title: 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.
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 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.
authors.affiliation: University of Colorado Health Sciences Center, and National Jewish Medical and Research Center, Denver, Colorado 80262, USA. michael.falta@uchsc.edu
authors.forename: Michael T
authors.initials: MT
authors.lastname: Falta
authors.forename: Andrew P
authors.initials: AP
authors.lastname: Fontenot
authors.forename: Edward F
authors.initials: EF
authors.lastname: Rosloniec
authors.forename: Frances
authors.initials: F
authors.lastname: Crawford
authors.forename: Christina L
authors.initials: CL
authors.lastname: Roark
authors.forename: Jerome
authors.initials: J
authors.lastname: Bill
authors.forename: Philippa
authors.initials: P
authors.lastname: Marrack
authors.forename: John
authors.initials: J
authors.lastname: Kappler
authors.forename: Brian L
authors.initials: BL
authors.lastname: Kotzin
mesh_terms: 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*
chemical_list: D001324:Autoantigens; D010455:Peptides; D011948:Receptors, Antigen, T-Cell