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:we generated DPHL v2 from 1608 DDA-MS data acquired using Orbitrap mass spectrometers. The data included 586 DDA-MS newly acquired from 17 tissue types, while 1022 files were derived from DPHL v1. DPHL v2 thus comprises data from 24 sample types, including several cancer types . We generated four variants of DPHL v2 to include semi-tryptic peptides and protein isoforms.
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:Broadly neutralizing antibodies (bnAbs) to the HIV envelope (Env) V2-apex region are important leads for HIV vaccine design. Most V2-apex bnAbs engage Env with an uncommonly long heavy chain complementarity-determining region 3 (HCDR3), suggesting that rarity of bnAb precursors poses a challenge for vaccine priming. We created precursor sequence definitions for V2-apex HCDR3-dependent bnAbs and searched for related precursors in human antibody heavy chain ultradeep sequencing data from 14 HIV-unexposed donors. We found potential precursors in a majority of donors for only two long-HCDR3 V2-apex bnAbs, PCT64 and PG9, identifying these bnAbs as priority vaccine targets. We then engineered ApexGT Env trimers that bind inferred germlines for PCT64 and PG9 and have higher affinities for bnAbs; determined cryo-EM structures of ApexGT trimers bound to inferred germline and bnAb forms of PCT64 and PG9; and developed an mRNA-encoded cell-surface trimer for our lead ApexGT candidate. The methods and immunogens developed here have promise to assist the development of an HIV vaccine.
Project description:Deciphering cis-regulatory logic underlying cell type identity is a fundamental question in biology. Single-cell chromatin accessibility (scATAC-seq) data has enabled training of sequence-to-function deep learning models allowing decoding of enhancer logic and design of synthetic enhancers. Training such models requires large amounts of high-quality training data across species, organs, development, aging, and disease. To facilitate the cost-effective generation of large scATAC-seq atlases for model training, we developed a new version of the open-source microfluidic system HyDrop with increased sensitivity and scale: HyDrop v2. We generated HyDrop-v2 atlases for the mouse cortex and Drosophila embryo development and compared them to atlases generated on commercial platforms. HyDrop-v2 data integrates seamlessly with commercially available chromatin accessibility methods (10x Genomics). Differentially accessible regions and motif enrichment across cell types are equivalent between HyDrop-v2 and 10x atlases. Sequence-to-function models trained on either atlas are comparable as well in terms of enhancer predictions, sequence explainability, and transcription factor footprinting. By offering accessible data generation, enhancer models trained on HyDrop-v2 and mixed atlases can contribute to unraveling cell-type specific regulatory elements in health and disease.
Project description:To define the genome-wide activity of Cas9-sgRNA directed at the gamma-globin promoter genes, we performed CHANGE-seq and identified 678 off-target sites from plerixafor-mobilized normal donors of African American ancestry (pre-GMP engineering runs, n = 3). Most of these off-targets are in intergenic and intronic regions of the human genome, as expected based on their relative genomic proportions. We selected the 277 CHANGE-seq candidate sites reproducibly identified in 2 or more biological donors and in silico predicted sites for multiplexed targeted amplicon sequencing (rhAmp-seq, IDT).
Project description:This study investigates the dynamic alterations in high vaginal fluid (HVF) proteome and its correlation with physiological changes during progression of term pregnancy. The HVF samples were collected at three time points as defined as V1 (6-12 weeks), V2 (18-20 weeks) and V3 (26-28 weeks) and SWATH-MS strategy were applied to profile changes in protein expression at early and middle stage of pregnancy. Using in-house generated HVF-specific protein library, 61 proteins (>1.5 fold at V2/V1 or V3/V1, q-value <0.05) changed as a function of gestational age. The stage-specific expression pattern of these proteins was mainly associated with the biology of cervical remolding, fetal development and microbial defense.