Resource Labs

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Tetramer Resource Lab

The Tetramer Resource Lab generates fluorescently labeled peptide:MHC multimers for the purpose of detecting polyclonal populations of antigen-specific T cells.

The understanding of adaptive immunity requires the study of T cells, which mediate cellular immune responses against protein-derived antigens.  T cells express antigen receptors (TCR) that bind complexes of peptide and MHC molecules (pMHC) displayed on the surface of antigen-presenting cells.  To study a T cell response to a particular antigen, one must identify T cells expressing TCRs that can bind to the pMHC derived from that antigen.

Many elaborate experimental systems have been developed to identify such antigen-specific T cells, but the most direct, and perhaps powerful approach involves the detection of these cells using a soluble reagent comprised of the pMHC ligand itself.  With such a reagent, one can stain for all the T cells within a population that are specific for a given pMHC epitope.  This allows for the comprehensive study of polyclonal T cell responses arising from an endogenous T cell repertoire rather than the more traditional approach of tracking a monoclonal response from a transferred population of TCR transgenic cells.  These types of studies will be essential in the future as the field of immunology demands more physiological experimental systems.

The binding affinity between a TCR and cognate pMHC complex is very low, which makes the use of a monomeric soluble pMHC complex inadequate.  However, multimerization of pMHC molecules creates a soluble protein that can interact with multiple TCRs on the surface of a T cell, resulting in a significant increase in overall avidity that allows for stable binding to the cell.  While fluorescently labeled pMHC tetramers have been in use for several years, there have been many technical problems related to their construction, particularly with pMHCII, that have prevented their widespread use.  We have incorporated several technical refinements into the design and production of pMHC tetramers to generate high quality reagents suitable for use in the tracking of antigen-specific T cells, which would be applicable to almost any study involving the immune system.

Director:

  • James J. Moon, Ph.D.

 

Technician:

  • Andre Han, BS

 

The Tetramer Resource Lab is currently under development under the auspices of Dr. James Moon.  When available, it will have the capacity to generate custom high quality pMHC class I and class II multimers labeled with a choice of fluorochromes.

The lab will contain the necessary infrastructure to design, clone, express, and purify recombinant pMHCI and pMHCII molecules, and to incorporate them into fluorescently labeled multimeric complexes (tetramers).  This will include basic equipment for routine molecular biology and protein purification applications, as well as specialized equipment for the growing of recombinant pMHCI and pMHCII from bacterial and insect cell expression systems, respectively.

The Tetramer Resource Lab will initially generate PE- and APC-labeled tetramers for some commonly used mouse I-A and human DR MHC class II-restricted peptide antigens.  When resources permit, custom tetramer orders for specifically requested antigens and/or MHC molecules will be considered, with first priority going to investigators within the CIID.

The generation of high quality pMHC tetramers requires numerous resources in the form of supplies, reagents, and labor.  A fee schedule to compensate these expenses will be determined for both stock and custom order tetramers.

Interested investigators should contact Dr. James Moon.