Perforin deficiency within CD8+ T cells in sentinel lymph nodes of urothelial bladder cancer
Ciputra Adijaya Hartana1, Robert Rosenblatt2, A. Ali Zirakzadeh1, Emma Ahlén Bergman1, David Krantz1, Malin E Winerdal1, Laszlo Szekely1, Hans Glise1, Amir Sherif2, Ola Winqvist1
1Karolinska Institutet, Department of Medicine Solna, Unit of Immunology and Allergy, Stockholm, Sweden 2Department of Surgical and Perioperative Sciences, Urology and Andrology, Umeå University Hospital, Umeå, Sweden
CD8+ T cells have the capability to directly kill tumor cells with their cytotoxic constituents, such as granzymes and perforin. Sentinel lymph nodes (SN) is the first site of interaction between CD8+ T cells and tumor microenvironment, allowing naïve CD8+ T cells to be immunized and ready to attack the tumor. Therefore, cytotoxicity of CD8+ T cells from SN of urothelial bladder cancer (UBC) patients is important to be understood as it might be compromised due to tumor immune escape mechanism. 24 patients diagnosed with UBC were recruited in this study. CD8+ T cells from peripheral blood (PBMC), SN, and tumor (TIL) were analyzed for their cytotoxicity by flow cytometry, RT-qPCR, and ELISA. CD8+ T cells were cultured for reactivation by tumor extract stimulation or stimulated in a Th1 pro-inflammatory environment. CD8+ T cells from SN showed perforin deficiency when compared to CD8+ T cells from PBMC or TIL. Perforin-deficient CD8+ T cells demonstrated low expression of the perforin gene (PRF1) and the production could not be reactivated by tumor extract stimulation. Furthermore, PD-1 expression was elevated with low expression of T-bet in perforin-deficient CD8+ T cells, whereas high GATA-3 transcript expression was present in SN-derived CD8+ T cells, consistent with CD8+ T cells from tumor. Perforin expression could be rescued by providing a Th1 pro-inflammatory environment using IL-12 and anti-IL-4. In conclusion, our preliminary data suggested that low expression of perforin in SN-derived CD8+ T cells maybe a tumor immune escape mechanism. By demonstrating that perforin expression can be rescued, it may be beneficial for improving sentinel node-based adoptive cellular immunotherapy.
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