Name: assessment of lymphocyte migration in an ex vivo transmigration system
Uploaded: 2019-09-20T14:30:00
Description: Watch this Scientific Journal Video about Assessment of Lymphocyte Migration in an Ex Vivo Transmigration System at JoVE.com

This work was funded by the American Lung Association (K.J.W.), the Memorial Eugene Kenney fund awarded to T.A.W. and K.J.W., generous start-up support from the University of Utah for K.J.W., and a Department of Veterans Affairs award to T.A.W. (VA I01BX0003635). T.A.W. is the recipient of a Research Career Scientist Award (IK6 BX003781) from the Department of Veterans Affairs. The authors wish to acknowledge editorial assistance from Ms. Lisa Chudomelka. The authors thank the UNMC Flow Cytometry core for their support in collecting the flow cytometry data generated for this manuscript.

All methods described here were reviewed and approved by the Institutional Animal Care and Use Committees at the University of Nebraska Medical Center (UNMC) and the University of Utah.
1. Setup and Preparation of Reagents
<ol>
	<li>Prepare complete RPMI (Roswell Park Memorial Institute) media.
	<ol>
		<li>Add 10 mL of heat-inactivated fetal bovine serum (FBS) to 90 mL of RPMI.</li>
		<li>Add 1 mL of 100x Penicillin-Streptomycin-Glutamine to 100 mL of 10% FBS RPMI.</li>
	</o...

<ol>
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Herein, we present a well-established method for assessing chemokine-induced migration of lymphocytes in an ex vivo transmigration system. There are several critical steps in the protocol, the first of which is verifying the expression of the correct chemokine receptor on the immune cells in the experiment. In our hands, we chose CCR4 because of the body of literature that highlights the importance of CCR4 on Th2 helper T cells in allergic inflammation. Ovalbumin-induced inflammation was shown previously to be limited by...

This original transmigration method was presented by Stephen Boyden in 1962 in the Journal of Experimental Medicine1. Much of what we know about chemotaxis and chemokinetics would not be possible without the development of the Boyden chamber. Prior to the discovery of the first chemokine in 1977, ex vivo transmigration systems were used to learn about serum-factors that could arrest cellular movement in macrophages while amplifying cellular motility in neutrophils1,2. A massive wealth of knowledge has been developed regarding immune cell migration, and to date, 47 chemokines have now been discovered with 19 corresponding receptors3,4. In addition, multitudes of inhibitors/enhancers of these chemokine pathways have undergone development for therapeutic purposes5,6,7,8. Many of those compounds have been tested in similar transmigration chambers to understand direct interactions between the compounds and immune cell responsiveness to a given chemokine9.
Transmigration, or diapedesis, into inflamed tissue is an essential process to a healthy inflammatory response to clear infection10,11. A Boyden chamber, transmigration system, or transwell apparatus are generally composed of two chambers separated by a porous membrane1,12. The bottom chamber most often holds media containing the chemokine of interest, while leukocytes are placed in the top chamber. The size of the pore in the membrane can be selected based on the size of the cell of interest. For this project, we selected a 3 &#181;m porous membrane, as lymphoid cells are 7-20 &#181;m in size, depending on the stage of cellular development. This pore size ensures that these cells are not passively falling through the pores, but that they are actively migrating in response to the chemokine gradient.
The major advantage of this protocol is its cost effectiveness. In vivo transmigration is difficult because it requires extensive training in animal handling and surgery, and often involves high-powered microscopy that is not always available to a researcher. Cost effective screening of compounds thought to enhance or inhibit transmigration can be accomplished in advance of in vivo imaging. Because the transmigration system is tightly controlled, cells may be treated initially then added to the transwell apparatus, or, vice versa, the chemokine may be treated first with a chemokine inhibitor then cells added to the transwell apparatus. Lastly, endothelial cells and/or basement membrane proteins can be added to the bottom of the transwell insert 1-2 days prior to the transmigration experiment to understand the involvement of these barrier cells in chemokinetics. Again, these manipulations of the system provide a powerful means of determining important information about the effectiveness of a given compound in advance of more complicated in vivo studies.
Utilizing a transmigration chamber system is an effective way to assess lymphocyte mobility under various in vivo and in vitro conditions12,13,14. Herein, we describe an optimized method for assessing ex vivo lymphocyte responsiveness to chemokines in a transmigration chamber. In this example experiment, CD4+ T cells and group 2 innate lymphoid cells (ILC2) were isolated from male and female, BALB/c mice following OVA-allergen exposure. A hypothesis was generated that CCR4+ CD45+ Lineage- (LIN-) ILC2 from allergen-challenged mice would migrate more efficiently towards CCL17 and CCL22 than CCR4+ CD4+ T helper cells. CCL17 and CCL22 are chemokines commonly produced by dendritic cells and macrophages of the M2 (allergic) phenotype, among other cells, in allergy15,16. CCL17 and CCL22 can be thought of as biomarkers of allergic inflammation as they are readily detected in the lungs during airway exacerbations16,17,18. Importantly, CCR4 expression is elevated in comparison to untreated controls, as revealed in bioinformatic data generated from ILC2 isolated from house dust mite treated animals, and similarly ILC2 from na&#239;ve animals treated ex vivo with IL-33 (allergen-promoting innate cytokine) upregulates CCR419,20. Furthermore, according to data for ILC2 in the Immunological Genome Project database (www.immgen.org), CCR4 mRNA is highly expressed in these innate immune cells. To date, little is known regarding trafficking of ILC2 into tissues, but it is likely that the ILC2 and CD4+ T cells use similar chemokines and receptors for chemotaxis and chemokinetics as they express similar transcription factors and receptors. Thus, we compared CCL17 versus CCL22 responsiveness, of ILC2 and CD4+ T lymphocytes, from both male and female, OVA-challenged animals.

