Direct ex vivo detection of HLA-DR3-restricted cytomegalovirus- and Mycobacterium tuberculosis-specific CD4+ T cells

In order to detect epitope-specific CD4(+) T cells in mycobacterial or viral infections in the context of human class II major histocompatibility complex protein human leakocyte antigen (HLA)-DR3, two HLA-DR3 tetrameric molecules were successfully produced. One contained an immunodominant HLA-DR3-restricted T-cell epitope derived from the 65-kDa heat-shock protein of Mycobacterium tuberculosis, peptide 1-13. For the other tetramer, we used an HLA-DR3-restricted T-cell epitope derived from cytomegalovirus (CMV) pp65 lower matrix protein, peptide 510-522, which induced high levels of interferon (IFN)-gamma-producing CD4(+) T cells in three of four HLA-DR3-positive CMV-seropositive individuals up to 0.84% of CD4(+) T cells by intracellular cytokine staining. In peripheral blood mononuclear cells from M. tuberculosis-exposed, Mycobacterium bovis bacille Calmette-Guerin (BCG)-vaccinated, or CNW-seropositive individuals, we were able to directly detect with both tetramers; epitopespecific T cells up to 0.62% and 0.45% of the CD4(+) T-cell population reactive to M. tuberculosis and CNW, respectively. After a 6-day culture with peptide p510-522, the frequency of CMV-specific tetramer-binding T cells was expanded up to 9.90% tetramer(+) CFSElow (5,6-carboxyfluorescein diacetate succinimidyl ester) cells within the CD4(+) T-cell population, further confirming the specificity of the tetrameric molecules. Thus, HLA-DR3/peptide tetrameric molecules can be used to investigate HLA-DR3-restricted antigen-specific CD4(+) T cells in clinical disease or after vaccination. Human Immunology 66, 950-961 (2005). (c) American Society for Histocompatibility and Immunogenetics, 2005. Published by Elsevier Inc.