract

The present invention pertains to the field of T Cell receptors (TCR) identification and clonotyping, and especially concerns particular TCRs identified by clonotyping of a HIV-specific TCR repertoire, or fragments thereof. the invention relates especially to TCRs recognizing Gag peptide located between positions 293-312 in the GAG protein of HIV-1. The present invention further relates to nucleic acid constructs suitable as means for cloning or expressing nucleic acid molecules or TCRs of the invention, such as plasmids, vectors, especially lentiviral transfer vectors. The invention is of particular interest in the context of therapeutic treatment of human beings seropositive for HIV.

2014 - HIV Controller CD4+ T cells preferentially express a public TCR clonotype that confers high avidity responses against Gag [Poster]
Benati, Daniela; Galperin, Moran; Lambotte, Olivier; Lim, Annick; Lemercier, Brigitte; Mukhopadhyay, Madhura; Claireaux, Mathieu; Hendou, Samia; Boufassa, Faroudy; Delfraissy, Jean-Francois; Arenzana-Seisdedos, Fernando; Chakrabarti, Lisa A.
abstract

Background: HIV Controllers are rare patients who spontaneously control HIV replication to levels below 50 copies viral RNA/ml in the absence of antiretroviral therapy. We previously reported that HIV Controllers harbor a pool of memory CD4+ T cells able to respond to minimal amounts of virus, due to the expression of T cell receptors (TCRs) with high avidity for immunodominant Gag epitopes. We set to characterize these high avidity TCRs at the molecular and functional levels. Methods: HIV Controllers from the ANRS CO21 CODEX cohort (n=8) were compared to efficiently treated patients (n=8). Primary CD4+ T cell lines were generated by stimulating PBMCs with decreasing doses of the immunodominant Gag293 peptide. Specific CD4+ T cells were labeled with Gag293-loaded MHC class II tetramers and sorted. The TCR repertoire of tetramer+ cells was evaluated by CDR3 length polymorphism analysis (Immunoscope) and sequencing. Highly represented TCR Vα and Vβ chains were cloned in bicistronic lentiviral vectors and tested for CD69 induction and cytokine production. Results: Stimulation with low Gag293 peptide doses generated IFNγ-positive CD4+ T cell lines for HIV Controllers, but rarely for treated patients, confirming the predominance of high avidity cells in the Controller group. Immunoscope analysis revealed a major amplification of TCR Vα24 chains with a 10 a.a. CDR3 in sorted tetramer+ cells from Controllers, while this amplification was rarely detected in treated patients (P< 0.05). Sequencing revealed the presence of a public TCR Vα24-J17 clonotype shared by 6 Controllers and 2 treated patients. TCRs comprised of the public Vα24 chain and of highly expressed Vβ chains conferred high avidity Gag293 recognition and polyfunctionality to transduced T cells. One of these TCRs could achieve Gag293 recognition in the context of 4 distinct HLA-DR molecules, possibly explaining its public nature. Conclusions: We identified a highly prevalent TCR sequence preferentially expressed by Gag-specific CD4+ T cells from HIV Controllers. This public clonotype confers high avidity polyfunctional responses to Gag, raising the possibility that it could be used as molecular marker of efficient responses against HIV.

2013 - Altered Responses to Homeostatic Cytokines in Patients with Idiopathic CD4 Lymphocytopenia [Articolo su rivista]
Bugault, Florence; Benati, Daniela; Mouthon, Luc; Landires, Ivan; Rohrlich, Pierre; Pestre, Vincent; Thèze, Jacques; Lortholary, Olivier; Chakrabarti, Lisa A.
abstract

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 (R = 0.74, p<0.005) and with lower CD4+ T cell counts (R = 0.69, 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 (R = 0.75, 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 (R = -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. © 2013 Bugault et al.

