evaluation-of-lung-function-in-an-ex-vivo-murine-model

Evaluation of Lung Function in an <em>Ex vivo</em> Murine Model

Respiratory mechanics are a key area of study in defining and treating lung pathologies by assessing functional lung capacity. Lung mechanics can be ...

Medical University of South Carolina

The solid tumor microenvironment is replete with factors that present a stress to infiltrating immune cells. Endoplasmic reticulum (ER) stress sensor PKR-like ER kinase (PERK) is primed to sense and respond to the burden of misfolded proteins in the ER lumen induced by cell stressors. PERK has documented roles as a master regulator of acute and chronic responses to cell stress as well as in the regulation of cell metabolism. Here, we provide an overview of the roles of PERK based on what is known and remains to be tested in immune cells in tumors and impacts on tumor control. PERK is one of several ER kinases able to preferentially induce activating transcription factor 4 (ATF4) as a response to cell stress. ATF4 orchestrates the oxidative stress response and governs amino acid metabolism. We discuss the tested role of ATF4 in tumor immunity and provide insight on the dueling protective and deleterious roles that ATF4 may play in the stress of solid tumors.

Stress relief for cancer immunotherapy: implications for the ER stress response in tumor immunity.

Protein synthesis supports robust immune responses. Nutrient competition and global cell stressors in the tumor microenvironment (TME) may impact protein translation in T cells and antitumor immunity. Using human and mouse tumors, we demonstrated here that protein translation in T cells is repressed in solid tumors. Reduced glucose availability to T cells in the TME led to activation of the unfolded protein response (UPR) element eIF2α (eukaryotic translation initiation factor 2 alpha). Genetic mouse models revealed that translation attenuation mediated by activated p-eIF2α undermines the ability of T cells to suppress tumor growth. Reprograming T-cell metabolism was able to alleviate p-eIF2α accumulation and translational attenuation in the TME, allowing for sustained protein translation. Metabolic and pharmacological approaches showed that proteasome activity mitigates induction of p-eIF2α to support optimal antitumor T-cell function, protecting from translation attenuation and enabling prolonged cytokine synthesis in solid tumors. Together, these data identify a new therapeutic avenue to fuel the efficacy of tumor immunotherapy.

Stress-Mediated Attenuation of Translation Undermines T-cell Activity in Cancer.

Lymphodepleting pre-conditioning is a nearly universal component of T cell adoptive transfer protocols. The side effects of pre-conditioning regimens used in adoptive cell therapy are clinically significant and include pan-cytopenia, immune suppression, and reactive myelopoiesis. We conducted studies to test the hypothesis that the mechanisms underlying effective engraftment are cell autonomous and not dependent on a lymphodepleted host immune status. These studies leveraged mouse models to examine the role of Stat5 signaling during T cell adoptive transfer. We observed that, by transiently expressing a constitutively active mutamer of Stat5b during the process of adoptive transfer, we could completely obviate the need for lymphodepletion prior to adoptive transfer. Using several functional assays, we benchmark the function of the engrafted T cells against T cells transferred after conventional lymphodepletion. These studies identify a cell-autonomous mechanism driven by transient Stat5b signaling with lasting effects on T cell phenotype and function. Furthermore, the results presented suggest that adoptive T cell therapy could be improved by removing lymphodepletion protocols entirely and replacing them with RNA transfection of T cells with transcripts encoding active Stat5.

Efficient T cell adoptive transfer in lymphoreplete hosts mediated by transient activation of Stat5 signaling.

In this chapter, the methodologies are outlined for generating CAR-T from PBMCs using transposon engineering. Additionally, some methods and guidance related to basic functional and phenotypic analysis are described. This methodology can be applied to manufacture and assess chimeric antigen receptors for preclinical applications targeting a variety of molecules.

Article	Year
Stress relief for cancer immunotherapy: implications for the ER stress response in tumor immunity. Cancer immunology, immunotherapy : CII\| PubMed ID: 33104836	2021
Stress-Mediated Attenuation of Translation Undermines T-cell Activity in Cancer. Cancer research\| PubMed ID: 36126165	2022
Efficient T cell adoptive transfer in lymphoreplete hosts mediated by transient activation of Stat5 signaling. Molecular therapy : the journal of the American Society of Gene Therapy\| PubMed ID: 37481703	2023
Transposon-Based Manufacturing of Human CAR-T Cells. Methods in molecular biology (Clifton, N.J.)\| PubMed ID: 38070116	2024

Megan Tennant

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