Structural and functional analysis of monomorphic determinants recognized by monoclonal antibodies reacting with the HLA class I alpha 3 domain.
To identify mAb reacting with the HLA class I alpha 3 domain, 14 mAb recognizing monomorphic determinants expressed on HLA-A, B, and C Ag or restricted to HLA-B Ag were screened in indirect immunofluorescence with mouse L cells expressing HLA-B7/H-2Kb chimeric Ag. mAb CR1S63, CR10-215, CR11-115, and W6/32 were found to react with the HLA class I alpha 3 domain in addition to the alpha 2 domain. mAb Q1/28 and TP25.99 were found to react only with the HLA class I alpha 3 domain. The determinants recognized by the six mAb were mapped on the HLA class I alpha 3 domain by indirect immunofluorescence staining of L cells expressing H-2Kb Ag containing different segments of the HLA-B7 alpha 3 domain chimerized with the H-2Kb alpha 3 domain. mAb TP25.99 reacts with chimeric Ag containing the HLA-B7 184 to 199 stretch, mAb CR10-215 and CR11-115 react with chimeric Ag containing the HLA-B7 184 to 246 stretch, mAb CR1S63 and Q1/28 react with chimeric Ag containing the HLA-B7 184 to 256 stretch, and mAb W6/32 reacts with chimeric Ag containing the whole HLA-B7 alpha 3 domain. Functional analysis using human CD8 alpha-bearing mouse H-2Kb-specific T cell hybridoma cells (HTB-Leu2) showed that only mAb TP25.99 inhibited IL-2 production by HTB-Leu2 cells stimulated with L cells expressing KbKbB7 Ag. This inhibition may occur because of the spatial proximity of the determinant defined by mAb TP25.99 to the CD8 alpha binding loop and/or because of change(s) in the conformation of the CD8 alpha binding loop induced by the binding of mAb TP25.99 to the HLA class I molecule. Furthermore, mAb TP25.99 inhibited the cytotoxicity of CD8-dependent and CD8-independent CTL clones. These results indicate that mAb TP25.99 has unique specificity and functional characteristics. Therefore it represents a valuable probe to characterize the role of the HLA class I alpha 3 domain in immunologic phenomena.
Tanabe
M
M
Department of Tumor Biology, University of Tokyo, Japan.
Sekimata
M
M
Ferrone
S
S
Takiguchi
M
M
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D015704:CD4 Antigens / Q000032:analysis; D016827:CD8 Antigens / Q000032:analysis; D000939:Epitopes / Q000032:analysis*; D015395:Histocompatibility Antigens Class I / Q000276:immunology* / Q000502:physiology; D006825:Hybridomas / Q000276:immunology; D007376:Interleukin-2 / Q000096:biosynthesis; D051379:Mice; D008969:Molecular Sequence Data; D013602:T-Lymphocytes, Cytotoxic / Q000276:immunology
D000911:Antibodies, Monoclonal; D015704:CD4 Antigens; D016827:CD8 Antigens; D000939:Epitopes; D015395:Histocompatibility Antigens Class I; D007376:Interleukin-2