Abstract
Chemokines are molecules that regulate the positioning of cells during homeostasis and inflammation. CXCL10 is an interferon-induced chemokine that attracts cells that express the chemokine receptor CXCR3 on their surface. CXCL10 expression is often induced upon inflammation and guides lymphocytes, such as T and NK cells, into the injured tissues. Notably, CXCL10 binding to CXCR3 induces receptor internalization and, therefore, low CXCR3 levels in cells positive for CXCR3 expression can be indicative of chemokine signaling.Here, we describe an in vitro method to evaluate the ability of murine CD8+ T cells to migrate towards recombinant murine CXCL10; and a flow cytometry assay to measure CXCR3 expression levels at the surface of T cells, after exposure to different doses of chemokine.
Keywords: Chemokines, Lymphocytes, Migration, Transwell, Receptor-internalization
Background
Chemokine-mediated T cell trafficking is an important process during homeostasis and inflammation. Activated CD8+ T cells express chemokine receptors, such as CXCR3, allowing them to migrate towards the chemokines CXCL9, 10 and 11, often upregulated at the injured tissue. The evaluation of molecular cues that modulate T cell migration is important to understand the biology behind their functions but the complex mechanisms operating in vivo are sometimes hard to deconvolve. Here, we provide detailed information on an in vitro method to evaluate chemokine functions on CD8+ T cells, focusing on CXCL10-mediated chemo-attraction and CXCR3 internalization. We use antigen-specific transgenic CD8+ T cells that can be easily expanded and activated in vitro, therefore providing enough number of phenotypically identical lymphocytes (e.g., high chemokine-receptor expression on their surface), required to perform an assay using enough replicates for biologically significant observations and statistical analysis. The cells used per assay originate from one single animal, therefore accounting for reduction of animal usage. This assay is combined with flow cytometry analysis, permitting simultaneous evaluation of 1) number of migrating CD8+ T cells; and 2) phenotypic characterization of chemokine receptor levels on their surface.
Materials and Reagents
Equipment
Software
Procedure
Data analysis
After exclusion of debris and dead cells (Aqua+ cells) the flow cytometry plots are visualized as represented in Figure 1. For calculation of the number of T cells that migrated into the lower chamber, gate on CD8+ cells (red gate) and on fluorescent beads (blue gate) and follow the instruction for calculation of cell numbers by the provider of the beads. For evaluation of CXCR3 levels on the surface of T cells, plot gated CD8+ T cells as histograms (Figure 2). You can obtain the values of mean fluorescence intensity (MFI) on the FlowJo software and plot them as graphs as well. It is recommended to run at least 3 technical replicates, per experiment. Figure 1. Example of flow cytometry plots corresponding to the cells collected from the receiving chambers of the transwell plate, after exposure to migration media (left plot) or migration media supplemented with 1,000 ng/ml of recombinant murine CXCL10 (right plot). Cells were stained and fluorescent counting beads (gated in blue) were added to the cells as described in the Procedure B section. Debris and Aqua+ (dead) cells were excluded before this representation. Live CD3+CD8+ T cells are gated in red. APC: Allophycocyanin. Figure 2. Example of flow cytometry histograms representing CXCR3 staining on CD3+CD8+ T cells collected from the receiving chambers of the transwell plate, after exposure to migration media (black) or migration media supplemented with 1,000 ng/ml of recombinant murine CXCL10 (red). % of max: percentage of maximum staining intensity; PE: R-Phycoerythrin.
Recipes
Acknowledgments
When using this protocol, please refer to Barreira da Silva et al. (2015) Nat Immunol. Funding was provided by the Pasteur-Roux post-doctoral fellowship (RBdS), the Ligue Contre le Cancer and the Fondation ARC pour la recherche sur le cancer (MLA) and the French government’s Invest in the Future Program, managed by the Agence Nationale de la Recherche (LabEx Immuno-Onco [RBdS, MLA]). Animal experimental protocols were approved by the comité d’éthique pour l’expérimentation animale (The ethics committee for animal experimentation) Paris.
References
If you have any questions/comments about this protocol, you are highly recommended to post here. We will invite the authors of this protocol as well as some of its users to address your questions/comments. To make it easier for them to help you, you are encouraged to post your data including images for the troubleshooting.