Induction and Monitoring of Adoptive Delayed-Type Hypersensitivity in Rats

Summary

Delayed type hypersensitivity (DTH) is an inflammatory reaction mediated by CCR7- effector memory T (TEM) lymphocytes. Here we demonstrate how to activate antigen-specific TEM cells, induce adoptive DTH in Lewis rats and monitor the inflammatory response.

Abstract

Delayed type hypersensitivity (DTH) is an inflammatory reaction mediated by CCR7- effector memory T lymphocytes that infiltrate the site of injection of an antigen against which the immune system has been primed. The inflammatory reaction is characterized by redness and swelling of the site of antigenic challenge. It is a convenient model to determine the in vivo efficacy of immunosuppressants. Cutaneous DTH can be induced either by adoptive transfer of antigen-specific T lymphocytes or by active immunization with an antigen, and subsequent intradermal challenge with the antigen to induce the inflammatory reaction in a given skin area. DTH responses can be induced to various antigens, for example ovalbumin, tuberculin, tetanus toxoid, or keyhole limpet hemocyanin. Such reactions can also be induced against autoantigen, for example to myelin basic protein (MBP) in rats with experimental autoimmune encephalomyelitis induced with MBP, an animal model for multiple sclerosis (1). Here we demonstrate how to induce an adoptive DTH reaction in Lewis rats. We will first stimulate ovalbumin-specific T cells in vitro and inject these activated cells intraperitoneally to naive rats. After allowing the cells to equilibrate in vivo for 2 days, we will challenge the rats with ovalbumin in the pinna of one ear, while the other ear wil receive saline. The inflammatory reaction will be visible 3-72 hours later and ear thickness will be measured as an indication of DTH severity.

Protocol

1. Stimulation of the ovalbumin-specific T lymphocytes

Resting ovalbumin-specific T cells are stimulated with ovalbumin in the presence of irradiated Lewis rat thymocytes (30 Gy) as antigen presenting cells (APCs), in Stimulation Medium (Prepare in DMEM + Pen/Strep/L-Glut + NEAA + RPMI Vitamins + 2ME + Rat Serum + Ovalbumin, at the final concentrations noted in the materials list.)

Rat serum preparation

1. We take serum from the rats we sacrifice as thymus donors.
2. Rats are deeply anesthetized by halothane inhalation and as much blood as possible is drawn by cardiac perfusion (5-10 ml per rat) using ....

Discussion

The antigenic challenge can also be performed at other sites, for example in the skin of the back (2). We however find that challenging. Using the pinna of the ear, as shown in this video, allows for the most precise measurement of the DTH reaction.

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Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
DMEM	GIBCO, by Life Technologies	12430-062	500ml
Sodium pyruvate	Sigma-Aldrich	S8636	1 mM final, in Stimulation medium
Penicillin – Streptomycin – L-Glutamine	Cambrex/BioWhittaker	17-718R	100 u/ml - 100 μg/ml - 4 mM respectively, final, in Stimulation Medium
Non essential amino acids	Sigma-Aldrich	R7131	NEAA, 1% final, in Stimulation Medium
RPMI vitamins	Sigma-Aldrich	R7256	1% final, in Stimulation medium
beta-mercapt–thanol	Sigma-Aldrich	M6250	2ME, 50 μM final, in Stimulation Medium
Syngeneic rat serum			1% final. See explanation in Protocol.
Ovalbumin	Sigma-Aldrich	A5503	10 μg/ml final, in Stimulation Medium.