Immunomonitoring for R3-specific CD8⁺ T lymphocytes in patient no. 8 with multiple myeloma. (A) The dot plots show the percentage of HLA-A2/R3-tetramer⁺/CD8⁺ T lymphocytes. Six-color staining for R3-specific CD8⁺ T lymphocytes revealed an increase of CD8⁺/HLA-A2/R3-tetramer⁺ T lymphocytes over 4 vaccinations and a decrease thereafter (i represents before; ii, after first; iii, after second; iv, after third; and v, 3 weeks after fourth/last vaccination). The HLA-A2/R3-tetramer*PE–positive CD8⁺ T lymphocytes could not be counterstained by WT1-tetramers demonstrating their specificity for the peptide R3. The staining for CCR7 and CD45RA demonstrated an increase of CD8⁺/HLA-A2/R3 tetramer⁺/WT1 tetramer⁻/CCR7⁻/CD45RA⁺ effector T cells from 47% to 81%. The frequency of CD8⁺/HLA-A2/R3 tetramer⁺/WT1 tetramer⁻/CCR7⁻/CD45RA⁺/CD27⁻ effector T cells rose from 63% up to 78%. The CD8⁺/HLA-A2/R3 tetramer⁺/WT1 tetramer⁻/CCR7⁻/CD45RA⁺/CD28⁻ effector T cells increased over 4 vaccinations, but decreased 3 weeks thereafter. Numbers on plots are percentages of all lymphocytes. (B) The results of the ELISpot assay for granzyme B correlated with the FACS data showing an increase and subsequent decrease of R3-specific CD8⁺ T-lymphocyte function during the course of vaccination. All assays were performed in triplicate. Error bars indicate the SD.