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

Summary

Tail-skin transplantation is a powerful model for studying T cell-dependent rejection and tolerance induction during allogeneic immune responses in mice. The advantages of this protocol are minor invasive surgery, and ease of monitoring with no need to sacrifice the recipient mouse.

Abstract

The study of T cell responses and their consequences during allo-antigen recognition requires a model that enables one to distinguish between donor and host T cells, to easily monitor the graft, and to adapt the system in order to answer different immunological questions. Medawar and colleagues established allogeneic tail-skin transplantation in mice in 1955. Since then, the skin transplantation model has been continuously modified and adapted to answer specific questions. The use of tail-skin renders this model easy to score for graft rejection, requires neither extensive preparation nor deep anesthesia, is applicable to animals of all genetic background, discourages ischemic necrosis, and permits chemical and biological intervention.

In general, both CD4⁺ and CD8⁺ allogeneic T cells are responsible for the rejection of allografts since they recognize mismatched major histocompatibility antigens from different mouse strains. Several models have been described for activating allogeneic T cells in skin-transplanted mice. The identification of major histocompatibility complex (MHC) class I and II molecules in different mouse strains including C57BL/6 mice was an important step toward understanding and studying T cell-mediated alloresponses. In the tail-skin transplantation model described here, a three-point mutation (I-A^bm12) in the antigen-presenting groove of the MHC-class II (I-A^b) molecule is sufficient to induce strong allogeneic CD4⁺ T cell activation in C57BL/6 mice. Skin grafts from I-A^bm12 mice on C57BL/6 mice are rejected within 12-15 days, while syngeneic grafts are accepted for up to 100 days. The absence of T cells (CD3^-/- and Rag2^-/- mice) allows skin graft acceptance up to 100 days, which can be overcome by transferring 2 x 10⁴ wild type or transgenic T cells. Adoptively transferred T cells proliferate and produce IFN-γ in I-A^bm12-transplanted Rag2^-/- mice.

Introduction

Transplantation of solid organs such as skin, heart and kidneys is now a standard procedure in medical practice worldwide¹. Successfully transplanted organs can be rejected by activation of the recipient immune system, which recognizes the major histocompatibility antigens of the donor. Therefore transplanted patients need treatment with immunosuppressive drugs². Allogeneic skin transplantation in mice was established by Medawar and colleagues in 1955 and was helpful for identifying the targeted molecules later described as major histocompatibility complex (MHC) class I and II. Since then, the skin transplantation model has been continuously modi....

Protocol

In this video publication and protocol, all animal procedures were conducted in accordance to the animal protocol approved by the Cantonal Authority Basel-Stadt, Switzerland. Perform all procedures in sterile conditions where possible.

1. Preparation of Surgery

1. Autoclave all surgical instruments and gauze before use.
2. Heat the warm pad and organize surgical instruments on the table (Table of Materials/Methods).
3. Open the finger strip bandages. Apply petroleum jelly on the wound pad with a cotton swab (make sure the gauze is completely covered).
4. Weigh the mice and administer analgesia ....

Results

In a first approach C57BL/6 mice were transplanted with I-A^bm12 allografts and I-A^b syngrafts. After bandage removal, grafts manifest signs of wound healing without closure of the contact zone in C57BL/6 mice (Figure 1A). After bandage removal, CD4⁺ T cell-mediated inflammation led to the appearance of necrotic areas (red spots) and rejection of I-A^bm12 allografts in C57BL/6 mice within 13 days after transplantation (Figure 1B-C, filled symbols.......

Discussion

Skin transplantation is a widely used method for studying rejection and tolerance dependent on T cells. Since the skin transplantation model was established, several adaptations and changes have been applied. In the described procedure, I-A^bm12 tail-skin transplantation is performed using anesthetic gas (isofluorane). The use of gas anesthesia decreases the time of execution and mice recovery, which diminishes the stress on transplanted mice. The procedure employs tissue adhesive to fix the tail-skin, which te.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Betadine standard solution	Mundipharma
Cotton swab	Carl Roth GmbH	31025060
Dafalgan , UPSA	Bristol Myers Squibb UPSA
Hansaplast finger strips	Beierdorf AG	REF.76861
Histoacryl tissue adhesive	Braun	REF.1050052
Leukotape classic , 2 cm x 10 m	BSN Medical SAS	REF.02204-00
PBS, Phosphate Buffered Saline, pH 7.4	Invitrogen	10010015	GIBCO
Sterile gauze, 5 x 5 cm, 8 ply	MaiMed GmbH	21010
Narrow pattern forceps	FST	11003-12
Fine iris scissor curved	FST	14095-11
Fine iris scissor	FST	14094-11
Mayo scissors	FST	14010-15
Artery scissors ball tip 11.5 cm	FST	14080-11
Tissue forceps	FST	11021-14
Surgical blade No. 20	Swann-Morton LTD	3006	Carbon steel
Surgical blade handles	Swann-Morton LTD
Syringe, 1 ml	ARTSANA		Disposable
Temgesic, buprenorphine	ESSEX Chemie AG		0.3 mg/ml
Tissue Culture dishes 10 cm, 60.1 cm2	TPP
Vaseline	Vifor SA
Warm pad	Solis		Type 223