Identification of Rare Antigen-Specific T Cells from Mouse Lungs with Peptide:Major Histocompatibility Complex Tetramers

Our lab studies the development of CD4 T cells in different contexts of tolerance and immunity. Across our projects, we are primarily interested in how antigen exposure drives the development of phenotypic subsets of T cells that establish immune memory that can enhance tolerance to itself or immunity against pathogens. Recently, we have focused on the development of regulatory and memory T-cell subsets that reside in lung tissue.

These tissue resident T cells play an important role in reinforcing tolerance to cell antigens during tissue injury, or promoting inflammatory responses to pathogens during reinfection. Our lab specializes in the direct study of antigen-specific T cells, with custom peptide MHC tetramer reagents. This allows us to study these T cells without the use of TCR transgenic models or other experimental manipulations that are known to cause experimental artifacts.

Although we can readily identify antigen-specific T cells with tetramers, these T cells are often present at very low frequencies, particularly when they're in a naive or memory state. It is also challenging to isolate these T cells from non-lymphoid tissues where they're often reside following an immune response. While there are several established protocols for isolating T cells from mouse lungs, the protocol we present here has been optimized for the subsequent detection of low-frequency antigen-specific T cells by tetramer staining.

Within this protocol, we have also included two options for tissue dissociation to better accommodate live resources. Our protocol will help researchers expand the use of tetramers to study antigen-specific T cells in lung tissue. They'll lead to more powerful in vivo studies of tissue resident memory T cell development and function, which are topics of intense interest in the field.

Inject one to three milligrams of fluorophore-conjugated anti-mouse CD45 antibody diluted in 100 milliliters of normal saline intravenously into each anesthetized mouse. Begin by placing four milliliters of ice cold HEPES buffer prepared in a specialized tissue dissociated tube on ice for each mouse. After euthanizing the mouse, place the resected lungs in their respective tissue dissociation tube chilled on ice.

Place the tubes onto the automated tissue dissociation and run the first preset program for lung tissue. Then add 500 microliters of Liberase stock solution and 500 microliters of aminoguanidine stock solution to each tube containing the dissociated lung tissue. Incubate it on a notating mixer for 30 minutes at 37 degrees Celsius.

Afterward, place the tubes back on the dissociator and run the second preset program for lung tissue. Now pour the single cell suspension from the tissue dissociator tube through a 100 micrometer cell strainer into a 50 milliliter conical tube. Rinse the tissue dissociating tube with five milliliters of complete EHAA and pass through the strainer into the 50 milliliter tube.

After removing the strainer, raise the volume of the cell suspension to 50 milliliters with complete EHAA. Instead of using specialized tissue dissociated tubes, place the resected lungs in their respective 1.5 milliliter microfuge tubes chilled on ice. Once all the lungs have been harvested, transfer each lung to a fresh microfuge tube without any cell media.

Using scissors, cut the lungs into small fragments within the microfuge tube. Add one milliliter of Liberase TM and DNase I digestion cocktail to each tube. Incubate for 30 minutes at 37 degrees Celsius in a water bath.

Then pour the sample over a 100 micrometer cell strainer placed on top of a 50 milliliter conical tube. Pipet out remaining sample onto the strainer. Mash the tissue against the mesh with the rubber end of the plunger from a sterile one milliliter syringe.

Rinse the strainer with cold sorter buffer, collecting a final volume of seven milliliters in the tube. Whether using the automated or manual dissociation method, centrifuge the cell suspension obtained. Carefully aspirate the supernatant, leaving behind approximately 100 microliters of supernatant and the cell pellet.

Finally, add approximately 100 microliters of FC block solution to bring the total volume up to 200 microliters and vortex vigorously to resuspend the pellet. To begin, add 50 microliters of anti-mouse CD90.2 microbeads to the isolated lung cells. After vortexing the mixture, incubate it for 10 minutes at four degrees Celsius.

Next, place a paramagnetic cell separation column onto a one or four position cell separation magnet. Position an open 15 milliliter conical tube under each column to capture the flow through. Prime the column with three milliliters of cold sorter buffer, allowing the column to drain by gravity into the conical tube below.

After the cells have incubated with the microbeads for 10 minutes, add cold sorter buffer to a volume of one milliliter and transfer the mixture through a 100 micrometer cell strainer placed atop the column. Collect the column flow through into a fresh 15 milliliter conical tube placed below the column. Once the cell suspension has completely drained through the column, add an additional three milliliters of cold sorter buffer to the original tube and apply it to the column through the mesh before removing the strainer.

When the sample has completely drained into the column and no further flow through is exiting, remove the column from the magnet and place it atop of fresh 15 milliliter conical tube. Add five milliliters of cold sorter buffer to the column. To elute the bound cells, push the column plunger down in one continuous motion, forcing the buffer out of the bottom into a new tube.

Centrifuge the eluted samples at 400 x g for five minutes at four degrees Celsius. Carefully aspirate the supernatant, leaving approximately 100 microliters of the supernatant and the cell pellet. On average, the enrichment process recovers more than 99%of all CD90.2+T cells from the lungs with greater than 90%purity.

After enriching the lung sample for T cells, add one milliliter of 10 millimolar dasatinib to each sample. Vortex the mixture and incubate for five minutes at room temperature. Add PE or APC-conjugated peptide MHC tetramer to a final concentration of 10 nanomolar.

After vortexing the mixture, incubate it in the dark for one hour at room temperature. While the sample is stained with peptide MHC tetramers, prepare a master mix of antibodies to stain the cell surface markers. With 15 minutes remaining in the tetramer staining incubation period, add the surface antibody master mix at a one to 100 dilution.

Add 50, 000 flow cytometry count beads and cold sorter buffer to reach a final volume of approximately five milliliters. Then add cold sorter buffer to a volume of 15 milliliters and centrifuge the tube at 400 x g for five minutes at four degrees Celsius. Carefully aspirate the supernatant, leaving approximately 100 microliters of the supernatant and the cell pellet.

Now resuspend the sample with an additional 200 microliters of sorter buffer and transfer it to a five milliliter FACS tube.