Rapid and Robust Analysis of Cellular and Molecular Polarization Induced by Chemokine Signaling

Podsumowanie

Chemokine signaling elicits marked alterations of cellular morphology and some important redistributions of intracellular proteins. Here, a rapid and detailed protocol is provided to study these events.

Streszczenie

Cells respond to chemokine stimulation by losing their round shape in a process called polarization, and by altering the subcellular localization of many proteins. Classic imaging techniques have been used to study these phenomena. However, they required the manual acquisition of many cells followed by time consuming quantification of the morphology and the co-localization of the staining of tens of cells. Here, a rapid and powerful method is described to study these phenomena on samples consisting of several thousands of cells using an imaging flow cytometry technology that combines the advantages of a microscope with those of a cytometer. Using T lymphocytes stimulated with CCL19 and staining for MHC Class I molecules and filamentous actin, a gating strategy is presented to measure simultaneously the degree of shape alterations and the extent of co-localization of markers that are affected by CCL19 signaling. Moreover, this gating strategy allowed us to observe the segregation of filamentous actin (at the front) and phosphorylated Ezrin-Radixin-Moesin (phospho-ERM) proteins (at the rear) in polarized T cells after CXCL12 stimulation. This technique was also useful to observe the blocking effect on polarization of two different elements: inhibition of actin polymerization by a pharmacological inhibitor and expression of mutants of the Par6/atypical PKC signaling pathway. Thus, evidence is shown that this technique is useful to analyze both morphological alterations and protein redistributions.

Wprowadzenie

Chemokines are small soluble proteins that attract cells to specialized locations¹. Therefore, they participate in the correct positioning of cells in tissues, a crucial function in development and physiology. The immune system is no exception to this rule as it relies on the action of many different cell types which act in concert to mount an effective immune response. By controlling the specific location of one immune cell type in a given state, chemokines are pre-required before foreign antigens can be detected and neutralized.

In T lymphocytes in particular, chemokines bind to specific surface receptors which, upon engagement, elicit many intracellular signals (calcium rise, ERK phosphorylation, Rho GTPases activation, increase in integrins affinity and cytoskeletal alterations) that favor T cell motility^2,3. At the cellular level, one can observe morphological alterations elicited upon chemokine stimulation. These changes in cell shapes are especially dramatic in T cells: resting T cells have a bead-like round morphology when travelling in the blood stream. However, the sensing of the presence of chemokines in inflammatory sites or at proximity of lymphoid organs is going to change the shape of T cells which now adopt a typical “hand-mirror” morphology consisting of a bipolar form: a leading edge at the front and a trailing edge, or uropod, at the back⁴. In addition, intracellular components can segregate into these two opposite regions of a polarized T cell to sustain migration. For example, actin filaments polymerization increases upon chemokine stimulation⁵ and polymerized actin accumulates at the front of a polarized T cell². On the other hand, several proteins, such as phosphorylated proteins of the Ezrin-Radixin-Moesin (ERM) family which link the plasma membrane to the cortical F-actin cytoskeleton, re-localize at the uropod of polarized T cells⁶. Interestingly, we and others have shown that this polarization process is required for T cell migration. Indeed, any treatment that interferes with polarization will inhibit cellular motility. For instance, inhibition of the activity of the members of the atypical protein kinases C (PKC) family, PKCζ and PKCι blocks T cell polarization and their migratory scanning process of dendritic cells⁷. T cell polarization is also regulated by Rho GTPases. We have shown that the modulation of RhoA activity by the recently described Fam65b protein interferes with T cell changes in morphology and their capacity to migrate in a Transwell assay⁶. As polarization is a prerequisite step for cellular motility, it is thus crucial to be able to quantify it as a main readout of chemokine responses. Cell shape alterations were previously measured manually⁸. However, this kind of quantification is very time-consuming, so that usually only a few tens of cells are taken into account.

Here, a new method is presented to rapidly quantify the degree of shape alterations of T lymphocytes exposed to chemokine stimulation. An imaging flow cytometry technology (see Table of Specific Reagents/Equipment) is used, which combines the advantages of a flow cytometer and a microscope⁹ to quantify efficiently the amount of polarized cells in different conditions of chemokine stimulation. In addition to the quantification of the morphological alterations that one can measure robustly with this technology, it is also possible to evaluate changes in the subcellular localization of some proteins upon chemokine signaling.

Protokół

1. Preparation of T Lymphocytes

1. Prepare primary mouse or human T cells as described^10,11.
2. Cultivate human T cells in complete RPMI medium with 10% human serum AB at a density of 2 - 3 x 10⁶ cells/ml.
   NOTE: The use of fetal calf serum (FCS) instead of human serum can elicit spontaneous polarization of a large fraction of human T cells in culture. Keep these cells in culture for 4 - 5 days and further process them at any time during this period.
3. Maintain mouse T cells in complete RPMI medium supplemented with 10% FCS at a similar cell density. Use right away or the next day, after an O/N culture at 37 °C in the presence of 10 ng/ml IL7.
4. Cultivate the CEM human T cell line in complete RPMI supplemented with 10% FCS.

