Multiparameter Flow Cytometric Analysis of the Conventional and Monocyte-Derived DC Compartment in the Murine Spleen

Abstract

Dendritic cells (DCs) are present in almost all tissues, where they act as sentinels involved in innate recognition and the initiation of adaptive immune responses. The DC family consists of several cell lineages that are heterogenous in their development, phenotype, and function. Within these DC lineages, further subdivisions exist, resulting in smaller, less characterized subpopulations, each with its unique immunomodulatory capabilities. Given the interest in utilizing DC for experimental studies and for vaccination purposes, it becomes increasingly crucial to thoroughly classify and characterize these diverse DC subpopulations. This understanding is vital for comprehending their relative contribution to the initiation, regulation, and propagation of immune responses. To facilitate such investigation, we here provide an easy and ready-to-use multicolor flow cytometry staining panel for the analysis of conventional DC, plasmacytoid DC, and monocyte-derived DC populations isolated from mouse spleens. This adaptable panel can be easily customized for the analysis of other tissue-specific DC populations, providing a valuable tool for DC research.

Keywords: dendritic cell; dendritic cell subpopulations; macrophages; monocytes; multicolor flow cytometry; myeloid cells; spleen.

Figure 2

Flow cytometry analysis of the steady-state splenic myeloid cell network. Splenocytes from 8-week-old mice were stained with the described DC panel (Table 1 and Supplementary Table S1). (A) Cells were initially gated on single cells using FSC-A and SSC-A gating to eliminate doublets and to exclude debris. Subsequently, living CD45⁺ leukocytes were selected using viability-negative gating, followed by the exclusion of T cells (CD90.2⁺ cells), B cells (CD19⁺ cells), and NK/NKT cells (NK1.1⁺, CD49b⁺ cells). (B) Within this lineage-negative cell population, the expression of F4/80 and CD64 marked splenic red pulp macrophages. (C) Subsequently, cDCs are characterized as cells expressing MHC class II (MHC-II) and CD11c. (D) pDCs were identified as positive for PDCA1, while displaying intermediate levels of CD11c. Additionally, pDCs displayed variable expression of CD8α, which might indicate differences in the activation status of these cells. Splenic cDCs can either be separated into CD4⁺CD8α⁻, CD4⁻CD8α⁺ and double-negative DCs (E, left), or, alternatively, into conventional cDC type 1 (cDC1; XCR1⁺; SIRPα⁻) and type 2 (cDC2; XCR1⁻; SIRPα⁺) subsets (E, right). (I) Monocytes were identified as CD11c⁻CX3CR1⁺CD1b⁺ cells among the remaining cells and comprise Ly6C⁺ (‘pro-inflammatory’) and Ly6C⁻ (‘patrolling’) populations (J). (F) Terminally differentiated cDC1s expressed CD8α and (G) consisted of Langerin⁺ and Langerin⁻ subpopulations. (H) cDC2s comprised two dominant lineages, cDC2As and cDC2Bs, based on the mutually exclusive expression of ESAM and Clec12A, respectively. (K) Next to cDC and pDC the newly characterized non-canonical transitional DC (tDC) are identified as CD11b⁻CX3CR1⁺ cells. tDC are further divided into Ly6C positive (‘pDC-like’) and Ly6C negative (‘cDC-like’) populations, each with different functional characteristics. Depicted is one exemplary gating strategy. Data acquisition was performed using a BD FACSymphony flow cytometer, and data analysis was conducted using FlowJo software.

Figure 1

Identification of the major leukocyte and cDC populations in the steady-state murine spleen using a conserved 26-color core-marker FACS panel. (A) Unbiased dimensionality reduction analysis via t-stochastic neighborhood embedding (t-SNE) of all living CD45⁺ splenocytes categorizes the major distinct lymphocyte populations (CD4⁺ and CD8⁺ T cells, B cells), myeloid cell populations (monocytes, macrophages), and DC populations (cDC1s, cDC2s, pDCs). (B) Mapping of XCR1⁺ cDC1s and SIRPα⁺ cDC2s demonstrates a clear separation of the two main cDC populations in the spleen and illustrates the heterogeneity within each population. Data acquisition was performed using a BD FACSymphony flow cytometer, and data were evaluated using FlowJo software. For t-SNE, CD11c⁺MHC-II⁺ cDCs of three wild-type mice were concatenated in FLowJo with 4.000 events per sample. All markers shown in Table 1, as well as FSC-A and SSC-A, were utilized.

Figure 3

Phenotypic characterization of cDC1 and cDC2 subpopulations in the murine spleen. (A) Expression of cDC1 markers CD24, CD26, CD103, CD205, and MHC-II on either Langerin⁺ (filled, light blue) or Langerin⁻ (dark blue lines) cDC1s. (B) Expression of cDC2 markers CD4, CD11b, CD26, CX3CR1, Ly6C, and MHC-II on either ESAM⁺ (dark green lines) or ESAM⁻ (filled, light green) cDC2s. Single-cell suspensions from the spleen were prepared as described in Section 2.2 and stained as described in Figure 1 and Figure 2. Isotype-matched control antibody stainings are indicated with gray lines in the histogram plots. Data acquisition was performed using a BD FACSymphony flow cytometer, and data analysis was carried out using FlowJo software.

Figure 4

Phenotypic characterization of MoDCs and Inf-cDC2s in the steady-state murine spleen. Frequency of Ly6C⁺CD64⁺ MoDCs (A) and MAR-01⁺CD64⁺ Inf-cDC2s (B) in the murine spleen. DCs were pre-gated as CD11c^hiMHCII⁺ cells. The gating strategy is based on the expression of either Ly6C⁺CD64⁺ or MAR-01⁺CD64⁺ on macrophages (CD11c^intMHCII⁺ cells). The FACS plots show one representative mouse/group. Data acquisition was performed using a BD FACSymphony flow cytometer, and data analysis was performed using FlowJo software.

Figure 5

Characterization of distinct DC populations. This figure summarizes the differentiation pathways and defining markers of the various DC subsets. Conventional DCs (cDCs) differentiate into two main populations: cDC1s and cDC2s, originating from the common DC precursor-derived pre-DC1s and pre-DC2s, respectively. Monocyte-derived DCs (MoDCs) develop from Ly6C^hi monocytes. In lymphoid organs, cDC1s are characterized by the expression of CD8α, CD24, and XCR1. Within the cDC1 population, a subset expressing CD103 and Langerin can be identified. The cDC2 population constitutes at least 2 distinct subpopulations: ESAM⁺ cDC2As and Clec12a⁺ cDC2Bs. The exact relationship between inflammatory cDC2s (Inf-cDC2s) and other DCs remains unclear. Key lineage-defining markers for each DC population are highlighted and identifiable through the outlined protocol.