Induction and Monitoring of Active Delayed Type Hypersensitivity (DTH) in Rats

Summary

Delayed type hypersensitivity (DTH) is an inflammatory reaction mediated by CCR7- effector memory T lymphocytes. Here we demonstrate how to induce active 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 (KLH).

Here we demonstrate how to induce an active DTH reaction in Lewis rats. We will first prepare a water-in-oil emulsion of KLH, our antigen of interest, in complete Freund’s adjuvant and inject this emulsion subcutaneously to rats. This will prime the immune system to develop memory T cells directed to KLH. Seven days later we will challenge the rats intradermally on the back with KLH on one side and with ovalbumin, an irrelevant antigen, on the other side. The inflammatory reaction will be visible 16-72 hours later and the red and swollen area will be measured as an indication of DTH severity.

Protocol

1. Preparation of the emulsion The emulsion is a 1:1 mixture of antigen (DTH can be induced to various antigens: ovalbumin, tetanus toxoid, keyhole limpet hemocyanin….) in aqueous solution added to complete Freund’s adjuvant. It is very important to add the antigen to the adjuvant and not the other way round!!! There is a lot of waste when making and injecting an emulsion, so always prepare 1.5 or 2 times more than what you need. Dissolve antigen in saline to prepare …

Discussion

For a more precise measurement of the DTH reaction we recommend challenging the rats with the antigen in the pinna of the ear. The reaction can then be measured as a difference in ear swelling between the relevant antigen-challenged ear and the ear challenged with either saline alone or with the irrelevant antigen. A demonstration of the challenge in the ear is shown in 1.

Materials

Material Name	Tipo	Company	Catalogue Number	Comment
Complete Freund’s adjuvant H37Ra	Reagent	Difco	231131	Distributed by Fisher under catalog # DF 311-360-5
Keyhole Limpet hemocyanin	Reagent	Sigma	H-5654
Ovalbumin	Reagent	Sigma	A-5503
5 ml glass syringes	Tool	Fisher	14-823-10 B	Need two 5 ml syringes for each batch of 5 ml of emulsion
18G metal bridge	Tool	Fisher	14-825-17L
Lewis rats	Animal	Harlan Sprague Dawley		Female rats 7-9 weeks old
23G 1″ needles	Tool	Fisher	14-826 AA	It is best to change needles between rats
3 ml Luer Lock syringes	Tool	Fisher	05-561-63
DPBS	Reagent	Gibco/Invitrogen	14040-182
1 ml regular tip syringes	Tool	Fisher	22-257-154
27G 1/2″ needles	Tool	Fisher	14-826-48
Electric cordless trimmer	Tool	Braintree Scientific	CLP-32130	To shave the rats before antigen challenge in the back.

Use sterile solutions reagents and syringes. The glass syringes and 18G bridges can be autoclaved. Read material data sheet before handling a reagent for the first time and wear appropriate protective equipment.