Generation of functional HLA-DR*1101 tetramers receptive for loading with pathogen or tumour derived synthetic peptides

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

Research output: Contribution to journalArticlepeer-review

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.

Original languageEnglish
Article number24
JournalBMC Immunology
Volume6
DOIs
Publication statusPublished - Dec 5 2005

ASJC Scopus subject areas

  • Medicine(all)
  • Immunology

Fingerprint Dive into the research topics of 'Generation of functional HLA-DR*1101 tetramers receptive for loading with pathogen or tumour derived synthetic peptides'. Together they form a unique fingerprint.

Cite this