Project description:Functional discovery of tumor-reactive T cell receptors by massively parallel library synthesis and screening: NKIRTIL063 titration screen
| PRJNA1068301 | ENA
Project description:Functional discovery of tumor-reactive T cell receptors by massively parallel library synthesis and screening: NKIRTIL063 neoantigen screen
| PRJNA1068303 | ENA
Project description:Functional discovery of tumor-reactive T cell receptors by massively parallel library synthesis and screening: validation in CD4 T cells and OVC190 TCR screen
Project description:Purpose: Determine the change in expression of genes in AR deficient CD8 T cells Methods: C57Bl/6J splenocytes were isolated, total CD8 T cells magnetically enriched and electroporated with non-targeting gRNA or AR-specific gRNAs. Cells were stimulated with aCD3/28 for three days before isolating RNA and library prep. Sequencing was performed by the Massively Parallel Sequencing Shared Resource (MPSSR) at OHSU. Results: AR regulated genes in mouse CD8 T cells.
Project description:The screening of a previously reported fluorescein labelled 10,000 member PNA encoded peptide library allowed information on the interaction between the peptide-ligands and the cell surface receptors to be extracted, identified new peptide ligands for cell surface receptors, and gave crucial information about consensus sequences. A novel indirect amplification of the PNA signal by amplification of the PNA-complementary DNA library was developed to screen PNA-encoded peptide library against D54, HEK293T, and HEK293T-CCR6 cells. This work generates a new approach to biological discovery and an expansion of modern microarray techniques. In addition, the microarray approach facilitates screening for differences in surface-receptor ligands and/or receptor expression between various cell types including diseased and normal cells.
Project description:Here we developed a massively parallel in-library ligation methodology to simultaneously perturb four pre-designed targets in CRISPR/Cas9 screening. Thousands of pairs of sequences precisely ligated with their counterparts in library, which enabled simultaneous expression of four gRNAs from each single vector. We demonstrated this novel method with 6,236 4-gene combinations targeting 1,599 immune response related genes, and generated a plasmid library with 1,400x coverage. The library performance was evaluated in a canonical T cell activation experiment, and combinations involved in TCR signaling pathway or TCR complex were successfully identified as positive regulators. Novel combination that is reflecting a potential pathway crosstalk was also verified. This new methodology expands the capacity of the perturbation in CRISPR screening and provided a powerful tool for researches in broad fields to study the combinatorial outcomes from coordinated gene behaviors.
Project description:Acral lentiginous melanoma (ALM) is the most common melanoma subtype in non-Caucasians. Despite advances in cancer immunotherapy, current immune checkpoint inhibitors remain unsatisfactory for ALM. Hence, we conducted comprehensive immune profiling using single-cell phenotyping with reactivity screening of the T cell receptors of tumor-infiltrating T lymphocytes (TILs) in ALM. Compared with cutaneous melanoma, ALM showed a lower frequency of tumor-reactive CD8 clusters and an enrichment of regulatory T cells with direct tumor recognition ability, suggesting a suppressive immune microenvironment in ALM. Tumor-reactive CD8 TILs showed heterogeneous expression of coinhibitory molecules, including KLRC1 (NKG2A), in subpopulations with therapeutic implications. Overall, our study provides a foundation for enhancing the efficacy of immunotherapy in ALM.
Project description:The emergence of antibiotic resistance necessitates the discovery of novel bacterial targets and antimicrobial agents. Here, we present a bacterial target discovery framework that integrates phenotypic screening of cysteine-reactive fragments with competitive activity-based protein profiling to map and functionally characterize the targets of screening hits. Using this approach, we identify FabH -ketoacyl synthase and MiaA tRNA prenyltransferase as primary targets of a hit fragment, 10-F05, that confer bacterial stress resistance and virulence in Shigella flexneri. Mechanistic investigations elucidate that covalent C112 modification in FabH, an enzyme involved in bacterial fatty acid synthesis, results in its inactivation and consequent growth inhibition. We further demonstrate that irreversible C273 modification at the MiaA RNA-protein interaction interface abrogates substrate tRNA binding, attenuating resistance and virulence through decreased translational accuracy. Our findings underscore the efficacy of integrating phenotypic and activity-based profiling of electrophilic fragments to accelerate the identification and pharmacologic validation of new therapeutic targets.