Transplantation of Tail Skin to Study Allogeneic CD4 T Cell Responses in Mice

The overall goal of the following procedure is to perform tail skin transplantation. To study T-cell mediated rejection or tolerance induction in a mouse model, this is accomplished by first harvesting the tail skin from a donor mouse. In the second step, the transplantation site is prepared on the recipient animal.

Next, the graft is transplanted and fixed with tissue adhesive and a bandage in the final step. The bandage is removed on day seven post transplantation and the graft is scored. Ultimately, the T-cell mediated alloimmune response can be studied.

This tail skin transplantation technique provides a powerful model for studying T-cell dependent rejection and tolerance induction during allogenic immune responses in mice. Before beginning the procedure, open the finger strip bandages and apply petroleum jelly onto the wound pads with a cotton swab, making sure the gauze is completely covered. Next, disinfect the tail of a euthanized donor mouse with disinfection solution.

Then position the tail on a clean plastic board. Turn the upper black stripe downside and make a longitudinal superficial incision through the skin along the midline of the tail. Use gripping forceps to peel off the skin and then place the tissue in a 10 centimeter cell culture dish filled with 10 milliliters of HBSS.

Shave the dorsal site of an anesthetized recipient mouse and use a dry piece of gauze to remove any contaminating hairs. After disinfecting the shaved transplantation site with aseptic solution, pinch the skin with forceps in the middle right side of the dorsum and use curved scissors to make a round incision of about one by 0.6 centimeters in diameter. Next, use a scalpel to cut a graft from the excised donor tail skin, trimming the corners to round the tissue, load the graft onto the scalpel, and blot the excess HBSS with a sterile cotton swab.

Then place the dried tissue onto the graft bed of the recipient mouse. Avoid overlapping the donor and host skin applying tissue adhesive onto the contact zone exclusively. Then wrap a plaster without creases around the waist of the mouse and apply an adhesive non elastic bandage to affix the plaster to the mouse to prevent the mouse catching its teeth on the bandage.

During recovery, make a three millimeter incision in the upper ventral side of the material. After full recovery on a warmed heat pad, return the transplanted animal to its cage and assess its mobility. To ensure that the bandage is not too tight, treat the mouse with paracetamol syrup in the animal's water for seven days.

After six to seven days, use sterile artery scissors to cut through and carefully remove the bandages taking care to avoid stretching the transplant site. Finally, monitor the transplanted mice for clinical signs and score for the rejection of the skin grafts. In this experiment, C 57 BL six mice were transplanted with IABM 12 allografts and IAB sin grafts.

After removing the bandages, the graft exhibited signs of wound healing without closure of the contact zone in the C 57 BL six mice. One to two weeks later, a CD four positive T-cell mediated inflammation led to the appearance of necrotic areas inside the graft. Rejection of the I A BM 12 allografts was observed in the C 57 BL six mice within 13 days of the transplantation.

While the syn grafts were tolerated for up to 100 days. Rejection of MHC Class two mismatched tails skin depends on the presence of allogeneic CD four positive T cells. For example, T-cell deficient CD three E knockout mice and t and B-cell deficient RAG two knockout mice exhibit complete wound healing and tolerate I a BM 12 grafts for up to 100 days.

Acceptance of the IABM 12 mismatched tail skin allows the study of allogeneic CD four positive T cells and their effector functions in a mouse model without severe disease pathologies. The advantage of this tail skin transplantation model is that functional analysis of CD four positive T cells can be easily performed by adoptive transfer of antigen specific CD four positive T cells In this representative experiment, the transfer of two times 10 to the fourth antigen specific transgenic A BM cells was sufficient to induce the rejection of IABM 12 graphs in rag two knockout mice within 13 days. The V alpha two V beta eight TCR chain expressing A BM cells were observed to proliferate extensively by the EFL six 70 dilution assay and to produce IFN Gamma upon PMA stimulation taken.Together.

These data demonstrate that only a few allogeneic CD four positive T cells are required to reject one A BM 12 skin grafts rendering this in ideal model for the analysis of allogeneic T cells in the absence of severe pathology. After watching this video, you should have a good understanding of how to perform tail skin transplantation and successfully monitor graft rejection.