人源化小鼠样本中多重人和小鼠细胞因子的评估

Humanized mice represent a critical model for the evaluation of advanced therapeutics targeting cancer, including chimeric antigen receptor T cell (CAR-T) therapies. Myriad humanized mouse models exist, each with varying levels of immunodeficiency present in the murine immune system and varying levels of immunocompetence engendered by the human graft cells. Furthermore, CAR-T cells themselves can produce human cytokines once transferred into a mouse, and murine stromal cells can release cytokines in response to cancerous cells and therapies . As such, a significant challenge to evaluating immunological responses in humanized mice is detecting and differentiating human and murine cytokines within relatively small sample volumes. We evaluated the ability of the MILLIPLEX® Humanized Mouse Magnetic Bead Panel to detect 17 human or mouse cytokines in murine serum and plasma. Briefly, human or murine PBMCs were isolated from whole blood and stimulated overnight in the presence of lipopolysaccharide, eBioscience TM cell stimulation cocktail, and Rapid-Act T Cell Activation Kit (mouse or human). Resulting cell culture supernatants were diluted into murine serum and plasma individually and in combination to generate murine serum and plasma samples rich in human, murine, or human and murine cytokines ex vivo . These samples were then evaluated for murine IL-2, IL-5, IL-6, GM-CSF, IFNgamma, IL-1beta, IL-17A, TNFalpha and human IL-2, IL-5, IL-6, GM-CSF, IFNgamma, IL-1beta, IL-17A, TNFalpha, and IL-10 using the MILLIPLEX® Humanized Mouse Magnetic Bead Panel on the Bio-Plex 200 platform. The MILLIPLEX® Humanized Mouse Magnetic Bead Panel was able to detect human and murine cytokines in samples spiked with human and/or murine PBMC supernatant. Additionally, the kit was able to distinguish between human and murine cytokines within spiked samples. On average, the level of human cytokines detected in serum spiked with human supernatant was 694-fold higher than human cytokine levels detected in serum spiked with murine supernatant alone, while the level of murine cytokine detected in serum spiked with murine supernatant alone was 261-fold higher than serum samples spiked with human supernatant alone. Similar results were seen in plasma samples. These data demonstrate the utility of the MILLIPLEX® Humanized Mouse Magnetic Bead Panel to evaluate human and murine cytokines within humanized mouse samples. These data demonstrate this kit can detect and distinguish key mediators of inflammation between human and mouse, including IL-2, IFNgamma, and TNFalpha, within small sample volumes. Collectively, this work suggests that this platform is suitable for evaluating human and murine cytokine responses within murine samples, allowing for reliable cytokine analysis across a variety of humanized mouse formats and experimental designs.