Project description:Murine chimeric antigen receptor transduced T cells (CAR-T cells) deficient in perforin recapitulate hemophagocytic lymphohistiocytosis (HLH)-like toxicities occuring in human CAR-T recipients. We used microarray to describe gene expression profiles of wild-type and perforin-deficient CAR-T cells.
Project description:Gene expression analysis of whole heart samples obtained from CAR wild type and knockout mouse E10.5 embryos. We used the microarray to detect any gene expression changes in the E10.5 embronic heart due to the global deletion of CAR.
Project description:The constitutive androstane receptor (CAR, NR1I3) modulates the transcription of numerous genes involving drug metabolism, energy homeostasis, and cell proliferation. Most functions of CAR however were defined from animal studies. Given the known species difference of CAR and the significant cross-talk between CAR and the pregnane X receptor (PXR), it is extremely difficult to decipher the exact role of human CAR (hCAR) in gene regulation, relying predominantly on pharmacological manipulations. Here, utilizing a newly generated hCAR-knockout (KO) HepaRG cell line, we carried out RNA-seq analysis of the global transcriptomes in wild-type (WT) and hCAR-KO HepaRG cells treated with CITCO, a selective hCAR agonist, phenobarbital (PB), a dual activator of hCAR and hPXR, or vehicle control. Real-time PCR assays in separate experiments were used to validate RNA-seq findings. Our results indicate that genes encoding drug-metabolizing enzymes are among the main clusters altered by both CITCO and PB. Specifically, CITCO significantly changed the expression of 135 genes in an hCAR-dependent manner, while PB altered the expression of 227 genes in WT cells of which 94 were simultaneously modulated in both cell lines reflecting the dual effects of PB on hCAR/PXR. Notably, we found that many genes promoting cell proliferation and tumorigenesis were up-regulated in hCAR-KO cells, suggesting that hCAR may play an important role in cell growth that differs from mouse CAR. Together, our results reveal both novel and known targets of hCAR and support the role of hCAR in maintaining the homeostasis of metabolism and cell proliferation in the liver.
Project description:Steroids enriched within the lung tumor microenvironment suppress the cytotoxic activity of tumor-infiltrating NK cells and intensify hypoxic stress. To model these conditions in vitro, CEACAM5-specific CAR-NK cells and NR3C1-knockout CAR-NK cells were exposed to hydrocortisone (1 µM), hypoxia-mimicking CoCl₂ (100 µM), or their combination. NK cells were treated for 24 hours, followed by a 6-hour co-culture with A549 lung cancer cells to activate NK-cell effector programs. After co-culture, NK cells were isolated to high purity and subjected to bulk RNA sequencing to characterize transcriptional changes driven by steroid signalling, hypoxia, and glucocorticoid receptor deficiency. This dataset includes multiple treatment groups: untreated CAR-NK cells, hypoxia-treated CAR-NK cells, combined hypoxia + hydrocortisone–treated CAR-NK cells, and corresponding NR3C1-knockout CAR-NK cell groups under identical conditions.
Project description:In this study, we delved into deciphering the glycosylation patterns and precise localization of glycosylation sites within CAR-T proteins. Specifically, we focused on samples derived from human T cells that underwent gene knockout of AAVS1 and SPPL3, two critical genes that could potentially influence CAR-T function. To ensure accuracy and precision, we employed optimal tribrid orbitrap mass spectrometry settings for glycosylation landscapes of the CAR-T proteins. By analyzing these data, we aimed to gain a deeper understanding of how AAVS1 and SPPL3 knockout impacted the glycosylation profiles and ultimately the function and efficacy of CAR-T cells in immunotherapy applications.
Project description:CEACAM5-specific CAR NK-92 cells were engineered to target CEACAM5-expressing lung tumor cells. To study their transcriptional response during tumor engagement, parental NK-92 cells, CEACAM5-CAR NK-92 cells, hydrocortisone-treated CAR NK-92 cells, NR3C1-knockout (cortisol-resistant) CAR NK-92 cells, and hydrocortisone-treated NR3C1-knockout CAR NK-92 cells were co-cultured with CEACAM5⁺ A549 lung cancer cells for 16 hours. Following co-culture, NK-92–derived effector cells were isolated by flow cytometry and processed for bulk RNA sequencing. This dataset captures transcriptional programs associated with CAR activation, hydrocortisone exposure, and glucocorticoid receptor deficiency in NK-92–based effector cells responding to CEACAM5⁺ tumor targets.
Project description:This study searched for cell-type specific effectors of endocytic escape in dendritic cells and identified a pore-forming protein, perforin-2 (encoded by Mpeg1), as a dedicated effector exclusive to cross-presenting cells. Whole cell proteomics identified differences in the abundance of tryptic and semi-tryptic (cleaved) perforin-2 peptides between control MutuDCs and cells treated with BafA, CpG or with knockout of asparagine endopeptidase (AEP). This revealed how perforin-2 undergoes proteolytic cleavage releasing its pore forming domain into the organellar lumen.