Induction of Graft Versus Host Disease in Mouse Models via Allogeneic Bone Marrow and Splenic Cell Transplantation

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

1. GvHD induction

1. Day 0: Total body irradiation of the recipient mice
   1. Use female CD45.2+ H2kd+ BALB/c mice that are at least 10 weeks old as recipients.
   2. Weigh and record the weight of the recipient mice prior to GvHD induction.
      NOTE: Ensure that the recipient has a minimal body weight of 20 g. The starting body weight will serve as the reference value to calculate the weight loss of the individual mouse over the course of GvHD induction and progression.
   3. Place up to five mice in the container of the X-ray irradiator.
   4. Irradiate the mice by total body irradiation with a single dose of 8 Gy of X-ray. Use Cs137 as a radiation source.
2. Day 1: Reconstitution of the irradiated mice with T-cell-depleted bone marrow
   NOTE: The transplantation of allogeneic bone marrow cells, as outlined and detailed under section 1.2, should take place within 24 hours after irradiation. Perform the procedures described below in a sterile tissue culture hood and use filtered reagents. Use allogeneic CD45.1/Ly5.1 B6.SJL-Ptprca Pepcb/BoyCrl mice as donors for T-cell-depleted bone marrow.
   1. Euthanize CD45.1/Ly5.1 B6.SJL mice that will donate allogeneic bone marrow cells in accordance with the institutional guidelines, 12-24 h after the irradiation of the recipient BALB/c mice. For this, anesthetize the CD45.1/Ly5.1 B6.SJL mice by inhalation of 5% isoflurane. Continue with the isoflurane exposure over 1 min after breathing arrest and confirm euthanasia by cervical dislocation.
   2. Disinfect the fur and skin of the mouse thoroughly with 70% ethanol.
   3. Position the mouse onto a clean working sheath so that the mouse is in a prone position, with its hindquarters facing the experimenter. Lift the fur at the Achilles' heel with the tip of Semken forceps with a length of 13 cm and serrated curved tips. Incise the skin between the forceps and the heel with hardened 8.5 cm fine scissors with straight tips.
   4. Elongate the incision cranially (i.e., from the heel over the lower leg and thigh to the hip region). Remove the skin and fur from the hind limb with the help of the forceps.
   5. Perform the same procedure on the other hind limb.
   6. Remove the hind limbs by cutting through the adjacent hip joints.
   7. Cut away the rear paws. Cut through the knee joint. Store the thigh and shank of every hind limb together in a 92 mm Petri dish filled with phosphate-buffered saline (PBS) solution on ice.
   8. Clean the femur and tibia bones carefully by removing as much muscle tissue as possible from every thigh and shank. Transfer the femur and tibia bones to a 50 mL tube filled with sterile RPMI 1640 medium and place it on wet ice until all tibia and femur bones collected in step 1.2.7 are cleaned from muscle tissue.
   9. To isolate the bone marrow, transfer one femur or tibia bone at a time (which has been cleaned as described in step 1.2.8) from the 50 mL tube to a Petri dish (with a diameter of 92 mm) filled with RPMI 1640 medium. Cut off the ends of the femur or tibia with a scalpel to get access to the cavity of the bone containing the bone marrow.
   10. Prepare a 50 mL collection tube with 5 mL of RPMI 1640 medium. Insert a 26 G needle attached to a 1 mL syringe filled with 1 mL of RPMI 1640 medium into the bone cavity and flush the bone marrow out of the cavity into the collection tube by pushing the plunger.
   11. Repeat step 1.2.10 until the bone appears light and shiny (i.e., the bone cavity is visibly devoid of reddish bone marrow). Discard the empty bone.
   12. Repeat steps 1.2.9 – 1.2.11, using all femur and tibia bones from step 1.2.8, and collect all recovered bone marrow pieces in the same 50 mL collection tube from step 1.2.10. Keep the collection tube on wet ice until all bones from step 1.2.8 have been processed accordingly.
   13. Create a single-cell suspension by softly pipetting the flushed, crumbly bone marrow pieces up and down. Filter the bone marrow single-cell suspension through a 40 µm mesh screen cell strainer placed on a new 50 mL collection tube.
   14. Centrifuge the 50 mL collection tube containing the bone marrow single-cell suspension at 450 x g for 5 min at 4 °C. Discard the supernatant.
