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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. CONCLUSION: 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.
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https://www.hal.inserm.fr/inserm-00089280
Contributor : Delphine Autard <>
Submitted on : Wednesday, August 16, 2006 - 5:16:45 PM
Last modification on : Tuesday, May 26, 2020 - 6:50:23 PM
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Monica Moro, Virginia Cecconi, Chiara Martinoli, Eliana Dallegno, Barbara Giabbai, et al.. Generation of functional HLA-DR*1101 tetramers receptive for loading with pathogen- or tumour-derived synthetic peptides.. BMC Immunology, BioMed Central, 2005, 6, pp.24. ⟨10.1186/1471-2172-6-24⟩. ⟨inserm-00089280⟩

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