Dextramers: new generation of fluorescent MHC class I/peptide multimers for visualization of antigen-specific CD8+ T cells.
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.
Batard
Pascal
P
Division of Clinical Onco-Immunology, Ludwig Institute for Cancer Research, Lausanne Branch, University Hospital (CHUV), Switzerland.
Peterson
Daniel A
DA
Devêvre
Estelle
E
Guillaume
Philippe
P
Cerottini
Jean-Charles
JC
Rimoldi
Donata
D
Speiser
Daniel E
DE
Winther
Lars
L
Romero
Pedro
P
D000951:Antigens, Neoplasm; D018414:CD8-Positive T-Lymphocytes / Q000276:immunology*; D003911:Dextrans / Q000737:chemistry / Q000276:immunology*; D018984:Epitopes, T-Lymphocyte / Q000276:immunology*; D005434:Flow Cytometry; D005456:Fluorescent Dyes / Q000737:chemistry*; D015789:HLA-A2 Antigen / Q000737:chemistry / Q000276:immunology*; D006801:Humans; D058965:MART-1 Antigen; D008856:Microscopy, Fluorescence; D008970:Molecular Weight; D009363:Neoplasm Proteins / Q000737:chemistry / Q000276:immunology; D010750:Phosphoproteins / Q000737:chemistry / Q000276:immunology; D013602:T-Lymphocytes, Cytotoxic / Q000276:immunology*; D015534:Trans-Activators / Q000737:chemistry / Q000276:immunology; D014763:Viral Matrix Proteins / Q000737:chemistry / Q000276:immunology; D014764:Viral Proteins / Q000737:chemistry / Q000276:immunology
D000951:Antigens, Neoplasm; D003911:Dextrans; D018984:Epitopes, T-Lymphocyte; D005456:Fluorescent Dyes; D015789:HLA-A2 Antigen; D058965:MART-1 Antigen; C548667:MLANA protein, human; D009363:Neoplasm Proteins; D010750:Phosphoproteins; C090236:SM protein, Human herpesvirus 4; D015534:Trans-Activators; D014763:Viral Matrix Proteins; D014764:Viral Proteins