title: Use of MHC class II tetramers to investigate CD4+ T cell responses: problems and solutions.
abstract: MHC-class I tetramers technology enabled the characterization of peptide-specific T cells at the single cell level in a variety of studies. Several laboratories have also developed MHC-class II multimers to characterize Ag-specific CD4+ T cells. However, the generation and use of MHC-class II multimers seems more problematic than that of MHC-I multimers. We have generated HLA-DR*1101 tetramers in a versatile empty form, which can be loaded after purification with peptides of interest. We discuss the impact of critical biological and structural parameters for the optimal staining of Ag-specific CD4+ T cells using HLA-DR*1101 tetramers, such as: (i) activation state of CD4+ T cells; (ii) membrane trafficking in the target CD4+ T cells; (iii) binding characteristics of the loaded CD4 epitope. Our data indicate that reorganization of TCR on the plasma membrane upon CD4+ T cell activation, as well as an homogenous binding frame of the CD4 epitopes to the soluble HLA-DR monomer, are critical for a stable TCR/MHC-class II tetramer interaction. These factors, together with the low frequencies and affinities of specific CD4+ T cells, explain the need for in vitro expansion or ex vivo enrichment of specific T cells for the optimal visualization with MHC-class II tetramers.
authors.affiliation: Experimental Immunology Unit, Cancer Immunotherapy and Gene Therapy Program, DIBIT, San Raffaele Scientific Institute, Milan 20132, Italy.
authors.forename: Virginia
authors.initials: V
authors.lastname: Cecconi
authors.forename: Monica
authors.initials: M
authors.lastname: Moro
authors.forename: Sara
authors.initials: S
authors.lastname: Del Mare
authors.forename: Paolo
authors.initials: P
authors.lastname: Dellabona
authors.forename: Giulia
authors.initials: G
authors.lastname: Casorati
mesh_terms: D000818:Animals; D000941:Antigens / Q000276:immunology; D015496:CD4-Positive T-Lymphocytes / Q000276:immunology*; D004330:Drosophila; D018604:Epitope Mapping; D006684:HLA-DR Antigens / Q000276:immunology; D059811:HLA-DRB1 Chains; D000949:Histocompatibility Antigens Class II / Q000737:chemistry* / Q000276:immunology*; D006801:Humans; D008213:Lymphocyte Activation; D021962:Membrane Microdomains; D010455:Peptides / Q000276:immunology; D055503:Protein Multimerization; D015203:Reproducibility of Results; D013696:Temperature
chemical_list: D000941:Antigens; D006684:HLA-DR Antigens; D059811:HLA-DRB1 Chains; C509455:HLA-DRB1*11:01 antigen; D000949:Histocompatibility Antigens Class II; D010455:Peptides