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 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 beta-chain variable region (V(beta)10), and sequencing of the TCR junctional regions showed diversity in the third complementarity-determining region.


CONCLUSIONS
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.
Falta
Michael T
MT
University of Colorado Health Sciences Center, and National Jewish Medical and Research Center, Denver, Colorado 80262, USA. michael.falta@uchsc.edu
Fontenot
Andrew P
AP
Rosloniec
Edward F
EF
Crawford
Frances
F
Roark
Christina L
CL
Bill
Jerome
J
Marrack
Philippa
P
Kappler
John
J
Kotzin
Brian L
BL
D000818:Animals; D000915:Antibody Affinity / Q000276:immunology*; D001172:Arthritis, Rheumatoid / Q000276:immunology*; D001324:Autoantigens / Q000276:immunology; D015496:CD4-Positive T-Lymphocytes / Q000276:immunology*; D005802:Genes, MHC Class II / Q000276:immunology*; D006825:Hybridomas / Q000276:immunology; D051379:Mice; D010455:Peptides / Q000276:immunology; D011948:Receptors, Antigen, T-Cell / Q000276:immunology*
D001324:Autoantigens; D010455:Peptides; D011948:Receptors, Antigen, T-Cell