Induction and Scoring of Graft-Versus-Host Disease in a Xenogeneic Murine Model and Quantification of Human T Cells in Mouse Tissues using Digital PCR

This protocol outlines how to induce xenograft-versus-host disease, or xenoGVHD, and how to blind and standardize clinical scoring to ensure consistent results. The xenoGVHD model provides an in vivo method for testing immunosuppressive therapies against human rather than murine T cells, improving the translation of these studies to the clinic. Demonstrating the procedure will be Amara Seng, a graduate student from my laboratory.

One day before the injection, place eight-to 12-week-old, non-obese diabetic scid gamma, or NSG, mice in a sterilized pie cage, and irradiate the mice in a cesium-137 source with a total dose of 150 centigrays, with a slow rotation to ensure an even irradiation. Then, place the mice into clean cages in a sterile biosafety cabinet overnight. The next morning, collect enough healthy human blood to isolate 1.1 times 10 to the seven peripheral blood mononuclear cells, or PBMCs, per mouse, and dilute the heparinized blood in an equal volume of 2%fetal bovine serum, or FBS, in PBS.

Carefully overlay up to 25 milliliters of the diluted blood onto 15 milliliters of lymphocyte separation density gradient medium in a 50-milliliter conical tube for density gradient centrifugation. At the end of the separation, harvest the PBMC at the interface, and wash the cells in 10 milliliters of PBS in a new 50-milliliter conical tube. Aspirate the supernatant, and flick the tube to loosen the pellet.

Resuspend the PBMCs in 10 milliliters of fresh PBS for another centrifugation, followed by resuspension in five milliliters of fresh PBS for counting. Then, collect the cells with a final centrifugation, and resuspend the pellet at a one times 10 to the eight PBMCs per milliliter of PBS concentration. For retro-orbital delivery of the isolated human PBMC into the mouse eye, load one one-milliliter syringe equipped with a 28-gauge needle with 100 microliters of cells.

Confirm a lack of response to toe pinch, and gently restrain the mouse with the thumb and middle finger. Insert the needle bevel-side down, lateral into the medial canthus, through the conjunctival membrane until the back of the eye is reached. Slightly retract the needle, and slowly inject the entire volume of cells.

Then, fully retract the needle for proper disposal of the syringe and needle. Close the eyelid, and apply mild pressure to the injection site with a gauze sponge. Then, examine the injection site for swelling or other visible trauma, and allow the mouse to regain consciousness in a sterile cage lined with paper towels and monitoring before moving the animal to its home cage.

To measure the GVHD score, place the cage in a laminar flow hood, and remove the food and water from the cage. To score the activity, observe the behavior of the mice for five minutes. To obtain a weight loss score, weigh each mouse in a glass beaker.

While the mouse is still in the beaker, inspect the animal for posture, fur texture, and skin integrity. After harvesting the tissues of interest, cut an approximately three-by-0.5-millimeter piece of tissue from the organ, and weigh the sample on a balance. Transfer the weighted tissue into a sterile 1.5-milliliter tube, and snap-freeze the sample in liquid nitrogen for overnight storage at minus 80 degrees Celsius.

The next morning, thaw the samples before lysis, according to the instructions from a genomic DNA isolation kit. For human T cell quantification by digital polymerase chain reaction, or PCR, prepare digital PCR reactions according to the protocol for DNA binding dyes for the digital PCR machine being used and using the appropriate primers for human CD3 epsilon genomic DNA. Then, carry out the digital PCR reaction under the appropriate reaction conditions.

Sublethally irradiated eight-to 12-week old NSG mice of both sexes that receive human PBMC begin displaying clinical signs of GVHD around day 10 post-injection compared to negative control mice treated with PBS only. XenoGVHD mice have a median survival of 23.5 days. Using digital PCR, CD3 epsilon-positive human T cells can be detected in the lung and liver samples of mice that receive human PBMCs.

It is critical that the scoring is performed consistently and that the researcher scoring the mice is blinded to their treatment. Following this procedure, serum can be collected from euthanized mice for peripheral cytokine expression analysis, and immune cells can be collected from the spleen for analysis via flow cytometry.