2013 - Molecular characterization of high avidity CD4+ T cells in HIV Controllers [Relazione in Atti di Convegno]
Benati, Daniela; Galperin, Moran; Lambotte, Olivier; Lim, Annick; Lemercier, Brigitte; Hendou, Samia; Boufassa, Faroudy; Zucman, David; Delfraissy, Jean-Francois; Arenzana-Seisdedos, Fernando; Chakrabarti, and Lisa A.
abstract

Background: HIV Controllers spontaneously control HIV replication to levels undetectable by standard assays in the absence of antiretroviral treatment. We previously reported that HIV Controllers harbour a pool of memory CD4+ T cells able to respond to the immunodominant Gag293 peptide with particularly high TCR avidity. We set to functionally analyze high avidity CD4+ T cells and to characterize their TCRs at the molecular level. Methods: HIV Controllers from the ANRS CODEX CO21 cohort (n=8) were compared to efficiently treated patients (HAART group, n=8). Primary CD4+ T cell lines were generated by stimulating PBMCs with decreasing doses of Gag293 peptide. Next, Gag293-specific CD4+ T cells were analyzed for cytokine production by IFNγ ELISPOT and for Gag293/MHC-classII tetramer binding ability. TCR diversity in sorted Gag293/Tetramer+ cells was evaluated by immunoscope analysis. Results: Stimulation with low Gag293 peptide doses generated IFNγ-positive CD4+ T cell lines in HIV Controllers (response rate: 6/8 at 10-9M, 2/8 at 10-11M), but not in HAART patients, confirming the presence of high-avidity Gag-293-specific cells in the HIC group. The immunoscope analysis revealed major amplifications of certain TCR Vα and Vβ chains in the sorted tetramer+ Gag293-specific population, indicating the presence of dominant clonotypes. Conclusion: We identified a number of TCR Vα and Vβ chains preferentially expressed by the high-avidity population of Gag293-specific CD4+ T cells. Transfer of these TCRs to heterologous cells will help determine whether they are sufficient to confer the efficient Gag-specific responses characteristic of HIV Controllers.

2012 - HIV controllers maintain a population of highly efficient Th1 effector cells in contrast to patients treated in the long term [Articolo su rivista]
Vingert, Benoît; Benati, Daniela; Lambotte, Olivier; de Truchis, Pierre; Slama, Laurence; Jeannin, Patricia; Galperin, Moran; Perez-Patrigeon, Santiago; Boufassa, Faroudy; Kwok, William W; Lemaître, Fabrice; Delfraissy, Jean-François; Thèze, Jacques; Chakrabarti, Lisa A
abstract

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 Hepatites 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-gamma) 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-gamma-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-gamma 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.

2011 - Characterization of high avidity CD4+ T cells in HIV Controllers [Poster]
Benati, Daniela; Vingert, Benoit; Lambotte, Olivier; Lim, Annick; Lemercier, Brigitte; Nehar-Belaïd, Djamel; Boufassa, Faroudy; Delfraissy, Jean-Francois; Thèze, Jacque; Chakrabarti, Lisa A.
abstract

Background: HIV Controllers spontaneously control HIV replication to very low levels (< 50 copies HIV RNA/mL) in the absence of antiretroviral treatment. We previously reported that HIV Controllers harbor a pool of memory CD4+ T cells with the capacity to recognize the immunodominant Gag293 peptide with a particularly high TCR avidity. Since this property may allow the persistence of an activated cellular response against minimal amounts of virus, we set to further characterize the nature of these high avidity memory CD4+ T cells. Methods: HIV Controllers from the ANRS CO18 cohort (n=8) with a viral load < 50 copies/mL for over 10 years were compared to efficiently treated patients with a similarly undetectable viral load for over 10 years (HAART group, n=8). Primary CD4+ T cell lines were generated by stimulating PBMC with decreasing doses of the Gag293 peptide. Cytokine production was monitored by IFN-γ ELISPOT and intracellular cytokine staining assays. TCR diversity was evaluated by staining with a panel of 24 anti-TCR Vβ chain antibodies and by Immunoscope analysis. Results: After stimulation with a high dose of Gag293 peptide (10-5 M), IFN-γ-positive CD4+ T cell lines were obtained for all patients studied (response rate of 8/8 in both groups). However, at low peptide doses, specific cell lines were obtained only for HIV Controllers (response rate of 6/8 at 10-9 M and 3/8 at 10-11 M) but not for treated patients. The Vβ repertoire appeared more restricted in cell lines stimulated with low peptide doses, suggesting that only a subset of HIV Controller memory CD4+ T cells were of high avidity. Conclusion: The presence of a high avidity subset within the pool of Gag293-specific memory CD4+ T cells is unique to HIV Controllers.

