title: Detection of autoreactive myelin proteolipid protein 139-151-specific T cells by using MHC II (IAs) tetramers.
abstract: Detection of autoreactive T cells using MHC II tetramers is difficult because of the low affinity of their TCR. We have generated a class II tetramer using the IA(s) class II molecule combined with an autoantigenic peptide from myelin proteolipid protein (PLP; PLP(139-151)) and used it to analyze myelin PLP(139-151)-reactive T cells. Using monomers and multimerized complexes labeled with PE, we confirmed the specificity of the reagent by bioassay and flow cytometry. The IA(s) tetramers stimulated and stained the PLP(139-151)-specific 5B6 TCR transgenic T cells and a polyclonal cell line specific for PLP(139-151), but not a control T cell line specific for PLP(178-191). We used this reagent to optimize conditions to detect low affinity autoreactive T cells. We found that high pH ( approximately 8.0) and neuraminidase treatment enhances the staining capacity of PLP(139-151) tetramer without compromising specificity. Furthermore, we found that induction of calcium fluxing by tetramers in T cells may be used as a sensitive measure to detect autoreactive T cells with a low affinity. Taken together, the data show that the tetrameric reagent binds and stimulates PLP(139-151)-reactive T cells with specificity. This tetrameric reagent will be useful in studying the evolution of PLP(139-151)-specific repertoire in naive mice and its expansion during the autoimmune disease experimental autoimmune encephalomyelitis.
authors.affiliation: Center for Neurologic Diseases, Brigham and Women's Hospital, Harvard Medical School, Boston, MA 02115, USA.
authors.forename: Jayagopala
authors.initials: J
authors.lastname: Reddy
authors.forename: Estelle
authors.initials: E
authors.lastname: Bettelli
authors.forename: Lindsay
authors.initials: L
authors.lastname: Nicholson
authors.forename: Hanspeter
authors.initials: H
authors.lastname: Waldner
authors.forename: Mei-Huei
authors.initials: MH
authors.lastname: Jang
authors.forename: Kai W
authors.initials: KW
authors.lastname: Wucherpfennig
authors.forename: Vijay K
authors.initials: VK
authors.lastname: Kuchroo
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001324:Autoantigens / Q000276:immunology*; D016367:Baculoviridae / Q000235:genetics / Q000276:immunology; D001692:Biological Transport / Q000276:immunology; D015496:CD4-Positive T-Lymphocytes / Q000737:chemistry / Q000276:immunology / Q000378:metabolism; D002118:Calcium / Q000378:metabolism; D018984:Epitopes, T-Lymphocyte / Q000032:analysis / Q000276:immunology*; D005260:Female; D000949:Histocompatibility Antigens Class II / Q000032:analysis / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology*; D051379:Mice; D008815:Mice, Inbred Strains; D008822:Mice, Transgenic; D008969:Molecular Sequence Data; D018991:Myelin Proteolipid Protein / Q000032:analysis / Q000276:immunology*; D009439:Neuraminidase / Q000032:analysis; D010446:Peptide Fragments / Q000032:analysis / Q000276:immunology*; D011108:Polymers / Q000032:analysis / Q000378:metabolism; D013194:Staining and Labeling; D016176:T-Lymphocyte Subsets / Q000737:chemistry / Q000276:immunology* / Q000378:metabolism; D017945:Theilovirus / Q000235:genetics / Q000276:immunology
chemical_list: D001324:Autoantigens; D018984:Epitopes, T-Lymphocyte; D000949:Histocompatibility Antigens Class II; D018991:Myelin Proteolipid Protein; D010446:Peptide Fragments; D011108:Polymers; C435023:myelin proteolipid protein (139-151); D009439:Neuraminidase; D002118:Calcium