A 39-color full spectrum flow cytometric panel to deeply characterize human thymopoiesis

We report the development of a 39-color (43-parameter) full spectrum flow cytometry panel designed and optimized to deeply characterize the intrathymic development of human conventional and unconventional T cells. The panel was designed using strategies dictated by best practices for full spectrum and multiparametric flow cytometry, and was validated using appropriate negative and positive controls. By including several markers that are variably expressed during T cell development, this panel allows the definition of T cell maturation stages and the investigation of possible deviation from normal thymopoiesis at unprecedented resolution, thus representing a valuable tool for understanding immune dysregulation associated with altered thymopoiesis, as occurring in immune deficiencies, thymic lesions, and immunosenescence. Notably, because most of the molecules targeted in this panel are also commonly used as activation markers or immune checkpoints on mature T cells, this 39-color panel can also be applied for a comprehensive profiling of peripheral T cells, particularly in those peripheral tissues where unconventional T cells, including Vδ1, Vδ2, and Vδ3 T cell subsets and MAIT cells, interact with αβ T cells to shape the local microenvironment.