Tu84 - Diverse Functional Profiles of Individual Human Gamma Delta T and iNKT Cells Revealed With a 32 Color Intracellular Cytokine Staining Spectral Flow Cytometry Panel

Abstract Text: Populations of innate T cell subsets such as gamma delta T cells and iNKT cells are unique in their ability to shift effector function profiles from inflammatory (Th1/Th17) to immunoregulatory/suppressive (Th2/Treg). The nature of innate T cell lineage commitment versus functional plasticity is unclear to date. Previous limitations in flow cytometry panel size disallowed for proper analysis of higher numbers of cytokines and other effector functions from these unique T cell populations with the single cell resolution required to address these questions. However, with the advancement of spectral cytometry, the design and optimization of larger scale flow cytometry panels is now feasible, thus allowing for the expansion of emission detectors and a wider optical spectral range to accommodate panels with 30 or more colors. Using the full optical spectrum, a 32-color Intracellular Cytokine Staining (ICS) panel was successfully optimized, enabling measurement of 11 cytokines, as well as Ki67, granzyme B, and CD107a from several immune cell subsets in PBMCs (CD4+ T cells, CD8+ T cells, NKT cells, iNKT cells, Vd1 gd T cells, Vd2 gd T cells, regulatory T (Treg) cells, and NK cells). Use of this panel revealed differences in polyfunctionality of gamma delta T cells between younger versus older subjects, and that individual innate-like T cells were found to express a ‘Th0’ phenotype, producing both Th1 and Th2 lineage cytokines after short term mitogenic stimulation. The application of large ICS panels will help elucidate innate T cell functional signatures in the context of different diseases and disorders.