Gene expression in self-activating CAR-T cells with CD28 or 4-1BB costimulatory domain and G4S or Whilow218 linker.
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE276932 | GEO | 2024/09/16
REPOSITORIES: GEO
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Project description:We observed self-activation of SKM-CAR-T cells which target novel mesothelioma antigen. CAR-T with CD28 costimulatory domain showed signs of exhaustion while those with 4-1BB costimulatory domain did not. Changes in the gene expression by substituting CD28 co-stimulatory domain to 4-1BB co-stimulatory domain was investigated.
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Project description:We observed self-activation of SKM-CAR-T cells which target novel mesothelioma antigen. CAR-T with CD28 costimulatory domain showed signs of exhaustion while those with 4-1BB costimulatory domain did not. To elucidate molecules responsible for self-activation-induced exhaustion, we performed RNA-seq analysis of SKM-CAR-T cells with CD28 or 4-1BB and CD19-CAR-T cells with CD28 or 4-1BB.
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2024-05-08 | GSE254546 | GEO
Project description:Chimeric Antigen Receptor T cells express an extracellular domain consisting of a single-chain fragment variable (scFv) targeting a surface tumor-associated antigen (TAA), it is important to select scFv regarding safety profile through evaluation of the efficacy/toxicity balance, especially in cases where the target antigen is also expressed on healthy cells. Here, we developed five CAR T cells targeting CD123 (CD123 CAR-T) by substituting only the scFv, looking for differences in terms of efficacy and on-target/off-tumor effects. Using in vitro models, we found that three of the five CD123 CAR-T were more cytotoxic against leukemic cells, without increasing lysis of monocytes or endothelial cells. Using the Incucyte system, we confirmed the low cytotoxicity of CD123 CAR-T on endothelial cells. Hematotoxicity evaluated by progenitor culture and CD34 cell lysis showed that two of the five CD123 CAR-T were less cytotoxic against HSC, and we confirmed in a humanized mouse model that CD123- cells were not eliminated by the CD123 CAR-T. These two CD123 CAR-T reduced tumor infiltration and increased overall survival of mice in three in vivo models of BPDCN. Moreover, in BPDCN aggressive model, bulk RNA-sequencing analysis showed that these CD123 CAR-T upregulated genes associated with cytotoxicity and activation/exhaustion a few days after the injection. Finally, we were able to produce CD123 CAR-T selected previously from patient T cells. Together, these results emphasize the importance of screening different scFv for the development of CAR-T constructs, to select the one with the optimal risk-benefit ratio for clinical development.
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Project description:mRNA vaccines have demonstrated efficacy against COVID-19. However, concerns regarding waning immunity and breakthrough infections have motivated the development of next-generation vaccines with enhanced efficacy. In this study, we investigated the impact of 4-1BB costimulation on immune responses elicited by mRNA vaccines in mice. We first vaccinated mice with an mRNA vaccine encoding the SARS-CoV-2 spike antigen like the Moderna and Pfizer-BioNTech vaccines, followed by administration of 4-1BB costimulatory antibodies at various times post-vaccination. Administering 4-1BB costimulatory antibodies during the priming phase did not enhance immune responses. However, administering 4-1BB costimulatory antibodies after 96 hours elicited a significant improvement in CD8 T cell responses, leading to enhanced protection against breakthrough infections. A similar improvement in immune responses was observed with multiple mRNA vaccines, including vaccines against common cold coronavirus, human immunodeficiency virus (HIV), and arenavirus. These findings demonstrate a time-dependent effect by 4-1BB costimulation and provide insights for developing improved mRNA vaccines.
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Project description:The activation of TLR-MyD88 (Toll like receptor- Myeloid differentiation factor 88) signaling within T cells functions as a potent costimulatory signal that boosts antitumor and antiviral responses. However, the molecular mechanisms underlying the costimulatory processes are poorly understood. We compared microarray gene analysis data between TLR1-TLR2 stimulated and unstimulated T cell receptor transgenic âpmelâ and MyD88-/-pmel CD8+ T cells and identified changes in the expression levels of several TNF family members. In particular, TLR-stimulation increased 4-1BB levels in pmel but not in MyD88-/-pmel T cells. A link between 4-1BB and TLR1-TLR2 signaling in CD8+ T cells was highlighted by in fact that 4-1BB-/- T cells exhibited suboptimal responses to TLR1-TLR2 agonist, but responded normally to CD28 or OX40 costimulation. Moreover, blocking 4-1BB signaling with antibodies also hindered the costimulatory effects of the TLR1-TLR2 agonist. The elevated levels of 4-1BB transcripts in TLR1-TLR2âstimulated cells were not due to increased mRNA stability nor increased histone activation but instead were associated with increased binding of p65 and c-Jun to two distinct 4-1BB promoter sites. Combining TLR1-TLR2 ligand with an agonistic anti-4-1BB antibody enhanced the antitumor activity in mice with established melanoma tumors. These studies reveal that the costimulatory effects of TLR1-TLR2 signaling in CD8+ T cells are in part mediated by 4-1BB and are important for mounting an effective antitumor immune response. CD8+ T cells from the B6.Cg-Thy1/Cy Tg(TcraTcrb)8Rest/J mice, referred to as âpmelâ T cells or from MyD88 knockout pmel mice (MyD88â/âpmel) were sorted. pmel and MyD88â/âpmel T cells were activated using MyD88â/â CD8 T cell-depleted splenocytes pulsed with 10ng/ml of mgp100. This was with or without 10µg/ml of Pam3CSK4. pmel or MyD88â/âpmel CD8 T cells were enriched and used for the extraction of RNA used for genomic analysis.
