[Application of MHC-Ig/peptide polymer in detecting antigen-specific cytotoxic T lymphocytes in mice with experimental autoimmune uveitis].
2010
Objective To explore the application of MHC-Ig/peptide polymer technique for detecting antigen-specific cytotoxic T lymphocytes(CTLs)in mice with experimental autoimmune uveitis(EAU).Methods B10RⅢ mice were immunized with an interphotoreceptor retinal-binding protein(IRBP) synthetic peptide(IRBP161-180).The in vivo primed T cells were separated and stained with MHC-Ig polymer combined with a panel of truncated peptides derived from IRBP161-180.The level of IRBP-specific CTLs cells was determined by FACS analysis.The CD8+T cells were isolated from the primed T cells and stimulated with complex polymers containing MHC-Ig and various IRBP-derived peptides.The proliferation of CD8+T cells was measured by 3H thymidine incorporation.The production of interferon-(IFN-) in the cell suspensions was measured by ELISA.Results The IRBP-specific CTLs were detected by MHC-Ig/peptide polymers.The MHC-Ig/IRBP168-177 peptide polymer dtected 12.3% specific CTLs,showing greater ability in stimulating proliferation of CTLs and production of IFN-than the other MHC-Ig/peptide polymers(P0.01).The truncated 10-mer peptide,IRBP168-177,was the major antigenic epitope for the IRBP-specific CTLs.The MHC-Ig/IRBP168-177 peptide polymer detected the highest level(4.9%±1.1%)of specific CTLs from peripheral blood mononuclear cells(PBMCs) at the acute stage of EAU.Conclusion The MHC-Ig polymer technique is an effective instrument for detecting antigen-specific CTLs,with good sensitivity and specificity in EAU studies.
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