Detecting Senescence in Immune Cell Populations during Natural Aging using γH2AX via Multiparameter Flow Cytometry
Childhood cancer survivors who receive chemotherapy treatment have a significantly increased risk of hospitalization due to infection. Doxorubicin (DOX) is a common chemotherapeutic that targets cancerous cells by inhibiting topoisomerase II. Due to the lack of specificity of DOX to cancerous cells, it can cause adverse effects on healthy cells. It has been well documented that DOX treatment causes immunosenescence through direct dysregulation of immune cell populations as well as increased inflammation, altogether believed to directly drive increased susceptibility to infectious diseases. Currently, it is unknown whether treatment with DOX induces cellular senescence in immune cell populations and it is unclear how DOX induces immune dysfunction. The tools available to define senescent, exhausted, and immunosenescent cells are limited. Currently, we are optimizing a protocol to detect and define senescent immune cells by γH2AX expression via flow cytometry. It has been previously established that senescent B cells in the ileum of mice increase with age and have an increased expression of γH2AX measurable using flow cytometry. Additionally, it has been previously established that aged virtual memory CD8+ T cells have a senescent-like phenotype and have an increased expression of γH2AX, that is detectable using flow cytometry. We can recapitulate these findings that virtual memory T cells from old mice have an increased expression of γH2AX. We analyzed γH2AX in multiple immune cells from lymphoid and non-lymphoid tissues to address whether γH2AX was increased with age across all tissues. Future experiments will address whether DOX treatment increases γH2AX in immune cell subsets. These data may provide a new approach to detect senescence in heterogenous cell populations, such as immune cells, via flow cytometry.