<table>
	<tbody>
		<tr>
			<td>0.4% Trypan Blue</td>
			<td>Sigma-Aldrich</td>
			<td>15250061</td>
		</tr>
		<tr>
			<td>1 mL syringe</td>
			<td>BD Bioscience</td>
			<td>329424</td>
			<td>U-100 Syringes Micro-Fine 28G 1/2&#34; 1cc</td>
		</tr>
		<tr>
			<td>100x Penicillin-Streptomycin, L-Glutamine</td>
			<td>Gibco</td>
			<td>10378-016</td>
			<td>Dilute to 1x in RPMI media</td>
		</tr>
		<tr>
			<td>15 mL conical tubes</td>
			<td>Olympus Plastics</td>
			<td>28-101</td>
			<td>polypropylene tubes</td>
		</tr>
		<tr>
			<td>3 um transwell inserts</td>
			<td>Genesee Scientific</td>
			<td>25-288</td>
			<td>24-well plate containing 12 transwell inserts</td>
		</tr>
		<tr>
			<td>3x stabilizing fixative</td>
			<td>BD Pharmigen</td>
			<td>338036</td>
			<td>Prepare 1x solution according to manufacturers protocol</td>
		</tr>
		<tr>
			<td>5 mL polystyrene tubes</td>
			<td>STEM Cell Technologies</td>
			<td>38007</td>
		</tr>
		<tr>
			<td>50 mL conical tubes</td>
			<td>Olympus Plastics</td>
			<td>28-106</td>
			<td>polypropylene tubes</td>
		</tr>
		<tr>
			<td>8-chamber easy separation magnet</td>
			<td>STEM Cell Technologies</td>
			<td>18103</td>
		</tr>
		<tr>
			<td>ACK Lysing Buffer</td>
			<td>Life Technologies Corporation</td>
			<td>A1049201</td>
		</tr>
		<tr>
			<td>Advanced cell strainer, 40 um</td>
			<td>Genesee Scientific</td>
			<td>25-375</td>
			<td>nylon mesh, 40 micron strainers</td>
		</tr>
		<tr>
			<td>Aluminum Hydroxide, Reagent Grade</td>
			<td>Sigma-Aldrich</td>
			<td>239186-25G</td>
			<td>20 mg/mL</td>
		</tr>
		<tr>
			<td>anti- mouse CCR4; APC-conjugated</td>
			<td>Biolegend</td>
			<td>131211</td>
			<td>0.5 ug/test</td>
		</tr>
		<tr>
			<td>anti-mouse CD11b</td>
			<td>BD Pharmigen</td>
			<td>557396</td>
			<td>0.5 ug/test</td>
		</tr>
		<tr>
			<td>anti-mouse CD11c; PE eFluor 610</td>
			<td>Thermo-Fischer Scientific</td>
			<td>61-0114-82</td>
			<td>0.25 ug/test</td>
		</tr>
		<tr>
			<td>anti-mouse CD16/32, Fc block</td>
			<td>BD Pharmigen</td>
			<td>553141</td>
			<td>0.5 ug/test</td>
		</tr>
		<tr>
			<td>anti-mouse CD19; APC-eFluor 780 conjugated</td>
			<td>Thermo-Fischer Scientific</td>
			<td>47-0193-82</td>
			<td>0.5 ug/test</td>
		</tr>
		<tr>
			<td>anti-mouse CD3; PE Cy 7-conjugated</td>
			<td>BD Pharmigen</td>
			<td>552774</td>
			<td>0.25 ug/test</td>
		</tr>
		<tr>
			<td>anti-mouse CD45; PE conjugated</td>
			<td>BD Pharmigen</td>
			<td>56087</td>
			<td>0.5 ug/test</td>
		</tr>
		<tr>
			<td>anti-mouse ICOS (CD278)</td>
			<td>BD Pharmigen</td>
			<td>564070</td>
			<td>0.5 ug/test</td>
		</tr>
		<tr>
			<td>anti-mouse NK1.1 (CD161); FITC-conjugated</td>
