Lymphocyte populations

A. Lymphocytes were gated based on CD45 versus Side Scatter and singlets were gated based on Side Scatter-Width. CD3- positive T cells can be identified. B. Identification and analysis of CD4/CD8 positive T cells. The CD8-positive cells are then separated into naïve/memory state by CCR7/CD45RA staining. In the CD8 T cells, a CD38-positive subset can also be identified, and some of those cells express HLA-DR or CD32. C. Analysis of the CD4-positive T cells. Regulatory T-cells can be identified in a staining with CD127 and CD25. Also, in the CD4 subset, naïve/memory cells can be separated using CCR7/CD45RA staining. The last two plots show the expression of four chemokine receptors, CXCR3, CCR10, CCR4, and CCR6 for the CD4 T cells. With these parameters, the different T helper cell subsets can be identified.

Monocyte, B-cell, and Dendritic cell populations

A. Monocytes were gated based on CD45 versus Side Scatter, and singlets were gated based on Side Scatter-Width. Classic monocytes are CD14-positive, but we can also identify a CD16-high intermediate population, and a CD14-neg/CD16-high non-classical monocyte population. The three populations also differ in the expression levels of HLA-DR and CD64, which is depicted in the last plot. B. B-cells were identified as CD19-positive cells from the lymphocyte gate. They can be separated into naive/memory state by staining with anti-IgD surface immunoglobulin expression and CD27. The class-switched memory B-cells have lost all IgD expression and are CD25-high. These cells also contain the actively antibody secreting cells (plasmablasts), which can be identified in the last plot as CD38-high/CD20-low cells. C. Dendritic cells (DC) can be identified by first gating on CD3/CD19/CD56 triple negative cells in the lymphocyte gate. In the next step, the gating is done on HLA-DR positive events and excludes any CD14-positive cells (contaminating monocytes in the lymphocyte gate). The last plot shows those cells stained with CD123 and CD11c to identify plasmacytoid DC (CD 123-positive) and myeloid DC (CD11c-positive).

NK and Regulatory T-cell populations

A. NK-cells can be identified by a CD56/CD57 stain in the cells that are CD3 and CD19 negative. There are three NK-cell populations visible: CD56dim, CD56high and the CD56dim/CD57 pos fraction. Those cells clearly differ in their expression patterns for HLA-DR and CD16, shown in the three adjacent plots. B. Regulatory T cells are not a homogeneous population. Three populations can be identified in a plot of HLA-DR versus CD45RA. Also the expression of chemokine receptors CXCR3 and CCR4, as well as PD-1 and CD38, reveal distinct subsets of those cells.

Characterization of human B cells using the B cell subset panel.

Representative analysis of PBMCs from healthy human donors (N = 2). Lymphocytes were first identified based on light scatter properties (not shown). A) From the CD3⁻CD19⁺ B cell gate, CD38^highCD24⁺ transitional B cells and CD38^dim/⁻CD24⁺ cells were identified. From the CD38^highCD24⁻ gate, antibody-secreting cells (ASC) or plasmablasts were defined as CD38^highCD27^highCD20⁻ cells. From the CD38^dim/⁻CD24⁺ gate, naïve (blue), non-class-switched (orange) and class-switched memory (red) B cells were identified based on differential expression of CD27 and IgD. B) Expected expression patterns for IgM and IgG throughout B cell differentiation were observed. Samples were acquired on a 5-laser, 18-color BD LSRFortessa™ X-20 Flow Cytometer. Data analysis was performed using FlowJo™ v10 Software. A panel enabling resolution of the same B cell subsets was also designed for and tested on a 3-laser, 12-color BD FACSCelesta™ Flow Cytometer.

Identification of differentiated, senescent and exhausted human T cell subsets using the T cell senescence and exhaustion panel

Representative analysis of CD8⁺ T cells from PBMCs isolated from healthy human subjects (N = 3). Lymphocytes were first identified based on light scatter properties (not shown). A) From the lymphocyte gate, CD3⁺ total T cells and CD8⁺ subset thereof were then defined. From the CD8⁺ T cells gate, central memory (CM), effector memory (EM) and effector memory RA (EMRA) subsets were identified based on differential expression of CD45RA and CD197 (CCR7). From the CD45RA⁺CCR7⁺ gate, CD95⁺ stem cell memory and CD95– naïve T cells were further identified. B-C) The expression of CD27, CD28, CD57 and PD-1 was assessed within each indicated T cell subset. As expected, T cell differentiation was marked by a progressive downregulation of CD27 and CD28 (B), and upregulation of CD57 and PD-1 (C). Samples were acquired on a 5-laser, 18-color BD LSRFortessa™ X-20 Flow Cytometer. Data analysis was performed using FlowJo™ v10 Software. A panel enabling resolution of the same T cell subsets was also designed for and tested on a 3-laser, 12-color BD FACSCelesta™ Flow Cytometer.

