BD ACCURI NEWS

 

September 2018


Spotlight

Detecting malaria parasites in blood cells

Sptlight - Malleret Thumb

Benoît Malleret is an assistant professor of Microbiology and Immunology at the National University of Singapore. His research focuses on the physiopathology and cell biology of malarial parasites as part of his lab’s focus on erythrocytic immunobiology. Dr. Malleret shared with us the advantages of his new lab developed assay for detecting these parasites in blood cells and explained why he wanted to adapt it for a BD Accuri™ personal flow cytometer.

Read the interview »

Noteworthy

Our final issue

Noteworthy - Accuri Small

Although this is the last issue of BD Accuri News, we will continue to generate exciting new data sheets and updates on the BD Accuri C6 Plus, which we will share on our website. We thank you all for your ongoing support in making the BD Accuri C6 Plus one of the leading personal research cytometers.

Go to the BD Accuri C6 Plus web page »
Browse the BD Accuri News archive »

Application Highlight

Evaluating multiple inhibitory receptors associated with T-cell exhaustion

T-cell exhaustion is a dysfunctional state caused by persistent antigen stimulation and defined by loss of antigen-specific CD8+ T-cell effector function. One hallmark of T-cell exhaustion is increased expression of inhibitory receptors, which normally serve as immune checkpoints transiently expressed by activated CD8+ T cells that play a key role in maintaining immune homeostasis and preventing autoimmunity. However, persistent stimulation can induce a sustained upregulation of inhibitory receptors that can be exploited by tumor cells.

The role of inhibitory receptors in T-cell exhaustion can be challenging to study because there are so many of them. For example, cytotoxic T-lymphocyte-associated protein 4 (CTLA-4, CD152) and programmed cell death 1 (PD-1, CD279) are two well-characterized inhibitory receptors. Both are associated with T-cell exhaustion, but exert their inhibitory function through different mechanisms. Because the immune system has developed multiple, nonredundant inhibitory mechanisms to ensure self-tolerance, effective studies of T-cell exhaustion should involve a combinatorial analysis of multiple inhibitory receptors. Multicolor flow cytometry is exceptionally well suited for a comprehensive immunophenotypic analysis of exhausted T cells because of its unique ability, in a single-tube assay, to simultaneously analyze the expression of multiple parameters at the single-cell level.

A new BD white paper presents a 12-color panel (Table 1) for the simultaneous analysis of six different inhibitory receptors associated with T-cell exhaustion within distinct T-cell subsets on a 3-laser BD FACSCelesta™ flow cytometer. The panel follows multicolor design principles that take into account antigen co-expression and relative expression, fluorochrome brightness and spillover. High-performing antibodies and fluorochromes, analyzed on the highly sensitive BD FACSCelesta system, ensure optimal resolution and quantitation of all the populations of interest.

App Highlight - Table 1

Table 1. 12-color inhibitory receptor panel for the BD FACSCelesta BVR configuration The panel analyzes six inhibitory receptors within fresh or cultured T-cell subsets and follows best panel-design principles.

We first delineated the expression patterns of the six inhibitory receptors in fresh, unstimulated peripheral blood mononuclear cells (PBMCs) from a healthy donor. CD3 was included in the panel to exclude contaminants such as monocytes and NK cells. CD4 and CD8 were used to broadly identify helper and cytotoxic T cells, respectively. CD45RA, CD197 and CD95 enabled the identification of naïve (Tn), stem cell memory (Tscm), central memory (Tcm), effector memory (Tem) and effector memory RA (Temra) T-cell subsets. Finally, CTLA-4, LAG-3, BTLA, PD-1, TIGIT and TIM-3 were the six inhibitory receptors whose expression patterns were analyzed within different T-cell subsets as well as combinatorially. No viability dye was included as fresh, unstimulated PBMCs are mostly viable.

To simulate persistent stimulation conditions, we magnetically enriched T cells from the same PBMC preparation and stimulated them in vitro with CD3/CD28 and IL-2 for nine days. This panel was identical except that CD3 was replaced with the viability dye 7-AAD to exclude dead cells resulting from stimulation and in vitro culture. Because pre-enrichment of the T cells and nonpermissive culture conditions eliminated potential contaminants (non-T cells), CD3 could be omitted.

App Highlight - Fig 2

Figure 1. Expression of multiple inhibitory receptors within T-cell subsets

App Highlight - Fig 2 Zoom
Figure 1. Expression of multiple inhibitory receptors within T-cell subsets

Fresh PBMCs were isolated from a healthy donor, stained with the 12-color panel (including CD3) described in Table 1 and acquired and analyzed on a BD FACSCelesta flow cytometer (BVR configuration). See white paper for method and gating details. Results: A. Representative analysis of TIGIT expression within total CD8+ T cells and subsets. B. Distribution of cells expressing each inhibitory receptor, showing unique expression patterns throughout CD8+ T-cell differentiation.

