Evaluation of TSTEM-like CAR-T cells
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE226037 | GEO | 2023/02/25
REPOSITORIES: GEO
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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:<p>The adoptive transfer of autologous T cells genetically modified to express a CD19-specific, 4-1BB/CD3zeta-signaling chimeric antigen receptor (CAR; CTL019) has shown remarkable activity in patients with B acute lymphoblastic leukemia. Similar therapy can induce long-term remissions for relapsed/refractory chronic lymphocytic leukemia (CLL) patients, but in only a small subset of subjects. The determinants of response and resistance to CTL019 therapy of CLL are not fully understood. We employed next generation sequencing of RNA (RNA-seq) to identify predictive indicators of response to CTL019 treatment. We performed RNA-seq on leukapheresis and manufactured infusion product T cells from patients with heavily pre-treated and high-risk disease. To characterize potency, we also performed RNA-seq on the cellular infusion product after CAR-specific stimulation. Our findings indicate that durable remission in CLL is associated with gene expression signatures of early memory T cell differentiation (e.g., STAT3), while T cells from poorly- or non-responding patients exhibited elevated expression of key regulators of late memory as well as effector T cell differentiation, apoptosis, aerobic glycolysis, hypoxia and exhaustion. These gene expression signatures, along with additional immunological biomarkers, may be used to identify which patients are most likely to respond to cellular therapies and suggest manufacturing modifications that might potentiate the generation of maximally efficacious infusion products.</p>
| phs001707 | dbGaP
Project description:Chimeric antigen receptor (CAR) T cells are known to be potent in recognizing and eliminating tumor cells. They induce marked responses in patients with refractory leukemia and lymphoma, but exhaustion is an inevitable obstacle important barrier for to persistant curative effect. CD19-targeted CAR incorporating the 4-1BB costimulation domain showed a lower tendency toward exhaustion during ex vivo expansion, and downstream differentiation was still observed with a prolonged culture time. We explored the transcriptional changes during the culture process using 4-1BB CD19-targeted CAR-T cells . In contrast to T cells (cultured 5 days in vitro) and short-term cultured (5 days) CAR-T cells, the pathway for regulation of the cytosolic calcium ion concentration was significantly upregulated in the long-term cultured (11 days) group.
2022-04-05 | GSE178570 | GEO
Project description:CAR T cells mediate antitumor effects in a small subset of cancer patients, but dysfunction due to T cell exhaustion is an important barrier to progress. To investigate the biology of exhaustion in human T cells expressing CAR receptors, we used a model system employing a tonically signaling CAR, which induces hallmarks of exhaustion described in other settings. Exhaustion was associated with a profound defect in IL-2 production alongside increased chromatin accessibility of AP-1 transcription factor motifs, and overexpression of numerous bZIP and IRF transcription factors that have been implicated in inhibitory activity. Here we demonstrate that engineering CAR T cells to overexpress c-Jun, a canonical AP-1 factor, enhanced expansion potential, increased functional capacity, diminished terminal differentiation and improved antitumor potency in numerous in vivo tumor models. We conclude that a functional deficiency in c-Jun mediates dysfunction in exhausted human T cells and that engineering CAR T cells to overexpress c-Jun renders them exhaustion-resistant, thereby addressing a major barrier to progress for this emerging class of therapeutics.
2019-10-01 | GSE136891 | GEO
Project description:CAR T cells mediate antitumor effects in a small subset of cancer patients, but dysfunction due to T cell exhaustion is an important barrier to progress. To investigate the biology of exhaustion in human T cells expressing CAR receptors, we used a model system employing a tonically signaling CAR, which induces hallmarks of exhaustion described in other settings. Exhaustion was associated with a profound defect in IL-2 production alongside increased chromatin accessibility of AP-1 transcription factor motifs, and overexpression of numerous bZIP and IRF transcription factors that have been implicated in inhibitory activity. Here we demonstrate that engineering CAR T cells to overexpress c-Jun, a canonical AP-1 factor, enhanced expansion potential, increased functional capacity, diminished terminal differentiation and improved antitumor potency in numerous in vivo tumor models. We conclude that a functional deficiency in c-Jun mediates dysfunction in exhausted human T cells and that engineering CAR T cells to overexpress c-Jun renders them exhaustion-resistant, thereby addressing a major barrier to progress for this emerging class of therapeutics.
