Development of a 30-parameter flow cytometry panel for functional characterization of unconventional T cells and innate lymphoid cells

Abstract Unconventional T cells, which include mucosal-associated invariant T (MAIT) cells, invariant natural killer T (iNKT) cells, and γδ T cells, are defined by their recognition of antigens and microbial products, such as vitamin B metabolites, presented on non-classical MHC molecules. These “innate-like” cells have garnered attention for their unique potential for bridging the gap between innate and adaptive immunity, although their exact roles during infection is still not well-understood. To evaluate the phenotypic and functional characteristics of these cells, we developed and optimized a comprehensive, 30-parameter flow cytometry panel which delineates these cell subsets on the basis of surface marker expression. In addition, we simultaneously assess effector functions through the measurement of intracellular markers for cytokines, activation molecules, and cytotoxic molecules, following stimulation. We also included markers for the evaluation of natural killer (NK) cells and innate lymphoid cells (ILCs), as they play distinct roles at this junction of innate and adaptive immunity. We optimized our panel in combination with innate, pan-T cell (IL-12/IL-18) and subset-specific (e.g. HMBPP for Vγ9 T cells) stimulation reagents to evaluate responses to distinct innate-like and adaptive immune stimulation modes. We will use this flow cytometry panel to assess the immune profiles of various cohorts, including individuals who are cytomegalovirus (CMV) positive and human immunodeficiency virus (HIV) positive, in order to further elucidate how different acute and chronic infections can alter the frequencies, phenotypes, and functional characteristics of these cells.