Separately, T cells were magnetically enriched from the same PBMC preparation using the BD IMag™ Human T Lymphocyte Enrichment Set-DM and cultured for 9 days in the presence of Dynabeads® Human Activator CD3/CD28 (1:1 ratio; Invitrogen) and human recombinant IL-2 (25 U/ml; Roche). Cells were stained with the panel in Table 1 (including 7-AAD), acquired and analyzed. See white paper for method and gating details. Results: C, D. Unique patterns of expression were observed for each inhibitory receptor, with overall upregulation induced by activation as compared to fresh PBMCs, except for BTLA and TIGIT. The data shows the utility of a multicolor flow cytometry assay for the assessment of expression and distribution of multiple inhibitory receptors within distinct T-cell subsets.

Partial results are shown in Figure 1. Figure 1A shows a representative expression histogram for the inhibitory receptor TIGIT in unstimulated PBMCs. TIGIT is expressed by a discrete subset of total CD8+ T cells. Within different subsets, TIGIT was not expressed on naïve (Tn) cells, while its expression was observed in a subset of Tscm cells and thereafter remained constant throughout differentiation.

Figure 1C shows that persistent stimulation suppressed TIGIT expression for all cell subsets. Notice that naïve (Tn) cells do not appear in Figure 1C since stimulation resulted in complete depletion of these cells..

Figures 1B and 1D graph the expression patterns for all six inhibitory receptors by unstimulated and stimulated T-cell subsets, respectively. Notably, CTLA-4, LAG-3 and TIM-3 were upregulated upon persistent stimulation, although with different levels of expression in different CD8+ T-cell subsets.

For details on gating of T-cell subsets, effects of stimulation on subset distribution and bivariate co-expression and combinatorial expression patterns for both CD4+ and CD8+ T cells, see the white paper.

With a choice of four different laser configurations including up to three lasers and 12 fluorescence parameters, the BD FACSCelesta flow cytometer is designed to help you extract a deeper level of biological information from your cell types of interest. The ability to simultaneously analyze six inhibitory receptors within five differentiation subsets of both CD4+ and CD8+ cells in a single tube assay, while retaining optimal resolution of all the populations of interest, makes the BD FACSCelesta a powerful tool for efficiently determining the differentiation and exhaustion state of chronically stimulated T cells.

Download the new T-cell exhaustion white paper »
Download the BD FACSCelesta brochure »


 

Tips & Tricks

Use software templates to simplify and streamline acquisition and analysis

Free, downloadable templates are available on bdbiosciences.com/accuri for many BD kits for popular applications such as stem cell pluripotency and differentiation, immunophenotyping, apoptosis, cell cycle, microbial counting and intracellular cytokines. Figure 2 shows how a BD Stemflow™ stem cell template appears when it is first opened, and again when data has been collected into its pre-gated, pre-labeled plots. Once the template is loaded, you can modify the preset gates and other settings to fit the cells being analyzed.

Tips and Tricks - Fig 3 Software Thumb

Figure 2. Free software templates speed acquisition and analysis

Tips and Tricks - Fig 3 Software Zoom
Figure 2. Free software templates speed acquisition and analysis

Free downloadable software templates, such as this one for the BD Stemflow™ Human and Mouse Pluripotent Stem Cell Analysis Kit (Cat. No. 560477), include gates, labels, run criteria, compensation settings and other workspace elements. The overlay (bottom right) shows how acquired data arrives in the template, ready for analysis.

If there is a particular assay that is run often on a BD Accuri flow cytometer, save time by creating a custom software template and saving it for future use. A template contains a predefined workspace for quick and easy setup and analysis. All markers, regions, gates, parameter names, sample names and compensation settings can be predefined.

Browse kits and templates for the BD Accuri C6 Plus »
Browse kits and templates for the BD Accuri C6 »


 

Publication Picks

This section highlights interesting recent articles that describe research using BD Accuri flow cytometers.

siRNA therapy for breast cancer

Parmar MB, Meenakshi Sundaram DN, KC RB, et al. Combinational siRNA delivery using hyaluronic acid modified amphiphilic polyplexes against cell cycle and phosphatase proteins to inhibit growth and migration of triple-negative breast cancer cells. Acta Biomater. 2018;66:294-309. PubMed

Dexmedetomidine effects on apoptosis

Zhang Y, Tan X, Xue L. The alpha2-adrenoreceptor agonist dexmedetomidine protects against lipopolysaccharide-induced apoptosis via inhibition of gap junctions in lung fibroblasts. Biochem Biophys Res Commun. 2018;495:92-97. PubMed

Amazon plant extract effects on squamous carcinoma cells

Ciani F, Tafuri S, Troiano A, et al. Anti-proliferative and pro-apoptotic effects of Uncaria tomentosa aqueous extract in squamous carcinoma cells. J Ethnopharmacol. 2018;211:285-294. PubMed

Bacterial changes from palm oil plant discharge

Sharuddin SS, Ramli N, Mohd-Nor D, et al. Shift of low to high nucleic acid bacteria as a potential bioindicator for the screening of anthropogenic effects in a receiving river due to palm oil mill effluent final discharge. Ecol Indicators. 2018;85:79-84. Abstract


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