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Isolation of adoptively transferred CD8⁺ T cells in mouse tumor tissues for lipid peroxidation detection.

ABSTRACT: The lipid peroxidation level of tumor-infiltrating CD8⁺ T cells is crucial for its activity and longevity. Here, we describe a protocol for effective and epitope-preserving dissociation of mouse tumors and subsequent leukocyte purification and lipid peroxidation staining of adoptively transferred CD8⁺ T cells. We use BODIPY 581/591 C11 to monitor the cellular lipid peroxidation level and detect its fluorescent change by flow cytometry, followed by analysis in FlowJo. This protocol is adaptable to intrinsic CD8⁺ T cells in tumors as well. For complete details on the use and execution of this protocol, please refer to Xiao et al. (2022)¹ and Ma et al. (2021).².

SUBMITTER: Xiao L

PROVIDER: S-EPMC9792557 | biostudies-literature | 2023 Mar

REPOSITORIES: biostudies-literature

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Isolation of adoptively transferred CD8+ T cells in mouse tumor tissues for lipid peroxidation detection.

Xiao Liuling L Yi Qing Q

STAR protocols 20221215 1

The lipid peroxidation level of tumor-infiltrating CD8+ T cells is crucial for its activity and longevity. Here, we describe a protocol for effective and epitope-preserving dissociation of mouse tumors and subsequent leukocyte purification and lipid peroxidation staining of adoptively transferred CD8+ T cells. We use BODIPY 581/591 C11 to monitor the cellular lipid peroxidation level and detect its fluorescent change by flow cytometry, followed by analysis in FlowJo. This p ...[more]

PMID: 36525345

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Project description:BackgroundAdoptive T cell transfer (ACT) therapy improves outcomes in patients with advanced malignancies, yet many individuals relapse due to the infusion of T cells with poor function or persistence. Toll-like receptor (TLR) agonists can invigorate antitumor T cell responses when administered directly to patients, but these responses often coincide with toxicities. We posited that TLR agonists could be repurposed ex vivo to condition T cells with remarkable potency in vivo, circumventing TLR-related toxicity.MethodsIn this study we investigated how tumor-specific murine CD8+ T cells and human tumor infiltrating lymphocytes (TILs) are impacted when expanded ex vivo with the TLR9 agonist CpG.ResultsHerein we reveal a new way to reverse the tolerant state of adoptively transferred CD8+ T cells against tumors using TLR-activated B cells. We repurposed the TLR9 agonist, CpG, commonly used in the clinic, to bolster T cell-B cell interactions during expansion for ACT. T cells expanded ex vivo from a CpG-treated culture demonstrated potent antitumor efficacy and prolonged persistence in vivo. This antitumor efficacy was accomplished without in vivo administration of TLR agonists or other adjuvants of high-dose interleukin (IL)-2 or vaccination, which are classically required for effective ACT therapy. CpG-conditioned CD8+ T cells acquired a unique proteomic signature hallmarked by an IL-2RαhighICOShighCD39low phenotype and an altered metabolic profile, all reliant on B cells transiently present in the culture. Likewise, human TILs benefitted from expansion with CpG ex vivo, as they also possessed the IL-2RαhighICOShighCD39low phenotype. CpG fostered the expansion of potent CD8+ T cells with the signature phenotype and antitumor ability via empowering a direct B-T cell interaction. Isolated B cells also imparted T cells with the CpG-associated phenotype and improved tumor immunity without the aid of additional antigen-presenting cells or other immune cells in the culture.ConclusionsOur results demonstrate a novel way to use TLR agonists to improve immunotherapy and reveal a vital role for B cells in the generation of potent CD8+ T cell-based therapies. Our findings have immediate implications in the clinical treatment of advanced solid tumors.

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Isolation of adoptively transferred CD8⁺ T cells in mouse tumor tissues for lipid peroxidation detection.

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Isolation of adoptively transferred CD8<sup>+</sup> T cells in mouse tumor tissues for lipid peroxidation detection.

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Isolation of adoptively transferred CD8+ T cells in mouse tumor tissues for lipid peroxidation detection.

Publications

Isolation of adoptively transferred CD8&lt;sup&gt;+&lt;/sup&gt; T cells in mouse tumor tissues for lipid peroxidation detection.

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OmicsDI is part of the ELIXIR infrastructure

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Isolation of adoptively transferred CD8⁺ T cells in mouse tumor tissues for lipid peroxidation detection.

Isolation of adoptively transferred CD8<sup>+</sup> T cells in mouse tumor tissues for lipid peroxidation detection.