Boody, Christopher M.. Characterizing chronic lymphocytic leukemia for novel biomarkers of CD40 responsiveness using intracellular flow cytometry. Retrieved from https://doi.org/doi:10.7282/T35B04B7
DescriptionChronic Lymphocytic Leukemia (CLL) is the most common form of leukemia. CLL is characterized by a slow progression that generally worsens over time and currently is without a cure. A number of prognostic indicators are currently utilized to identify CLLs with a more progressive disease course. One such prognostic indicator is the responsiveness of the CLL to CD40 stimulation. CLLs that do not demonstrate a functional response to CD40L show a more aggressive clinical outcome than those that are responsive. We sought to establish a system to classify CLL cases as CD40 responsive or unresponsive using novel biomarkers by intracellular flow cytometry. This allowed us to analyze a very small number of cells from each CLL. Altogether, our work addressed optimizing in vitro culturing conditions for proliferation and viability. Also, this work analyzed a number of CD40 stimulation conditions to determine the optimal approach to analyzing CD40-mediated events in signaling. Finally, this established a system to evaluate early intracellular signaling in the NF-κB canonical pathway with p65 phosphorylation and IκBα degradation as well as upregulation of CD80 following extended stimulation. Using IκBα we were able to discern CD40 activation in CLLs that were previously characterized as unresponsive with p65 and CD80 as markers. Thus, our work extends the definition of “CD40 responsiveness” and suggests that CD40-mediated CD80 responses, which are linked to co-stimulation, may be a better predictor of CLL prognostic outcomes than early NF-B responses.