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.
