Pro-inflammatory cytokine responses coupled with the expression of granzyme B and CD107a (LAMP-1), are valuable biomarkers in the immuno-oncology space1-3. Cell-based assays that can quantify the expression of these targets in tumor-derived effector lymphocytes provide insights into the mechanism of action of drug candidates. The Preclinical Oncology Group offers a new off-the-shelf assay that utilizes the Cytokine/Cytotoxicity standard flow cytometry panel to quantify these endpoints in T cells, natural killer (NK) and natural killer T cell (NKT) subsets. This technology spotlight demonstrates how the Cytokine/Cytotoxicity™ panel can be incorporated into in vivo studies and, combined with conventional immunophenotyping, help build robust data sets during preclinical drug development.
Cytokines including IFNγ, TNFα, and IL-2 have anti-tumor activity by promoting innate and adaptive immune cell activation and T cell proliferation in the tumor microenvironment. In vivo therapies that inhibit tumor growth by way of T cell activation, through direct or indirect mechanisms, often increase the frequency of tumor-infiltrating T cells that are primed to respond vigorously to ex vivo stimulation. Similar effects have been demonstrated to occur in NK and NKT cells with respect to priming and subsequent IFNγ/TNFα production. In addition to cytokine production, in vivo treatment can induce effector lymphocytes to acquire heightened cytotoxic properties that enable tumor cell lysis by direct cell-to-cell contact mechanisms. This process is mediated by the expression and release of perforins and granzymes from cytolytic granules stored inside the immune cells. These toxins disrupt the cell membrane and induce apoptosis in the target tumor cell. Furthermore, degranulation coincides with the expression of CD107a on the surface of the effector cell and can thus be used as a marker for cytotoxic activity.
Together, the signatures described above can be profiled to examine the activation state of lymphocyte subsets in the tumor microenvironment. The Cytokine/Cytotoxicity panel enables these measurements by combining cell surface immunophenotyping using T cell, NK, and NKT cell-specific antibodies with intracellular staining for cytokines and granzyme B expression (Table 1).
Table 1: Cytokine/Cytotoxicity™ Panel