Identification of classical human immune cell subsets circulating in human blood

T cells, B cells, DCs and NK cells were gated on CD45+ cells by excluding 7AAD+ inviable cells and doublets. Classical T cells were identified as either CD4+, CD8+ or TCRγδ+, followed by identification of well-characterized T cell subsets based on the expression of CD62L and CD45RA or CD45RO (central memory and TN/SCM naïve and stem cell memory T cells), CD45RA and CCR7 (naïve, central memory, effector memory and terminally differentiated effector memory cells), CD95 and CCR7 (stem memory T cells TSCM), CD127 and CD25 (Tregs), and CD185 and CD45RA (T follicular helper cells). PD1 expression was assessed in CD8+ naïve, central memory, effector memory and terminally differentiated effector memory cells, and KLRG1 expression was assessed in CD8+ TEMRA cells. Classical DCs were identified as CD3- CD19- CD20- CD16- CD14- CD56- HLA-DR+, followed by identification of the pDC subset exclusively as CD303+CD123+. Basophils were identified as CD3- CD19- CD20- CD16- CD14- CD56- HLA-DR- CD123+. NK cells were identified as CD3- CD19- CD20- CD14- CD123- HLA-DR-. Mature and immature NK cells were distinguished based on the expression of CD16 and CD56 followed by identification of KIR-NK cells as CD57+CD158+ mature NK cells. NK cells and non-NK cells were assessed for the expression of CD122. B cells were identified as CD19+ followed by identification of mature B cells as CD27-IgD+. Plasmablasts (CD27+ CD20-) were identified by assessing IgD- B cells. IgG expression was compared between IgD- B cells and mature B cells. Data was acquired on a BD FACSymphony™ A5 SE Cell Analyzer and analyzed with FlowJo™ v10.9.0 Software

Characterization of human B cells using the B cell subset panel.

Representative analysis of PBMCs from healthy human donors (N = 2). Lymphocytes were first identified based on light scatter properties (not shown). A) From the CD3⁻CD19⁺ B cell gate, CD38^highCD24⁺ transitional B cells and CD38^dim/⁻CD24⁺ cells were identified. From the CD38^highCD24⁻ gate, antibody-secreting cells (ASC) or plasmablasts were defined as CD38^highCD27^highCD20⁻ cells. From the CD38^dim/⁻CD24⁺ gate, naïve (blue), non-class-switched (orange) and class-switched memory (red) B cells were identified based on differential expression of CD27 and IgD. B) Expected expression patterns for IgM and IgG throughout B cell differentiation were observed. Samples were acquired on a 5-laser, 18-color BD LSRFortessa™ X-20 Flow Cytometer. Data analysis was performed using FlowJo™ v10 Software. A panel enabling resolution of the same B cell subsets was also designed for and tested on a 3-laser, 12-color BD FACSCelesta™ Flow Cytometer.

Identification of differentiated, senescent and exhausted human T cell subsets using the T cell senescence and exhaustion panel

Representative analysis of CD8⁺ T cells from PBMCs isolated from healthy human subjects (N = 3). Lymphocytes were first identified based on light scatter properties (not shown). A) From the lymphocyte gate, CD3⁺ total T cells and CD8⁺ subset thereof were then defined. From the CD8⁺ T cells gate, central memory (CM), effector memory (EM) and effector memory RA (EMRA) subsets were identified based on differential expression of CD45RA and CD197 (CCR7). From the CD45RA⁺CCR7⁺ gate, CD95⁺ stem cell memory and CD95– naïve T cells were further identified. B-C) The expression of CD27, CD28, CD57 and PD-1 was assessed within each indicated T cell subset. As expected, T cell differentiation was marked by a progressive downregulation of CD27 and CD28 (B), and upregulation of CD57 and PD-1 (C). Samples were acquired on a 5-laser, 18-color BD LSRFortessa™ X-20 Flow Cytometer. Data analysis was performed using FlowJo™ v10 Software. A panel enabling resolution of the same T cell subsets was also designed for and tested on a 3-laser, 12-color BD FACSCelesta™ Flow Cytometer.

Assessment of activation-induced markers using the activated T cell panel.

