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Transfer of antigen target into CAR T cells via trogocytosis

ABSTRACT: To characterize transfer of molecules from target cells into CAR T cells via trogocytosis we cultured NALM-6 leukemia cell line expressing a CD19-mCherry fusion protein with CAR T cells. NALM6-CD19-mCherry were loaded with heavy amino acid and cocultured with CAR T cells for 1 hour. CAR T cells were next sorted into two fractions, mCherry-positive (TrogPos), and -negative (TrogNeg). Proteomics analysis revealed the presence of targeted antigen (CD19) in the TrogPos only.

OTHER RELATED OMICS DATASETS IN: GSE126753

INSTRUMENT(S): Q Exactive

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): T Cell

DISEASE(S): Acute Leukemia

SUBMITTER: Zhuoning Li

LAB HEAD: Michel Sadelain

PROVIDER: PXD012833 | Pride | 2019-03-29

REPOSITORIES: Pride

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			Action	DRS
	D1_NALM6_Fr1.raw	Raw
	D1_NALM6_Fr2.raw	Raw
	D1_NALM6_Fr3.raw	Raw
	D1_NALM6_Fr4.raw	Raw
	D1_NALM6_Fr5.raw	Raw

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Publications

CAR T cell trogocytosis and cooperative killing regulate tumour antigen escape.

Hamieh Mohamad M Dobrin Anton A Cabriolu Annalisa A van der Stegen Sjoukje J C SJC Giavridis Theodoros T Mansilla-Soto Jorge J Eyquem Justin J Zhao Zeguo Z Whitlock Benjamin M BM Miele Matthew M MM Li Zhuoning Z Cunanan Kristen M KM Huse Morgan M Hendrickson Ronald C RC Wang Xiuyan X Rivière Isabelle I Sadelain Michel M

Nature 20190327 7750

Chimeric antigen receptors (CARs) are synthetic antigen receptors that reprogram T cell specificity, function and persistence1. Patient-derived CAR T cells have demonstrated remarkable efficacy against a range of B-cell malignancies1-3, and the results of early clinical trials suggest activity in multiple myeloma4. Despite high complete response rates, relapses occur in a large fraction of patients; some of these are antigen-negative and others are antigen-low<su ...[more]

PMID: 30918399

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CAR T cells inhibit B cell activating phosphorylation in their targets

Project description:Adoptive cell therapy, a subset of cancer immunotherapy, is collection of therapeutic approaches which aim to redirect the immune system by reprogramming patient T-cells to target antigenic molecules differentially and specifically expressed in certain cancers. One promising immunotherapy technique is CAR T-cell therapy, where cancer cells are targeted through the expression a chimeric antigen receptor (CAR), a synthetic trans- membrane receptor that functionally compensates for the T-cell receptor (TCR) but targets a tumor associated antigen on the cancer cell surface. While CAR T-cell therapy is promising with two clinically approved second-generation CARs (Kymriah and Yescarta), few studies have investigated the mechanism of signal propagation in T-cells and no studies have investigated the potential signaling response in the target cells. To gain further insight to CAR-based signaling, we stimulated third generation CD19 CAR-expressing Jurkat T-cells by co-culture with SILAC labeled CD19HI Raji B-cells and used two phosphoenrichment strategies coupled with liquid chromatography-tandem mass spec- trometry (LC-MS/MS) to detect and analyze global phosphorylation changes in both cell populations. Analysis of the phosphopeptides originating from the CD19-CAR T cells revealed an increase in many phosphorylation events necessary for canonical TCR signaling. We also observed for the first time a significant decrease in B-cell receptor- related phosphopeptide abundance in CD19HI Raji B-cells after co-culture with CD19-targetted CAR T-cells.

