Induction of Autoimmune Disease by Depletion of Regulatory T Cells

Steve Simmonds1, Don Mason1

1 University of Oxford, Oxford, United Kingdom
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 15.12
DOI:  10.1002/0471142735.im1512s30
Online Posting Date:  May, 2001
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Abstract

Organ‐specific autoimmune diseases can be induced in rodents that do not normally spontaneously develop autoimmunity by using procedures that render the animals partially T cell deficient. Using a protocol of adult thymectomy followed by four doses of sublethal gamma irradiation, insulin‐dependent diabetes can be induced in normal PVG.RT1u rats, an inbred congenic strain that has the same major histocompatibility complex (MHC) allotype as the spontaneously diabetic BB rat. Onset of the disease ranges from 3 to 18 weeks after the final dose of irradiation, with 98% of male and 70% of female animals becoming diabetic. This unit describes the induction of insulin‐dependent diabetes in the rat. A modified protocol allows for the induction of a more severe form of the disease.

     
 
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Table of Contents

  • Basic Protocol 1: Induction of Diabetes in Young Adult Rats by Thymectomy
  • Alternate Protocol 1: Induction of Diabetes in Three‐Week‐Old Rats by Thymectomy
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Induction of Diabetes in Young Adult Rats by Thymectomy

  Materials
  • PVG.RT1u rats, 6 weeks old (Harlan Bioproducts for Science)
  • Glucose hexokinase HK reagent (Sigma)
  • Methoxyflurane
  • 70% (v/v) ethanol
  • Small animal clippers
  • Dissecting board
  • Blunt‐ended iris scissors, sterile
  • Blunt‐ended forceps, sterile
  • 7‐cm alms‐type retractor (John Weiss), sterile
  • Metric 3 braided silk suture (Ethicon)
  • 9‐mm wound clips (e.g., Clay Adams autoclips) and applicator, sterile
  • γ irradiator (Cs137 Gammacell, Atomic Energy of Canada, or equivalent)
  • Additional reagents and equipment for bleeding (unit 1.7), anesthesia with methoxyflurane (unit 1.4), and determination of blood glucose levels (see manufacturer's instructions for glucose hexokinase HK reagent)

Alternate Protocol 1: Induction of Diabetes in Three‐Week‐Old Rats by Thymectomy

  • PVG.RT1u rats, 3 weeks old, 40 to 55 g (Harlan Bioproducts for Science)
  • 1‐ml syringe barrel
  • Fine‐pointed forceps, sterile
  • Curved forceps, sterile
  • Glass suction cannula: Pasteur pipet cut off at 5 to 6 cm from the tip (i.e., just above the taper, leaving ∼4‐mm opening) and fire polished
  • Water aspirator
  • Heat lamp
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Figures

Videos

Literature Cited

   Bendelac, A., Carnaud, C., Boitard, C., and Bach, J.F. 1987. Syngeneic transfer of autoimmune diabetes from diabetic NOD mice to healthy neonates. Requirement for both L3T4+ and Lyt2+ T cells. J. Exp. Med. 166:823‐832.
   Crisa, L., Mordes, J.P., and Rossini, A.A. 1992. Autoimmune diabetes mellitus in the BB rat. Diabetes Metab. Rev. 8:4‐37.
   Fowell, D. and Mason, D. 1993. Evidence that the T cell repertoire of normal rats contains cells with the potential to cause diabetes. Characterization of the CD4+ T cell subset that inhibits this autoimmune potential. J. Exp. Med. 177:627‐636.
   Fowell, D., McKnight, A.J., Powrie, F., Dyke, R., and Mason, D.W. 1991. Subsets of CD4+ T cells and their roles in the induction and prevention of autoimmunity. Immunol. Rev. 123:37‐64.
   Penhale, W.J. and Young, P.R. 1988. The influence of the normal microbial flora on the susceptibility of rats to Experimental Autoimmune Thyroiditis. Clin. Exp. Immunol. 72:288‐292.
   Penhale, W.J., Farmer, A., McKenna, R.P., and Irvine, W.J. 1973. Spontaneous thyroiditis in thymectomized and irradiated Wistar rats. Clin. Exp. Immunol. 15:225‐236.
   Penhale, W.J., Irvine, W.J., Inglis, J.R., and Farmer, A. 1976. Thyroiditis in T cell‐depleted rats: Suppression of the autoallergic response by reconstitution with normal lymphoid cells. Clin. Exp. Immunol. 25:6‐16.
   Penhale, W.J., Stumbles, P.A., Huxtable, C.R., Sutherland, R.J., and Pethick, D.W. 1990. Induction of diabetes in PVG/c strain rats by manipulation of the immune system. Autoimmunity 7:169‐179.
Key Reference
   Fowell et al., 1991. See above.
  Characterization of the CD4+ T cell subset that prevents development of autoimmune disease.
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