OMIP-069: Forty-Color Full Spectrum Flow Cytometry Panel for Deep Immunophenotyping of Major Cell Subsets in Human Peripheral Blood

Abstract

This 40-color flow cytometry-based panel was developed for in-depth immunophenotyping of the major cell subsets present in human peripheral blood. Sample availability can often be limited, especially in cases of clinical trial material, when multiple types of testing are required from a single sample or timepoint. Maximizing the amount of information that can be obtained from a single sample not only provides more in-depth characterization of the immune system but also serves to address the issue of limited sample availability. The panel presented here identifies CD4 T cells, CD8 T cells, regulatory T cells, γδ T cells, NKT-like cells, B cells, NK cells, monocytes and dendritic cells. For each specific cell type, the panel includes markers for further characterization by including a selection of activation and differentiation markers, as well as chemokine receptors. Moreover, the combination of multiple markers in one tube might lead to the discovery of new immune phenotypes and their relevance in certain diseases. Of note, this panel was designed to include only surface markers to avoid the need for fixation and permeabilization steps. The panel can be used for studies aimed at characterizing the immune response in the context of infectious or autoimmune diseases, monitoring cancer patients on immuno- or chemotherapy, and discovery of unique and targetable biomarkers. Different from all previously published OMIPs, this panel was developed using a full spectrum flow cytometer, a technology that has allowed the effective use of 40 fluorescent markers in a single panel. The panel was developed using cryopreserved human peripheral blood mononuclear cells (PBMC) from healthy adults (Table 1). Although we have not tested the panel on fresh PBMCs or whole blood, it is anticipated that the panel could be used in those sample preparations without further optimization. @ 2020 Cytek Biosciences, Inc. Cytometry Part A published by Wiley Periodicals LLC on behalf of International Society for Advancement of Cytometry.

Keywords: Aurora; OMIP; PBMCs; broad immunophenotyping; full spectrum; high-dimensional flow cytometry; spectral.

Figure 1

A. Manual gating strategy. The gating strategy used to identify the main cellular subsets is presented. Arrows are used to visualize the relationships across plots, and numbers are used to call attention to populations described here. After doublets and dead cells were excluded, basophils (1) were delineated as CD45⁺CD123⁺HLA‐DR^‐. Lymphocytes and monocytes (2) were gated based on FSC‐A/SSC‐A properties. Monocytes (3) were then classified by CD14 and CD16 expression as non‐classical (CD14⁻CD16⁺), intermediate (CD14⁺CD16^+/low), and classical (CD14⁺CD16^‐). From the lymphocyte gate (2), the following populations were identified: CD3⁻TCRγδ⁻, CD3⁺TCRγδ⁺, and CD3⁺TCRγδ⁻ (4). The CD3⁺TCRγδ⁺ population (5) was characterized based on CD45RA and CCR7 expression. The CD3⁺TCRγδ⁻ population was divided in CD3⁺CD56⁺ (NKT‐like) and CD3⁺CD56⁻ subsets (6). The inclusion of CD2 and CD8 enables further classification of the NKT‐like cells (7). CD4⁺, CD8⁺, CD4⁺CD8⁺ and CD4⁻CD8⁻ T cells were identified from the CD3⁺CD56⁻ gate (8). T_regs were identified from the CD4⁺ population using CD127 and CD25 expression (CD127^lo/‐CD25^hi) and CD39 and CD45RA were used to further classify these cells (9). CCR7, CD45RA, CD27, and CD28 allowed for further classification of memory/effector CD4 and CD8 T cell subsets (10, 11). CD19⁺ and/or CD20⁺ cells (B cells) were gated out of the CD3⁻TCRγδ⁻ population (12). CD19⁺CD20^+/− cells were further gated as IgD⁺CD27⁻, IgD⁺CD27⁺, or IgD⁻CD27^+/−; the IgD⁻CD27^+/− subset was divided into plasmablasts or IgD⁻ memory B cells based on CD20 expression and IgG and IgM expression were assessed within the IgD^‐ memory B cells (13). NK cells were defined as CD3⁻TCRγδ⁻HLA‐DR⁻ and classified as early NK (CD56⁺CD16⁻), mature NK (CD56⁺CD16⁺), and terminal NK (CD56⁻CD16⁺) cells (14). Dendritic cells (DCs, 15) were identified first by gating on CD3⁻CD19⁻CD56⁻CD14⁻HLA‐DR⁺ and from there CD123⁺ (pDCs) and CD11c⁺ DCs were identified. CD11c⁺ DCs were further divided into CD16⁻ and CD16⁺. CD1c and CD141 were then used to further classify the CD11c⁺CD16⁻ and CD11c⁺CD16⁺ DCs. Finally, innate lymphoid cells (ILCs, 16) were identified as CD3⁻CD19⁻CD20⁻CD14⁻CD123⁻CD127⁺ and subsetted based on CD2 and CD4 expression. All data presented is derived from frozen PBMCs of one healthy donor (donor ID 4559). B. High‐dimensional data analysis on PBMCs from four donors displaying FlowSOM clusters projected on to two UMAP dimensions to show concordance between manual and automated analysis techniques. the overlay plot shows concatenated events from all four samples, while the density plots show differences in population distribution between the individual samples. As expected with a combination of high‐resolution and high‐dimensional data, several clusters contain events that evade a canonical definition. These populations are displayed in gray.