2022-01-28 | PXD028109 | Pride

Generation of clinical grade CD19-specific CAR-modified CD8+ memory stem cells for the treatment of B-cell malignancies

Project description:Long-lived, self-renewing, multipotent T memory stem cells (TSCM) can trigger profound and sustained tumor regression but their rareness poses a major hurdle to their clinical application. Presently, clinically compliant procedures to generate relevant numbers of this T cell population are undefined. Here, we provide a strategy for deriving large numbers of clinical grade tumor-redirected TSCM cells starting from naÃ¯ve precursors. CD8+CD62L+CD45RA+ naÃ¯ve T cells enriched by streptamer-based serial positive selection were activated by CD3/CD28 engagement in the presence of IL-7, IL-21 and the glycogen synthase-3Î² inhibitor TWS119, and genetically engineered to express a CD19-specific chimeric antigen receptor (CD19-CAR). These conditions allowed for the generation of CD19-CAR modified TSCM cells that were phenotypically, functionally and transcriptomically equivalent to their naturally occurring counterpart. Compared with T cell products currently under clinical investigation, CD19-CAR modified TSCM cells exhibit enhanced metabolic fitness, persistence and anti-tumor activity against systemic acute lymphoblastic leukemia xenografts. Based on these findings, we have initiated a phase 1 clinical study to evaluate the activity of CD19-CAR modified TSCM in patients with B-cell malignancies refractory to prior allogeneic hematopoietic stem cell transplantation. Three healthy human blood donors provided lymphocyte-enriched apheresis blood for this study after informed consent. From all samples, total RNA was isolated using an miRNeasy Mini Kit (Qiagen), processed by Ambionâ??s WT expression kit, fragmented and labeled with a WT Terminal Labeling Kit (Affymetrix), hybridized to WT Human Gene 1.0 ST arrays (Affymetrix) and stained on a Genechip Fluidics Station 450 (Affymetrix), all according to the respective manufacturer's instructions. Samples represent exon-level and gene-level analyses.

2016-06-29 | E-GEOD-68003 | biostudies-arrayexpress

Monocyte-derived IL-1 and IL-6 are differentially required for cytokine release syndrome and neurotoxicity by CAR-T cells.

Project description:Single-cell transcriptome profiling using a 3' droplet-based platform (Chromium,10x Genomics) of human CD45+ leukocytes isolated from leukemic HuSGM3 mice infused with CD19.28z CAR-T cells, two days after cytokine release syndrome (CRS) onset and 5 days later.

2018-05-24 | E-MTAB-6505 | biostudies-arrayexpress

An immunoproteomic approach to characterize the CAR interactome and signalosome

Project description:Adoptive transfer of chimeric antigen receptor (CAR)-T cells is expected to become the first line of treatment for multiple malignancies, following the enormous success of anti-CD19 therapies. However, their mechanism of action is not fully understood, and clear guidelines for the design of safe and efficient receptors are missing. We hereby describe a systematic analysis of the CAR “interactome” in human primary T cells, which allowed us to identify molecular traits that influence CAR-T cell efficacy. Interactome analysis was based on immunoprecipitation of CARs followed by protein identification by mass spectrometry.

2019-11-07 | PXD007085 | Pride

Targeted scRNA-seq and AbSeq of human CAR-T cell infusion product from 28 cancer patients

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

Cas13d experimental viral genome-wide screen identifies regions of RNA vulnerability in SARScov2

Project description:We performed an experimental Cas13d-SARScov2 genome-wide screen to identify gRNAs that would allow Cas13d to degrade the viral RNA. We built mCherry reporter plasmids that express mCherry with a 3kb 3'UTR deriving from the SARScov2 genome. In total we designed 11 reporters covering the entire plus strand of the viral genome and 11 other reporters covering the entire minus strand. Each of the 22 mCherry reporter plasmids carries a U6 expression cassette containing a Cas13d gRNA that targets the 3'UTR of the mCherry reporter. Each reporter is represented by a pool of reporters each containing a different gRNA that targets mCherry 3'UTR for a total average of ~300 gRNA per 3'UTR. The entire pool of 22 reporters, each with a pool of ~300 different gRNAs constitutes a comprehensive set ~6,500 reporters (~ 6,500 different gRNAs) that allowed us to interrogate the entire SARScov2 plus and minus strand viral RNA for regions of vulnerability and targetability. In order to specifically interrogate Cas13d activity an remove the biases that would be introduced in the reporter expression by the presence of a large 3kb 3'UTR we used a case (presence of Cas13d) control (absence of Cas13d) design. Briefly, the ~6,500 reporters were lentiviral transduced in RKO cells, the cells were split in 2 populations, 1 population was transduced with Cas13d and the other serving as control did not. The population expressing Cas13d was FACS sorted in low mCherry (efficient gRNAs) and high mCherry (un-efficient gRNAs) in 2 biological replicates and the genomic DNA of these populations was extracted, gRNAs were PCR amplified and sequenced. For the population that did not express Cas13d, a low mCherry, one high mCherry and unsorted population were sequenced as control libraries.

