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

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 11 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 tetra-mer, 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 P-chain variable region (V(beta)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.