In Situ Detection of Autoreactive CD4 T Cells in Brain and Heart Using Major Histocompatibility Complex Class II Dextramers

Ethics Statement:

All animal procedures were conducted in accordance with the guidelines for the Care and Use of Laboratory Animals and approved by the University of Nebraska-Lincoln, Lincoln, NE.

1. Induction of EAE

1. To induce EAE, prepare the peptide/complete Freund’s adjuvant (CFA) emulsion as described in ¹¹ with the following modifications: Step 1.1: prepare PLP 139-151 at a concentration of 1 mg/ml in 1x phosphate buffered saline (PBS). Step 1.4: use 200 µl of CFA emulsion containing 100 µg of PLP 139-151 per animal. Step 1.4.1: to immunize ten animals, prepare 2 ml of emulsion by mixing 1 ml of PLP 139-151 (1 mg/ml in 1x PBS) and an equal volume of CFA. Step 1.5: inject 200 μl of emulsion in split doses subcutaneously in the two inguinal regions (~75 μl each) and sternal region (~50 μl) of five-to-six-week-old female SJL/J mice. Steps 1.7 and 1.8 are not applicable for induction of EAE.

2. Clinical Scoring of EAE Mice and Harvesting of Cerebrums

1. Monitor the animals for appearance of clinical signs, and score the disease-severity daily from day 9 postimmunization as follows: 0 – healthy; 1 – limp tail or hind limb weakness but not both; 2 – limp tail and hind limb weakness; 3 – partial paralysis of hind limbs; 4 – complete paralysis of hind limbs; 5 – moribund or dead ¹².
2. Euthanize the mice when they reach a neurological score of 3 or 4 using CO₂. To euthanize the mice, pre-fill the euthanasia chamber first with CO₂ by turning the regulator of the CO₂ tank not exceeding 6 psi and allow animals to stay within the chamber for approximately 5 min to achieve complete asphyxia. Soak the euthanized animals in 70% alcohol. Immediately, place the animals on an absorbent pad and using a pair of sterile forceps and scissors expose the hearts by cutting through the thoracic cavity. Puncture the right auricles and perfuse by injecting 10 ml of cold 1x PBS into the left ventricles of the hearts.
3. Excavate the skull by snipping the cranium in a circular fashion using a pair of curved scissors, and carefully, flip the skull bones with forceps. Harvest the brain by blunt dissection and using a clean scalpel, separate cerebrum from the cerebellum. Place the cerebrum in a tube containing cold 1x PBS and leave the tube on ice until processing.

3. In Situ Staining of Cerebral Sections with MHC Class II (IA^s)/PLP 139-151 Dextramers

1. Prepare 4% PBS-buffered low-melting agarose, melt by heating at 50 °C and leave it in a 40 °C water bath until use.
2. Place the cerebrum in a disposable deep base mold histology cassette kept over ice, and pour molten (40 °C) agarose. Immediately, press the cerebrum gently with forceps to submerge the tissue. Leave the cassette on ice for 15-20 min until the solidification is complete.
3. Fill the vibratome bath with chilled 1x PBS. Fill the space around vibratome bath with ice cubes to maintain the temperature of PBS between 0 and 2 °C. Fix a clean razor blade into the vibratome and set the blade angle to 15°.
4. Placement of tissue in vibratome. Trim excess agarose from all the sides of the embedded tissue and slightly expose the ventral surface of the cerebrum.
   1. Smear vibratome tissue block with super glue and transfer the agarose-embedded tissue over the glued surface by placing the exposed ventral surface of the cerebrum over the glue for transverse sectioning. Do not move the tissue, once the tissue is set over the block.
   2. Place the vibratome block containing the agarose-embedded tissue on ice, and allow the glue to dry for 15 min.
5. In the meantime, place the tissue chambers one per well of a 24-well plate. Fill the wells containing the tissue chambers with 1 ml of cold 1x PBS, and leave the plate on ice.
   1. Prepare the tissue chambers by attaching a thin nylon mesh to the cut-end of a 25 ml polypropylene pipette using super glue; after trimming the excess mesh around the pipette, cut the pipette to a length of 1 cm from the mesh.
6. Lower the vibratome blade to the level of the top surface of tissue; set the vibratome speed to 0.22 mm/sec and start sectioning with a forward movement to make 200 µm thick sections. Transfer one cerebral section per well using a watercolor soft brush into the tissue chambers placed within the wells of a 24-well plate. Keep the plate on ice until sectioning is complete to prevent tissue degradation. Note: Follow these steps to obtain in total, three paired sections from three different regions of the brain dorsal to the ventral surface (Figure 2). One section from each pair (in total three sections) placed in three individual tissue chambers are designated for staining with specific dextramers (IA^s/PLP 139-151) and anti-CD4. Likewise, another set of three sections that are placed in three individual tissue chambers are designated for staining with control dextramers (IA^s/TMEV 70-86) and anti-CD4.
7. Transfer the three tissue chambers containing the sections designated for staining with specific dextramers into three wells in a 24-well plate with one chamber per well in which, 300 µl of a cocktail containing allophycocyanin (APC)-conjugated PLP 139-151 dextramers (2.5 μg/ml) and anti-CD4-phycoerythrin (PE; 5 µg/ml) in blocking solution (1x PBS with 2% normal goat serum) was previously added. Cover the wells with a lid.
8. Similarly, transfer the three tissue chambers containing the sections designated for staining with control dextramers into three wells in a 24-well plate with one chamber per well in which, 300 µl of a cocktail containing APC-conjugated TMEV 70-86 dextramers (2.5 μg/ml) and anti-CD4- PE (5 µg/ml) in blocking solution was previously added. Cover the wells with a lid. Note: Refer to Massilamany et al., ⁸ for preparation of dextramers, and the dextran molecules to prepare dextramers were kindly provided by Immudex, Copenhagen, Denmark.
9. Label the wells, and wrap the plate with aluminum foil and place the plate on a rocking platform set to gentle rotations for 1.5 hr in the dark at RT.
10. After the incubation period, wash tissue sections on the rocking platform, by transferring the tissue chambers to a fresh well containing 1 ml of cold 1x PBS, and avoid dribbling the contents of one well into the other. Repeat washing thrice, allowing at least 10 min per wash. Ensure that the tissue sections do not get dried as they can stick to the sides of the chamber.
11. Fix the sections by transferring the tissue chambers into a fresh well in 24-well plate containing 1ml of filtered 4% PBS-buffered paraformaldehyde (pH 7.4) on a rocking platform for 2 hr with gentle rotations.
12. Repeat washing thrice as described in step 3.10.
13. Place the stained and fixed sections on a clean microscopic slide using a watercolor soft brush. Wipe excess water without touching the section, and mount the section using mounting medium. Cover with a microscopic coverslip and allow the slides to dry at RT O/N in the dark room.
14. Store the slides in a light-proof slide storage box at 4 °C.

