The human immune system is a highly complex, tightly regulated component of the body that is a key player in protection from infectious diseases and foreign antigens. It has a role in virtually every disease of man and dysregulation of this system can have severe consequences not only in terms of infection, but also in the forms of autoimmune disease or cancer. While this system is widely accepted as being highly complex, approaches for its study have historically been very simplistic. Quite often a specific cell type e.g. dendritic cells, B cells, or T regulatory cells) is studied in great detail in the context of one disease or a limited number of diseases, or a very cursory examination is performed of multiple cell types in a similar setting. Studies of the immune system in mice, while at times more comprehensive, have limitations in their applicability to human immunology (Davis, 2008
). Thus a new paradigm is beginning to emerge in which a comprehensive, more systems-based approach is used to study human immunology in both its basal state and upon perturbation by disease, therapy, or vaccination.
Comprehensive assessment of the immune system utilizes a multitude of technologies to examine as many facets as possible. Assays for these studies may include gene expression arrays, SNP genotyping, proteomics, ELISPOT assays, serologic studies, a vast assortment of functional assessments, serum cytokine analyses and flow cytometric analysis of a large number of cell surface markers, as well as intracellular antigens. In order to truly provide a comprehensive, detailed view of the immune system in toto, the flow cytometry assays must be far more in depth and comprehensive than any previously attempted, spanning all lineages but at the same time providing detailed information regarding all known leukocyte subsets. Previous approaches to immunophenotyping have selected either depth (detailed immunophenotyping of a single lineage or subset) or breadth (immunophenotyping of multiple lineages without significant depth), whereas the current approach seeks both depth and breadth. We term this comprehensive leukocyte immunophenotyping (CLIP). This comprehensive approach for immunophenotyping, in addition to all known leukocyte subsets, should also include activation markers, intracellular cytokines, phosphorylated signaling proteins, and in certain instances, tetramer analysis. In many aspects, this approach more closely resembles gene profiling studies than it does immunophenotyping of the past.
For such a panel to be developed in a meaningful manner necessitates the use of high dimensional immunophenotyping where as many of these markers as possible are studied concomitantly. To this end, our laboratory is developing an in-depth and comprehensive panel to immunophenotype as many components of the immune system as possible, with the end goal of better defining immune perturbations in both health and disease. This panel utilizes 15-color, 17-parameter flow cytometry to study known lineages and subsets within these lineages as well as permitting ‘data mining’ to characterize previously undiscovered subsets. The construction of the panel is centered on developing individual tubes within lineages that, when combined, will yield a comprehensive phenotype of all lineages.
Optimally, this panel possesses recurring markers within the tubes of a given lineage in order to permit a deep level of inter-tube concatenation of immunophenotypes. Currently in its infancy, this panel consists of ten 15-color, 17-parameter tubes, but could well reach sixty or more in the next several years. Herein we present details concerning the development of this comprehensive panel and the progress to date in this effort.