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Skin Allograft Rejection

Hugh I. McFarland1,  Amy S. Rosenberg1

1Food and Drug Administration, Bethesda, Maryland

Publication Name: 
Current Protocols in Immunology
Unit Number: 
Unit 4.4
DOI: 
10.1002/0471142735.im0404s84
Online Posting Date: 
February, 2009
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Abstract

Skin allograft rejection is a test of the competence of T lymphocytes to mediate in vivo tissue destruction, which in turn reflects their role in critical functions such as anti-viral and tumor immunity. The tail-skin graft procedure described in this unit is useful predominantly because of the ease of preparation and resistance to ischemic (nonspecific) necrosis. Additionally, it is not necessary to sacrifice the donor mouse. However, rejection of tail-skin grafts should not be used to test for genetic homogeneity in breeding experiments or to detect minor histocompatibility (minor-H) antigens because tail skin is less sensitive than trunk skin in detecting such differences. Curr. Protoc. Immunol. 84:4.4.1-4.4.13. © 2009 by John Wiley & Sons, Inc.

Keywords: transplantation; skin; allograft; rejection; T lymphocyte; histocompatibility; MHC; grafting; mouse

     
 
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Table of Contents

  • Basic Protocol
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

 Basic Protocol
 Materials
  • Allogeneic donor and recipient (host) mice, <1 year old and preferably female (see Critical Parameters and Troubleshooting)
  • Isoflurane or ketamine/xylazine anesthesia (see recipe)
  • Oxygen source
  • 70% ethanol
  • Phosphate-buffered saline (PBS; appendix 2A), sterile
  • 10% povidone/iodine
  • Isoflurane vaporizer (Ohio or Cyprane style, VetEquip)
  • Nose cone
  • Thermal barrier (Vetko)
  • Dry (bead) sterilizer
  • Balance for weighing animals (Ohaus triple beam balance, 700 series, with animal basket)
  • Sterile gauze pads
  • Microdissecting forceps (4-in., half-curved, serrated; 5-in., straight, serrated or nonserrated)
  • Watchmaker's or jeweler's forceps (4½-in., 0.17-mm wide, 0.10-mm thick; Dumont no. 7)
  • 100 × 20–mm plastic petri dishes
  • 9.0-cm Whatman no. 1 filter paper discs
  • Scalpel blades (Bard Parker no. 10)
  • Electric animal clippers
  • Microdissecting scissors (4½ in., curved, with very sharp points; 4 in., straight)
  • Operating board
  • 3 × 18–in. Vaseline petrolatum gauze
  • 3/4-in. sheer Band-Aid-type sheer bandage (CVS, Curad)
  • Good light source, preferably one that can be focused (e.g., a multipiped fiberoptic light)
  • Autoclip (automatic wound clip–applying forceps), with wound clips and autoclip remover
  • Additional reagents and equipment for anesthesia (unit 1.4), intraperitoneal injection of mice (unit 1.6), and animal euthanasia (unit 1.8)
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Figures

  • Figure 4.4.1
    (A) Removing tail skin. Following anesthetization of the donor, the tail is severed at the base with a scalpel blade and the dorsal skin is incised. The skin is then stripped off the cartilaginous base using forceps. (B) Cutting graft pieces. The tail skin is placed raw-side down in a petri dish and the edges spread flat. Graft pieces are cut with a sharp scalpel blade. (C) Harvesting trunk skin. Following sacrifice of the donor, the trunk is shaved from the hind legs to forelegs. Two circumferential cuts are connected by a dorsal longitudinal cut and the skin is peeled away with forceps. (D) Pigmented nevus. Pigmented or thickened areas of skin should not be used as grafts. (E) Fat and subcutaneous tissue is removed by scraping with watchmaker's forceps. (F) Appearance of skin ready for grafting.