2011 - The shc family protein adaptor, Rai, negatively regulates T cell antigen receptor signaling by inhibiting ZAP-70 recruitment and activation [Articolo su rivista]
Ferro, Micol; Savino, Maria Teresa; Ortensi, Barbara; Finetti, Francesca; Genovese, Luca; Masi, Giulia; Ulivieri, Cristina; Benati, Daniela; Pelicci, Giuliana; Baldari, Cosima T.
abstract

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. © 2011 Ferro et al.

2010 - Opposite effects of simvastatin on the bactericidal and inflammatory response of macrophages to opsonized S. aureus [Articolo su rivista]
Benati, Daniela; Ferro, Micol; Savino, Maria Teresa; Ulivieri, Cristina; Schiavo, Ebe; Nuccitelli, Annalisa; Pasini, Franco Laghi; Baldari, Cosima T.
abstract

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 FcγR 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 FcγR 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. © Society for Leukocyte Biology.

2010 - The strepsipteran endoparasite Xenos vesparum alters the immunocompetence of its host, the paper wasp Polistes dominulus [Articolo su rivista]
Manfredini, Fabio; Benati, Daniela; Beani, Laura
abstract

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. © 2009 Elsevier Ltd. All rights reserved.

2008 - SRC family kinases as potential therapeutic targets for malignancies and immunological disorders [Articolo su rivista]
Benati, Daniela; Baldari, Cosima T
abstract

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.

2008 - Simvastatin impairs humoral and cell-mediated immunity in mice by inhibiting lymphocyte homing, T-cell activation and antigen cross-presentation [Articolo su rivista]
Ulivieri, Cristina; Fanigliulo, Daniela; Benati, Daniela; Pasini, Franco Laghi; Baldari, Cosima T.
abstract

Statins block the activity of HMG-CoA reductase, which catalyses the production of mevalonate, an intermediate in cholesterol biosynthesis, which is also a precursor of isoprenoids. In addition to lowering circulating cholesterol, these drugs display anti-inflammatory and immunomodulatory activities in vitro; however, their effects on the development of adaptive immune responses in vivo, as well as the underlying mechanisms, are as yet largely unknown. Here we investigated the outcome of simvastatin treatment on a number of processes, which together orchestrate adaptive immunity to specific antigen. Simvastatin treatment resulted in a marked reduction of T and B cells in spleen, lymph nodes and peripheral blood in mice. This effect could be ascribed principally to an impairment of lymphocyte homing to secondary lymphoid organs. In addition, simvastatin was found to strongly inhibit T-cell responses to the MHCI restricted hen ovalbumin peptide antigen SIINFEKL and to impair ovalbumin uptake and cross-presentation by MHCI. Simvastatin also suppressed antibody responses to immunization with ovalbumin and delayed-type hypersensitivity to allergens. These activities could be largely accounted for by the simvastatin-dependent inhibition of HMG-CoA reductase. The data provide novel mechanistic insight into the activities of simvastatin in the highly complex context of the immune response. © 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

2007 - Bcr-Abl stabilizes β-catenin in chronic myeloid leukemia through its tyrosine phosphorylation [Articolo su rivista]
Coluccia, Addolorata Maria Luce; Vacca, Angelo; Dũach, Mireia; Mologni, Luca; Redaelli, Sara; Bustos, Victor H.; Benati, Daniela; Pinna, Lorenzo A.; Gambacorti-Passerini, Carlo
abstract

