Methods of MHC class II epitope mapping, detection of autoimmune T cells and antigens, and autoimmune treatment
View Patent ↗The present invention provides of using multimeric MHC class II/peptide complexes. In one aspect, methods provided for identifying MHC class II-restricted immune epitopes of a predetermined polypeptide antigen. Methods for identifying an immunostimulatory epitope for a predetermined polypeptide antigen are provided. In a related aspect, methods for screening a therapeutic polypeptide agent for an MHC class II epitope are provided. In other aspects, methods for modulating T cells and for determining or monitoring an MHC class II-restricted immune status of a patient are also provided.
1. A method for identifying an MHC class II epitope of a predetermined polypeptide antigen, comprising;
preparing a library of at least two peptide pools, each peptide pool comprising at least two peptides, the peptides comprising a fragment of the predetermined polypeptide antigen and sharing a region of overlapping amino acid sequence identity with at least one other peptide in the library;
forming pools of MHC class II/peptide tetramers from the pools of peptides and soluble human MHC class II molecules, each MHC class II/peptide tetramer comprising four MHC class II molecule/peptide pairs, wherein the peptides are bound to the MHC class II molecules, said forming of MHC class II/peptide tetramers comprising loading the human MHC class II molecules with the peptide pools;
contacting the pools of MHC class II/peptide tetramers with human T cells;
identifying at least one pool of MHC class II/peptide tetramers that binds to the T cells; and
contacting T cells with MHC class II/peptide tetramers Conned with individual peptides from the pool of MHC class II/peptide tetramers that binds to the T cells to identify at least one epitope in the peptide pool.
2. The method of claim 1 , wherein the identifying is by fluorescence activated cell sorting, T cell proliferation assay or cytokine secretion capture assay.
3. The method of claim 1 , wherein the T cells are peripheral blood mononuclear cells.
4. The method of claim 1 , further comprising confirming the identification of the pool of MHC class II/peptide tetramers that bind to the T cells by T cell proliferation assay or cytokine secretion capture assay.
5. The method of claim 4 , wherein the cytokine secretion capture assay detects IFN gamma production.
6. The method of claim 1 , wherein the soluble MHC Class II molecules are loaded by contacting the soluble MHC class II molecules with about a 10 to about a 25 fold molar excess of the peptide pools.
7. The method of claim 1 , wherein the peptides are about 10 to about 20 amino acids in length.
8. The method of claim 1 , wherein each peptide pool comprises between about 3 to about 8 different peptides.
9. The method of claim 8 , wherein each peptide pool has about 5 different peptides.
10. The method of claim 1 , wherein the soluble human MHC class II molecules comprise α subunits and β subunits, the α subunits being HLA-DPα, HLA-DQα or HLA-DRα, and the β subunits being HLA-DPβ, HLA-DQβ or HLA-DRβ.
11. The method of claim 1 , wherein the soluble human MHC class II molecules comprise separate α and β subunits, each subunit having a leucine zipper domain and a conformationally flexible linker region.
12. The method of claim 1 , wherein the soluble human MHC class II molecules further comprise a ligand, and the MHC class II tetramers are formed by interaction of the ligand with a polyvalent binding partner.
13. The method of claim 12 , wherein the polyvalent binding partner is streptavidin.
14. The method of claim 12 , wherein the binding partner is labeled.
15. The method of claim 14 , wherein the label is a radioactive molecule, a luminescent molecule, a fluorescent molecule, an enzyme, or biotin.
16. The method of claim 14 , wherein the label is attached to the binding partner by a spacer.