2019-10-01 | GSE136853 | GEO
Project description:CAR T cells mediate antitumor effects in a small subset of cancer patients, but dysfunction due to T cell exhaustion is an important barrier to progress. To investigate the biology of exhaustion in human T cells expressing CAR receptors, we used a model system employing a tonically signaling CAR, which induces hallmarks of exhaustion described in other settings. Exhaustion was associated with a profound defect in IL-2 production alongside increased chromatin accessibility of AP-1 transcription factor motifs, and overexpression of numerous bZIP and IRF transcription factors that have been implicated in inhibitory activity. Here we demonstrate that engineering CAR T cells to overexpress c-Jun, a canonical AP-1 factor, enhanced expansion potential, increased functional capacity, diminished terminal differentiation and improved antitumor potency in numerous in vivo tumor models. We conclude that a functional deficiency in c-Jun mediates dysfunction in exhausted human T cells and that engineering CAR T cells to overexpress c-Jun renders them exhaustion-resistant, thereby addressing a major barrier to progress for this emerging class of therapeutics.
2019-10-01 | GSE136796 | GEO
Project description:Whether or not human Tregs are susceptible to exhaustion, and if so, if this would limit their therapeutic effect, was unknown. We studied how a tonic-signaling CAR, which induce Tconv exhaustion, affect human Tregs. We thus sorted naïve Tregs and naïve conventional CD4s and transduced them with a non-TS CAR (CD19) or a TS-CAR (HA).
2023-03-15 | GSE209835 | GEO
Project description:Purpose: Compared the differences in the transcriptome of T cells versus EpCAM CAR-T cells and EpCAM CAR-T cells versus rapamycin-pretreated EpCAM CAR-T cells.Methods: 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 T cells and EpCAM CAR-T cells，and between EpCAM CAR-T cells and rapamycin-pretreated EpCAM CAR-T cells. Results: RNA-seq showed that CAR-T cells significantly upregulated the expression of exhaustion markers compared to T cells. Gene Set Enrichment Analysis (GSEA) analysis showed that CAR-T cells cells were enriched for signatures of apoptosis. Compared with T cells, Kyoto Encyclopedia of Genes and Genomessignaling(KEGG) pathway analysis showed that the PI3K/AKT/mTOR signaling pathway was significantly enriched. Likewise, GSEA showed that the mTOR signaling pathway was significantly upregulated in CAR-T cells. Next, we analyzed the RNA-seq data between CAR-T cells and rapamycin-pretreated CAR-T cells. RNA-seq showed that rapamycin-treated CAR T down-regulated the expression of exhaustion markers compared with untreated CAR-T cells. GSEA analysis showed that the apoptosis-related gene set was enriched in untreated CAR-T cells.Conclusions:In vitro culture promotes the terminal differentiation of CAR-T cells.CAR-T cells present a highly activated mTOR phenotype. Rapamycin pretreatment prevents CAR-T cells terminal differentiation.
2021-12-05 | GSE162796 | GEO
Project description:Zebley et al. show that CD8+ CD19-CAR T cells undergo genome-wide DNA methylation changes during an anti-tumor response in patients with B-cell acute lymphoblastic leukemia (ALL). Post-infusion CAR T cell differentiation involves acquisition of DNA methylation programs associated with effector function, repression of memory potential, and transition toward exhaustion.
2021-11-08 | GSE188325 | GEO
Project description:Dysfunction in T cells limits the efficacy of cancer immunotherapy. We profiled the epigenome, transcriptome, and enhancer connectome of exhaustion-prone GD2-targeting HA-28z chimeric antigen receptor (CAR) T cells and control CD19-targeting CAR T cells, which present less exhaustion-inducing tonic signaling, at multiple points during their ex vivo expansion. We found widespread, dynamic changes in chromatin accessibility and 3D chromosome conformation preceding changes in gene expression, notably at loci proximal to exhaustion-associated genes such as PDCD1, CTLA4, and HAVCR2, and increased DNA motif access for AP-1 family transcription factors, which are known to promote exhaustion. Although T cell exhaustion has been studied in detail in mouse, we find that the regulatory networks of T cell exhaustion differ between the species and involve distinct loci of accessible chromatin and cis-regulated target genes in human CAR T cell exhaustion. Deletion of exhaustion-specific candidate enhancers of PDCD1 suppress the expression of PD-1 in an in vitro model of T cell dysfunction and in HA-28z CAR T cells, suggesting enhancer editing as a path forward in improving cancer immunotherapy.
2021-05-01 | GSE168881 | GEO