2. Chemokine Stimulation

1. Wash 5 x 10⁵ cells per experimental condition in warm HBSS medium supplemented with 10 mM Hepes. For some experiments, co-transfect primary human T cells the day before with 2 µg pmaxGFP and 8 µg pcDNA3.1⁺ (empty vector, control), PKCζ kinase dead, or N-terminal Par6 plasmids.
2. Resuspend the cell pellet in warm HBSS-Hepes medium (use 0.3 ml x number of experimental conditions).
3. Distribute the cells in 1.5 ml microcentrifuge tubes.
   NOTE: Therefore, one tube corresponding to a single experimental condition will contain 5 x 10⁵ cells in 0.3 ml HBSS-Hepes medium. For some experiments, add 500 nM Latrunculin A or vehicle (DMSO) and incubate the cells for 30 min before chemokine stimulation.
4. Add 10 to 500 ng/ml CCL19 or CXCL12 chemokine in each tube.
   NOTE: We usually use 10 - 200 ng/ml chemokine for human T cells. CEM cells do not express the CCR7 receptor that binds CCL19. Therefore, CEM cells will only be able to respond to a CXCL12 stimulation. Moreover, use higher chemokine concentrations (300 - 500 ng/ml) to polarize mouse T cells.
5. Flip the tubes several times and incubate them in a 37 °C water bath for 8 or 10 min for primary T cells or CEM, respectively.
6. Stop the polarization process by adding to each tube 0.3 ml of a warm solution containing 2% paraformaldehyde (PFA) and 10 mM Hepes in PBS. Flip the tubes and incubate them at 37 °C for 5 min.

3. Stainings

1. Transfer the cells from the microcentrifuge tubes to flow cytometry tubes.
2. Fill the tubes completely with a RT solution containing 1% bovine serum albumin (BSA), 0.5 mM EDTA and 10 mM Hepes in PBS.
3. Centrifuge the tubes at 460 x g for 4 min.
4. Discard the supernatant and resuspend the cells in 100 µl of a RT solution containing 5% FCS in PBS.
5. Add 5 µl of FITC-anti-HLA-ABC in each tube, vortex them, and incubate them for 30 min at RT, away from light.
6. Wash cells once with PBS containing 5% FCS, and once with PBS only.
7. At RT: add 500 µl 1% PFA, incubate for 10 min, then wash cells with a solution containing 1% BSA, 0.5 mM EDTA and 10 mM HEPES in PBS.
8. Discard the supernatant, fill the tubes completely with a solution of PBS containing 0.2% BSA, 0.1% saponin, 0.5 mM EDTA and 10 mM Hepes (permeabilization buffer).
9. Wash cells twice in this medium.
10. Flip the tubes to remove the medium and vortex them to dissociate the cell pellet.
11. Add 0.25 µg/ml TRITC-Phalloidin and/or a 1:100 dilution of anti-P-ERM antibody to each tube, vortex them and incubate for 30 min at RT.
12. Wash cells with the permeabilization buffer. Incubate with a fluorescent secondary antibody if necessary.
13. Resuspend the cells in 200 µl PBS and transfer them to microcentrifuge tubes.
    NOTE: Cells are ready for acquisition. Alternatively, while keeping a maximum total volume of 200 µl per tube, add concentrated PFA to a final 1% concentration in order to preserve the stainings if the cells are not to be used the same day for further processing.

