Our technique allows the precise imaging of lung-accumulated human immune cells in in vivo inflammatory conditions providing exciting insights into the process of T cell lung homing. This protocol supports translational research on T cell lung homing and may thereby help in the identification of new targets for an optimized therapy of inflammatory or allergic pulmonary diseases. In particular, the ability to consider the influence of disease-specific immune cell properties, pulmonary tissue organization, and inflammatory milieu on T cell lung homing makes this method highly advantageous.
Overall, our technique is of high value for immunological research in the field of chronic inflammatory lung diseases, which are often driven by an overwhelming tissue accumulation of activated immune cells. After confirming a lack of response to toe pinch in an at least 16-gram, six-week-old, anesthetized C57 black 6J mouse, use a micropipet to slowly deliver 10 microliters of freshly prepared papain solution into one nostril of the mouse once a day for three consecutive days. The day after the last papain administration, layer 10 milliliters of Ficoll medium under human peripheral blood from a healthy volunteer pre-diluted at a one to two ratio in PBS for density gradient separation by centrifugation.
Transfer the peripheral mononuclear blood cell or PBMC interphase layer into a new 50-milliliter tube and collect the cells by centrifugation. Next, perform magnetic microbead isolation of CD4 positive cells according to the manufacturer's protocol. Re-suspending the bead isolated CD4 positive T cells at an up to one times ten to the seventh T cells per at least 500 microliters of PBS concentration.
Label the cells with an appropriate red light excitable cell proliferation dye for 10 minutes at 37 degrees Celsius. At the end of the incubation, arrest the labeling reaction with five volumes of RPMI medium supplemented with 10%FBS for five minutes on ice followed by three washes in medium. For adoptive transfer of the human CD4 positive T cells into papain-exposed recipient mice, re-suspend the fluorescently labeled T cells to a one times 10 to the seventh cells per milliliter concentration in PBS and load 100 microliters of cells into one one-milliliter syringe equipped with a 30-gauge needle per recipient animal, then inject the entire volume of cells from one syringe into the tail vein a papain-treated animal maintaining the needle tip within the vein for an additional five seconds after cell delivery to prevent discharge of the cell suspension.
Three hours after adoptive cell transfer, puncture the right ventricle of each euthanized mouse with a 21-gauge cannula connected to a catheter and make an incision in the left ventricle. Slowly perfuse the lung with 20 milliliters of ice-cold PBS supplemented with five millimolar EDTA followed by two milliliters of ice-cold 4%paraformaldehyde. When all of the fixative has been flushed through the tissue, remove the salivary glands and cut the sternal hyoid muscle to allow a forceps to be slid under the trachea to expose the airtube tissue.
Perform an initial puncture of the exposed trachea with a 30-gauge needle before replacing the needle with a blunt 30-gauge catheter to prevent further damage to the tissue. Use a needle holder to seal the junction between the inserted catheter and the trachea, and carefully fill the airways with approximately 37 degrees Celsius 0.75%agarose until complete unfolding of the lung. A tight junction between catheter and trachea is critical for expanding the lung to its physiological size and maintaining tissue architecture for imaging.
When the agarose has completely solidified, remove the catheter and carefully transfer the lung into a darkened, two-milliliter tube filled with 4%paraformaldehdye. Next, dehydrate the tissue with sequential ethanol incubations under continuous rotation at 31 rotations per minute and four degrees Celsius as indicated for four hours per incubation. After complete dehydration, transfer the sample into ethyl cinnamate for an overnight incubation at room temperature until the tissue appears translucent.
For a light-sheet fluorescent microscopy, use a small drop of organic solvent stable glue to stick the lung to the sample holder of the microscope and set the lung lobe onto the holder in an upright position. Place the sample holder into its chamber and select the appropriate lasers for the experiment in the instrument software. Next, use the sliders under Optics to set a sheet width that numerical aperture that uniformly reveal the whole organ or a specific section of interest.
Use the slider to set the laser intensity under laser transmission control for each selected laser and click Apply. Use Advanced measurement settings to merge the left and right light sheets to create a homogeneously illuminated image. Define the start and end positions of the Z stack to be measured under scan range and set the step size to five micrometers then select autosave to save the files automatically and begin the measurement to capture the images.
When all of the images have been acquired, activate the Surpass button in the 3D post-imaging analysis software and load the first image from one Z stack to initiate the opening and automatic 3D reconstruction of all other images of the selected Z stack, then under Display Adjustment, adjust the intensity, black level and contrast for each channel. To compare the accumulation of human cells within the pulmonary tissue across different samples, select Edit and Crop 3D to define a lung cube with a specific volume. To quantify the labeled cells within the defined lung cube, open Add New Spots in the toolbar, click Skip automatic creation, edit manually, and select the 640 nanometer channel.
Set the radius scale to 10.00 microns, click Select, and under Edit, Shift click with the left mouse button to individually mark each fluorescing cell with a dot. The overall number of counted cells will be displayed in the statistics. To capture an image, use the snapshot tool and or use the animation tool to capture a video.
Microbead-based cell enrichment and subsequent fluorescence labeling as demonstrated typically result in a CD4 positive purity of at least 95%and a successful labeling of these cells as determined by flow cytometry. Ethyl cinnamate-based tissue clearing achieves a high level of organ transparency indicating a successful refractive index matching. The auto-fluorescent signal of the lung tissue offers a helpful tool to image the anatomic structure of the lung by light-sheet microscopy which can be displayed as a mean intensity projection or in surface mode.
Compared to conventional microscopy, light-sheet microscopy offers the advantage of whole organ imaging and subsequent 3D reconstruction allowing the identification and visualization of proliferation dye-labeled, transferred human T cells in the context of the whole organ, moreover, the preference of transferred human T cells for selective accumulation within the inflamed lung tissue of papain-exposed animals is further supported by the fact that human CD4 positive T cells cannot be retrieved from the intestinal mucosa of the recipient animals. For the quantification and comparison of the pulmonary accumulation of human CD4 positive T cells between different conditions, the labeled cells can also be counted in several lung cubes of defined volume as demonstrated. Careful tissue preparation including lung perfusion, inflation and tissue clearing is highly important for an optimized generation of high resolution light-sheet microscopic images.
To further confirm the successful diapedesis of transferred human lymphocytes, this protocol can easily be combined with the flow cytometric quantification of human lymphocytes within the murine bronchoalveolar lavage. The ability to monitor immune cells derived from individual patients in in vivo conditions supports the identification of functional alterations in immune cells imprinted by a particular disease or therapy.
