Human Immune Gene Expression Profile in activated and expanded NK cells, TIGITKO NK cells, GD2.CAR NK cells and TIGITKOGD2.CAR NK cells
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PROVIDER: PRJNA1216524 | ENA |
REPOSITORIES: ENA
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Project description:The purpose of this experiment to examine how the gene expression profile and differential gene expression in NK cells, TIGITKO NK cells, TIGITKOGD2.CAR NK cells or GD2.CAR NK cells changes after 72 hours of neuroblastoma tumor exposure.
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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.
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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.
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Project description:Diffuse midline glioma (DMG) is a lethal pediatric brain cancer with limited treatment options. We developed a human brainstem organoid model to study H3.3K27M-driven DMG, enabling formation of patient-like tumors with molecular heterogeneity. To characterize this model, we performed a time-course transcriptomic analysis of healthy organoid development. We then introduced a DMG-inducing plasmid cocktail via electroporation and profiled the resulting tumors using single-cell RNA sequencing. Tumors were treated with GD2 CAR T cells, which were subsequently analyzed by single-cell RNA sequencing to capture treatment responses. Finally, we assessed the role of brain-resident myeloid cells by performing sort-seq on PMP-DMGO cocultures with or without CAR T cell treatment. This platform supports extended in vitro experimentation and reveals immune dynamics relevant for therapy development.
2025-08-28 | E-MTAB-15147 | biostudies-arrayexpress
Project description:Purpose: Next-generation sequencing (NGS) has revolutionized the systems-based analysis of cellular pathways. The goals of this study are to compare NGS-derived transcriptome profiling (RNA-seq) between two generations of CAR-T cells aimed at integrating their fitness to overcome exhaustion and stress in tumor microenvironments Methods: mRNA profiles of CAR-T cells co-cultured with target cells for 48 hours were generated by deep sequencing. Human CD45 enrichment was performed to decrease tumor contamination. Results: Gene Set Enrichment Analysis (GSEA) of the RNA expression profile of T cells obtained 48 hours post co-culture with target cells demonstrated the enrichment of genes with memory T cell phenotype in B7H3 CAR-T compared to GD2-B7H3 T cells. However, B7H3 CAR-T cells were enriched in genes associated with glycolysis and apoptosis pathways suggesting a committed terminal fate for these T cells. There was significantly greater expression of genes attributed to exhaustion in B7H3 CAR-T cells than GD2-B7H3 T cells. In particular, B7H3 CAR-T cells showed higher expression of inhibitory receptors LAG3, HAVCR2 (TIM3), and BTLA genes, along with exhaustion-related transcription factor genes TBX21 (T-bet), PRDM1 (Blimp-1), and IKZF2 (Helios). B7H3 CAR-T cells also expressed genes that encode transcription factors reported to be associated with activated and memory T cells, such as KLF6, JUN, and JUNB. Conclusions: Our study represents the first analysis of transcriptome comparing SynNotch gated GD2-B7H3 CAR-T cells versus the conventional B7H3 CAR-T cells. GD2-B7H3 T cells exhibited superior resistance to exhaustion and greater metabolic fitness in comparison to conventional CAR-T cells. Metabolic preference (glycolytic versus oxidative) has an enormous impact on T cell fate. After eradicating NBL cells, GD2-B7H3 T cells had an oxygen consumption rate similar to UT cells, suggesting that the gated T cells can revert to their naïve metabolic state. Improved metabolic plasticity and reprogramming in favor of oxidative rather than glycolytic phosphorylation supports the hypothesis that the gated CAR-T cells likely have intact expansion potential, similar to unmanipulated naïve T cells. We observed significant enrichment of glycolytic genes in conventional B7H3 CAR-T cells compared to GD2-B7H3 T cells, supporting our metabolic studies. Our data suggest that SynNotch gated CAR-T cells have a more favorable oxidative metabolic profile than 4-1BB CAR-T cells.
2020-11-30 | GSE161942 | GEO
Project description:The purpose of the experiment was to compare the transcriptional profile of T-cells expressing a GD2-CAR construct or a GD2-CAR.C7R construct (where the GD2-CAR and the C7R construct are co-expressed), after the T-cells had been exposed to two serial tumor cell co-cultures. C7R is an engineered IL-7 receptor that has been rendered constitutively active. The sample replicates were generated using peripheral blood mononuclear cells (PBMCs) obtained from healthy human donors.
