Establishment of an expression cloning system for CD4+ T cell epitopes.
We previously reported an epitope presenting vector, pCI, a derivative of a human invariant chain (Ii) expression vector, in which the class II associated invariant chain peptide (CLIP, Ii p89-101) could be substituted with antigenic peptides. In the current study, we used this vector to develop a new expression cloning system to identify CD4+ T cell epitopes. We inserted double-stranded oligo DNAs of randomized sequences into this vector and prepared an epitope-presenting library which loads randomized 13-mer peptides onto HLA class II molecules coexpressed in COS-7 cells. Utilizing this library, we isolated a cross-reactive epitope recognized by a glutamic acid decarboxylase (GAD) 65-autoreactive T cell clone established from a patient with insulin-dependent diabetes mellitus. Although the newly identified epitope (PVQLSNQWHVVGATF) was far different from the original epitope, GAD65 p116-128 (NILLQYVVKSFDR), it did have the capacity to stimulate the T cell clone comparable to that of the original GAD epitope. Our system may be applicable not only for identifying of cross-reactive epitopes for CD4+ T cells of known specificity, but also for detection of epitopes stimulatory for CD4+ T cells the epitopes of which are unknown.
Fujii
S
S
Division of Immunogenetics, Department of Neuroscience and Immunology, Kumamoto University Graduate School of Medical Sciences, Honjo 2-2-1, Kumamoto, 860-0811, Japan.
Uemura
Y
Y
Iwai
L K
LK
Ando
M
M
Senju
S
S
Nishimura
Y
Y
D000595:Amino Acid Sequence; D000944:Antigens, Differentiation, B-Lymphocyte / Q000276:immunology; D001483:Base Sequence; D015496:CD4-Positive T-Lymphocytes / Q000276:immunology*; D003001:Cloning, Molecular; D003429:Cross Reactions; D003920:Diabetes Mellitus / Q000276:immunology; D000939:Epitopes / Q000235:genetics* / Q000276:immunology; D005822:Genetic Vectors; D005968:Glutamate Decarboxylase / Q000276:immunology*; D000949:Histocompatibility Antigens Class II / Q000276:immunology; D006801:Humans; D007527:Isoenzymes / Q000276:immunology*; D008969:Molecular Sequence Data; D019151:Peptide Library
D000944:Antigens, Differentiation, B-Lymphocyte; D000939:Epitopes; D000949:Histocompatibility Antigens Class II; D007527:Isoenzymes; D019151:Peptide Library; C045084:invariant chain; D005968:Glutamate Decarboxylase; C401141:glutamate decarboxylase 2