Project description:The role of T cell immunity in protection against COVID-19 in immunocompromised patients (ICp) who failed to mount serological responses remains ill-defined. Intradermal skin test (IDT) with mRNA vaccines may represent a simple, reliable and affordable tool to measure T cell response in seronegative patients.We compared anti-SARS-CoV-2 antibodies and cellular responses in vaccinated ICp (n=58), healthy seronegative naive controls (NC, n=8), and healthy seropositive vaccinated controls (VC, n=32) by Luminex, spike-induced IFN-γ ELIPSOT and an IDT 3 to 6 months after vaccination. ICp regrouped 18 transplant recipients, 33 individuals with autoimmune diseases, and eight patients with primary immunodeficiencies. In three vaccinated volunteers, we performed a skin biopsy 24h after IDT and single-cell RNAseq of the skin-infiltrating CD45+ cells. Twenty-five percent of seronegative NC had a positive ELIPSOT (2/8) and IDT (1/4), compared to 95% (20/21) and 93% (28/30) in seropositive VC, respectively. Single-cell RNA seq data of positive IDT consistently showed a mixed population of helper and cytotoxic T cells composed of memory T cells in 87%. The TCR repertoire of infiltrating skin lymphocytes revealed 18/1218 clonotypes with known specificities against SARS-CoV-2, among which six were spike-specific. Seronegative ICp with positive Elispot and IDT were in the majority treated with B cell-depleting reagents only, while those with negative IDT were all transplant recipients. Our results indicate that local reaction to IDR is mainly composed of memory T cells and includes SARS-CoV-2-specific T cells, opening the perspective to use IDT as a correlate of protection in immunosuppressed patients.
Project description:Pediatric patients affected by B-cell precursor ALL and enrolled in the AIEOP-BFM treatment protocol in Italian centers were analysed at diagnosis for mutations in cohesin genes. The experiment was performed designing a custom panel of 40 genes involved in leukaemia (IDT probe).
Project description:By CHANGE-seq-BE, we identified a total of 81 potential off-targets and confirmed 95.4% frequencies of on-target editing with no evident off-targets above the detection threshold compared to controls (rhAmp-seq, IDT).
Project description:Studies of AML patient samples have shown that specific combinations of AML disease alleles confer an adverse outcome, however, in vivo models do not exist for the majority of common, poor-prognosis genotypes. Here we show that TET2/FLT3 mutations can cooperate to induce AML in vivo using a genetically engineered mouse model, and that this model has a defined stem-cell population with a characteristic transcriptional and epigenetic profile. TET2 and FLT3 mutations cooperate to induce site-specific changes in DNA methylation and gene expression, including at loci that regulate hematopoietic differentiation. We demonstrate that re-expression of genes that are silenced in TET2/FLT3-mutant AML restores normal differentiation, demonstrating that the epigenetic program of TET2/FLT3-mutant AML cells can be reversed in vitro and in vivo. Using ERRBS, we profiled genome-wide DNA methylation patterns of the hematopoietic stem cells (LSK) population in Wide-type, Flt3-IDT, Tet2-/-, and Tet2-/-Flt3-IDT mice, each in triplicates
Project description:Pediatric patients with Down Syndrome, affected by B-cell precursor ALL and enrolled in the AIEOP-BFM treatment protocol in Italian centers were analysed at diagnosis and at remission for mutations in JAK2 kinase and in RAS pathway. The experiment was performed designing a custom panel of 40 genes involved in leukaemia (IDT probe), by the Illumina Nextera Flex for Enrichment protocol on NexSeq550 (2x150).
Project description:Organelles like lysosomes and synaptic vesicles are acidified by V-ATPases, which consist of a cytosolically-oriented V1 complex that hydrolyzes ATP and a membrane-embedded VO complex that pumps protons. In yeast, V1-VO association is facilitated by the RAVE complex, but how higher eukaryotes assemble V-ATPases remains unclear. Here, we identify a metazoan RAVE complex (mRAVE) whose structure and composition are notably divergent from the ancestral counterpart. mRAVE consists of DMXL1 or DMXL2, WDR7, and the central linker ROGDI. DMXL1/2 interacts with subunits A and D of the inactive, isolated V1. Upon dissipation of proton gradients, mRAVE binds to V1 and VO, forming a supercomplex on the membrane. mRAVE then catalyzes V1-VO assembly, enabling lysosomal acidification, neurotransmitter loading into vesicles, and ATG16L1 recruitment for LC3/ATG8 conjugation onto single-membranes (CASM). Our findings provide a molecular basis for neurological disorders caused by mRAVE mutations.