   15. Resuspend the pellet in 5 mL of ACK lysing buffer (1 mM Na²EDTA; 10 mM KHCO₃; 144 mM NH₄Cl; pH 7.2) for red blood cell lysis. Incubate the cell suspension for 3 min at room temperature. Afterward, immediately add 10 mL of PBS solution to the cell suspension.
   16. Centrifuge the suspension at 450 x g for 5 min at 4 °C. Discard the supernatant and resuspend the cells in 2 mL of PBS solution.
   17. Count the bone marrow cells, using a hemocytometer. Preserve an aliquot of 6 x 10⁶ cells for flow cytometry analysis to check the purity of the cells after cell purification (see step 1.2.20).
   18. For the depletion of T cells from the bone marrow single-cell suspension, use a commercially available cell purification kit. Magnetically deplete CD90.2+ cells (i.e., lymphocytes) from the total bone marrow cells, following the manufacturer's protocol.
   19. Count the T-cell-depleted bone marrow cells that have been isolated as described in step 1.2.18, using a hemocytometer. Preserve an aliquot of 1 x 106 cells for flow cytometry analysis to be performed later on, as described in step 1.2.20.
       NOTE: The on-average cell yield derived from one donor mouse is usually from 2 x 107 to 3.4 x 107 T-cell-depleted bone marrow cells.
   20. Confirm a successful T-cell depletion by flow cytometry. Stain 1 x 10⁶ cells, preserved from steps 1.2.17 (bone marrow cells before magnetic cell separation) and 1.2.19 (T-cell-depleted bone marrow cells after magnetic cell separation), with the following antibodies: α-CD45.1 (A20), α-CD3 (17A2), α-CD4 (GK1.5), and α-CD8α (53-6.7). Use the remaining cells from step 1.2.17 as an unstained control and for single-staining controls, to set up the flow cytometer.
       1. Transfer 1 x 10⁶ cells from each sample to a well of a 96-well plate with a V-bottom to perform the flow cytometric staining procedure.
       2. Spin down the cells within the 96-well plate (step 1.2.20.1) at 450 x g for 5 min at 4 °C. Discard the supernatant and resuspend the cells in 100 µL of FACS buffer (PBS supplemented with 3% filtered bovine serum).
       3. Centrifuge the cells within the 96-well plate at 450 x g for 5 min at 4 °C. Discard the supernatant.
       4. Add an appropriate amount of the antibody of choice (compare with step 1.2.20) to the samples for single staining in 100 µL of FACS buffer.
       5. Prepare a mixture of all antibodies (master mix) containing the appropriate amounts sufficient to separately stain the bone marrow cell aliquots preserved from prior (see step 1.2.17) and after (see step 1.2.19) magnetic T-cell depletion. Add 100 µL of the master mix to both aliquots.
       6. Add only 100 µL of FACS buffer to the unstained sample.
       7. Incubate the cells within the 96-well plate for 20 min at 4 °C in the dark.
       8. Add 100 µL of FACS buffer and centrifuge the cells within the 96-well plate at 450 x g for 5 min at 4 °C. Discard the supernatant and resuspend the cells in 100 µL of FACS buffer.
       9. Centrifuge the cells at 450 x g for 5 min at 4 °C. Discard the supernatant and resuspend the cells in 250 µL of FACS buffer.
       10. Transfer the samples to a 5 mL polystyrene round-bottom tube and analyze them with a flow cytometer instrument that is able to detect the fluorescent molecules that were used to label the antibodies employed to characterize the cell samples (see step 1.2.20).
       11. Compare the cell composition from before and after the magnetic cell separation.
           NOTE: A purity of approximately 95% CD45.1+CD3- (i.e., T-cell-depleted bone marrow) cells within the live gate is usually achieved.
   21. Wash the T-cell-depleted bone marrow cell suspensions 2x with PBS solution (450 x g for 5 min at 4 °C) and, finally, resuspend the cells in PBS solution, adjusting the cell concentration to 5 x 10⁷ cells/mL. Keep the cells on wet ice until the injection.
   22. Inject T-cell-depleted bone marrow cells into the recipient mice, which were irradiated the day before (see section 1.1).
       1. Place the experimental mouse in a leak-proof chamber and induce anesthesia by inhalation of up to 4% isoflurane, until the mouse is unconscious. Confirm analgesia and loss of consciousness by testing the loss of the righting reflex and the subsequent loss of the pedal withdrawal reflex.
       2. Inject 5 x 10⁶ T-cell-depleted bone marrow cells (i.e., 100 µL of 5×10⁷ cells/mL) containing PBS solution using a 1mL syringe equipped with a 30 G needle intravenously into the retrobulbar space containing the venous sinus.
