Gene expression profile of CAR T cells compared to control ΔCAR T cells
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE113387 | GEO | 2018/04/20
REPOSITORIES: GEO
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Project description:The objective of this experiment was to compare the transcriptional profile of T cells modified to express CAR, TBBR and 4/7 ICR (i.e. SmarT-cells) against control 1G CAR T cells expressing CD3z endodomain. RNA samples were isolated from trangenic T cells generated from three independent donors.
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Project description:The objective of this experiment was to compare the transcriptional profile of 4/7 ICR (IL4R ectodomain fused to IL7R endodomain) vs control ΔIL4R (truncated version that lacks an endodomain) modified T cells. RNA samples were isolated from trangenic T cells generated from three independent donors.
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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
Project description:The objective of this experiment was to compare the transcriptional profile of T cells modified to express CAR, TBBR and 4/7 ICR (i.e. SmarT-cells) against control T cells expressing the delta constructs (i.e. ΔCAR, ΔTGFbRII and ΔIL4R). RNA samples were isolated from trangenic T cells generated from three independent donors.
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Project description:An obstacle with continued clinical development of CAR T cells is the limited understanding of CAR T cell biology and its mechanisms of anti-tumor immunity. We and others have shown that CARs with a CD28 co-stimulatory domain drive high levels of T cell activation that also lead to exhaustion and shortened persistence. This led us to hypothesize that by incorporating null mutations of CD28 subdomains (YMNM, PRRP, or PYAP) we could optimize CAR T cell signaling and reduce exhaustion. In vivo we found that mice given CAR T cells with a PYAP CD28 endodomain had a significant survival advantage. We also observed that mutant CAR T cells had significantly less expression of PD1 and were increased in the bone marrow and spleen. In addition, these mutant CAR T cells had optimized signaling resulting in a reduction of exhaustion related transcription factors and genes. Our results demonstrate that CAR T cells with a mutant CD28 endodomain have better survival and decreased exhaustion and is the result of decreased CAR dependent NFAT/NR4A1 transcription factors. This work allows for development of enhanced CAR T cell therapies by optimizing CAR T cell signaling.
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Project description:The adoptive transfer of chimeric antigen receptor- (CAR) modified T cells is revolutionizing the treatment of B cell malignancies and has the potential to be applied to other diseases. CARs redirect T cell specificity by linking an antigen recognition domain to T cell signaling modules comprised of CD3z to provide signal 1, and CD28 or 4-1BB to provide costimulation. CD28/CD3z and 4-1BB/CD3z CARs confer differences in effector function and cell fate that affect clinical efficacy and toxicity. These differences may result from activation of divergent transcriptional programs. To gain this insight, we analyzed changes in gene expression in stimulated and resting CD28/CD3z or 4-1BB/CD3z CAR T cells. CD28/CD3z CAR stimulation initiated more marked early transcriptional changes with greater fold increases in the expression of effector molecules including GZMB, IFNG, IL2, TNF, and IL6. Direct comparison of CD28/CD3z and 4-1BB/CD3z samples stimulated for 6 hours identified 1,673 differentially expressed genes. Of these, the memory T cell-associated genes KLF2, IL7R, and FAM65B were expressed at lower levels in CD28/CD3z CAR T cells. KLF2 and IL7R are FOXO transcription factor family targets and we found that FOXO4 expression was similarly reduced in CD28/CD3z CAR T cells. CD28/CD3z CAR stimulation induces an effector T cell-like transcriptional profile that may underlie the decreased persistence and increased risks of toxicities observed with CD28/CD3z CAR T cells in early clinical trials.
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Project description:CAR-T cell therapy against MM currently shows promising results, but usually with serious toxicities. CAR-NK cells may exert less toxicity when redirected against resistant myeloma cells. CARs can be designed through the use of receptors, such as NKG2D, which recognizes a wide range of ligands to provide broad target specificity. Here, we test this approach by analyzing the anti-tumor activity of activated and expanded NK cells (NKAE) and CD45RA- T cells from MM patients that were engineered to express an NKG2D-based CAR. NKAE cells were cultured with irradiated Clone9.mbIL21 cells. Then, cells were transduced with an NKG2D-4-1BB-CD3z-CAR. CAR-NKAE cells exhibited no evidence of genetic abnormalities. Although memory T cells were more stably transduced, CAR-NKAE cells exhibited greater in vitro cytotoxicity against MM cells, while showing minimal activity against healthy cells. In vivo, CAR-NKAE cells mediated highly efficient abrogation of MM growth, and two of the treated mice remained disease-free. Overall, these results demonstrate that it is feasible to modify autologous NKAE cells from MM patients to safely express a NKG2D-CAR. Additionally, autologous CAR-NKAE cells display enhanced anti-myeloma activity demonstrating that they could be an effective strategy against MM supporting the development of NKG2D-CAR NK cell therapy for MM.
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Project description:To identify the CAR-, PXR- and PPARα-specific genome-wide expression changes, hepatocyte cultures from six individual donors were treated with the prototypical ligands for CAR (CITCO), PXR (rifampicin) and PPARα (WY14,643) as well as DMSO (vehicle control). Afterwards, the mRNA expression in these samples was determined utilizing Affymetrix® microarrays. Primary human hepatocytes (PHH) from 6 donors were treated for 24 h with 10µM of rifampicin, 50µM of WY14,643, 1µM of CITCO and 0.1% of DMSO (vehicle).
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Project description:CD19-specific CARs that comprise CD28 and CD3z signaling domains program highly performing effector functions that mediate potent tumor elimination, but they impart a relatively limited T cell lifespan. Increasing functional T cell persistence without reducing effector potency is therefore likely to further enhance the therapeutic success of 1928z CAR T cells. We demonstrate that the number and position of ITAMs in 1928z CAR T cells influence functional, phenotypic and transcriptional programs, resulting in profound effects on antitumor efficacy. Improved therapeutic potency of CAR T cells can thus be achieved by calibrating activation strength, thereby retaining memory functions and preventing exhaustion, without compromising effector functions. Our transcriptional analysis underscores the potential of ITAM dosage and position to direct different T cell fates. We were able to identify a novel CAR design, termed 1XX, which programs a favorable balance of effector and memory signatures, inducing increased persistence of highly functional CARs with the replicative capacity of long-lived memory cells and potent effector functions.
2018-12-17 | GSE121226 | GEO