RESUMO
Roquin and Regnase-1 proteins bind and post-transcriptionally regulate proinflammatory target messenger RNAs to maintain immune homeostasis. Either the sanroque mutation in Roquin-1 or loss of Regnase-1 cause systemic lupus erythematosus-like phenotypes. Analyzing mice with T cells that lack expression of Roquin-1, its paralog Roquin-2 and Regnase-1 proteins, we detect overlapping or unique phenotypes by comparing individual and combined inactivation. These comprised spontaneous activation, metabolic reprogramming and persistence of T cells leading to autoimmunity. Here, we define an interaction surface in Roquin-1 for binding to Regnase-1 that included the sanroque residue. Mutations in Roquin-1 impairing this interaction and cooperative regulation of targets induced T follicular helper cells, germinal center B cells and autoantibody formation. These mutations also improved the functionality of tumor-specific T cells by promoting their accumulation in the tumor and reducing expression of exhaustion markers. Our data reveal the physical interaction of Roquin-1 with Regnase-1 as a hub to control self-reactivity and effector functions in immune cell therapies.
Assuntos
Autoimunidade , Citotoxicidade Imunológica , Imunoterapia Adotiva , Melanoma Experimental/terapia , Proteínas Repressoras/metabolismo , Ribonucleases/metabolismo , Neoplasias Cutâneas/terapia , Linfócitos T/transplante , Ubiquitina-Proteína Ligases/metabolismo , Animais , Feminino , Células HEK293 , Células HeLa , Humanos , Imunidade Humoral , Masculino , Melanoma Experimental/genética , Melanoma Experimental/imunologia , Melanoma Experimental/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mutação , Fenótipo , Ligação Proteica , Proteínas Repressoras/genética , Ribonucleases/genética , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/imunologia , Neoplasias Cutâneas/metabolismo , Linfócitos T/imunologia , Linfócitos T/metabolismo , Microambiente Tumoral , Ubiquitina-Proteína Ligases/genéticaRESUMO
BACKGROUND: pO2 and pH are physiological parameters relevant for different processes in health and disease, including wound healing and cancer progression. Head and neck squamous cell carcinomas (HNSCC) and oesophageal squamous cell carcinomas (ESCC) have a high rate of local recurrence that is partly related to treatment-resistant residual tumour cells. Hence, novel diagnostic tools are required to visualise potential residual tumour cells and thereby improve treatment outcome for HNSCC and ESCC patients. We developed a device to spatiotemporally measure oxygen consumption rates (OCR) and extracellular acidification rates (ECAR) to distinguish HNSCC and ESCC cells from healthy cells in vitro, exploiting general metabolic differences between cancer cells and healthy cells. METHODS: OCR and ECAR were measured via a newly developed device named STO2p-Q (SpatioTemporal O2 and pH Quantification) using the VisiSens technology based on ratiometric fluorescence imaging, facilitating spatiotemporal resolution. Results were confirmed using extracellular flux analyses (Seahorse technology). RESULTS: STO2p-Q is described and used to measure OCR and ECAR in HNSCC and ESCC cell lines and normal fibroblast and epithelial cells as components of the tumour microenvironment. OCR measurements showed differences amongst HNSCC and ESCC cell lines and between HNSCC/ESCC and normal cells, which on average had lower OCR than HNSCC/ESCC cells. Both OCR and ECAR measurements were independently verified using the Seahorse technology. Additionally, using STO2p-Q, HNSCC/ESCC, and normal cells could be spatially resolved with a resolution in the low millimetre range. CONCLUSIONS: We developed a method to spatiotemporally measure OCR and ECAR of cells, which has many potential in vitro applications and lays the foundation for the development of novel diagnostic tools for the detection of cancerous tissue in HNSCC and ESCC patients in vivo.