Antigen-specific CD8-expressing T cells play a crucial role in the host's defense against viral disease and malignancy. Epitope-specific CD8(+)T cell responses to malignant and viral disease can be accurately measured using tetramers (tHLA) of HLA class I molecules loaded with antigenic peptides. In addition, tHLA have been used to evaluate immune responses to antigen-specific immunization. tHLA bind specifically to complementary T-cell receptor (TCR) structures on the surface of T cells expressing the CD8 coreceptor. Surprisingly, however, CD8(-) cells binding tHLA are often observed. This study uses four-color flow cytometry to show that HLA-A*0201-tHLA-stained CD8(-) cells can be divided into two subsets: 87% represent B-lymphocytes (CD19(+), CD45RA(+), HLA-DR(+), and CD20(+)), and 13% represent T-helper cells (CD3(+), CD4(+), CD45RA(+), and CD27(+)). This phenomenon is not HLA-restricted because it could be observed even in peripheral blood mononuclear cells (PBMC) from a non HLA-A*0201-expressing healthy donor. In addition, no T-cell receptor was detected on the B-lymphocytes. Retrospective enumeration of vaccine-induced CD8(-) tHLA cells in 243 PBMC samples from 36 patients with melanoma undergoing peptide vaccination revealed that tHLA staining is not dependent on immunization status or the presence of CD8(+) tHLA(+) T cells. These findings, suggest that the nonspecific binding of tHLA to non-TCR-expressing T cells requires a careful interpretation of results and further steps in preparation of sample for tHLA-based sorting of epitope specific T cells.
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