			<td>BD Pharmigen</td>
			<td>553164</td>
			<td>0.25 ug/test</td>
		</tr>
		<tr>
			<td>anti-mouse ST2 (IL-33R); PerCP Cy5.5 conjugated</td>
			<td>Biolegend</td>
			<td>145311</td>
			<td>0.5 ug/test</td>
		</tr>
		<tr>
			<td>Automated Cell Counter</td>
			<td>BIORAD</td>
			<td>1450102</td>
		</tr>
		<tr>
			<td>Automated Dissociator</td>
			<td>MACS Miltenyi Biotec</td>
			<td>130-093-235</td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin, Lyophilized Powder</td>
			<td>Sigma-Aldrich</td>
			<td>A2153-10G</td>
			<td>0.5% in serum-free RPMI</td>
		</tr>
		<tr>
			<td>Cell Counter Clides</td>
			<td>BIORAD</td>
			<td>1450015</td>
		</tr>
		<tr>
			<td>Chicken Egg Ovalbumin, Grade V</td>
			<td>Sigma-Aldrich</td>
			<td>A5503-10G</td>
			<td>500 ug/mL</td>
		</tr>
		<tr>
			<td>Collagenase, Type 1, Filtered</td>
			<td>Worthington Biochemical Corporation</td>
			<td>CLSS-1, purchase as 5 X 50 mg vials (LS004216)</td>
			<td>25 U/mL in RPMI</td>
		</tr>
		<tr>
			<td>compensation beads</td>
			<td>Affymetrix</td>
			<td>01-1111-41</td>
			<td>1 drop per contol tube</td>
		</tr>
		<tr>
			<td>Dissociation Tubes</td>
			<td>MACS Miltenyi Biotec</td>
			<td>130-096-335</td>
		</tr>
		<tr>
			<td>FACS Buffer</td>
			<td>BD Pharmigen</td>
			<td>554657</td>
			<td>1x PBS + 2% FBS, w/ sodium azide; stored at 4 &#176;C</td>
		</tr>
		<tr>
			<td>Heat Inactivated-FBS</td>
			<td>Genesee Scientific</td>
			<td>25-525H</td>
			<td>10% in complete RPMI &#38; ILC2 Expansion Media</td>
		</tr>
		<tr>
			<td>mouse CCL17</td>
			<td>GenScript</td>
			<td>Z02954-20</td>
			<td>50 ng/mL</td>
		</tr>
		<tr>
			<td>mouse CCL22</td>
			<td>GenScript</td>
			<td>Z02856-20</td>
			<td>50 ng/mL</td>
		</tr>
		<tr>
			<td>mouse CD4+ T cell enrichment kit</td>
			<td>STEM Cell Technologies</td>
			<td>19852</td>
		</tr>
		<tr>
			<td>mouse IL-2</td>
			<td>GenScript</td>
			<td>Z02764-20</td>
			<td>20 ng/mL</td>
		</tr>
		<tr>
			<td>mouse ILC2 enrichment kit</td>
			<td>STEM Cell Technologies</td>
			<td>19842</td>
		</tr>
		<tr>
			<td>mouse recombinant IL-33</td>
			<td>STEM Cell Technologies</td>
			<td>78044</td>
			<td>20 ng/mL</td>
		</tr>
		<tr>
			<td>RPMI</td>
			<td>Life Technologies Corporation</td>
			<td>22400071</td>
		</tr>
		<tr>
			<td>Separation Buffer</td>
			<td>STEM Cell Technologies</td>
			<td>20144</td>
			<td>1 X PBS + 2% FBS; stored at 4C</td>
		</tr>
		<tr>
			<td>small animal nebulizer and chamber</td>
			<td>Data Sciences International</td>
		</tr>
		<tr>
			<td>sterile saline</td>
			<td>Baxter</td>
			<td>2F7124; NDC 0338-0048-04</td>
			<td>0.9% Sodium Chloride</td>
		</tr>
	</tbody>
</table>

