CDK6 antibody-captured chimeric band in Jurkat cell line
Ontology highlight
ABSTRACT:
INSTRUMENT(S): LTQ Orbitrap XL
ORGANISM(S): Homo Sapiens (ncbitaxon:9606)
SUBMITTER: 
Young-Ki Paik
PROVIDER: MSV000083822 | MassIVE | Sun May 19 22:56:00 BST 2019
REPOSITORIES: MassIVE
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Project description:SLAMF6 is a homotypic receptor of the Ig-superfamily associated with progenitor exhausted T cells. In humans, SLAMF6 has three splice isoforms involving its V-domain.We KO isoform 1 of SLAMF6 in a Jurkat cell line and harvest this cell line RNA using Bulk-RNA-Seq after activation in 5 time points. Comparing the results to WT Jurkat cell line, we analyzed the transcriptional landscape and showed that after activation SLAMF6 isoform 3 is the prominent expressed isoform, and that the KO Jurkat cells have a stronger cytotoxic phenotype.
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Project description:By performing 10x 3’ scRNA-seq on Jurkat cells post TCR acivation in CRISPR/Cas9 screening, we want to investigate the compatibility of the A/G mixed capture sequence on multiple single cell RNA-seq platforms. By mimicking the adenylated endogenous mRNA, gRNA transcripts could be directly captured by poly(dT) primer during the reverse transcription, and serve as perturbation index in high identification rate.
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Project description:Extracellular vesicles (EVs) mediate cell-cell communication including the intercellular transfer of miRNAs, which alter gene expression in target cells. Previously, we have shown that CD47 is also present on EVs and alters their effects on target cells, suggesting that specific surface markers define functionally distinct EVs. This hypothesis will be addressed by comparing total cellular versus EVs miRNA contents between Jurkat and JinB8 (CD47 deficient) T cells. The Jurkat T cell line (E6.1) was purchased from ATCC. The cells were maintained using RPMI 1640 containing 10% FBS, glutamine, penicillin, and streptomycin (Gibco). The Jurkat and JinB8 T cells used for experiments were maintained less than 6 weeks in culture. The cultured Jurkat were washed with IXPBs followed by a second wash with HITES medium (DMEM/F12 supplemented with, bovine serum albumin, hydrocortisone, insulin, transferrin, and trace elements). EVs were isolated from Jurkat T cells were cultured overnight using HITES medium. The conditioned media from Jurkat T cells were collected, and cell debris were removed using centrifugation at 300 g for 5 minutes and 2500 g for 10 minutes respectively. Centrifuged media were concentrated using Ultra-4 centrifugal filters to about 1 ml volume and further centrifuged at 10,000 g for 10 minutes. EVs were isolated using an Exo-Quick kit (SBI). miRNaseazy (Qiagen) kits were used to extract total RNA from Jurkat and JinB8 T cells and their EVs according to the manufacturer's instructions.
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Project description:Background: T cells that are genetically modified with chimeric antigen receptor (CAR) hold promise for immunotherapy of cancer. Currently, there are intense efforts to improve the safety and efficacy of CAR T cell therapies against liquid and solid tumors. Earlier we designed a novel CAR backbone (FiCAR) where the spacer is derived from immunoglobulin (Ig) -like domains of the signal-regulatory protein alpha (SIRPα). However, the analysis of novel CAR using primary T cells is slow and laborious. Methods: To explore the versatility of the CAR backbone, we designed a set of variant FiCARs with different spacer lengths and targeting antigens. To expedite the analysis of the novel CARs, we transduced the FiCAR genes using lentiviruses into Jurkat reporter T cells carrying fluorescent reporter genes. The expression of fluorescent markers in response to FiCAR engagement with targets was analyzed by flow cytometry, and cytotoxicity was evaluated using killing assays. Furthermore, the killing mechanisms that are employed by FiCAR-equipped Jurkat T cells were investigated by flow cytometry, and the intracellular pathways involved in signaling by FiCAR were analyzed by phosphoproteomic analysis using mass spectrometry. Results: Seven different CARs were designed and transduced into Jurkat reporter cells. We show that the SIRPα derived FiCARs can be detected by flow cytometry using the SE12B6A4 antibody recognizing SIRPα. Furthermore, FiCAR engagement leads to robust activation of NFκβ and NFAT signaling, as demonstrated by the expression of the fluorescent reporter genes. Interestingly, the Jurkat reporter system also revealed tonic signaling by a HER-2 targeting FiCAR. FiCAR-equipped Jurkat T cells were cytotoxic in cocultures with target cells and target cell engagement lead to an upregulation of CD107a on the Jurkat reporter T cell surface. Phosphoproteomic analyses confirmed signal transduction via the intracellular CD28/CD3ζ sequences upon the interaction of the FiCAR1 with its antigen. In addition, downstream signaling of CD3ζ/ZAP70- SLP-76-PLCγ, PI3K-AKT-NFκB pathways and activation of NFAT and AP-1 were observed. Conclusion: We conclude that the FiCAR backbone can be shortened and lengthened at will by engineering it with one to three SIRPα derived Ig-like domains, and the FiCARs are functional when equipped with different single chain variable fragment target binding domains. The Jurkat reporter system expedites the analysis of novel CARs as to their expression, signaling function, evaluation of tonic signaling issues and cytotoxic activity.
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