New design of MHC class II tetramers to accommodate fundamental principles of antigen presentation.
Direct identification and isolation of Ag-specific T cells became possible with the development of MHC tetramers, based on fluorescent avidins displaying biotinylated peptide-MHC complexes. This approach, extensively used for MHC class I-restricted T cells, has met very limited success with class II peptide-MHC complex tetramers (pMHCT-2) for the detection of CD4(+)-specific T cells. In addition, a very large number of these reagents, although capable of specifically activating T cells after being coated on solid support, is still unable to stain. To try to understand this puzzle and design usable tetramers, we examined each parameter critical for the production of pMHCT-2 using the I-A(d)-OVA system as a model. Through this process, the geometry of peptide-MHC display by avidin tetramers was examined, as well as the stability of rMHC molecules. However, we discovered that the most important factor limiting the reactivity of pMHCT-2 was the display of peptides. Indeed, long peptides, as presented by MHC class II molecules, can be bound to I-A/HLA-DQ molecules in more than one register, as suggested by structural studies. This mode of anchorless peptide binding allows the selection of a broader repertoire on single peptides and should favor anti-infectious immune responses. Thus, beyond the technical improvements that we propose, the redesign of pMHCT-2 will give us the tools to evaluate the real size of the CD4 T cell repertoire and help us in the production and testing of new vaccines.
Landais
Elise
E
Department of Immunology and Microbial Sciences, Scripps Research Institute, La Jolla, CA 92037, USA.
Romagnoli
Pablo A
PA
Corper
Adam L
AL
Shires
John
J
Altman
John D
JD
Wilson
Ian A
IA
Garcia
K Christopher
KC
Teyton
Luc
L
D000818:Animals; D017951:Antigen Presentation / Q000235:genetics / Q000276:immunology*; D019921:Biotinylation / Q000235:genetics; D002645:Chickens; D000949:Histocompatibility Antigens Class II / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology; D006825:Hybridomas; D051379:Mice; D008807:Mice, Inbred BALB C; D008822:Mice, Transgenic; D010047:Ovalbumin / Q000008:administration & dosage / Q000276:immunology; D010446:Peptide Fragments / Q000008:administration & dosage / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology; D015202:Protein Engineering; D013601:T-Lymphocytes / Q000276:immunology / Q000378:metabolism
D000949:Histocompatibility Antigens Class II; C111389:I-Ad antigen; C111390:OVA 323-339; D010446:Peptide Fragments; D010047:Ovalbumin