4. Induction of EAM and Collection of Hearts

1. Induce EAM as described in reference ¹¹.
2. Euthanize the mice on day 21 postimmunization using clean chamber fitted with CO₂ gas cylinder, and soak the animals in 70% alcohol. Immediately, place the animals on an absorbent pad and using a pair of forceps and scissors expose the hearts by cutting through the thoracic cavity. Puncture the right auricles and perfuse by injecting 10 ml of cold 1x PBS into the left ventricles of the hearts. Collect the hearts and place in tubes containing cold 1x PBS.

5. In Situ Staining of Heart Sections with MHC Class II (IA^k)/Myhc 334-352 Dextramers

1. Prepare 4% PBS-buffered low-melting agarose, melt by heating at 50 °C and leave it in a 40 °C water bath until use.
2. Place the heart in a disposable deep base mold histology cassette kept on ice, and pour molten 40 °C agarose until the tissues are completely submerged as described in step 3.2.
3. Follow step 3.3.
4. Trim excess agarose from all the sides of the embedded tissue and slightly expose the base of the heart. Smear vibratome tissue block with super glue and transfer the agarose-embedded tissue over the glued surface by placing the exposed surface of the heart over the glue. Do not move the tissue, once the tissue is set over the block.
   1. Place the vibratome block containing the agarose-embedded tissue on ice, and allow the glue to dry for 15 min.
5. Follow steps 3.5 and 3.6.
6. Transfer the heart sections using a watercolor soft brush into the tissue chambers placed within the wells of a 24-well plate. Keep the plate on ice until sectioning is complete to prevent degradation of tissue. Note: Follow these steps to obtain in total, eight paired sections from the apex to base of the heart (Figure 2). Place all sections in tissue chambers, allocating up to 3 sections per chamber. One section from each pair (in total eight sections) is designated for staining with specific dextramers (IA^k/Myhc 334-352) and anti-CD4. Likewise, another set of eight sections is designated for staining with control dextramers (IA^k/RNase 43-56) and anti-CD4.
7. Transfer the three tissue chambers containing the sections designated for staining with specific dextramers into three wells in a 24-well plate with one chamber per well in which, 300 µl of a cocktail containing APC-conjugated Myhc 334-352 dextramers (2.5 μg/ml) and anti-CD4-PE (5 µg/ml) in blocking solution was previously added. Cover the wells with a lid.
   1. Similarly, transfer the three tissue chambers containing the sections designated for staining with control dextramers into three wells in a 24-well plate with one chamber per well in which, 300 µl of a cocktail containing APC-conjugated RNase 43-56 dextramers (2.5 μg/ml) and anti-CD4- PE (5 µg/ml) in blocking solution was previously added. Cover the wells with a lid.
8. Label the wells, and wrap the plate with aluminum foil and place the plate on a rocking platform set to gentle rotations for 1.5 hr in the dark at RT.
9. Follow steps, 3.10 to 3.11
10. Repeat washing thrice as described in step 3.10.
11. Place the stained and fixed sections (up to three sections/slide) on a clean microscopic slide using a watercolor soft brush. Wipe excess water without touching the sections, and mount the sections using mounting medium. Cover with a microscopic coverslip and allow the slides to dry at RT O/N in the dark room.
12. Store the slides in a light-proof slide storage box at 4 °C.

