Tetramers - Technical Background
The major histocompatibility complex (MHC) plays a central role in the immune system. MHC-encoded class I molecules serve as peptide binding, transport and display proteins on the cell surface. Class I molecules consist of two noncovalently associated subunits: a highly polymorphic heavy chain and a conserved ß2-microglobulin (ß2-M) light chain. Two of the three extracellular domains of the heavy chain are folded into a 'groove' structure which anchors an antigenic peptide for presentation to T cells.
Human class I molecules have been refolded from Escherichia coli-produced heavy chain and ß2-M subunits in association with synthetic peptides (1). Recombinant MHC-peptide molecules have all the characteristics of the native complexes, i.e., they can elicit cytolytic T cell responses and stimulate the proliferation of antigen-specific CD8+ T cells (2).
T-cell receptors (TCR) have an intrinsic low affinity for their cognate MHC-peptide ligand. To produce soluble MHC-peptide complexes in a form that would bind stably and could be used to identify, count and isolate CD8 T cells having the appropriate TCR, multimeric MHC-peptide complexes, i.e. dimerized molecules and tetramers were prepared (3,4). This work builds on the assembly of recombinant MHC peptide complexes using a mutant human ß2-M chain. With biotin added to the surface-exposed SH-group, the assembled MHC-peptide monomers form tetramers with avidin linked to a fluorochrome (5). This strategy permits the production of MHC class I tetramers, without the necessity of genetic engineering each heavy chain, a significant advantage in view of the enormous variety and polymorphism of MHC heavy chains (6).
CD8+ T cells recognize virally infected cells or tumor cells and use the perforin / granzyme pathway to kill the target cell. T cells that are stimulated by antigen are developmentally programmed to divide at least 7 - 10 times and to differentiate into effector cytotoxic T lymphocytes and long lived, functional memory CD8+ T cells.
Tetramers provide a powerful and general tool for the study of antigen-specific T cells. Availability of these reagents will permit clinical researchers to investigate T cell responses to a variety of natural pathogens. FACS based T cell sorting of specific T cell populations, labeled with these reagents, will allow specific expansion of high-avidity cytotoxic T cells for adoptive immunotherapy. Finally, analysis of antigen-specific T cell populations could provide a direct diagnostic method to assay CMV and EBV responses in transplant patients.
References:
1. HLA-A2-peptide complexes: refolding and crystallization of molecules expressed in E.coli and complexed with single antigenic peptides.
Garboczi DN, Hung DT, Wiley DC
PNAS, 1992, 89, 3429-3433
2. Stimulation of human cytotoxic T cells with HIV-1-derived peptides presented by recombinant HLA-A2 peptide complexes.
Walter JB, Brander C, Mammen M, Garboczi DN, Kalams SA, Whitesides GM, Walker BD, Eisen HN
Int. Immunology, 1997, 9, 451-459
3. A soluble divalent class I major histocompatibility complex molecule inhibits alloreactive T cells at nanomolar concentrations.
Dal Porto J, Johansen TE, Catipovic B, Parfiit DJ, Tuveson D, Gether U, Kozlowski S, Fearon DT, Schneck JP
PNAS, 1993, 90, 6671-6675
4. Phenotypic analysis of antigen-specific T lymphocytes.
Altman JD, Moss PA, Goulder PJ, Barouch DH, McHeyzer-Williams MG, Bell JI, McMichael AJ, Davis MM
Science, 1996, 274, 94-96
5. US Patent: 6,248,564 B1; Mutant MHC Class I Molecules;
Walter JB, and Garboczi DN
6. A mutant human beta 2-microglobulin can be used to generate multimeric class I peptide complexes as specific probes for T cell receptors.
Walter JB, Garboczi DN, Fan QR, Zhou X, Walker BD, Eisen HN
Journal of Immunological Methods, 1998, 214, 41-50