    View Image
  • Figure 4.4.2
    (A) Preparing the graft bed. The skin is pinched up by the watchmaker's forceps and cut by the microdissecting scissors. (B) Graft bed prior to removing the intact square of skin. (C) Removing skin from the graft-bed site. (D) Panniculus carnosus. Note the skein of blood vessels, which are branches of the lateral thoracic and mammary arteries. (E) Skin graft placed in graft bed.

    View Image
  • Figure 4.4.3
    Applying the bandage. (A) A square of Vaseline petroleum gauze is placed over the graft. (B) The mouse is placed graft-down on the elastic bandage. (C) The elastic bandage is wrapped around the mouse. (D) The mouse is placed such that the graft is over the sterile pad on a Band-Aid-type sheer strip. (E) The sheer strip is affixed to the elastic bandage.

    View Image
  • Figure 4.4.4
    Skin graft appearance. (A) Completely healed skin graft without evidence of rejection. (B) Acute rejection in progress. (C) Complete acute graft rejection (scab). (D) Chronic graft rejection. (E) Scar.

    View Image

Literature Cited

Literature Cited
    Benichou, G., Kant, C.D., Madsen, J., and Tocco, G. 2007. Modulation of alloreactivity to MHC-derived peptides and transplantation tolerance. Front. Biosci. 12:4239-4247.
    Bergstresser, P.R., Fletcher, C.R., and Streilein, J.W. 1980a. Surface densities of Langerhans cells in relation to rodent epidermal sites with special immunologic properties. J. Invest. Dermatol. 74:77-80.
    Bergstresser, P.R., Toews, G.B., Gilliam, J.N., and Streilein, J.W. 1980b. Unusual numbers and distributions of Langerhans cells in skin with unique immunologic properties. J. Invest. Dermatol. 74:312-314.
    Billingham, R.E. 1954. "The storage of skin". In Preservation and Transplantation of Normal Tissues (G.E.W. Wolstenholme and M.P. Cameron, eds.) pp. 160-171. Churchill, London.
    Billingham, R.E. 1961. "Free skin grafting in mammals". In Transplantation of Tissues and Cells (R.E. Billingham and W.K. Silvers, eds.) pp. 1-29. Wistar Institute Press, Philadelphia.
    Billingham, R.E. and Brent, L. 1956. Further attempts to transfer transplantation immunity by means of serum. Br. J. Exp. Pathol. 37:566-569.
    Billingham, R.E. and Medawar, P.B. 1951. The technique of free skin grafting in mammals. J. Exp. Biol. 28:385-402.
    Billingham, R.E., Brent, L., and Medawar, P.B. 1954. Quantitative studies on tissue transplantation immunity. II. The origin, strength, and duration of actively and adoptively acquired immunity. Proc. R. Soc. Lond. B. Biol. Sci. 143:58-80.
    Cerny, A., Ramseier, H., Bazin, H., and Zinkernagel, R.M. 1988. Unimpaired 1st set and 2nd set skin graft rejection in agammaglobulinemic mice. Transplantation 45:1111-1113.
    Chen, H.-D. and Silvers, W.K. 1983. Influence of Langerhans cells on the survival of H-Y incompatible skin grafts in rats. J. Invest. Dermatol. 81:20-23.
    Cobbold, S.P., Adams, E., Graca, L., Daley, S., Yates, S., Paterson, A., Robertson, N.J., Nolan, K.F., Fairchild, P.J., and Waldmann, H. 2006. Immune privilege induced by regulatory T cells in transplantation tolerance. Immunol. Rev. 213:239-255.
    Converse, J.M. and Rapaport, F.T. 1956. The vascularization of skin autografts and homografts: An experimental study in man. Ann. Surg. 143:306-310.
    Counce, S., Smith, P., Barth, R., and Snell, G.D. 1956. Strong and weak histocompatibility gene differences in mice and their role in the rejection of homografts of tumors and skin. Ann. Surg. 144:198-204.