2017-10-05 | GSE102567 | GEO
Project description:Purpose: To compare cell states between CD19-28z and GD2-28z human CAR T cells on day 10 of cell culture. Methods: Human T cells were activated and lentivirally transduced with CD19-28z or GD2-28z CAR constructs and maintained in culture for 10 days, and then delivered to the Stanford Functional Genomics Facility for 3' single-cell RNA-sequencing on the 10X Genomics platform. Results: Comparison of transcription factor profiles by single cell RNA-seq analysis of CD8+ T cells expressing CD19-28z vs. GD2-28z CAR confirmed that the bZIP family members JUN, JUNB, JUND, and ATF4 were among the most differentially expressed and broadly connected in exhausted GD2-28z CAR T cells. Conclusions: This study provides insights into cell states that could explain the underlying differences between highly functional CD19-28z CAR T cells and exhaustion-prone GD2-28z CAR T cells on day 10 in culture.
2019-10-01 | GSE136874 | GEO
Project description:We generate macrophages from pluripotent stem cells (PSCs) with GD2 CARs integrated into AAVS1 locus. To produce CAR macrophages (CAR-M) we established a serum- and feeder-free differentiation protocol which generates macrophages through arterialized hemogenic endothelium.
2022-12-31 | GSE201174 | GEO
Project description:Diffuse intrinsic pontine glioma (DIPG) and other H3K27M-mutated diffuse midline gliomas (DMGs) are universally lethal central nervous system (CNS) tumors that occur most commonly in children and young adults1. Average life expectancy is ten months from diagnosis, and 5-year survival is less than 1%2. Palliative radiotherapy is the only established treatment3, and neither cytotoxic nor targeted pharmacological approaches have demonstrated anti-tumor responses or improved prognosis to date3,4. We previously discovered that the disialoganglioside GD2 is highly and uniformly expressed on H3K27M+ DMG cells and demonstrated that intravenously (IV) administered GD2.4-1BB.z chimeric antigen receptor (CAR) T-cells eradicated established DMGs in patient-derived orthotopic murine models5, thereby providing the rationale for a first-in-human/first-in-child Phase 1 clinical trial (NCT04196413). Because CAR T-cell-induced inflammation and edema of the brainstem can result in obstructive hydrocephalus, increased intracranial pressure, and dangerous tissue shifts, a number of neurocritical care precautions were incorporated in the clinical trial design and management plan. Here we present the clinical experience from the first four patients with H3K27M+ DMG treated with GD2-CAR T-cells at dose level 1 (DL1; 1e6 GD2-CAR T-cells/kg administered IV). Patients who exhibited clinical benefit were eligible for subsequent administrations of GD2-CAR T-cells. Given preclinical evidence for increased CAR T-cell potency6, and the potential for diminished immunogenicity with locoregional administration, second doses were administered intracerebroventricularly (ICV) through an Ommaya catheter to three patients. As predicted from preclinical models, toxicity was largely related to the neuroanatomical location of the tumors and was reversible with intensive supportive care. Although GD2 is expressed at low levels in normal neural tissue, no evidence of on-target, off-tumor toxicity was observed. Three of four patients exhibited clinical and radiographic improvement, underscoring the promise of this approach for H3K27M+ DMG therapy. Correlative studies of serum and CSF revealed marked proinflammatory cytokine production following GD2 CAR T cell administration and single cell transcriptomic analysis of 65,598 single cells from CAR T cell products and patient CSF has begun to reveal differences that correlate with the heterogeneity between subjects and routes of administration.
2022-02-07 | GSE186802 | GEO
Project description:Cord blood (CB)-derived chimeric antigen receptor (CAR)-natural killer (NK) cells targeting CD19 has been shown to be effective against B cell malignancies. While human CD56+ NK cells can be expanded in vitro, it is also known that NK cells can be differentiated from hematopoietic progenitor cells. It is still unclear whether CAR-NK cells are originated from mature NK cells or NK progenitor cells in CB. Here we found that CAR-NK cells are predominantly derived from the CD56- NK progenitor cells. We first found that substantial numbers of CD19 CAR-NK cells were produced from the CD56- CB mononuclear cells after in vitro culture for two weeks. Single cell RNAseq analysis of CD56-CD3-CD14-CD19- CB mononuclear cells revealed that those cells were subdivided into three subpopulations based on the expression of CD34 and HLA-DR. NK cells were predominantly produced from the CD34-HLA-DR- cells. In addition, in the CD34-HLA-DR- cells, only CD7+ cells could differentiate into NK cells. These results indicate that CD56-CD7+CD34- HLA-DR-lineage marker (Lin)- cells are the major origin of human CB-derived CAR-NK cells, indicating that we need to develop methods to enhance the quality and quantity of NK cells produced from these NK cell progenitor cells.
2024-12-18 | GSE253575 | GEO