Ontology highlight
ABSTRACT:
PROVIDER: PRJNA1102443 | ENA |
REPOSITORIES: ENA
Similar Datasets
Project description:Library-based single-cell analysis of CAR signaling reveals drivers of in vivo persistence
| PRJNA1103794 | ENA
Project description:In vivo persistence of chimeric antigen receptor (CAR) T cells correlates with therapeutic efficacy, yet CAR-specific factors that support persistence are not well resolved. Using a CAR containing a single chain variable fragment (scFv) specific for CD33 linked to a 4-1BB and CD3 zeta signaling domain that is currently in advanced clinical trials, we show that CAR-expression, in a ligand-independent manner, alters T cell differentiation during ex vivo expansion. CAR-transduced T cells displayed decreased naïve and stem memory populations and increased effector subsets relative to vector-transduced control cells, and this was associated with reduced in vivo persistence. Altered persistence was not due to antigen specificity or tumor presence, but was linked to tonic signaling through the CAR, most notably CD3 zeta ITAMs, prior to transfer. We identified the PI3K/AKT pathway in CD33 CAR T cells as responsible. Treatment with a PI3K inhibitor modulated the differentiation program of CAR T cells, preserved a less differentiated state without affecting T cell expansion, and improved in vivo persistence relative to control cells. These results help resolve mechanisms by which tonic signaling modulates CAR T cell fate, and identifies a novel pharmacologic approach to enhance the durability of CAR T cells for cell-based immunotherapy.
2018-05-17 | GSE93386 | GEO
Project description:Chimeric antigen receptor–T (CAR-T) cell therapies can eliminate relapsed and refractory tumors, but the durability of antitumor activity requires in vivo persistence. Differential signaling through the CAR costimulatory domain can alter the T cell metabolism, memory differentiation, and influence long-term persistence. CAR-T cells costimulated with 4-1BB or ICOS persist in xenograft models but those constructed with CD28 exhibit rapid clearance. Here, we show that a single amino acid residue in CD28 drove T cell exhaustion and hindered the persistence of CD28-based CAR-T cells and changing this asparagine to phenylalanine (CD28-YMFM) promoted durable antitumor control. In addition, CD28-YMFM CAR-T cells exhibited reduced T cell differentiation and exhaustion as well as increased skewing toward Th17 cells. Reciprocal modification of ICOS-containing CAR-T cells abolished in vivo persistence and antitumor activity. This finding suggests modifications to the costimulatory domains of CAR-T cells can enable longer persistence and thereby improve antitumor response.
2020-02-11 | GSE145007 | GEO
Project description:Due to the low-throughput process of systematically designing and functionally testing chimeric antigen receptors (CARs), only a small set of immune signaling domains have been thoroughly explored, despite their major role in T cell activation, effector function and persistence. SpeedingCARs presents an integrated method for engineering CAR T cells by signaling domain shuffling and functional screening by single-cell sequencing. Leveraging the inherent modularity of natural signaling domains, we generated a diverse library of 180 unique CAR variants, which were genomically integrated into primary human T cells by CRISPR-Cas9. Functional and pooled screening of the CAR T cell library was performed by co-culture with tumor cells, followed by single-cell RNA sequencing (scRNA-seq) and single-cell CAR sequencing (scCAR-seq), thus enabling high-throughput profiling of multi-dimensional cellular responses.
2022-10-03 | GSE214231 | GEO
Project description:Despite the revolutionary achievements of chimeric antigen receptor (CAR) T cell therapy in treating cancers, especially leukemia, several significant challenges still limit its therapeutic efficacy. Of particular relevance is the relapse of cancer in large part, as a result of exhaustion and short persistence of CAR-T cells in vivo. IL-2-inducible T cell kinase (ITK) is a critical modulator of the strength of TCR signaling, while its role in CAR signaling is unknown. By electroporation of Cas9 ribonucleoprotein (RNP) complex into CAR-T cells, we successfully deleted ITK in CD19-CAR-T cells with high efficiency. Bulk and single-cell RNA sequencing (scRNA-seq) analyses revealed down-regulation of exhaustion and up-regulation of memory gene signatures in ITK-deficient CD19-CAR-T cells. Our results further demonstrated a significant reduction of T cell exhaustion and enhancement of T cell memory, with significant improvement of CAR-T cell expansion and persistence both in vitro and in vivo. Moreover, ITK-deficient CD19-CAR-T cells showed better control of tumor relapse. Our work provides a promising strategy of targeting ITK to develop sustainable CAR-T products for clinical use.
2024-11-20 | GSE278601 | GEO
Project description:Despite the revolutionary achievements of chimeric antigen receptor (CAR) T cell therapy in treating cancers, especially leukemia, several significant challenges still limit its therapeutic efficacy. Of particular relevance is the relapse of cancer in large part, as a result of exhaustion and short persistence of CAR-T cells in vivo. IL-2-inducible T cell kinase (ITK) is a critical modulator of the strength of TCR signaling, while its role in CAR signaling is unknown. By electroporation of Cas9 ribonucleoprotein (RNP) complex into CAR-T cells, we successfully deleted ITK in CD19-CAR-T cells with high efficiency. Bulk and single-cell RNA sequencing (scRNA-seq) analyses revealed down-regulation of exhaustion and up-regulation of memory gene signatures in ITK-deficient CD19-CAR-T cells. Our results further demonstrated a significant reduction of T cell exhaustion and enhancement of T cell memory, with significant improvement of CAR-T cell expansion and persistence both in vitro and in vivo. Moreover, ITK-deficient CD19-CAR-T cells showed better control of tumor relapse. Our work provides a promising strategy of targeting ITK to develop sustainable CAR-T products for clinical use.
