Figure 3.
Tumors that relapse following T cell treatment functionally express antigen, resist a second T cell treatment, but are responsive to T cell treatment after serial transplantation. HLA-A2 tg mice bearing established tumors from B16:A2-YLEP clone were conditioned and treated with 7.5 × 106 T cell receptors (TCR) or mock T cells according to legend to Figure 1a. (a) Following T cell treatment, mice with regressing (treated with TCR T cells, group 1), progressing (treated with mock T cells, group 2), and relapsing (treated with TCR T cells, group 3) tumors were sacrificed at the indicated time points. Tumors were isolated, single cell suspensions were prepared and short-term cultures (4–7 days) were set up (n = 3–5 mice per group). (b) Tumor cell HLA-A2 surface expression was measured by flow cytometry and data are presented as mean % positive cells in viable gate + SEM. (c) IFN-γ production by TCR T cells upon a 20 hours exposure to tumor cells ex vivo was analyzed by enzyme-linked immunosorbent assay. Data are presented as mean ng/ml + SEM. Statistical significances were calculated with Student's t-tests: *P < 0.05, ***P < 0.0005. (d) HLA-A2 tg mice bearing relapsed B16:A2-YLEP tumors were treated with TCR T cells for a second time (conditioned at days 30–32 and treated with T cells at day 34). A second treatment with mock T cells and no second treatment were included as controls. (e) Relapsed tumors from mice were isolated, cultured for 4 days, stored frozen (to allow synchronization of multiple samples), and retransplanted upon thawing (0.5 × 106 viable cells) in naïve HLA-A2 tg mice (retransplanted tumors, n = 5). When tumors re-established, recipient mice were subjected to treatment with TCR T cells as described in legend to Figure 3a. HLA-A2 tg mice bearing established tumors from B16:A2-YLEP clone were used as controls (transplanted tumors, n = 4). Tumor sizes in Figure 3d,e were measured and expressed as described in legend to Figure 1b.