    Goldstein, D.R. 2006. Toll like receptors and acute allograft rejection. Transplant. Immunol. 17:11-15.
    Gould, D.S. and Auchincloss, H. 1999. Direct and Indirect recognition: The role of MHC antigens in graft rejection. Immunol. Today 20:77-82.
    Graff, R.J., Hildemann, W.H., and Snell, G.D. 1966a. Histocompatibility genes of mice. VI. Allografts of mice congenic at various non-H-2 histocompatibility loci. Transplantation 4:425-437.
    Graff, R.J., Silvers, W.K., Billingham, R.E., Hildemann, W.H., and Snell, G.D. 1966b. The cumulative effect of histocompatibility antigens. Transplantation 4:605-617.
    Hainz, U., Jürgens, B., and Heitger, A. 2007. The role of indolamine 2,3-diolygenase in transplantation. Transplant. Int. 20:118-127.
    Hildemann, W.H. and Cohen, N. 1967. "Weak histoincompatibilities: Emerging immunogenetic rules and generalizations". In Histocompatibility Testing (E.S. Curtoni, P.L. Mattiuz, and R.M. Tosi, eds.) pp. 13-20. Williams & Wilkins, Baltimore.
    Hildemann, W.H. and Walford, R.L. 1960. Chronic skin homograft rejection in the Syrian hamster. Ann. N.Y. Acad. Sci. 87:56.
    Kubai, L. and Auerbach, R. 1980. Regional differences in the growth of skin transplants. Transplantation 30:128.
    Long, E. and Wood, K.J. 2007. Understanding Foxp3: Progress towards achieving transplantation tolerance. Transplantation 84:459-461.
    Mathieson, B.J., Flaherty, L., Bennett, D., and Boyse, E.A. 1975. Differences in the rejection of trunk, skin, and tail-skin allografts involving weak histocompatibility loci. Transplantation 19:525-526.
    Medawar, P.B. 1944. The behavior and fate of skin autografts and skin homografts in rabbits. J. Anat. 78:176-196.
    Ramselaar, C.G. and Ruytenberg, E.J. 1981. The influence of the hair follicle cycle on skin allografts in the congenitally athymic “nude” mouse. Br. J. Exp. Pathol. 62:252-258.
    Rosenberg, A.S. and Singer, A. 1992. Cellular basis of skin allograft rejection: An in vivo model of immune-mediated tissue destruction. Ann. Rev. Immunol. 10:333-358.
    Rosenberg, A.S., Mizuochi, T., and Singer, A. 1986. Analysis of T cell subsets in rejection of Kb mutant skin allografts differing at class I MHC. Nature 322:829.
    Sena, J., Wachtel, S.S., and Murphy, G. 1976. A comparison of the survival of H-Y incompatible ear, tail, and body skin grafts. Transplantation 21:412-416.
    Silvers, W.K., Murphy, G., and Poole, T.W. 1977. Studies on the H-Y antigen in rats. Immunogenetics 4:85-100.
    Steinmuller, D. 1984. "Skin grafting". In Methods in Enzymology (G. Di Sabato, J.J. Langone, and H. Van Vunakis, eds.) p. 20. Academic Press, San Diego.
    Sykes, M., Auchincloss, H. Jr., and Sachs, D.H. 2003. "Transplantation immunology". In Fundamental Immunology, 5th ed. (W.E. Paul, ed.) pp. 1481-1555. Lippincott Williams and Wilkins, Philadelphia.
    Wachtel, S.S. and Silvers, W.K. 1971. Skin homografts: Tolerogenic versus immunogenic influences in mice. J. Exp. Med. 133:921-937.
    Winn, H.J. 1986. "Antibody-mediated rejection". In Kidney Transplantation Rejection (G.M. Williams, J.F. Burdick, and K. Solez, eds.) pp. 17-28. Marcel Dekker, New York.
    Wood, K.J. and Sakaguchi, S. 2003. Regulatory T cells in transplantation tolerance. Nat. Rev. 3:199-210.
 Key References
    Sykes et al., 2003. See above.

Comprehensive and easy-to-read overview of graft rejection on both the clinical and basic research levels.

    Steinmuller, 1984. See above.

A good “how to” including elegant refinements of some techniques.

     
 
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