Figure 5
Figure 5. Recognition of residual patient-derived T-cells in PB before CD4+ DLI by allo-reactive HLA-DPB1–specific CD4 T-cell clones. (A) Coexpression of HLA-DR and HLA-DP on CD3+ T cells in cryopreserved PBMCs from patient 1 before CD4+ DLI and on patient-derived CMV-specific T cells after 2 weeks of in vitro expansion and 24 hours after restimulation with CMV antigens was determined by flow cytometry. As a control, expression of HLA-DR and -DP is shown for healthy donor CD3+ T cells. (B-C) Activation of HLA-DPB1–specific CD4+ T-cell clones from patient 1 (B) and patient 2 (C) was measured upon stimulation with patient-derived T cells after direct ex vivo isolation from cryopreserved PBMCs (-▲-), patient-derived CMV-specific T cells after 2 weeks of in vitro expansion (-○-), and 24 hours after in vitro restimulation with CMV antigens (-●-) and against patient (-▪-) and donor (-□-) EBV-LCL. Activation was measured 24 hours after stimulation by flow cytometric staining for CD137. Percentages of activated CD137+ cells are depicted for different CD4+ T-cell clones at different responder/stimulator cell ratios. Responder/stimulator cell ratio 1/30 could not be tested for patient-derived T cells after ex vivo isolation from cryopreserved PBMCs because of insufficient material. In total, 4 HLA-DPB1*0101– and 4 HLA-DPB1*0301–specific CD4+ T-cell clones from patient 1 (B) and 3 HLA-DPB1*0301–specific CD4+ T-cell clones from patient 2 (C) were analyzed. No or only minimal activation of HLA-DPB1–restricted CD4+ T-cell clones was observed upon stimulation with patient-derived T cells after direct ex vivo isolation and patient-derived CMV-specific T cells after 2 weeks of in vitro expansion. Upon stimulation with patient-derived CMV-specific T cells 24 hours after in vitro restimulation with CMV antigens, however, 3 HLA-DPB1*0101–specific and 2 HLA-DPB1*0301–specific CD4+ T-cell clones from patient 1 (B, upper) and 1 HLA-DPB1*0301–specific CD4+ T-cell clone from patient 2 (C, upper) were activated, whereas the remaining HLA-DPB1–restricted CD4+ T-cell clones from patient 1 (B, lower) and patient 2 (C, lower) were not or were only minimally activated.

Recognition of residual patient-derived T-cells in PB before CD4+ DLI by allo-reactive HLA-DPB1–specific CD4 T-cell clones. (A) Coexpression of HLA-DR and HLA-DP on CD3+ T cells in cryopreserved PBMCs from patient 1 before CD4+ DLI and on patient-derived CMV-specific T cells after 2 weeks of in vitro expansion and 24 hours after restimulation with CMV antigens was determined by flow cytometry. As a control, expression of HLA-DR and -DP is shown for healthy donor CD3+ T cells. (B-C) Activation of HLA-DPB1–specific CD4+ T-cell clones from patient 1 (B) and patient 2 (C) was measured upon stimulation with patient-derived T cells after direct ex vivo isolation from cryopreserved PBMCs (-▲-), patient-derived CMV-specific T cells after 2 weeks of in vitro expansion (-○-), and 24 hours after in vitro restimulation with CMV antigens (-●-) and against patient (-▪-) and donor (-□-) EBV-LCL. Activation was measured 24 hours after stimulation by flow cytometric staining for CD137. Percentages of activated CD137+ cells are depicted for different CD4+ T-cell clones at different responder/stimulator cell ratios. Responder/stimulator cell ratio 1/30 could not be tested for patient-derived T cells after ex vivo isolation from cryopreserved PBMCs because of insufficient material. In total, 4 HLA-DPB1*0101– and 4 HLA-DPB1*0301–specific CD4+ T-cell clones from patient 1 (B) and 3 HLA-DPB1*0301–specific CD4+ T-cell clones from patient 2 (C) were analyzed. No or only minimal activation of HLA-DPB1–restricted CD4+ T-cell clones was observed upon stimulation with patient-derived T cells after direct ex vivo isolation and patient-derived CMV-specific T cells after 2 weeks of in vitro expansion. Upon stimulation with patient-derived CMV-specific T cells 24 hours after in vitro restimulation with CMV antigens, however, 3 HLA-DPB1*0101–specific and 2 HLA-DPB1*0301–specific CD4+ T-cell clones from patient 1 (B, upper) and 1 HLA-DPB1*0301–specific CD4+ T-cell clone from patient 2 (C, upper) were activated, whereas the remaining HLA-DPB1–restricted CD4+ T-cell clones from patient 1 (B, lower) and patient 2 (C, lower) were not or were only minimally activated.

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