6. Analysis of Dextramer⁺ (dext⁺) CD4⁺ T Cells by Laser Scanning Confocal Microscope (LSCM) (Common to Both Brain and Heart Sections)

1. Use Nikon A1-Eclipse 90i confocal microscope system for routine scanning and acquisition of images. To start Click open, the NIS Elements AR software (version 4.13).
   1. Choose “TRITC” and “Cy5” channels from the panel. The excitation/emission wavelengths respectively for TRITC and Cy5 channels, 561.5 nm / 553-618 nm and 640.7 nm / 663-738 nm appear by default.
   2. Assign the pseudocolors “green” and “red” for the TRITC (CD4-PE) and Cy5 (dextramer-APC) channels, respectively.
   3. Place the slide to be examined on the microscopic stage and focus manually under 100X magnification. Set the “HV” to 110 and offset to “0”. Click “Focus” and adjust the “voltage” to optimize the lasers.
   4. Click “Focus”; scan the section from top to bottom, and set the ‘Z’ level for image acquisition. Click “CH series” and acquire the image sequentially. Identify dext⁺ CD4⁺ T cells appearing as yellow punctate cells based on co-localization of signals generated from both the red (dextramers) and green (CD4) channels.
2. Use Olympus FV500-BX60 confocal microscope for easy quantitative enumeration of dext⁺ CD4⁺ T cells. To begin click open, the FLUOVIEW software (version 4.3).
   1. Select the dyes “Cy3” and “Cy5” from the drop-down menu, and click “apply” to load the respective lasers. Note: the excitation/emission wavelengths for Cy3 (543 nm/pseudocolored “green”; CD4-PE) and Cy5 (633 nm/pseudocolored “red”; dextramer-APC) channels.
   2. Place the slide to be examined on the microscopic slide, and focus it manually under low magnification (4X or 10X). Click “Focus” while the Cy3 laser is on, and focus the inflammatory focus.
   3. Change the objective to 100X magnification. Set the file size to “512/512” from the drop-down menu. Click “Focus” and adjust the values for “PMT”, “Gain” and “Offset” parameters to optimize the lasers separately for Cy3 and Cy5.
   4. Click “Z stage” and then click “Focus” while both the lasers are on. Set the thickness of the “Z stage” by clicking the “up” and “down” arrows. Set the “Z” interval to 2 µm.
   5. Click “Stop”, and choose “Seq123”. Then, click “XYZ” and start acquiring “Z” series images for the selected area within the inflammatory focus. Save the “Z serial images” as AVI files. To save image files, select the image from the “Z serial image”, and then save as “tiff” format.
3. Examine three paired sections from the cerebrum where one set of three sections was stained with IA^s/PLP 139-151 dextramers/anti-CD4 and the other set of three sections with IA^s/TMEV 70-86 dextramers/anti-CD4 to perform quantitative analysis.
   1. Similarly, analyze eight paired sections for heart, in which, one set of eight sections was stained with IA^k/Myhc 334-352 dextramers/anti-CD4 and another set of eight sections with RNase 43-56 dextramers/anti-CD4.
4. Examine 10 to 15 inflammatory foci per section in cerebrum, and in each focus, acquire a set of 15 to 25 serial images sequentially.
   1. Examine a total of 20 inflammatory foci from all the eight sections of heart and from each focus acquire 10 to 15 serial images sequentially. Take the ‘Z’ serial images by scanning the slides in a ‘horizontal-vertical up-horizontal-vertical down’ movement so that repetition of ‘Z’ series within the same focus is avoided.
   2. Once ‘Z’ serial images are captured, name the files by identifying the slide numbers, name of the sections, and the number of the ‘Z’ serial images taken.
      1. Use ‘ImageJ’ software to count dext⁺ CD4⁺ T cells in relation to the total number of CD4⁺ T cells. Select the ‘type of counter’ in ‘ImageJ, check ‘show all’ and start counting by clicking the center of each cell and continue counting in different ‘Z’ serial images by using side arrows. After counting all the CD4 T cells, choose another type of counter and count the CD4⁺ dext⁺ T cells. Click the results tab to get the total number of cells counted with the two different counters.
      2. To obtain the total number, add the number of cells in all the ‘Z’ serial images representing all the three cerebral sections or all the eight heart sections.