Class II major histocompatibility complex–peptide tetramer staining in relation to functional avidity and T cell receptor diversity in the mouse CD4+ T cell response to a rheumatoid arthritis–associated antigen
✍ Scribed by Michael T. Falta; Andrew P. Fontenot; Edward F. Rosloniec; Frances Crawford; Christina L. Roark; Jerome Bill; Philippa Marrack; John Kappler; Brian L. Kotzin
- Publisher
- John Wiley and Sons
- Year
- 2005
- Tongue
- English
- Weight
- 159 KB
- Volume
- 52
- Category
- Article
- ISSN
- 0004-3591
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✦ Synopsis
Abstract
Objective
Although studies have suggested that human cartilage (HC) gp‐39 may be an antigen recognized by autoreactive CD4+ T cells in rheumatoid arthritis, we previously failed to identify specific CD4+ T cells in patients' synovial fluid or blood using a class II major histocompatibility complex–peptide tetramer composed of the immunodominant HC gp‐39^263–275^ epitope covalently linked to DR4. We undertook this study to better understand the parameters for specific binding of this tetramer.
Methods
DR4‐transgenic mice were immunized with the HC gp‐39 peptide, and a set of peptide‐responsive hybridomas was derived. Hybridomas were stained with the DR4–gp‐39 tetramer and cultured with increasing amounts of peptide in the presence of DR4‐expressing antigen‐presenting cells to determine functional avidity.
Results
Great variability was apparent in the ability of the tetramer to stain the hybridomas, and there was a strong correlation between the intensity of tetramer staining and functional avidity. Importantly, nearly 30% of the hybridomas did not stain with tetramer, and these cells exhibited relatively low functional avidity. Although the addition of an anti–T cell receptor (anti‐TCR) monoclonal antibody during the staining procedure enhanced binding of the tetramer to a number of the hybridomas, a significant percentage remained unstainable. Analysis of TCR expression showed that >90% of the hybridomas expressed the same TCR β‐chain variable region (V~β~10), and sequencing of the TCR junctional regions showed diversity in the third complementarity‐determining region.
Conclusion
These results suggest that immune responses dominated by relatively low‐affinity TCR interactions, such as those that may occur in autoimmune disease, will be difficult to detect using standard tetramer techniques.