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

Open Access
Publisher Website
Google Scholar
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-39263–275 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 (Vβ10), 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.

This publication has 47 references indexed in Scilit: