WT1-TCB–mediated T-cell cytotoxicity and T-cell activation. (A) Induction of downstream TCR signaling in NFAT Jurkat Reporter cells upon recognition of RMF peptide–MHC complexes on primary AML cells. Statistical analysis, Mann-Whitney U test (n = 10). (B) Dose-dependent specific lysis of primary HLA-A*02+ AML cells by allogeneic (black solid line; n = 9) or autologous (black dashed line; n = 3) PBMCs after 48 hours in short-term cytotoxicity assays with WT1-TCB. Controls: HLA-A*02+ WT1– CD33+ HD cells (gray solid line; n = 6) and primary HLA-A*02− AML cells (gray dashed line; n = 10). CD25 (C) and CD69 (D) expression on CD3+ T cells after 48 hours in killing assays with allogeneic PBMCs and primary HLA-A*02+ AML cells (black solid line; n = 9), primary HLA-A*02− AML cells (gray dashed line; n = 10), or HLA-A*02+ WT1− CD33+ HD cells (gray solid line; n = 6). (E) T-cell proliferation after 96 hours in killing assays with allogeneic PBMCs and primary HLA-A*02+ AML cells (black solid line; n = 8), primary HLA-A*02− AML cells (gray dashed line; n = 7), or HLA-A*02+ WT1− CD33+ HD cells (gray solid line; n = 3). (F) Secretion of interferon-γ (IFN-γ) and granzyme B in allogeneic and autologous settings after 72 hours in a short-term cytotoxicity assay with primary HLA-A*02 AML cells. (G) Specific lysis of primary AML cells by allogeneic T cells (left: HLA-A*02− AML, n = 11; center: HLA-A*02+ AML, n = 19) or autologous T cells (right: HLA-A*02+ AML, n = 8) after 10 to 13 days in long-term cocultures and treatment with WT1-TCB*. (H) Representative dot plots from flow cytometry analysis showing CD2 and CD33 expression in long-term coculture samples. (I) Upregulation of activation and surrogate exhaustion markers on CD3+ T cells on days 3 to 4 in long-term cocultures with primary HLA-A*02+ and HLA-A*02− AML cells (n = 14-22). Bars represent the mean ± SEM. Statistical analysis: Kruskal-Wallis test and subsequent Dunn’s test. **P < .01, ****P < .0001. MFI, mean fluorescence intensity; n.s., not significant; RLU, relative luminescence units.
Figure 4.

WT1-TCB–mediated T-cell cytotoxicity and T-cell activation. (A) Induction of downstream TCR signaling in NFAT Jurkat Reporter cells upon recognition of RMF peptide–MHC complexes on primary AML cells. Statistical analysis, Mann-Whitney U test (n = 10). (B) Dose-dependent specific lysis of primary HLA-A*02+ AML cells by allogeneic (black solid line; n = 9) or autologous (black dashed line; n = 3) PBMCs after 48 hours in short-term cytotoxicity assays with WT1-TCB. Controls: HLA-A*02+ WT1– CD33+ HD cells (gray solid line; n = 6) and primary HLA-A*02− AML cells (gray dashed line; n = 10). CD25 (C) and CD69 (D) expression on CD3+ T cells after 48 hours in killing assays with allogeneic PBMCs and primary HLA-A*02+ AML cells (black solid line; n = 9), primary HLA-A*02− AML cells (gray dashed line; n = 10), or HLA-A*02+ WT1− CD33+ HD cells (gray solid line; n = 6). (E) T-cell proliferation after 96 hours in killing assays with allogeneic PBMCs and primary HLA-A*02+ AML cells (black solid line; n = 8), primary HLA-A*02− AML cells (gray dashed line; n = 7), or HLA-A*02+ WT1− CD33+ HD cells (gray solid line; n = 3). (F) Secretion of interferon-γ (IFN-γ) and granzyme B in allogeneic and autologous settings after 72 hours in a short-term cytotoxicity assay with primary HLA-A*02 AML cells. (G) Specific lysis of primary AML cells by allogeneic T cells (left: HLA-A*02− AML, n = 11; center: HLA-A*02+ AML, n = 19) or autologous T cells (right: HLA-A*02+ AML, n = 8) after 10 to 13 days in long-term cocultures and treatment with WT1-TCB*. (H) Representative dot plots from flow cytometry analysis showing CD2 and CD33 expression in long-term coculture samples. (I) Upregulation of activation and surrogate exhaustion markers on CD3+ T cells on days 3 to 4 in long-term cocultures with primary HLA-A*02+ and HLA-A*02− AML cells (n = 14-22). Bars represent the mean ± SEM. Statistical analysis: Kruskal-Wallis test and subsequent Dunn’s test. **P < .01, ****P < .0001. MFI, mean fluorescence intensity; n.s., not significant; RLU, relative luminescence units.

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