Biology subjects

Cavazzana, M.

Publications and source records attributed to Cavazzana, M..

5 recordsLinked to original sources

bioRxiv2025

Tracking HIV-1 DNA fate from Cell Culture to Humanized mice Tissues.

Unambiguous identification of HIV reservoirs is essential for their characterization and for developing curative strategies. A major obstacle to curing HIV is the persistence of silent viral genomes in host cells that evade immune detection and resist therapy. Here, we present a fluorescence microscopy-based method for direct in vivo visualization of full-length double-stranded HIV-1 DNA, an achievement not previously realized. To achieve this, we developed an advanced imaging platform adapted for replication-competent HIV-1. This system leverages a bacterial-derived two-component tagging strategy, where a fluorescent protein (OR-GFP), efficiently expressed in xenografted human immune cells, specifically binds to an engineered ANCH3 tag sequence integrated into the viral genome. The tagged virus infects CD4+ T cells both in vitro and in vivo, producing bright nuclear puncta corresponding to individual viral genomes. Multiple nucleation sites within ANCH3 enable a stable, shortened tag form for persistent and long-term tracking. Transcriptional profiling during acute infection revealed both transcriptionally active and silent genomes in spleen, lymph nodes, and bone marrow, with silent forms enriched in lymph nodes and marrow, supporting early reservoir establishment. This live-cell imaging strategy enables high-specificity detection of latent HIV, offering a transformative tool for studying reservoir dynamics and guiding future cure strategies.

microbiology↗

bioRxiv2022

Novel lentiviral vectors for gene therapy of sickle cell disease combining gene addition and gene silencing strategies

Sickle cell disease (SCD) is due to a mutation in the {beta}-globin (HBB) gene causing the production of the toxic sickle hemoglobin (HbS, a2{beta}S2). Transplantation of autologous hematopoietic stem/progenitor cells (HSPCs) transduced with lentiviral vectors (LVs) expressing an anti-sickling {beta}-globin ({beta}AS) is a promising treatment; however, it is only partially effective and patients still present elevated HbS levels. Here, we developed a bifunctional LV expressing {beta}AS3-globin and an artificial microRNA (amiR) specifically downregulating {beta}S-globin expression with the aim of reducing HbS levels and favoring {beta}AS3 incorporation into Hb tetramers. Efficient transduction of SCD HSPC by the bifunctional LV led to a substantial decrease of {beta}S-globin transcripts in HSPC-derived erythroid cells, a significant reduction of HbS+ red cells and effective correction of the sickling phenotype, outperforming {beta}AS gene addition and BCL11A gene silencing strategies. The bifunctional LV showed a standard integration profile and neither the HSPC viability, engraftment and multi-lineage differentiation nor the erythroid transcriptome and miRNAome were affected by the treatment, confirming the safety of this therapeutic strategy. In conclusion, the combination of gene addition and gene silencing strategies can improve the efficacy of current LV-based therapeutic approaches without increasing the mutagenic vector load, thus representing a novel treatment for SCD.

genetics↗

bioRxiv2020

CD28 costimulatory domain protects against tonic signaling-induced functional impairment in CAR-Tregs

The use of chimeric antigen receptor (CAR)-engineered regulatory T cells (Tregs) has emerged as a promising strategy to promote immune tolerance in transplantation. However, in conventional T cells (Tconvs), CAR expression is often associated with tonic signaling resulting from ligand-independent baseline activation. Tonic signaling may cause CAR-T cell dysfunction, especially when the CAR structure incorporates the CD28 costimulatory domain (CSD) rather than the 4-1BB CSD. Here, we explored the impact of tonic signaling on human CAR-Tregs according to the type of CSD. Compared to CD28-CAR-Tregs, 4-1BB-CAR-Tregs showed enhanced proliferation and greater activation of MAP kinase and mTOR pathways but exhibited decreased lineage stability and reduced abilities to produce IL-10 and be restimulated through the CAR. Although both CAR-Treg populations were suppressive in vivo, cell tracking with bioluminescence imaging found longer persistence for CD28-CAR-Tregs than for 4-1BB-CAR-Tregs. This study demonstrates that CD28-CAR best preserves Treg function and survival in the context of tonic signaling, in contrast with previous findings for Tconvs. SUMMARYLamarthee et al investigated the impact of chimeric antigen receptor (CAR) tonic signaling on CAR-engineered Tregs according to the incorporated costimulatory domain (either CD28 or 4-1BB). CD28 ameliorated Treg stability, long-term survival and function compared to 4-1BB.

immunology↗

bioRxiv2020

Single cell analysis of FOXP3 deficiencies in humans and mice unmasks intrinsic and extrinsic CD4+ T cell perturbations

ABSTRACTFOXP3 deficiency in humans with IPEX syndrome and mice results in fatal systemic autoimmunity by altering regulatory T cell (Treg) physiology, but actual cellular and molecular mechanisms of disease are unclear, part because Treg surface markers may be unreliable in disease states. We used deep profiling by flow cytometry, population and single-cell RNAseq to analyze Tregs and conventional (Tconv) CD4+ T lymphocytes in cohorts of IPEX patients with a range of genetic lesions, and in Foxp3-deficient mice. In all patients and mice, heterogeneous Treg-like cells with an active FOXP3 locus were observed, some differing very little from normal Tregs, others more distant. Tconv showed no widespread activation or Th bias. The dominant mark was a monomorphic signature equally affecting all CD4+ T cells, unexpectedly dampening tumor-Treg and cytokine-signaling modules. In mixed bone marrow chimeras, WT Tregs exerted dominant suppression, normalizing the states of mutant Treg and Tconv, extinguishing the disease signature, and revealing a small gene cluster truly regulated, cell-intrinsically, by FOXP3. These results suggest a two-step pathogenesis model, with therapeutic implications: limited downregulation of a few core Treg genes de-represses a systemic mediator(s), which imprints the disease signature on all T cells, and further dampens Treg function.Competing Interest StatementThe authors have declared no competing interest.View Full Text

immunology↗

bioRxiv2020

Heterozygous RELA mutations cause early-onset systemic lupus erythematosus by hijacking the NF-κB pathway towards transcriptional activation of type-I Interferon genes

Systemic Lupus Erythematosus (SLE) is an autoimmune and inflammatory disease characterized by uncontrolled production of autoantibodies and inflammatory cytokines such as the type-I interferons. Due to the lack of precise pathophysiological mechanisms, treatments are based on broad unspecific immunossupression. To identify genetic factors associated with SLE we performed whole exome sequencing and identified two RELA heterozygous activating mutations in 3 early-onset and familial SLE cases. The corresponding RELA/p65 mutant were abundant in the nucleus but poorly activate transcription of genes controlled by NF-{kappa}B consensus sequences. The co-expression of the mutant and wild-type RELA/p65 strongly activated the expression of genes controlled by the IFN-consensus sequences. These molecular mechanisms lead to the overproduction of type-I IFN in the patients cells. Our findings highlight a novel mechanism of autoimmunity where these new RELA mutants are transactivating the type-I IFN genes and are thus promoting type-I interferon production and early-onset SLE, thereby paving the way to the identification of new and specific therapeutic targets. SummaryHeterozygous RELA mutations are associated with Systemic Lupus Erythematosus, with increased expression of genes controlled by the IFN-consensus sequences.

immunology↗