title: Generation of functional HLA-DR*1101 tetramers receptive for loading with pathogen- or tumour-derived synthetic peptides.
abstract: BACKGROUND
  MHC class I-peptide tetramers are currently utilised to characterize CD8+ T cell responses at single cell level. The generation and use of MHC class II tetramers to study antigen-specific CD4+ T cells appears less straightforward. Most MHC class II tetramers are produced with a homogeneously built-in peptide, reducing greatly their flexibility of use. We attempted the generation of "empty" functional HLA-DR*1101 tetramers, receptive for loading with synthetic peptides by incubation. No such reagent is in fact available for this HLA-DR allele, one of the most frequent in the Caucasian population.
  
  
  RESULTS
  We compared soluble MHC class II-immunoglobulin fusion proteins (HLA-DR*1101-Ig) with soluble MHC class II protein fused with an optimised Bir site for enzymatic biotynilation (HLA-DR*1101-Bir), both produced in insect cells. The molecules were multimerised by binding fluorochrome-protein A or fluorochrome-streptavidin, respectively. We find that HLA-DR*1101-Bir molecules are superior to the HLA-DR*1101-Ig ones both in biochemical and functional terms. HLA-DR*1101-Bir molecules can be pulsed with at least three different promiscuous peptide epitopes, derived from Tetanus Toxoid, influenza HA and the tumour associated antigen MAGE-3 respectively, to stain specific CD4+ T cells. Both staining temperature and activation state of CD4+ T cells are critical for the binding of peptide-pulsed HLA-DR*1101-Bir to the cognate TCR.
  
  
  CONCLUSIONS
  It is therefore possible to generate a soluble recombinant HLA-DR*1101 backbone that is receptive for loading with different peptides to stain specific CD4+ T cells. As shown for other HLA-DR alleles, we confirm that not all the strategies to produce soluble HLA-DR*1101 multimers are equivalent.
authors.affiliation: Cancer Immunotherapy and Gene Therapy Program, Dept. of Oncology, DIBIT San Raffaele Scientific Institute, 20132 Milan, Italy. moro.monica@hsr.it
authors.forename: Monica
authors.initials: M
authors.lastname: Moro
authors.forename: Virginia
authors.initials: V
authors.lastname: Cecconi
authors.forename: Chiara
authors.initials: C
authors.lastname: Martinoli
authors.forename: Eliana
authors.initials: E
authors.lastname: Dallegno
authors.forename: Barbara
authors.initials: B
authors.lastname: Giabbai
authors.forename: Massimo
authors.initials: M
authors.lastname: Degano
authors.forename: Nicholas
authors.initials: N
authors.lastname: Glaichenhaus
authors.forename: Maria Pia
authors.initials: MP
authors.lastname: Protti
authors.forename: Paolo
authors.initials: P
authors.lastname: Dellabona
authors.forename: Giulia
authors.initials: G
authors.lastname: Casorati
mesh_terms: D000818:Animals; D017951:Antigen Presentation; D000951:Antigens, Neoplasm / Q000737:chemistry / Q000276:immunology*; D019921:Biotinylation; D015496:CD4-Positive T-Lymphocytes / Q000276:immunology; D002460:Cell Line; D018076:DNA, Complementary / Q000235:genetics; D004331:Drosophila melanogaster / Q000166:cytology; D000939:Epitopes / Q000276:immunology; D005803:Genes, Immunoglobulin; D005802:Genes, MHC Class II; D005813:Genes, Synthetic; D006684:HLA-DR Antigens / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D059811:HLA-DRB1 Chains; D019267:Hemagglutinin Glycoproteins, Influenza Virus / Q000737:chemistry / Q000276:immunology*; D006801:Humans; D007141:Immunoglobulin Fc Fragments / Q000235:genetics; D007074:Immunoglobulin G / Q000235:genetics; D016350:Leucine Zippers; D009363:Neoplasm Proteins / Q000737:chemistry / Q000276:immunology*; D010446:Peptide Fragments / Q000737:chemistry / Q000276:immunology*; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000276:immunology; D013745:Tetanus Toxoid / Q000276:immunology*; D014162:Transfection
chemical_list: D000951:Antigens, Neoplasm; D018076:DNA, Complementary; D000939:Epitopes; D006684:HLA-DR Antigens; D059811:HLA-DRB1 Chains; C509455:HLA-DRB1*11:01 antigen; D019267:Hemagglutinin Glycoproteins, Influenza Virus; D007141:Immunoglobulin Fc Fragments; D007074:Immunoglobulin G; C072013:MAGEA3 protein, human; D009363:Neoplasm Proteins; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D013745:Tetanus Toxoid; C509456:tetanus toxoid (830-845)