3. Day 2: Transfer of T cells
   NOTE: Perform the described procedures below in a sterile tissue culture hood and use filtered reagents. Use CD45.2 C57Bl/6 (wild-type; WT) mice as donors for alloreactive T cells. The usage of congenic marker systems allows the distinguishability between recipient (CD45.2+ H2kd+ Balb/c mice) and donor hematopoietic cell types (bone marrow cells: CD45.1+ H2kb+ B6.SJL mice; allogeneic T cells: CD45.2+ H2kb+ C57/Bl6 mice).
   1. Euthanize C57Bl/6 mice in accordance with the applying institutional guidelines and authorities. Therefore, anesthetize mice by inhalation with a concentration of 5% isoflurane. Continue the isoflurane exposure until 1 min after breathing arrest and confirm euthanasia by cervical dislocation. Disinfect the fur and skin of the mouse thoroughly with 70% ethanol.
   2. Place a strainer with a 40 µm mesh screen on a 50 mL collection tube. Remove the spleen and place it onto the strainer.
   3. With a syringe plunger, comminute the spleen in the strainer. Wash the strainer and syringe plunger with PBS to collect all splenocytes.
   4. Centrifuge the cells within the 50 mL collection tube at 450 x g for 5 min at 4 °C. Discard the supernatant.
   5. Resuspend the splenocytes in 3 mL of ACK lysing buffer to lyse red blood cells. Incubate the cell suspension for 3 min. Afterward, add 10 mL of PBS and centrifuge the cells at 450 x g for 5 min at 4 °C. Discard the supernatant and resuspend the splenocytes in PBS.
   6. Count the spleen cells, using a hemocytometer. Divert an aliquot of 6 x 10⁶ cells for flow cytometry analysis, to evaluate the magnitude of purification in step 1.3.9.
   7. For a total CD3+ T-cell isolation from total splenocytes, use a commercially available cell purification kit. Isolate the splenic T cells, following the manufacturer's protocol.
   8. Count the splenic CD3+ T cells isolated as described in step 1.3.7, using a hemocytometer. Preserve an aliquot of 1 x 10⁶ T cells for flow cytometry analysis.
      NOTE: The on-average yield of splenic T cells per donor mouse isolated by magnetic cell separation ranges from 1.3 x 10⁷ to 2 x 10⁷ cells.
   9. Confirm the success of the T-cell isolation by flow cytometry. For a detailed description of the staining protocol, confer and follow steps 1.2.20.1 – 1.2.20.10.
      1. Stain 1 x 10⁶ splenocytes of steps 1.3.6 (before the magnetic cell separation) and 1.3.8 (after magnetic cell separation) with the following antibodies: α-CD45.2 (104), α-CD3 (17A2), α-CD4 (GK1.5), and α-CD8α (53-6.7).
      2. Use the remaining cells from step 1.3.6 as an unstained control and for single staining with the respective antibodies, to set up the flow cytometer.
      3. Compare the cell composition before and after magnetic cell separation.
         NOTE: A purity of ≥95% of CD45.2+CD3+ T cells within the lymphocyte live gate should be accomplished.
   10. Wash the T cells 2x with PBS (450 x g for 5 min at 4 °C) and, finally, resuspend the cells in PBS solution, adjusting the cell concentration to 7 x 10⁶ cells/mL. Keep the cells on ice until the injection.
   11. Inject alloreactive, splenic T cells (see step 1.3.10) into irradiated BALB/c mice who have previously received CD45.1+ T-cell-depleted bone marrow cells (see step 1.2.22).
       1. Induce anesthesia by placing the experimental mouse in a leak-proof chamber in which it is exposed to up to 4% isoflurane until it loses consciousness. Confirm analgesia and loss of consciousness by testing the loss of the righting reflex and the subsequent loss of the pedal withdrawal reflex.
       2. Inject 0.7 x 10⁶ alloreactive CD3+ T cells using a 1 mL syringe equipped with a 30 G needle  (i.e., 100 µL of 7 x 10⁶ cells/mL) containing PBS solution (see step 1.3.10), intravenously into the retrobulbar space of the mice, to induce GvHD. Denominate these mice as the experimental group.
       3. Inject a different group of mice with 100 µL of PBS alone, intravenously into the retrobulbar space, and denominate this group as the control group, subsequently called "no-T-cell-transplanted (noT) mice".