Representative analysis of T cells magnetically enriched from PBMCs isolated from a healthy donor. T cells were cultured for 24 hours in the absence (resting, top blue histogram) or presence (activated, bottom red histogram) of Dynabeads Human T-Activator CD3/CD28 (Thermo Fisher Scientific). A) Lymphocytes were first identified based on light scatter properties (not shown). Live T cells were identified as CD3⁺ 7-AAD⁻ cells prior to gating of CD4⁺ and CD8⁺ T cells. B-C) Expression of activation markers was assessed in either resting (blue, top histogram) or activated (red, bottom histogram) CD4⁺ (B) and CD8⁺ (C) T cells. As expected, T cell activation induced upregulation of all the tested markers. The sample was acquired on a 5-laser, 18-color BD LSRFortessa X-20™ Flow Cytometer. Data were analyzed using FlowJo™ v10 Software.

This panel is available for purchase as Cat. No. 653848.

The BD Horizon™ Dri Memory T Cell Panel is a 7-color flow cytometry panel designed to identify major subsets of human CD4⁺ or CD8⁺ T cells, including naïve, stem cell memory, central memory, effector memory and terminally differentiated effector memory re-expressing CD45RA (EMRA) T cells. This panel is available as unit sized, preformulated and performance-optimized dried cocktail.

Identification of subsets of human monocytes using the BD Horizon™ Dri Monoset Panel.

Representative analysis of whole blood from healthy human subjects (N = 5). Samples were lysed with BD FACS™ Lysing Solution after surface marker staining using a lyse/no wash procedure. Cells were first gated as SSC^low CD14⁺/ ⁻, followed by gating of cells with high forward scatter. From the SSC^lowFSC^high gate, monocytes were identified as HLA-DR⁺ CD14^low/⁺ cells. From the monocytes gate, classical monocytes were identified as CD14⁺CD16⁻ cells. From the CD16⁺CD14^low/⁺ gate, CCR2^high intermediate and CCR2^low/ ⁻ non-classical monocytes were further detected. Samples were acquired on a 3-laser, 12-color BD FACSLyric™ Flow Cytometer. Data analysis was performed using FlowJo™ v10 Software.

Identification of subsets of human T helper cells using the CD4+ T cell subset panel.

Representative analysis of PBMCs isolated from human healthy subjects (N = 5). T cells were first gated based on scatter properties typical of lymphocytes (not shown). A) After gating on CD3⁺CD4⁺ T cells, regulatory T cells (Tregs) and conventional T cells (non-Tregs) could be identified based on differential expression of CD127 and CD25. From the non-Tregs gate, conventional memory Th cells and T follicular helper cells (Tfh) were defined as CD45RA⁻CXCR5⁻ and CD45RA⁻CXCR5⁺ cells, respectively. Tfh could be further dissected into CXCR3⁺CCR6⁻ Tfh1, CXCR3⁻CCR6⁻ Tfh2 and CXCR3⁻CCR6⁺ Tfh17 subsets. From the memory Th cells gate, Th1, Th2, Th17, Th22, Th9, ThG subsets were defined based on differential expression of CXCR3, CCR4, CCR6, CCR10 and CRTH2 using a gating strategy adapted from Wingender et al.3 B) From the Tregs gate, memory/effector Tregs and T follicular regulatory cells (Tfr) were defined as CD45RA⁻CXCR5⁻ and CD45RA⁻CXCR5⁺ cells, respectively. Memory/effector Tregs could be further dissected into CXCR3⁺CCR6⁻ Th1-like, CXCR3⁻CCR6⁻ Th2-like, CXCR3⁻CCR6⁺CCR4⁺CCR10⁻ Th17-like and CXCR3⁻CCR6⁺CCR4⁺CCR10⁺ Th22-like subsets. Samples were acquired on a 3-laser, 12-color BD FACSLyric™ Flow Cytometer. Data were analyzed using FlowJo™ v10 Software.

Figure 1. Clear separation of cytotoxic immune cell populations in healthy human peripheral blood.

Based on forward and side scatter cell features, an initial gate around lymphocytes excluded debris and most monocytes/granulocytes. Subsequent gates eliminated cell doublets. Then, FVS575V-labeled dead cells and other lineage cells (CD19+/CD14+/CD123+/CD141+) were also excluded enriching the samples with T cells and NK cells. Within live and lineage-negative cells, analysis of CD56 versus CD3 revealed various cell populations that were color coded as cytokine-producing NK cells (pink), cytotoxic NK cells (blue), CD56+ T cells containing NKT cells (brown), CD56+ T cells containing γδ T cells (green) and cytotoxic CD8+ T cells (purple).

