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.
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
Preparation of the antigen presenting cells
Stimulation
2. Adoptive transfer of the ovalbumin-specific T lymphocytes
3. Challenge with antigen in the ear
4. Measurement of DTH
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.
We thank Dr. Alexander Flugel (Martinsried, Germany) for providing us with the GFP-labeled, ovalbumin-specific T cell line used in this protocol.
Material Name | Type | Company | Catalogue Number | Comment |
---|---|---|---|---|
DMEM | Gibco/Invitrogen | 12430-062 | 500ml | |
Sodium pyruvate | Sigma | 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 | R7131 | NEAA, 1% final, in Stimulation Medium | |
RPMI vitamins | Sigma | R7256 | 1% final, in Stimulation medium | |
beta-mercaptoethanol | Sigma | M6250 | 2ME, 50 µM final, in Stimulation Medium | |
Syngeneic rat serum | 1% final. See explanation in Protocol. | |||
Ovalbumin | Sigma | A5503 | 10 µg/ml final, in Stimulation Medium. |