2024-11-20 | GSE278612 | GEO
Project description:CD19-targeted CAR therapies have successfully treated B cell leukemias, but many responders later relapse or experience toxicities. CAR ICDs are key to converting antigen recognition into anti-tumor effector functions. Despite the many possible immune signaling domain combinations that could be included in CARs, almost all CARs currently rely upon CD3, CD28, and/or 4-1BB signaling. To explore the signaling potential of CAR ICDs, we generated a library of 700,000 CD19 CAR molecules with diverse signaling domains and developed a high throughput screening platform to enable optimization of CAR signaling for anti-tumor functions. Our strategy identifies CARs with novel signaling domains that elicit distinct T cell behaviors from a clinically available CAR, including enhanced proliferation and persistence, lower exhaustion, potent cytotoxicity in an in vitro tumor rechallenge condition, and comparable tumor control in vivo. This approach is readily adaptable to numerous disease models, cell types, and selection conditions, making it a promising tool for rapidly improving adoptive cell therapies and expanding their utility to new disease indications.
2021-07-13 | GSE179767 | GEO
Project description:The anti-tumor function of engineered T cells expressing chimeric antigen receptors (CARs) is dependent on signals transduced through intracellular signaling domains (ICDs). Different ICDs are known to drive distinct phenotypes, but systematic investigations into how ICD architectures direct T cell function—particularly at the molecular level—are lacking. Here, we use single-cell sequencing to map diverse signaling inputs to transcriptional outputs, focusing on a defined library of clinically relevant ICD architectures. Informed by these observations, we functionally characterize transcriptionally distinct ICD variants across various contexts to build comprehensive maps from ICD composition to phenotypic output. We identify a unique tonic signaling signature associated with a subset of ICD architectures that drives durable in vivo persistence and efficacy in liquid, but not solid, tumors. Our findings work toward decoding CAR signaling design principles, with implications for the rational design of next-generation ICD architectures optimized for in vivo function.
2024-07-13 | GSE264681 | GEO
Project description:Adoptive T cell therapy with gene-modified T cells expressing chimeric antigen receptor (CAR) is a rapidly growing field of translational medicine and recently has shown dramatic therapeutic success in the treatment of B-cell malignancies. However, challenges to achieve similar response in patients harboring solid tumour are still considerable. To achieve anti-tumor efficacy, these cells must survive, expand and persist after infusion into patients. An important lesson has been derived from clinical trials using CAR technologies: the relevance of the CAR design to enhance signaling and sustain T cell proliferation and survival. Here, we prove that third generation CARs specific for GD2 antigen incorporating CD28.4-1BB costimulatory domains improves T cell immunotherapy in a neuroblastoma (NB) pre-clinical model, as compared to a CAR with the same specificity but including CD28.OX40 costimulation. Indeed, we test the anti-tumor activity of polyclonal T cells genetically modified with third generation CAR.GD2 incorporating either CD28.4-1BB or CD28.OX40 in frame with the safety switch inducible Caspase 9 (iC9). We prove a significant in vitro and in vivo amelioration of the approach by the presence of 4.1BB signaling in terms of: 1) less T cell exhaustion, 2) lower basal T cell activation, 3) higher in vivo tumor control and 4) T cell persistence. In addition, the fine tuning of T cell culture conditions with the use of IL7 and IL15 show to be synergic with the third generation CAR.GD2 design to optimize CAR T immunotherapy for NB. Our results provide a proof-of-concept for the need to optimize not only CAR construct design but also T cell culture conditions in order to boost T cell activity in vivo.
2018-06-27 | PXD005426 | Pride
Project description:Immunotherapy using CD19-directed chimeric antigen receptor (CAR)-T cells has shown excellent results for treatment of B-cell leukaemia and lymphoma. To produce CAR-T cells, the patient’s own T cells are isolated from the blood and modified in a laboratory with a genetic vector to express a tumor antigen-directed CAR on its surface. The CAR-T cells are then expanded in numbers and given back to the patient with the aim to eradicate the tumors. However, some patients display primary resistance to CAR-T treatment while others relapse quickly after CAR-T treatment. In this experiment, we seek to understand whether the quality of the individual CAR-T cell product the patients were given can predict outcome to the therapy. We investigate the transcriptional profile of the individual CAR-T infusion products using single-cell RNA sequencing. In this dataset, we identified a T cell subset correlating with response that could be used as an indicator for clinical outcome. Targeted RNA and protein single-cell libraries were obtained using the BD Rhapsody platform (BD Biosciences). In total four separate targeted libraries were produced with 6 patients per library. Sequencing was performed on NovaSeq 6000 S1 sequencer at the SNP&SEQ Technology Platform (Uppsala, Sweden). The raw scRNA-seq data was pre-processed by BD Biosciences using the Rhapsody Analysis pipeline to convert the raw reads into Unique Molecular Identifier (UMI) counts. UMIs are further adjusted within Rhapsody by applying BD’s Recursive Substitution Error Correction (RSEC) and Distribution-Based Error Correction (DBEC) in order to remove false UMIs caused by sequencing or library preparation errors. Pooled samples were deconvoluted using Sample-tag reads. The scRNA-seq and AbSeq counts were loaded, processed and used for clustering and differential gene expression with Seurat v. 4.0.0.
2023-07-24 | E-MTAB-12407 | biostudies-arrayexpress