A mutant human beta2-microglobulin can be used to generate diverse multimeric class I peptide complexes as specific probes for T cell receptors.
Antigen-specific receptors (TCR) on CD8 T lymphocytes form relatively short-lived complexes with their natural ligands: peptides in association with major histocompatibility complex (MHC) class I molecules, which consist of a polymorphic heavy chain and a conserved light chain, beta2-microglobulin (beta2-M). To produce soluble MHC-peptide complexes in a form that would bind more stably and could be used to identify, count, and isolate CD8 T cells having the appropriate TCR, we prepared multimeric MHC-peptide complexes. Our work builds on the assembly of recombinant MHC class I peptide complexes using a mutant human beta2-M chain (Tyr 67 > Cys) which can form stable heterodimers with diverse MHC heavy chains. With biotin added to the SH group, the assembled MHC-peptide monomers formed multimers with avidin linked to a fluorochrome. The specific reactivity of the multimeric reagents with human and mouse cytotoxic T cells (CTL) is described. The present approach permits the production of class I multimers, without the necessity of genetic engineering each heavy chain, a significant advantage in view of the enormous polymorphism of MHC heavy chains. Because human beta2-M forms stable heterodimers with diverse class I heavy chains from various species (human and non human primates, mouse, etc.), this procedure is a general method for producing multimers of MHC-peptide complexes as T cell receptor-specific probes.
Center for Cancer Research and Department of Biology, Massachusetts Institute of Technology, Cambridge 02139, USA. j.b.walter@tonline.de
J B
JB
Walter
D N
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Garboczi
Q R
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Fan
X
X
Zhou
B D
BD
Walker
H N
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Eisen
D000818:Animals; D001665:Binding Sites; D001710:Biotin / Q000737:chemistry; D005456:Fluorescent Dyes / Q000737:chemistry; D015395:Histocompatibility Antigens Class I / Q000737:chemistry*; D006801:Humans; D007143:Immunoglobulin Heavy Chains / Q000737:chemistry; D051379:Mice; D009154:Mutation; D010455:Peptides / Q000138:chemical synthesis*; D011948:Receptors, Antigen, T-Cell / Q000032:analysis* / Q000737:chemistry; D013379:Substrate Specificity; D001613:beta 2-Microglobulin / Q000737:chemistry* / Q000235:genetics
D005456:Fluorescent Dyes; D015395:Histocompatibility Antigens Class I; D007143:Immunoglobulin Heavy Chains; D010455:Peptides; D011948:Receptors, Antigen, T-Cell; D001613:beta 2-Microglobulin; D001710:Biotin