A comprehensive multiparameter flow cytometry panel for immune profiling and functional studies of frozen tissue, bone marrow, and spleen

Abstract

Flow cytometry (FC) is a highly informative technology that can provide valuable information about immune phenotype monitoring and immune cell states. However, there is a paucity of comprehensive panels developed and validated for use on frozen samples. Here, we developed a 17-plex flow cytometry panel to detect subtypes, frequencies, and functions of different immune cells that can be leveraged to study the different cellular characteristics in different disease models, physiological, and pathological conditions. This panel identifies surface markers to characterize T cells (CD8⁺, CD4⁺), natural killer (NK) cells and their subtypes (immature, cytotoxic, exhausted, activated),natural killer T (NKT) cells, neutrophils, macrophages (M1 (pro-inflammatory) and M2 (anti-inflammatory)), monocytes and their subtypes (classical and non-classical), dendritic cells (DC) and their subtypes (DC1, DC2), and eosinophils. The panel was designed to include only surface markers to avoid the necessity for fixation and permeabilization steps. This panel was optimized using cryopreserved cells. Immunophenotyping of spleen and bone marrow using the proposed panel was efficient in correctly differentiating the immune cell subtypes in inflammatory model of ligature-induced periodontitis, in which we found increased percentage of NKT cells, activated and mature/cytotoxic NK cells in the bone marrow of affected mice. This panel enables in-depth immunophenotyping of murine immune cells in bone marrow, spleen, tumors, and other non-immune tissues of mice. It could be a tool for systematic analysis of immune cell profiling in inflammatory conditions, systemic diseases, and tumor microenvironments.

Keywords: Immune profiling; Multi-color flow cytometry; Phenotypic analysis; Tumor immune cells.

Fig. 1.

Fluorescence minus one (FMO) compensation. FMOs were performed for each antibody and used for negative control when setting the gate for target populations to facilitate accurate sorting. The top row are the FMOs staining and the bottom row is the full staining. We can observe the absence of staining in FMOs.

Fig. 2.

Gating strategy for BM cells isolated from ligature-induced periodontitis mouse. Firstly, we identified the single cells and live lymphocytes with CD45 and Zombie Aqua^™ markers in the BM of healthy mice. A) We gated over TCRβ and from the TCRβ⁺ population, we identified CD4⁺ T cell, DN T cell, DP T cell, and CD8⁺ T cell based on the CD4 and CD8 surface markers. B) From CD45⁺Zombie Aqua^™- population, we gated over TCRβ and NK1.1 and identified NK (TCRβ⁻NK1.1⁺) and NKT (TCRβ⁺NK1.1⁺) cells. Further stratification of the NK cell population by level of CD11b is performed to differentiate cytotoxic NK cells (CD11bhigh NK) and immature NK cells (CD11blow NK). The activation and exhaustion of NK cells were marked by the presence of CD69 and TIGIT. C) From CD45⁺Zombie Aqua^™- a population we gated over various markers to stratify hematopoietic cells. Neutrophils are defined as Ly6G⁺CD11b⁺ and from the Ly6G⁻CD11b⁺ subset, we further stratified macrophages and monocytes. M1 macrophage is defined as CD80⁺CD206⁻ while M2 macrophage is defined as CD80⁻CD206⁺. Monocytes were stratified according to surface Ly6C expression into inflammatory monocytes (Ly6C⁺) and noninflammatory monocytes (Ly6C⁻). The eosinophils were defined as SigliecF⁺CD11b⁺.

Fig. 3.

The panel was able to demonstrate high capacity for detection of difference in the frequency of cell populatins including NKT cells, NK cells, and NK activation and mature states in BM of ligature induced periodontitis versus health. A) Representative images of current validated panel in the spleen (Naïve), bone marrow (health), and bone marrow isolated from ligature-induced periodontitis model. B.C·D) statistically significant increase in NKT, activated NK (CD69⁺), and mature NK (CD11b⁺) cells in BM in ligature-induced periodontitis model. Results are representative of 12 biolgical replicates (n = 6 per goup). Parametric Students' t-test was used for statistical analysis. ** indicates P < 0.01 and * indicates P < 0.05. ** indicates P < 0.01 and * indicates P < 0.05.