Details

Autor(en) / Beteiligte

• Moro, Monica
• Cecconi, Virginia
• Martinoli, Chiara
• Dallegno, Eliana
• Giabbai, Barbara
• Degano, Massimo
• Glaichenhaus, Nicholas
• Protti, Maria Pia
• Dellabona, Paolo
• Casorati, Giulia

Titel

Generation of functional HLA-DR1101 tetramers receptive for loading with pathogen- or tumour-derived synthetic peptides

Ist Teil von

• BMC immunology, 2005-12, Vol.6 (1), p.24-24, Article 24

Ort / Verlag

England: BioMed Central Ltd

Erscheinungsjahr

2005

Quelle

SpringerLink

Beschreibungen/Notizen

• 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. 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. 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.