2016-06-14 | E-GEOD-79475 | biostudies-arrayexpress
Project description:RNAseq analysis of human CAR-Tregs, bearing CD28 or 4-1BB as a costimulatory domains, initially isolated (before engineering) from peripheral blood of healthy donors
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Project description:CAR T-cell therapy for solid tumors has shown limited efficacy in early phase clinical studies. The majority of CARs encode CD28 and/or 41BB costimulatory endodomains and we explored here if MyD88 and CD40 (MC) costimulatory endodomains in CARs improve their antitumor activity. We generated CD28-, 41BB-, and MC-CAR T-cells and demonstrate that MC-CAR T-cells have greater proliferative capacity and antitumor activity in repeat stimulation assays and in tumor models in vivo. Transcriptomic analysis revealed that MC-CAR T-cells expressed higher levels of MYB and FOXM1, key cell cycle regulators, and are activated at base line. After stimulation, MC-CAR T-cells remain in a less differentiated state than CD28- and 41BB-CAR T-cells as judged by low levels of TBET and BLIMP1 expression, and lower cytolytic activity in comparison to CD28- and 41BB-CAR T-cells. Thus, including MyD88 and CD40 signaling domains in CARs may improve current CAR T-cell therapy approaches for solid tumors.
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Project description:Adoptive T cell therapy with gene-modified T cells expressing chimeric antigen receptor (CAR) is a rapidly growing field of translational medicine and recently has shown dramatic therapeutic success in the treatment of B-cell malignancies. However, challenges to achieve similar response in patients harboring solid tumour are still considerable. To achieve anti-tumor efficacy, these cells must survive, expand and persist after infusion into patients. An important lesson has been derived from clinical trials using CAR technologies: the relevance of the CAR design to enhance signaling and sustain T cell proliferation and survival. Here, we prove that third generation CARs specific for GD2 antigen incorporating CD28.4-1BB costimulatory domains improves T cell immunotherapy in a neuroblastoma (NB) pre-clinical model, as compared to a CAR with the same specificity but including CD28.OX40 costimulation. Indeed, we test the anti-tumor activity of polyclonal T cells genetically modified with third generation CAR.GD2 incorporating either CD28.4-1BB or CD28.OX40 in frame with the safety switch inducible Caspase 9 (iC9). We prove a significant in vitro and in vivo amelioration of the approach by the presence of 4.1BB signaling in terms of: 1) less T cell exhaustion, 2) lower basal T cell activation, 3) higher in vivo tumor control and 4) T cell persistence. In addition, the fine tuning of T cell culture conditions with the use of IL7 and IL15 show to be synergic with the third generation CAR.GD2 design to optimize CAR T immunotherapy for NB. Our results provide a proof-of-concept for the need to optimize not only CAR construct design but also T cell culture conditions in order to boost T cell activity in vivo.
2018-06-27 | PXD005426 | Pride
Project description:The activation of TLR-MyD88 (Toll like receptor- Myeloid differentiation factor 88) signaling within T cells functions as a potent costimulatory signal that boosts antitumor and antiviral responses. However, the molecular mechanisms underlying the costimulatory processes are poorly understood. We compared microarray gene analysis data between TLR1-TLR2 stimulated and unstimulated T cell receptor transgenic ‘pmel’ and MyD88-/-pmel CD8+ T cells and identified changes in the expression levels of several TNF family members. In particular, TLR-stimulation increased 4-1BB levels in pmel but not in MyD88-/-pmel T cells. A link between 4-1BB and TLR1-TLR2 signaling in CD8+ T cells was highlighted by in fact that 4-1BB-/- T cells exhibited suboptimal responses to TLR1-TLR2 agonist, but responded normally to CD28 or OX40 costimulation. Moreover, blocking 4-1BB signaling with antibodies also hindered the costimulatory effects of the TLR1-TLR2 agonist. The elevated levels of 4-1BB transcripts in TLR1-TLR2–stimulated cells were not due to increased mRNA stability nor increased histone activation but instead were associated with increased binding of p65 and c-Jun to two distinct 4-1BB promoter sites. Combining TLR1-TLR2 ligand with an agonistic anti-4-1BB antibody enhanced the antitumor activity in mice with established melanoma tumors. These studies reveal that the costimulatory effects of TLR1-TLR2 signaling in CD8+ T cells are in part mediated by 4-1BB and are important for mounting an effective antitumor immune response.
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