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

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.The direct ex-vivo visualization and quantification of antigen-specific T cells is key to the characterisation of complex immune responses. The antigen specificity of T cells is determined by the highly specific interaction between their TCR and the cognate MHC-peptide complex. The low affinity and fast off-rate of this interaction, however, precludes its exploitation to identify specific T cells [1-5]. Tetramer technology allows to circumvent this limitation, since the overall increased avidity of the MHC multimers compensates for the low affinity of the TCR-peptide/MHC interaction [6,7]. MHC class I tetramers are now indeed largely used to characterise CD8+ T cells in many basic or clinical settings [8-13]. By contrast, the generation of functional soluble peptide/MHC class II multimers, to characterise CD4+ T cell responses, seems somewhat less straightforwa