45-Color Full Spectrum Flow Cytometry Panel for Deep Immunophenotyping of the Major Lineages Present in Human Peripheral Blood Mononuclear Cells with Emphasis on the T cell Memory Compartment.

ABSTRACT The need for more in-depth exploration of the human immune system has moved the flow cytometry field forward with advances in instrumentation, reagent development and user-friendly implementations of data analysis methods. The increase in the number of markers evaluated simultaneously requires a careful selection of highly overlapping dyes to avoid introducing detrimental spread and compromising population resolution. In this manuscript, we present the strategy used in the development of a high-quality human 45-color panel which allows for comprehensive characterization of major cell lineages present in circulation including T cells, gamma delta T cells, NKT-like cells, B cells, NK cells, monocytes, basophils, dendritic cells, and ILCs, as well as more in-depth characterization of memory T cells. The steps taken to ensure that each marker in the panel was optimally resolved are discussed in detail. We highlight the outstanding discernment of cell activation, exhaustion, memory, and differentiation states of CD4+ and CD8+ T cells using this 45-color panel, enabling an in-depth description of very distinct phenotypes associated with the complexity of the T cell memory response. Furthermore, we present how this panel can be effectively used for cell sorting on instruments with a similar optical layout to achieve the same level of resolution. Functional evaluation of sorted specific rare cell subsets demonstrated significantly different patterns of immunological responses to stimulation, supporting functional and phenotypic differences within the T cell memory subsets. In summary, the combination of flow cytometry full spectrum technology, careful assay design and optimization, results in high resolution multiparametric assays. This approach offers the opportunity to fully characterize immunological profiles present in peripheral blood in the context of infectious diseases, autoimmunity, neurodegeneration, immunotherapy, and biomarker discovery. PURPOSE AND APPROPRIATE SAMPLE TYPES This 45-color flow cytometry-based panel was developed as an expansion of the previously published OMIP-069 1 and serves as an in-depth immunophenotyping of the major cell subsets present in human peripheral blood. The goal of this panel is to maximize the amount of high-quality data that can be acquired from a single sample, not only for more in-depth characterization of the immune system, but also to address the issue of limited sample availability. The panel’s development included identifying fluorochromes that could improve the performance of the original 40-color panel and expanding the number of markers for deeper delineation of memory status of T cell subpopulations. To increase the number of markers, it was critical that any expansion did not negatively impact the resolution and quality of the data. To achieve this, the fluorochrome combinations were carefully characterized to ensure optimal resolution of each marker. The panel allows for deep characterization of the major cell lineages present in circulation (CD4 T cells, CDS T cells, regulatory T cells, yo T cells, NKT-like cells, B cells, NK (Natural Killer) cells, monocytes, and dendritic cells), while also providing an in-depth characterization of the T cell compartment, with a combination of activation, inhibitory, exhaustion, and differentiation markers. The panel supports deep exploration of the memory status of CD4 + T cells, CDS + T cells, and NKT-like cells. The steps taken in the optimization of the panel ensured outstanding resolution of each marker within the multicolor panel and unequivocal identification of each cell subset. This panel design and optimization will enhance the ability to characterize immunological profiles present in peripheral blood in the context of oncology, infectious diseases, autoimmunity, neurodegeneration, immunotherapy, and biomarker discovery. The panel was developed using fresh and cryopreserved human peripheral blood mononuclear cells (PBMCs) from healthy adults. We have not tested the panel on whole blood or biopsies; hence it is anticipated that the panel might require further optimization to be used with other sample types.