assessment of lymphocyte migration in an ex vivo transmigration system

Herein, we present an efficient method that can be executed with basic laboratory skills and materials to assess lymphocyte chemokinetic movement in an ex vivo transmigration system. Group 2 innate lymphoid cells (ILC2) and CD4+ T helper cells were isolated from spleens and lungs of chicken egg ovalbumin (OVA)-challenged BALB/c mice. We confirmed the expression of CCR4 on both CD4+ T cells and ILC2, comparatively. CCL17 and CCL22 are the known ligands for CCR4; therefore, using this ex vivo transmigration method we examined CCL17- and CCL22-induced movement of CCR4+ lymphocytes. To establish chemokine gradients, CCL17 and CCL22 were placed in the bottom chamber of the transmigration system. Isolated lymphocytes were then added to top chambers and over a 48 h period the lymphocytes actively migrated through 3 &#181;m pores towards the chemokine in the bottom chamber. This is an effective system for determining the chemokinetics of lymphocytes, but, understandably, does not mimic the complexities found in the in vivo organ microenvironments. This is one limitation of the method that can be overcome by the addition of in situ imaging of the organ and lymphocytes under study. In contrast, the advantage of this method is that is can be performed by an entry-level technician at a much more cost-effective rate than live imaging. As therapeutic compounds become available to enhance migration, as in the case of tumor infiltrating cytotoxic immune cells, or to inhibit migration, perhaps in the case of autoimmune diseases where immunopathology is of concern, this method can be used as a screening tool. In general, the method is effective if the chemokine of interest is consistently generating chemokinetics at a statistically higher level than the media control. In such cases, the degree of inhibition/enhancement by a given compound can be determined as well.

CCR4 expression on CD4+ T cells and ILC2.
For the success of the ex vivo transmigration experiment, it is imperative to determine whether the lymphocytes are responsive to CCL17 and CCL22 through CCR4; therefore, we determined CCR4 expression on both CD4+ T cells and ILC2 by flow cytometry. While it is well known that OVA-specific CD4+ helper T cells express CCR4, less is known of the expression of CCR4 on ILC2. Figure 1 show...

