Figure 2
Figure 2. AML-reactive CD8+ T cells of patient J.C. specifically recognized the HLA-A*0101–restricted peptide YVDFREYEYY (YVD/A1) encoded by JC-FLT3-ITD. (A) Specificity of IVS-responder populations. JC-PBMCs collected in CR after induction chemotherapy were stimulated with peptide YVD/A1 in independent IVS. IVS responders were tested on day 13 in a 20-hour IFN-γ ELISPOT assay for recognition of unloaded, YVD/A1-loaded, wtFLT3 mRNA–transfected, or JC-FLT3-ITD mRNA–transfected K562/A1 cells (1 × 105 per well) as well as for recognition of autologous AML cells (1 × 105 per well). The results obtained with IVS JC#1, JC#2, and JC#4 are shown as representative examples. (B) Specificity of T-cell clones derived from IVS JC#1. JC#1 responders were cloned on day 14 by limiting dilution using HLA-I–compatible EF-DCs transfected with JC-FLT3-ITD mRNA as stimulators. T-cell clones were tested 20 days later against unloaded or YVD/A1-loaded K562/A1 cells (1 × 105 per well) as well as against autologous AML cells (1 × 105 per well) in a 20-hour IFN-γ ELISPOT assay. Three representative clones, JC#1.5, JC#1.6, and JC#1.12, are shown. (C) Reactivity against YVD/A1 detectable in ex vivo CD8+ T cells. CD8+ T cells were positively selected from JC-AML cells with immunomagnetic beads (Miltenyi Biotec, Bergisch Gladbach, Germany) and tested in a 30-hour IFN-γ and in a 30-hour GrB ELISPOT assay against unloaded or peptide-loaded untransfected K562 cells (1 × 105 per well) as well as against unloaded or peptide-loaded K562/A1 cells (1 × 105 per well). Peptides were YVD/A1- and known HLA-A1–binding peptides from HCMV pp65, tyrosinase, and influenza A basic polymerase 1 (pp65 364 to 373, SEHPTFTSQY; tyrosinase 146 to 156, SSDYVIPIGTY; PB1 591 to 599, VSDGGPNLY, respectively). The pp65 peptide served as a positive control; tyrosinase and influenza A peptides were negative controls. Data are means of duplicates. Notably, YVD/A1 recognition required the presence of HLA-A1 on K562 cells, because peptide-loaded but untransfected K562 cells did not induce spot formation in any test.

AML-reactive CD8+ T cells of patient J.C. specifically recognized the HLA-A*0101–restricted peptide YVDFREYEYY (YVD/A1) encoded by JC-FLT3-ITD. (A) Specificity of IVS-responder populations. JC-PBMCs collected in CR after induction chemotherapy were stimulated with peptide YVD/A1 in independent IVS. IVS responders were tested on day 13 in a 20-hour IFN-γ ELISPOT assay for recognition of unloaded, YVD/A1-loaded, wtFLT3 mRNA–transfected, or JC-FLT3-ITD mRNA–transfected K562/A1 cells (1 × 105 per well) as well as for recognition of autologous AML cells (1 × 105 per well). The results obtained with IVS JC#1, JC#2, and JC#4 are shown as representative examples. (B) Specificity of T-cell clones derived from IVS JC#1. JC#1 responders were cloned on day 14 by limiting dilution using HLA-I–compatible EF-DCs transfected with JC-FLT3-ITD mRNA as stimulators. T-cell clones were tested 20 days later against unloaded or YVD/A1-loaded K562/A1 cells (1 × 105 per well) as well as against autologous AML cells (1 × 105 per well) in a 20-hour IFN-γ ELISPOT assay. Three representative clones, JC#1.5, JC#1.6, and JC#1.12, are shown. (C) Reactivity against YVD/A1 detectable in ex vivo CD8+ T cells. CD8+ T cells were positively selected from JC-AML cells with immunomagnetic beads (Miltenyi Biotec, Bergisch Gladbach, Germany) and tested in a 30-hour IFN-γ and in a 30-hour GrB ELISPOT assay against unloaded or peptide-loaded untransfected K562 cells (1 × 105 per well) as well as against unloaded or peptide-loaded K562/A1 cells (1 × 105 per well). Peptides were YVD/A1- and known HLA-A1–binding peptides from HCMV pp65, tyrosinase, and influenza A basic polymerase 1 (pp65 364 to 373, SEHPTFTSQY; tyrosinase 146 to 156, SSDYVIPIGTY; PB1 591 to 599, VSDGGPNLY, respectively). The pp65 peptide served as a positive control; tyrosinase and influenza A peptides were negative controls. Data are means of duplicates. Notably, YVD/A1 recognition required the presence of HLA-A1 on K562 cells, because peptide-loaded but untransfected K562 cells did not induce spot formation in any test.

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