Dextramers: New generation of fluorescent MHC class I/peptide multimers for visualization of antigen-specific CD8 + T cells

Abstract

Direct identification as well as isolation of antigen-specific T cells became possible since the development of “tetramers” based on avidin–fluorochrome conjugates associated with mono-biotinylated class I MHC–peptide monomeric complexes. In principle, a series of distinct class I MHC–peptide tetramers, each labelled with a different fluorochrome, would allow to simultaneously enumerate as many unique antigen-specific CD8 + T cells. Practically, however, only phycoerythrin and allophycocyanin conjugated tetramers have been generally available, imposing serious constraints for multiple labeling. To overcome this limitation, we have developed dextramers which are multimers based on a dextran backbone bearing multiple fluorescein and streptavidin moieties. Here we demonstrate the functionality and optimization of these new probes on human CD8 + T cell clones with four independent antigen specificities. Their applications to the analysis of relatively low frequency antigen-specific T cells in peripheral blood, as well as their use in fluorescence microscopy, are demonstrated. The data show that dextramers produce a stronger signal than their fluoresceinated tetramer counterparts. Thus, these could become the reagents of choice as the antigen-specific T cell labeling transitions from basic research to clinical application.