Watch this Scientific Journal Video about Assessment of Lymphocyte Migration in an Ex Vivo Transmigration System at JoVE.com

Assessment of Lymphocyte Migration in an Ex Vivo Transmigration System

In this protocol, lymphocytes are placed in the top chamber of a transmigration system, separated from the bottom chamber by a porous membrane. Chemokine is added to the bottom chamber, which induces active migration along a chemokine gradient. After 48 h, lymphocytes are counted in both chambers to quantitate transmigration.

Herein, we present an efficient method that can be executed with basic laboratory skills and materials to assess lymphocyte chemokinetic movement in an ex ...

Lymphocyte transmigration is an important feature of any immune response. Therefore, this protocol is significant because it allows a researcher to assess the mobility of lymphocytes in a well-defined in vitro system. The main advantage of this technique is that various chemokines can be assessed to determine their effectiveness of inducing migration in recently defined cells, such as the group two innate lymphoid cells, or ILC2.A second advantage is that inhibitors or biologic therapies aimed at blocking particular chemokine pathways may be tested by this method prior to doing more expensive experiments with animals. Begin by excising lungs and spleens from euthanized ova-treated male and female mice using two to three animals per group. Place excised tissues in separate dissociation tubes according to tissue type and animal gender.Place the lung tissue in 500 microliters of lung dissociation media in a dissociation tube, place the tube in the automated tissue dissociator, and dissociate it using the lung protocol. Repeat these steps for a total of two times. To dissociate spleen tissue, place it in 500 microliters of complete RPMI media in the dissociator, and then use the spleen protocol.Working in a biological safety cabinet using sterile technique from now on, filter each dissociated tissue through a 40-micrometer cell strainer, and collect into 50-milliliter conical tubes. Rinse the dissociation tubes containing lung homogenates with five milliliters of additional lung dissociation media, tubes containing spleen homogenates with five milliliters of complete RPMI, and filter their contents through the filters used for their corresponding tissues. To further dissociate lung tissue, incubate lung homogenates for 15 to 30 minutes in a 37-degree Celsius incubator with 5%carbon dioxide.Then add five milliliters of complete RPMI to both the lung and spleen homogenates, and centrifuge. After discarding the supernatant, combine the pellets containing splenocytes and lung cells from the group of animals of the same sex into a single 50-milliliter conical tube. Add 10 milliliters of complete RPMI to each tube, and resuspend.Determine total cell counts using an automated cell counter. Adjust male and female cell suspensions to 100 million cells per milliliter in separation buffer, and then add to a five-milliliter polystyrene tube. After isolating group two innate lymphoid cells from 2/3 of the total cells as described in the manuscript, use the remaining cells for the CD4-positive T cell isolation procedure.To start CD4-positive T cell isolation, add rat serum to the CD4 T cell enrichment suspension. Then add isolation cocktail to the cell sample, and incubate for 10 minutes at room temperature. Vortex rapid spheres for 30 seconds, and add to the cell sample to achieve a dilution of 75 microliters per milliliter.Gently mix, and incubate for 2 1/2 minutes at room temperature. Top the samples with approximately two milliliters of separation buffer up to three milliliters, place the five-milliliter tubes into the eight-chamber easy separation magnet, and incubate for five minutes at room temperature. Then tip the magnet forward, and pipette each cell suspension into a clean five-milliliter tube.Add 1.5 milliliters of serum-free RPMI to each tube, and centrifuge. Pour off the supernatant, and resuspend the CD4-positive T cell pellet at 10 million cells per milliliter in serum-free RPMI. To begin this part of the protocol, determine the number of Transwell inserts needed for the experiment for both ILC2 and CD4-positive T cells as described in the manuscript.Gently move three-micron Transwell inserts from the middle rows of a 24-well plate to the top and bottom rows of the same 24-well plate with a pipette tip and gloved hands. Add 500 microliters of migration media with CCL17 to 1/3 of the wells in the middle row of the 24-well plate. Add 500 microliters of