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Volume 6 Supplement 3

AIDS Vaccine 2009

Open Access

P16-56 LB. Novel tetramer technology for the detection of high affinity CD8 T cells

  • M Hoffmann1,
  • S Hickling2,
  • A Filby3,
  • C Wilburg2,
  • D Cole4,
  • A Turner2,
  • S Sims2,
  • A McLean5,
  • A Sewell4,
  • P Klenerman2,
  • J Frater2 and
  • RE Phillips2
Retrovirology20096(Suppl 3):P409

Published: 22 October 2009


Cell Base VaccineBase VaccineIndividual Cell LevelMolecule ConformationImage Flow Cytometry


Defining the quality of HIV T cell responses is a major hurdle in the development of T cell based vaccines. A key determinant of viral control is the affinity of the T Cell Receptor (TCR) for the HLA/epitope complex. We report for the first time in HIV the development of Class I HLA tetramers, which allow detection of CD8 with high TCR affinity that may prove to be invaluable in assessing the quality of T cell immunity.


HLA Class I molecules A*0201 and B*0702 were mutated at positions D227K and T228A to nullify CD8 binding and refolded with HIV epitopes: SLYNTVATL (A*0201), ILKEPVHGV (A*0201) and GPGHKARVL (B*0702). BIACORE confirmed abolition of CD8 binding and HLA molecule conformation. Mutated HLA monomers were termed CD8null. Peripheral blood mononuclear cells from HLA-matched acute patients in the SPARTAC trial (n = 30) were stained with A*0201 or B*0702 CD8 wild-type and CD8null tetramers. Real-time Image Flow Cytometry, directly examined the CD8null and CD8 wild-type tetramer/TCR interaction on an individual cell level.


HLA Class I A*0201 and B*0702 CD8 null monomers had undetectable CD8 binding. Wild-type monomers had comparable CD8 binding capacity for A*0201 and B*0702 (KD = 5.9 × 10-4 and 6.0 × 10-4 M, respectively). Both CD8wild-type and CD8null monomers are bound by the anti-HLA w6/32 antibody with equal affinity (KD = 4.2 × 10-10 M). Ex vivo imaging showed slower internalisation of CD8null, compared to CD8wild-typetetramers, indicating prolonged HLA/TCR interaction. HIV patients stained with CD8null and CD8wild-type had distinct high affinity CD8 populations for A*0201 SLYNTVATL and ILKEPVHGV (p < 0.05) but not for B*0702 GPGHKARVL.


CD8null tetramers represent a novel technology that allow the direct ex vivo detection and characterisation of high affinity CD8 T responses. This represents a crucial new tool for assessing the quality of T cell responses to vaccination.

Authors’ Affiliations

Department of Infectious Diseases, Cantonal Hospital St Gallen, Switzerland
The University of Oxford, Oxford, UK
Cronus Technologies UK, London, UK
University of Cardiff, Cardiff, UK
James Martin 21st Century School, The University of Oxford, Oxford, UK


© Hoffmann et al; licensee BioMed Central Ltd. 2009

This article is published under license to BioMed Central Ltd.