Figure 4.
The HLA-A11-restricted TCR for dNPM1 does not target AML in mice. Male NSG mice were injected i.v. with 1 × 106 OCI-AML3.bm10 (HLA-A*02:01+, dNPM1) cells that were transduced with HLA-A11 and luciferase. CD8 T-cells from an HLA-A*02:01+ (HLA-A2) and HLA-A11+ healthy individual (donor 3944) were transduced with a TCR on day 2, purified on mouse TCR β chain expression on day 8 and injected on day 11. After 11 days of tumor engraftment, mice were infused i.v. with PBS or 6 × 106 CD8 T-cells transduced with the HLA-A11 dNPM1 or EBV TCR or the HLA-A2 dNPM1 TCR. Tumor growth was monitored for 2 weeks after T-cell injection, after which mice were sacrificed and bone marrow was harvested. (A) In vivo growth of luciferase-transduced OCI-AML3.bm10 cells was measured twice per week by bioluminescent imaging. Depicted is the tumor burden in individual mice (left panel) and per treatment group (right panel). No effect was observed in mice treated with T-cells with the HLA-A11 dNPM1 TCR (n = 8, red line) or EBV TCR (n = 7, blue line) or in mice receiving PBS (n = 3, black line), whereas mice receiving HLA-A2 dNPM1 T-cells (n = 5, green line) showed a reduction in tumor growth. Symbols represent mean ± SD; (B) T-cells were tested on the day of infusion for recognition of OCI-AML3.bm10 cells in vitro at different E:S ratios. IFN-γ secretion was measured after overnight incubation by ELISA. T-cells transduced with the HLA-A11 dNPM1 TCR (red bars) clearly recognized OCI-AML3.bm10 cells, although IFN-γ production was lower as compared to the HLA-A2 dNPM1 TCR (green bars) or EBV TCR (blue bars; OCI-AML3.bm10 loaded with 1 µM EBV peptide). Bars represent mean + SD of duplicate wells; (C) Mouse bone marrow samples harvested 2 weeks after T-cell infusion were thawed and cultured for 3 weeks, and samples that showed outgrowth of AML cells were tested for T-cell recognition at an E:S ratio of 1:7.5. IFN-γ release was measured by ELISA after overnight incubation. TCR-transduced T-cells that were frozen on the day of infusion were thawed, stimulated and expanded for 11 days before testing. AML cells were also pulsed with a mix of dNPM1 and EBV peptides at a concentration of 500 nM per peptide. All AML samples were recognized by T-cells with the HLA-A11 dNPM1 TCR (red bars) or HLA-A2 dNPM1 TCR (green bars) and by T-cells with the EBV TCR (blue bars) after peptide loading, but IFN-γ release by T-cells with the HLA-A11 dNPM1 TCR was lower. Bars represent mean + SD of duplicate wells.