Project description:A key mechanism of tumor resistance to immune cells is mediated by expression of peptide-loaded HLA-E in tumor cells, which suppresses natural killer (NK) cell activity via ligation of the NK inhibitory receptor CD94/NKG2A. To bypass HLA-E inhibition, we developed a way to generate highly functional NK cells lacking NKG2A. Constructs containing a single-chain variable fragment derived from an anti-NKG2A antibody were linked to endoplasmic reticulum-retention domains. After retroviral transduction in human peripheral blood NK cells, these NKG2A Protein Expression Blockers (PEBLs) abrogated NKG2A expression. The resulting NKG2Anull NK cells had higher cytotoxicity against HLA-E-expressing tumor cells.
Project description:We engineered a new m6A editing system consisting of GCN4 (SunTag)-dCas13b, anti-GCN4 single chain variable fragment (scFv)-FTO or ALKBH5-GBI and sgRNA to provide an applicable method to remove m6A modification at specific loci.
Project description:Oxidized phospholipids (OxPL) are pro-inflammatory and pro-atherogenic, but their roles in non-alcoholic steatohepatitis (NASH) are unknown. Here, we show that OxPL accumulate in human and murine NASH. Using a transgenic mouse that expresses a functional single chain variable fragment of E06, a natural antibody that neutralizes OxPL, we demonstrate the casual role of OxPL in NASH. Targeting OxPL in hyperlipidemic Ldlr-/- mice decreased multiple aspects of NASH, including steatosis, inflammation, fibrosis, hepatocyte death and progression to hepatocellular carcinoma. Mechanistically, we found that OxPL promote ROS accumulation to induce mitochondrial dysfunction in hepatocytes. Neutralizing OxPL in AMLN diet-fed Ldlr-/- mice reduced oxidative stress, improved hepatic and adipose tissue mitochondrial function and fatty acid oxidation. Since neutralizing OxPL also protects against atherogenesis, targeting OxPL may be an effective therapeutic strategy for both NASH and atherosclerosis.
Project description:We used single cell RNA sequencing (scRNAseq) to compare gene expression in human T cells following stimulation with antibodies directed against a CD3 subunit (CD3ε) versus the TCRβ chain variable (V) domain
Project description:A semi-automated method for enrichment of groups of peptides using individual or multiplexed combinations of single chain variable fragment antibodies was developed and tested in plasma.
Project description:Recombinant proteins containing disulfide bonds, like antibody fragments, are usually produced in the periplasm of E. coli, because in this compartment of the E. coli cell the formation of disulfide bonds is catalyzed. A recombinant protein is targeted to the periplasm with the help of an N-terminally fused signal sequence, which is clipped off from the recombinant protein upon translocation across the cytoplasmic membrane. The single-chain variable antibody fragment BL1 N-terminally fused to the DsbA signal sequence was produced in the E. coli Lemo21(DE3) recombinant protein production strain at conditions non-optimal (0 µM L-rhamnose) and optimal (500 µM L-rhamnose) for the production of the scFv BL1 in the periplasm. Lemo21(DE3) cells not producing a recombinant protein were used as a reference.
Project description:The human immunoglobulin heavy chain (IGH) locus is exceptionally polymorphic with high allelic diversity of variable (V) genes and structural variation affecting large regions of the locus. Thus, our germline IGHV gene and allele content is highly personal, which may influence how we respond to infections and vaccinations. Here, we coupled individualized IGHV genotyping with the isolation of monoclonal antibodies against the SARS-CoV-2 spike, focusing on the IGHV1-69 and IGHV3-30 group of genes, which were over-represented in spike-specific B cells. These genes are characterized by both allelic and copy number variations, making them ideal for expanding our understanding of inter-individual differences in antigen-specific antibody responses. We found that for the IGHV1-69*20-using CAB-I47 antibody, the allele usage was critical as germline reversion to other, highly similar, IGHV1-69 alleles abolished the neutralizing activity. Our results demonstrate that as little as single nucleotide differences between different alleles can greatly influence the biological response.
Project description:Production of an ostrich-derived phage display library to capture the ostrich-derived single-chain variable fragment (scFv) antibodies
Project description:Heterogeneity, shortage of material, and lack of progenitor-specific cell surface markers are major obstacles to elucidating the mechanisms underlying developmental processes. Here we report a proteomic platform that alleviates these difficulties and demonstrate its effectiveness in fractionating heterogeneous cultures of early endoderm derived from human embryonic stem cells. The approach, designated cell-capture antibody array, is based on highly parallel, comparative screening of live cell populations using hundreds of antibodies directed against cell-surface antigens. The results demonstrate the potential of the cell-capture antibody array as a powerful tool for detailed dissection of heterogeneous cellular systems. Genome-wide comparion of mRNA profile in hES-derived CD61+ cells versus CXCR4+ and CXCR4+/CD61- cells using Affymetrix arrays.
Project description:Cryobanking and transplantation of ovarian tissue is a promising approach to restore fertility in cancer patients. However, ischemic stress following avascular ovarian cortex grafting is known to induce stromal tissue fibrosis and alteration in follicular development. The aim of the study is to analyse the impact of freezing-thawing and grafting procedures on gene expression in human ovarian tissue. Frozen-thawed ovarian tissue from 4 patients was xenografted for 7 days in nude mice and one non-grafted fragment was used as control. Immediately after recovery, grafts were processed for RNA extraction and histological analysis. Their expression profile was screened by whole-genome oligonucleotide array (n=4) and validated by reverse-transcriptase polymerase chain analysis (n=10). 84 of the transcripts were significantly altered after 7 days of grafting including matrix metalloproteinase-9 and -14 and angiogenic factors such as Placental growth factor and C-X-C chemokine receptor type 4 (CXCR4). Major biological processes were related to tissue remodelling including secretory processes, cellular adhesion and response to chemical and hormone stimuli. Angiopoietin signaling, interleukin-8 pathway and peroxisome proliferator-activated receptors activation were shown to be differentially regulated. On day 7, overexpression was confirmed by PCR for interleukin-8, transforming growth factor-beta 1, matrix metalloproteinase-14 and CXCR4 compared to the nongrafted control. In conclusion, new as well as known genes involved in tissue restructuration and angiogenesis were identified after human ovarian tissue transplantation. This will facilitate the development of strategies to optimize grafting techniques. In this study, ovarian tissue was obtained from 4 patients. Each biopsy was divided into 2 fragments. One fragment was frozen/thawed and placed in TRIzol reagent (Invitrogen) for RNA extraction. The other fragment was frozen-thawed and grafted in the intraperitoneal cavity of a nude mice. After 7 days graft were recovered and total RNA was extracted.