Self-renewal of Bcr-Abl+ chronic myeloid leukemia (CML) cells is sustained by a nuclear activated serine/threonine-(S/T) unphosphorylated β-catenin. Although β-catenin can be tyrosine (Y)-phosphorylated, the occurrence and biological relevance of this covalent modification in Bcr-Abl-associated leukemogenesis is unknown. Here we show that Bcr-Abl levels control the degree of β-catenin protein stabilization by affecting its Y/S/T-phospho content in CML cells. Bcr-Abl physically interacts with β-catenin, and its oncogenic tyrosine kinase activity is required to phosphorylate β-catenin at Y86 and Y654 residues. This Y-phospho β-catenin binds to the TCF4 transcription factor, thus representing a transcriptionally active pool. Imatinib, a Bcr-Abl antagonist, impairs the β-catenin/TCF-related transcription causing a rapid cytosolic retention of Y-unphosphorylated β-catenin, which presents an increased binding affinity for the Axin/GSK3β complex. Although Bcr-Abl does not affect GSK3β autophosphorylation, it prevents, through its effect on β-catenin Y phosphorylation, Axin/GSK3β binding to β-catenin and its subsequent S/T phosphorylation. Silencing of β-catenin by small interfering RNA inhibited proliferation and clonogenicity of Bcr-Abl+ CML cells, in synergism with Imatinib. These findings indicate the Bcr-Abl triggered Y phosphorylation of β-catenin as a new mechanism responsible for its protein stabilization and nuclear signalling activation in CML.

2006 - Erratum: Simvastatin inhibits the MHC class II pathway of antigen presentation by impairing Ras superfamily GTPases (European Journal of Immunology (2006) vol. 36 (11) 10.1002/eji.200636567)) [Nota a Sentenza]
Ghittoni, R.; Napolitani, G.; Benati, D.; Ulivieri, C.; Patrussi, L.; Pasini, F. L.; Lanzavecchia, A.; Baldari, C. T.
abstract

2006 - SKI-606 decreases growth and motility of colorectal cancer cells by preventing pp60(c-Src)-dependent tyrosine phosphorylation of β-catenin and its nuclear signaling [Articolo su rivista]
Coluccia, Addolorata Maria Luce; Benati, Daniela; Dekhil, Hafedh; De Filippo, Annamaria; Lan, Cathy; Gambacorti-Passerini, Carlo
abstract

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 β-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 β-catenin tyrosine phosphorylation in colorectal cancer. Src-dependent activation of β-catenin was prevented by SKI-606, a novel Src family kinase inhibitor, which also abrogated β-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 β-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 μmol/L) and clonogenic potential of colorectal cancer cells. Protein levels of β-catenin remained substantially unchanged by SKI-606, which promoted instead a cytosolic/membranous retention of β-catenin as judged by immunoblotting analysis of cytosolic/nuclear extracts and cell immunofluorescence staining. The SKI-606-mediated relocalization of β-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 β-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 β-catenin by inhibiting its pp60(c-Src)-dependent tyrosine phosphorylation; this could constitute a new therapeutic target in colorectal cancer. ©2006 American Association for Cancer Research.

2006 - Simvastatin inhibits the MHC class II pathway of antigen presentation by impairing Ras superfamily GTPases [Articolo su rivista]
Ghittoni, Raffaela; Napolitani, Giorgio; Benati, Daniela; Uliveri, Cristina; Patrussi, Laura; Laghi Pasini, Franco; Lanzavecchia, Antonio; Baldari, Cosima T.
abstract

Statins are widely used hypocholesterolemic drugs that inhibit 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase, a rate-limiting enzyme of the mevalonate pathway whose biosynthetic endproduct is cholesterol. As a result of this activity, statins may perturb the composition of cell membranes, resulting in lipid raft disruption. Furthermore, by inhibiting protein prenylation, a process also dependent on mevalonate, statins block membrane targeting and activity of small GTPases. Antigen uptake, processing and presentation involve the interplay of Rab and Rho family GTPases. Furthermore, lipid rafts have been implicated both in antigen internalization by the BCR and in MHC class II clustering at the immunological synapse. Here we have addressed the effects of simvastatin on antigen processing and presentation by human B cells and dendritic cells. The results show that simvastatin potently suppresses tetanus toxoid processing and presentation to CD4+ T cells by HLA-DR by inhibiting protein antigen uptake through both receptor-mediated endocytosis and macropinocytosis. This effect can be largely accounted for by defective prenylation of Rho and Rab GTPases in the absence of any measurable perturbation of lipid rafts. In addition, simvastatin was found to preferentially affect the invariant chain-dependent MHC class II pathway, thereby identifying this route of antigen processing and presentation as a selective target of statins. © 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.