4. Images Acquisition and Analysis

1. Switch on the apparatus (see Table of Specific Reagents/Equipment) and let it calibrate itself according to the manufacturer’s instructions.
   NOTE: The INSPIRE software was used with a 40X objective (0.75 NA). The IDEAS 3.0 software was used for all the reported quantifications.
2. Prepare a series of acquisition windows which can be saved in a template for future experiments. Draw histograms that quantify the RMS gradient values. From the "In Focus" population, selected on the RMS gradient histogram, delimitate the "Single cell" population on a histogram of the Area. Once the microcentrifuge tube has been inserted in the machine, adjust the laser power, gate on individual cells and acquire 5,000 T lymphocytes.
3. In the Ideas software, open the acquisition file and create a histogram that will show the value of the gradient RMS value for bright field images (Channel 1) obtained for each individual cell. Draw a gate on the RMS gradient histogram that considers cells exhibiting RMS gradient value above 57.
   NOTE: The RMS gradient allows taking into account in the analysis only the T lymphocytes that are in the focal plane. We have determined empirically that individual cells exhibiting a RMS gradient value superior to 57 are in the focal plane, and can be accurately considered.
4. In the Analysis/Masks menu, create a new mask, called Erode(M01,2) from the morphology mask on the brightfield images M01, eroded of 2 pixels. Then, in the Analysis/Features menu, create a new feature of the Area, calculated on the Erode(M01,2) mask. From the cells selected in the previous gate, open a histogram showing the Area of cells using this newly created mask.
   NOTE: The morphology masks that are available by default are much bigger than the actual size of the cell. Thus, it is more precise to erode it up to 2 pixels.
5. Draw a gate on the individual T cells, excluding the calibration beads and debris (small Area) and the doublets (large Area). Adjust this gate at each acquisition.
   NOTE: Variations in cell size will affect the position of the gate. As mouse T cells are smaller than human T cells which are themselves smaller than CEM cells, the area of each cell type will be proportional to its size.
6. Considering the single, in-focus cells that were gated at the previous steps, open a dot plot consisting of the Raw Max Pixel on the HLA-ABC staining (Channel 2, x axis) as a function of the Raw Max Pixel on the phalloidin staining (Channel 4, y axis). Create a gate to exclude the unstained or saturated fluorescent events. Open two histograms showing the Raw Max Pixel for HLA-ABC (Channel 2) and phalloidin (Channel 4) stainings.
7. In the Analysis/Masks menu, create a new mask, called Erode(M02,2) from the morphology mask of the HLA-ABC stained cells (Channel 2), eroded of 2 pixels. Then, in the Analysis/Features menu, create a new feature consisting of the Circularity parameter, calculated on the Erode(M02,2).
   NOTE: This parameter measures the deviation of the cell shape from a circle. Therefore, a perfectly round T cell will have a high circularity value whereas elongated polarized cells will exhibit a low circularity index.
8. Subsequently, create another histogram that will report the values of the Circularity feature from the previously gated cells. Use this histogram to directly plot the distribution of an individual, in-focus cell population according to the value of the Circularity for each individual cell.
9. Looking at the shape of the histogram for non-stimulated cells, draw a gate for polarized cells, starting at the lowest circularity value up to the circularity limit value where most of the non-stimulated cells are plotted. Look at the statistics display for the percentage of polarized cells among the gated population (%Gated).
   NOTE: We have set this gate for Circularity index values below 13.
10. In order to look at the polarization of the actin staining in the cells, plot a histogram for the Bright Detail Similarity R3 value, comparing the HLA-ABC staining (Channel 2) and the phalloidin staining (Channel 4).
    NOTE: The bigger the value of this feature, the more superimposed the two stainings are.
11. Using the same gating strategy as before, plot a gate on the non-stimulated cells for the segregated staining that shows the percentage of cells with polarized actin staining.
12. When completed, the percentage of cells exhibiting a segregation in the distribution of the MHC Class I and phalloidin stainings, the percentage of polarized cells together with the mean values of the Circularity and Similarity indexes of all the cells ± SD are shown in the corresponding statistics panels.

Wyniki

The first example chosen here concerns the use of human primary T lymphocytes stimulated with CCL19. However, the same strategy can be used with primary mouse T cells, T cell lines or any other cell type responsive to chemokine stimulation. The gating strategy presented here includes a series of windows that select the focused events, then the single cells. Finally the range of fluorescence intensity is followed here as an example on two markers: the MHC Class I HLA-ABC and filamentous actin for resting (Figure 1...

Dyskusje

Using a recent technology of imaging flow cytometry, a rapid and informative gating strategy to analyze cellular and molecular events induced by chemokine stimulation is presented. From a single experiment, one can obtain two main types of information: the changes in cell morphology induced by chemokine stimulation and the subcellular distribution of different proteins during the polarization process. Interestingly, evidence is also provided for the possibility to analyze a large number of cells, which allows statistical...

Materiały

Name	Company	Catalog Number	Comments
RPMI	Gibco	61870-010
Human serum AB	PAA	C11-021	Pre-heat to inactivate the complement.
Fetal calf serum	PAN Biotech	P30-3300	Pre-heat to inactivate the complement.
Human T cell nucleofactor kit	Lonza	VCA-1002
Murine IL7	Peprotech	217-17
HBSS	Gibco	14025-050	Warm in 37 °C water bath before use.
Hepes	Gibco	15630-056
Murine CCL19	Peprotech	250-27B	Aliquots are thawed on ice before adding the chemokine to the cells.
Human CCL19	Peprotech	300-29B	Aliquots are thawed on ice before adding the chemokine to the cells.
Human CXCL12	Peprotech	300-28A	Aliquots are thawed on ice before adding the chemokine to the cells. This chemokine can also be used on mouse T cells.
PFA	Electron Microscopy Sciences	157-8-100	This is a 8% PFA solution in water. Mix volume to volume with 2x PBS to obtain a 4% PFA solution in PBS.
BSA	Sigma	A3059
Saponin	Fluka	84510
Alexa Fluor 594 phalloidin	Invitrogen	A12381
FITC-anti-HLA-ABC antibody	Beckman Coulter	IM1838	clone B9.12.1
Anti-P-ERM antibody	Cell Signaling Technology	3149P
ImageStreamx Mark II	Amnis