Fig. 5.

Detection of MAGE-A3_111–125 specific CD4⁺ T-cell clones with MAGE-A3 111–125/DPB1*04 multimer. a Specific (clones 131, 134, 48) and nonspecific (clone 30) clones from LAU 2091 generated against MAGE-A3_111–125 were labeled with DP4/MAGE-A3_111–125 -PE multimer and anti-CD4-FITC antibodies. Cells were gated on live CD3⁺ cells. b Fine specificity of DP4 tetramer shown by distinct staining of tetanus toxoid_947–960 and MAGE-A3_111–125 CD4⁺ T-cell clones (clone 4 and 13, respectively) with a combination of DP4/TT_947–960-APC and DP4/MAGE-A3_111–125-PE multimers. Cells were gated on live CD4⁺ T cells. c DP4/MAGE-A3_111–125 specific CD4⁺ T cells detected after 1, 2 and 3 rounds of in vitro stimulation of the LAU 2091 PBLs with MAGE-A3 overlapping peptide pools. Left dot plots PBLs were screened with the tetramer and gated on live CD4⁺ T cells. Right dot plots phenotype of CD4⁺ tetramer T-cell populations analyzed by CD45RA and CCR7 staining. All dot plots are gated on live cells