Biology subjects

Yung, E.

Publications and source records attributed to Yung, E..

4 recordsLinked to original sources

bioRxiv2025

Identification of Novel DNA Sequence Motifs that Modulate Transcription in T cells

Considerable progress has been made towards associating transcription factor binding sites (TFBS) with cell-type-specific gene expression, however, the full repertoire of DNA sequence motifs that regulate transcription remains unknown. Improving our understanding of transcriptional regulation is especially important in T cells, given the enormous potential of genetically engineered T cells as an emerging class of therapeutics. Here, we report results from a comprehensive and unbiased survey investigating whether there are novel motifs enriched in regulatory regions of genes with the highest constitutive and selective expression across diverse T- cell subsets. Using computational and experimental methods, we identified 2,036 novel motifs and 629 previously curated TFBS that are enriched, both individually and in specific combinations, in the regulatory regions of genes exhibiting T-cell-specific gene expression. We then used the self-transcribing active regulatory region sequencing (STARR-seq) assay to evaluate all possible three- way combinations of a subset of 18 candidate motifs to test their ability to modulate transcription in immortalized lymphoblastic cell lines of T-cell origin (Jurkat E6) versus myeloid origin (K562). Our results revealed novel motifs that modulate gene transcription in T cells, with some exhibiting stronger regulatory effects than TFBS for TFs with established roles in T cells. The regulatory activity of these novel motifs was influenced by the motifs orientation, position, and copy number. Overall, these results highlight our incomplete understanding of the relationship between sequence composition and T-cell gene regulation and indicate that previously annotated TFBS represent only a subset of motifs capable of modulating gene transcription in T cells.

bioinformatics↗

bioRxiv2023

A Synthetic Cytotoxic T cell Platform for Rapidly Prototyping TCR Function

Current tools for functionally profiling T cell receptors with respect to cytotoxic potency and cross-reactivity are hampered by difficulties in establishing model systems to test these proteins in the contexts of different HLA alleles and against broad arrays of potential antigens. We have implemented and validated a granzyme-activatable sensor of T cell cytotoxicity in a novel universal prototyping platform which enables facile recombinant expression of any combination of TCR-, peptide-, and class I MHC-coding sequences and direct assessment of resultant responses. This system consists of an engineered cell platform based on the immortalized natural killer cell line, YT-Indy, and the MHC-null antigen-presenting cell line, K562. These cells were engineered using contemporary gene-editing techniques to furnish the YT-Indy/K562 pair with appropriate protein domains required for recombinant TCR expression and function in a non-T cell chassis, integrate a fluorescence-based target-centric early detection reporter of cytotoxic function, and deploy a set of protective genetic interventions designed to preserve antigen-presenting cells for subsequent capture and downstream characterization. Our data show successful reconstitution of the surface TCR complex in the YT-Indy cell line at biologically relevant levels. We also demonstrate successful induction and highly sensitive detection of antigen-specific response in multiple distinct model TCRs, with significant responses (p < 0.05 and Cohens d >1.9) in all cases. Additionally, we monitored destruction of targets in co-culture and found that our survival-optimized system allowed for complete preservation after 24-hour exposure to cytotoxic effectors. With this bioplatform, we anticipate investigators will be empowered to rapidly express and characterize T cell receptor responses, generate new knowledge regarding the patterns of T cell receptor recognition, and optimize novel therapeutic T cell receptors for improved cytotoxic potential and reduced cross-reactivity to undesired antigenic targets.

immunology↗

bioRxiv2020

Recombinant T cell receptors specific for HLA-A*02:01-restricted neoepitopes containing KRAS codon 12 hotspot mutations

KRAS codon 12 mutations are among the most common hotspot mutations in human cancer. Using a functional screening platform we set out to identify {beta} T-cell receptors (TCRs) as potential targeting reagents for KRASG12D and/or KRASG12V neoepitopes presented by the prevalent HLA-A*02:01 allele. Here we describe isolation and characterization of three distinct CD8+ T cell clones from a pre-treated 76 year old patient with pancreatic ductal adenocarcinoma (PDAC). One clone was KRASG12V reactive and two clones were KRASG12D reactive. Tetramer staining showed high specificity of each T cell clone for its cognate HLA-A*02:01 restricted KRASG12V or KRASG12D neoepitope (>98% tetramer positive) without appreciable cross-reactivity to wild-type KRAS (<2% tetramer positive). We amplified and sequenced the full-length TCR alpha and beta chains from each of the three T cell clones and determined that these three TCRs comprised distinct combinations of two different TCR alpha chains and two distinct TCR beta chains. We resynthesized these TCR alpha and beta chain nucleotide sequences and reconstituted the original pairs in healthy donor CD8+ T cells by lentiviral transduction, substituting the human {beta} TCR constant gene segments with murine {beta} TCR constant gene segments to prevent mispairing with endogenous TCR subunits. Tetramer analysis and IFN-{gamma} ELISpot analysis confirmed the specificity of each reconstituted TCR for its cognate HLA-A*02:01 restricted KRAS neoepitope. To test cytolytic activity TCR-transduced healthy donor CD8+ T cells were co-cultured with KRASG12V, KRASG12D or KRASwt peptide-pulsed K562-HLA-A*02:01 antigen presenting cells at an effector to target cell ratio of 4:1. Under these conditions we observed neoepitope-specific killing of 16.5% to 19.0% of target cell populations. To assess in vivo activity we developed a KRASG12V/A*02:01 patient-derived xenograft (PDX) mouse model. Over a 56-day period, PDX bearing mice infused with human TCR-transduced T cells had significantly reduced tumor growth and longer survival compared to mice infused with non-transduced control T cells. In conjunction with other therapeutic approaches, immune effector cell therapies expressing these TCRs may improve outcomes for HLA-A*02:01 patients with KRASG12V and/or KRASG12D positive tumors.

immunology↗

bioRxiv2020

Selective B cell depletion upon infusion of replication-incompetent anti-CD19 CAR lentivirus

Anti-CD19 CAR-T therapy for B cell malignancies has shown clinical success, but a major limitation is the logistical complexity and high cost of manufacturing autologous cell products. If engineered for improved safety, direct infusion of viral gene transfer vectors to initiate in vivo CAR-T transduction, expansion and anti-tumor activity could provide an alternative, universal approach. To explore this approach we administered approximately 20 million replication-incompetent VSV-G lentiviral particles carrying an anti-CD19CAR-2A-GFP transgene comprising either an FMC63 (human) or 1D3 (murine) anti-CD19 binding domain, or a GFP-only control transgene, to wild-type C57BL/6 mice by tail vein infusion. The dynamics of immune cell subsets isolated from peripheral blood were monitored at weekly intervals. We saw emergence of a persistent CAR-transduced CD3+ T cell population beginning week 3-4 that reached a maximum of 13.5 +/-0.58% (mean +/-SD) and 7.8 +/-0.76% of the peripheral blood CD3+ T cell population in mice infused with ID3-CAR or FMC63-CAR lentivector, respectively, followed by a rapid decline in each case of the B cell content of peripheral blood. Complete B cell aplasia was apparent by week 5 and was sustained until the end of the protocol (week 8). No significant CAR positive populations were observed within other immune cell subsets or other tissues. These results indicate that direct IV infusion of conventional VSV-G pseudotyped lentiviral particles carrying a CD19 CAR transgene can transduce T cells that then fully ablate endogenous B cells in wild type mice.

immunology↗