2023-10-29 | E-MTAB-11111 | biostudies-arrayexpress

Bulk mRNA Seq CAR.CCR7 vs. CAR

Project description:The study aimed to elucidate the transcriptional changes introduced by genetic engineering of CAR T cells to stably express the homeostatic chemokine receptor CCR7. In vitro killing assays hinted to a higher cytolytic capacity that could be tied to an altered cytoskeletal rearrangement. Human CAR and CAR.CCR7 T cells were generated by retroviral transduction and enriched by FACS at the end of the expansion phase (day 10), either without or with a previous over-night co-culture with target Jeko1 tumor cells that express the CAR target antigen CXCR5. Enriched cells were immediately processed for bulk mRNA Seq

2023-06-30 | E-MTAB-12778 | biostudies-arrayexpress

Gene modification by fast-track recombineering for cellular localization and isolation of components of plant protein complexes

Project description:Genes encoding Arabidopsis subunit homologs of TFIIH kinase module were labelled with coding sequences of GFP and mCherry (green and red fluorescent proteins) using fast-track recombineering. To illustrate the applicability of recombineering for accelerating the isolation and identification of plant protein complexes, proteins associated with CDKD;2-GFP, CYCLIN-H-mCherry and DNA replication-dependent HISTONE H3.1-mCherry complexes were purified on GFP-Trap and RFP-TRAP and analysed by LC-MS/MS mass spectrometry. The results confirmed association of know TFIIH subunit homologs with CDKD;2 and CYCLIN H, and identified subunits of CAF1 (CHROMATIN ASSEMBLY FACTOR 1) and ASF1A/B histone chaperon in complex with HISTONE H3.1.

2019-08-01 | PXD013637 | Pride

Global phosphoproteomic analysis of signaling by distinct chimeric antigen receptors reveals kinetic and quantitative differences that influence cell function

Project description:Chimeric antigen receptors (CARs) are synthetic proteins that redirect T cell specificity by linking an extracellular ligand binding domain to intracellular T cell signaling domains. CAR-expressing T (CAR-T) cells have demonstrated significant efficacy for the treatment of refractory B cell malignancies and are being evaluated as immunotherapeutic reagents for many other cancers. CAR designs are based on the fundamental principles of TCR recognition and most CARs employ the T cell-activating CD3z endodomain alongside a costimulatory domain from CD28 or 4-1BB. However, emerging data suggest that CD28/CD3z and 4-1BB/CD3z signaling modules promote divergent metabolic pathways, gene expression programs, and cell fates. To determine how CAR phosphoprotein signaling drives these disparate cell fates, we analyzed CAR ligation-induced signaling networks in primary human T cells using shotgun mass spectrometry. We isolated CD8+CD62L+ T cells from healthy donors and introduced a CD28/CD3z or 4-1BB/CD3z CAR by lentiviral transduction. Transduced T cells were purified by FACS and expanded once in vitro. When the cells returned to a resting state, CD28/CD3z or 4-1BB/CD3z CAR-T cells were stimulated for 10 or 45 minutes with magnetic microbeads coated with a monoclonal antibody specific for a 9 amino acid tag in the CAR extracellular sequence. CAR-T cells were also left unstimulated for 10 or 45 minutes to serve as controls. Altogether, 8 unique conditions were tested in an experiment and three independent experiments were performed.

2018-08-13 | PXD007921 | Pride

Activating transcription factor 6 is necessary and sufficient for alcoholic fatty liver disease in zebrafish

Project description:ATF6 is a key regulator of the unfolded protein response. Through use of zebrafish and cultured cells we demonstrate that ATF6 drives fatty liver disease by interaction with fatty acid synthase (FASN). Total small RNA from livers of 5 dpf larval zebrafish were collected: 2 batches of Tg(fabp10:nls-mCherry) control larvae, 2 batches of ethanol-treated Tg(fabp10:nls-mCherry) larvae, and 1 batch of Tg(fabp10:nAtf6-cherry; cmlc2:GFP). Each batch was purified for preparation of high-throughput sequencing libraries.

2014-05-01 | E-GEOD-56498 | biostudies-arrayexpress

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