The development of methods to detect the different hallmarks of immunogenic cell death

Immunogenic cell death (ICD) is a form of regulated cell death (e.g. immunogenic apoptosis, necroptosis) that is able to activate both an innate and adaptive immune response. The induction of ICD is an interesting strategy in the development of future immunotherapies. So far, several markers of immunogenic cell death are known and summarized by Galluzzi L. et al. (Cell death and differentiation, 2018), including calreticulin (CRT), ATP, high-mobility group box I (HMGB-1), type I interferon (IFN) and annexin A1 (ANXA1). In this context, we developed and/or optimized different assays to detect these hallmarks using both flow cytometry based and enzyme-linked immunosorbent assays (ELISA). 624-mel, 938-mel (both human melanoma cells) and B16 (mouse melanoma cells) were treated with several known ICD-inducers like staurosporine and doxorubicine and analyzed with different in-house developed and/or optimized protocols. The presence of exposed CRT can be analyzed with a flow cytometry based assay. ATP can be detected after staining of treated cells with quinacrine, a dye that stains intracellular ATP vesicles. Using flow cytometry, it can be shown that the percentage of quinacrine positive cells is decreasing after induction of ICD. Using a reporter cell line, HEK293T cells transduced with a lentiviral vector expressing a reporter gene under the control of an IFN dependent promotor, the supernatant of treated cells can be screened for the presence of type I IFN. The assays for the detection of both HMGB-1 and ANXA1 are currently being optimized. In conclusion, these methods allow to analyze the different markers of ICD and make it possible to fingerprint the type of ICD in different experimental settings.