MHC Peptide Tetramer Core

Locations: Virginia Mason Research Center, Seattle, WA
The Diabetes Center at UCSF, San Francisco, CA

Overview | Investigators | Background | Resources

Overview

The ITN has established two core facilities related to MHC-peptide tetramer studies. First, new MHC Class II tetramer reagents and staining procedures are being developed in facilities at Virginia Mason Research Institute, Seattle, Washington. These new allele constructs will be produced at the ITN production facility located at the University of California, San Francisco and to the NIAID Tetramer Facility. The ITN production facility will perform large-scale production of MHC Class II tetramers and multiparameter flow cytometry analyses of samples from ITN patients.

The ITN MHC peptide tetramer core is being used to innumerate antigen specific T cells and class II-recognizing T cells (CD4 cells). At present the major application for this technology is for Type 1 diabetes where we are looking at T cell responses to GAD65 antigen. Major questions include, are therapies decreasing the number of T cells specific for antigen or is there no change, indicating T cells becoming functionally anergic. This is one of the fundamental questions that we hope to answer with this technique.

In the future, T cell responses to insulin and to some of the peptide antigens presumed to be involved in Multiple Sclerosis as well as antigens implicated in other autoimmune diseases will be investigated. A list of 38 MS and type 1 diabetes peptides have currently been identified that will form a the standard peptide list for ITN investigations.

Methods: Coding regions for DR or DQ alpha and beta chains are truncated and spliced to leucine zipper and biotinylation sequence motifs, to generate chimeric constructs, which are then subcloned into the Cu-inducible Drosophila expression vector pRmHa-3. These chimeric cDNAs in the Schneider expression vector pRmHa-3 together with the plasmid pUChsneo, which carries the neomycin resistance marker, are cotransfected into Schneider cells S-2 by standard calcium phosphate transfection techniques and selected with G418 at 2 mg/ml. Cells are expanded to a density of 1 ? 107 cells/ml and treated with 1 mM CuSO4 to induce the production of soluble class II molecules, which are purified by affinity chromatography using anti-class II Mab.

After purification, the chimeric class II molecules are biotinylated using the Bir A enzyme and loaded with peptide by incubation for 72 hours at 37°C with 10-fold molar peptide excess. Class II molecules are then incubated overnight at room temperature with PE-streptavidin (BioSource International, Camarillo, CA) at an 8:1 molar ratio to allow the formation of tetrameric class II–peptide complexes. (see J Clin Invest 104:R63-R67, 1999 for detailed methods).

Participating Investigators

	Gerry Nepom, Virginia Mason Research Center
	Jeffrey Bluestone, The Diabetes Center at UCSF
	Bill Kwok, Virginia Mason Research Center
	Emma Masteller, The Diabetes Center at UCSF

Background Articles

	MHC class II tetramers identify peptide-specific human CD4+ T cells proliferating in response to influenza A antigen - J Clin Invest [go]
	Defining antigen-specific responses with human MHC class II tetramers - J Allergy Clin Immunol [go]
	Detection of GAD65-specific T-cells by major histocompatibility complex class II tetramers in type 1 diabetic patients and at-risk subjects - Diabetes [go]

Resources & Interesting Links

	MHC Class II Tetramer Information - Virginia Mason [go]
	MHC Tetramer technology (overview) - Molecular Probes [go]