Figure 2. Comparative analysis of circulating cytotoxic cells.

Each colored histogram represents a cell population gated as depicted in Figure 1. A. The top row reveals the expression of key granzymes (GrzmK and GrzmB) and perforin. B. The bottom row shows the expression of two major activating (CD314, NKG2D) and inhibitory (CD159a, NKG2A) receptors and the death receptor CD95 (Fas). The fluorescence minus one (FMO) histograms were generated after gating on the total live and lineage negative cells.

Figure 3. Phenotyping of circulating cytotoxic cells using a 16-color panel.

The plots represent the analysis of cytolytic proteins in combination with various cell differentiation markers, enabling a deeper characterization of the cell populations gated in Figure 1. A. Overlay of NK cell subsets. B. Analysis of CD56dimCD3+ T cell subsets, highlighting cell subsets that express either GrzmB or GrzmK. C. Analysis of CD56dimCD3bright T cell subsets also showing GrzmB and GrzmK in the different subsets. D. Identification of activated CD8 T cells based on the expression of CD38 and HLA-DR. The HLA-DR FMO staining helped to determine the gating boundaries for proper detection of the double positive cells. The figure also depicts different CD8 T cell subsets based on the expression of GrzmB versus GrzmK or CD95 versus CD57.

Deep immunophenotyping of human dendritic cells using the dendritic cell subset panel.

Representative analysis of PBMCs isolated from healthy human subjects (N = 3). A) Identification of major dendritic cell (DC) subsets. After exclusion of CD3⁺, CD56⁺ and CD19⁺ lineage cells (not shown), DCs were identified and gated as HLA-DR⁺CD14⁻ cells. From this gate, CD141⁺ cDC1 (blue) and CD16⁺ DCs (green) were identified. From the CD141⁻CD16⁻ gate, Axl⁺ DCs were defined as Axl⁺Siglec-6⁺ cells. Axl⁺ DCs were further divided into CD123⁺ (violet) and CD11c⁺ (orange) Axl⁺ DCs. From the Axl⁻Siglec-6⁻ gate, CD123⁺CD11c⁻ pDCs (aqua) and CD11c⁺CD1c⁺ cDC2 (red) were identified. B) The 18-color panel further enabled the analysis of eight additional DC phenotyping markers and the identification of distinct expression patterns across the major DC subsets. Samples were acquired on a 5-laser, 28-color BD FACSymphony™ A3 Flow Cytometer. Data were analyzed using FlowJo™ v10 Software. .

Assessment of markers on the surface of ILC1 cells using a 21-color flow cytometry panel

Fresh PBMCs from the same healthy donor were stained with fluorochrome-conjugated antibodies. A. Gating strategy for analysis of ILC groups. Cells were gated based on conventional phenotypes, ILC1 (CD117⁻CD294⁻), ILC2 (CD117⁺/⁻CD294⁺),ILC progenitor cells ILCP/ILC3 (CD117⁺CD294⁻). Samples were acquired on the BD FACSymphony A5 Cell Analyzer. Lineage⁻CD3⁻CD56⁻CD127⁺CD117⁻CD294⁻ ILC1 cells were further gated into three subsets: CD4⁻CD8⁻ (blue), CD4⁺CD8⁻ (red) and CD4⁻CD8⁺ (green). B. Protein expression patterns across the three ILC1 subsets.

This set of backbone markers clearly resolves major lymphocyte cell subsets.

Human whole blood was stained with the Lymphocyte Backbone Panel and acquired on a 5-laser BD FACSymphony™ A3 Cell Analyzer. Performance was also tested on the BD FACSymphony™ A5 and BD FACSymphony™ A5 SE Cell Analyzers. Initial gating was done on the lymphocyte population. Plot A is gated from singlets. Clear resolution of all cell subsets was observed irrespective of the instrument used (not shown).

Washed human whole blood was stained with the Lymphocyte Backbone Panel Block and the B cell Maturation and Subset Panel Block and acquired on a 5-laser BD FACSymphony™ A3 Cell Analyzer.

Performance was also tested on the BD FACSymphony™ A5 and BD FACSymphony™ A5 SE Cell Analyzers. Clear resolution of the B cell subsets was observed irrespective of the instrument utilized (not shown). B cells were gated according to the Lymphocyte Backbone Panel Block. Furthermore, performance of the B Cell Panel Block was tested both with the B Cell Subset Panel Block as well as the T Cell Subset Panel Block with comparable results.