Membrane-anchored beta 2-microglobulin stabilizes a highly receptive state of MHC class I molecules.
The magnitude of response elicited by CTL-inducing vaccines correlates with the density of MHC class I (MHC-I)-peptide complexes formed on the APC membrane. The MHC-I L chain, beta2-microglobulin (beta2m), governs complex stability. We reasoned that genetically converting beta2m into an integral membrane protein should exert a marked stabilizing effect on the resulting MHC-I molecules and enhance vaccine efficacy. In the present study, we show that expression of membranal human beta2m (hbeta2m) in mouse RMA-S cells elevates MHC-I thermal stability. RMA-S transfectants bind an exogenous peptide at concentrations 10(4)- to 10(6)-fold lower than parental RMA-S, as detected by complex-specific Abs and by T cell activation. Moreover, saturation of the transfectants' MHC-I by exogenous peptide occurs within 1 min, as compared with approximately 1 h required for parental cells. At saturation, however, level of peptide bound by modified cells is only 3- to 5-fold higher. Expression of native hbeta2m only results in marginal effect on the binding profile. Soluble beta2m has no effect on the accelerated kinetics, but the kinetics of transfectants parallel that of parental cells in the presence of Abs to hbeta2m. Ab inhibition and coimmunoprecipitation analyses suggest that both prolonged persistence of peptide-receptive H chain/beta2m heterodimers and fast heterodimer formation via lateral diffusion may contribute to stabilization. In vivo, peptide-loaded transfectants are considerably superior to parental cells in suppressing tumor growth. Our findings support the role of an allosteric mechanism in determining ternary MHC-I complex stability and propose membranal beta2m as a novel scaffold for CTL induction.
Berko
Dikla
D
Laboratory of Immunology, MIGAL-Galilee Technology Center, Kiryat Shmona, Israel.
Carmi
Yaron
Y
Cafri
Gal
G
Ben-Zaken
Shimrit
S
Sheikhet
Helena Migalovich
HM
Tzehoval
Esther
E
Eisenbach
Lea
L
Margalit
Alon
A
Gross
Gideon
G
D000276:Adjuvants, Immunologic / Q000138:chemical synthesis / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D001666:Binding Sites, Antibody; D001667:Binding, Competitive / Q000276:immunology; D019496:Cancer Vaccines / Q000235:genetics / Q000276:immunology / Q000627:therapeutic use; D045744:Cell Line, Tumor; D015395:Histocompatibility Antigens Class I / Q000378:metabolism*; D006801:Humans; D019169:Jurkat Cells; D007700:Kinetics; D008546:Melanoma, Experimental / Q000235:genetics / Q000276:immunology / Q000517:prevention & control; D008565:Membrane Proteins / Q000138:chemical synthesis / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008810:Mice, Inbred C57BL; D010446:Peptide Fragments / Q000037:antagonists & inhibitors / Q000378:metabolism; D011485:Protein Binding / Q000276:immunology; D011993:Recombinant Fusion Proteins / Q000138:chemical synthesis / Q000235:genetics / Q000378:metabolism; D013816:Thermodynamics; D014162:Transfection; D001613:beta 2-Microglobulin / Q000138:chemical synthesis / Q000235:genetics / Q000276:immunology / Q000378:metabolism*
D000276:Adjuvants, Immunologic; D019496:Cancer Vaccines; D015395:Histocompatibility Antigens Class I; D008565:Membrane Proteins; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D001613:beta 2-Microglobulin