Improvement of antigen specific CD8 T cell assay with longer stimulation 3896

Abstract Description Induction of a robust cytotoxic CD8 T cell response is a core focus of HIV vaccine and therapy research as these cells are capable of eliminating virally infected cells. Effective strategies for identifying successful induction of HIV specific CD8 T cells remains a challenge for the field, in part, due to the low level of sensitivity with currently available assays. Immunomonitoring assays require further development to characterize this rare cell population needed to perform cross vaccine strategy comparisons. We are utilizing cutting edge technologies, including novel fluorophores and spectral enabled flow cytometry, to incorporate activation induced markers (AIM) with our cytokine induced staining (ICS) methodology to improve specificity and sensitivity. Incorporation of AIM markers, including 4-1BB, ICOS, OX40, etc., requires measuring temporal changes in expression post-simulation to optimize assay marker combinations. For these purposes, we standardized our positive control stimulations, CEFX (a broad mixture of common peptides) and Staphylococcal Enterotoxin B. CEFX served as a biological relevant stimulant that has shown to be sufficient to detect CD8 T cell responses as early as 6 hours post-stimulation. Our testing shows expression of target AIM is temporally controlled requiring further in-depth testing, including longer peptide stimulation strategies, to optimize a specific and sensitive flow based cytotoxic assay for use to compare HIV vaccination strategies. Funding Sources This work was supported by a cooperative agreement (W81XWH-18-2-0040) between the Henry M. Jackson Foundation for the Advancement of Military Medicine, Inc., and the U.S. Department of Defense (DOD). This research was funded, in part, by the U.S. National Institute of Allergy and Infectious Diseases. Topic Categories Technological Innovations in Immunology (TECH)