migration media with CCL22 to another 1/3 of the wells.And finally, add 500 microliters of serum-free RPMI containing no chemokine to the last 1/3 of the wells. Label the lid on the plate clearly with the name of the appropriate transmigration media placed in the bottom wells. Then, place the Transwell inserts back into the wells containing the various treatments.Gently add 100 microliters of CD4 T cells or ILC2 to the top well of each insert, making sure not to mix or pipette the cell suspension up and down in the Transwells. Label the lid on the plate clearly with the cell type placed in each well, and write the date and time of day that the cells were added. Repeat this process starting from removing the Transwell inserts from the plate until all the cells have been placed in Transwell inserts with media.Once the cells of interest are isolated, the most important steps are to keep track of the chemokines and cell types that are placed in particular wells, to gently add cells to the top chambers, and lastly to avoid unnecessary contact with the transmigration plate during the 48-hour incubation. Gently place the plate in a 37-degree Celsius incubator with 5%carbon dioxide, and incubate for 48 hours, making sure to minimize contact with the plate over the incubation period. After gently removing the plate from the incubator, remove all the Transwell inserts from the middle rows into the empty wells just above or below.Collect the cells from the top and bottom wells of the Transwell inserts into tubes labeled with top or bottom with CCL17, CCL22, or media, with the cell type, and with the replicate number. Rinse the bottom wells with 500 microliters of 1x PBS, collect it into the corresponding tubes, and do the same for the top wells. Centrifuge the tubes at 378 times g at room temperature for five minutes.Use a pipette to gently remove all of the supernatant, and then resuspend T cell and ILC2 pellets with 50 microliters of 1x PBS. After removing 10 microliters of the cell suspension, add 90 microliters of 0.4%trypan blue. Count the cells on the automated cell counter.And for each sample, record percentage of viability and cell count per milliliter, and determine the total number of cells per treatment in the top and the bottom chamber. When CCR4 expression was assessed on both CD4-positive T cells and ILC2 by flow cytometry, there were no differences between male and female hosts. However, the expression of CCR4 on a per-cell basis on ILC2 was higher in comparison to CD4-positive T cells.When the bottom chamber of the Transwell apparatus was filled with untreated cell culture media, media containing CCL22, or media containing CCL17, less than 14%of the cells migrated in media control conditions. In response to CCL22, both cell types, regardless of whether they were from male or female hosts, responded to CCL22. Also, CCL22 induced greater migration than CCL17 in male ILC2.On the other hand, CCL17 induced significant migration of the female CD4 T cells and ILC2 compared to media alone. CCL17 treatment was not different than media for male CD4-positive T cells or male ILC2. Under suboptimal conditions, when the plate with CD4 cells remained at room temperature for the first 24 hours and then moved into the incubator, there was no migration toward CCL22 and CCL17.Furthermore, the viability of the cells was notably low for the cells in the bottom chamber, showing the importance of using the correct temperatures and conditions described in this protocol to achieve optimal results. Once the transmigration procedure is completed, one should see significant migration to the chemokine of interest in comparison to the media control wells. If there is not significant migration seen, one can assume that the procedure did not work properly or that the lymphocytes are not responsive to the chemokine in question.This technique can be used broadly to understand lymphocyte migration when those lymphocytes have undergone a broad range of in vivo treatments.

Research

JoVE Journal

Immunology and Infection

Human T Lymphocyte Isolation, Culture and Analysis of Migration In Vitro

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Lymphocyte Isolation from Human Skin for Phenotypic Analysis and Ex Vivo Cell Culture

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The thymus, the primary organ for the generation of &#945;&#946; T cells and backbone of the adaptive immune system in vertebrates, has long been ...

Flow Cytometric Analysis of Particle-bound Bet v 1 Allergen in PM10

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