Fig 1.
Establishment of MLL/AF9 leukemia cells expressing a model tumor antigen.
(A) Scheme for establishment of MLL/AF9-HPC-OVA cells. (B) FACS analysis of MLL/AF9-HPC-OVA cells. Blue lines represent MLL/AF9-HPC cells. (C) 51Cr-release assay using activated CD8+ T cells from the OT-1 mouse as effector cells. MLL/AF9-HPC-OVA or MLL/AF9-HPC cells were used as targets. E:T ratio denotes effector-per-target ratio. (D) Kaplan–Meier curves for overall survival of wild-type mice that received 1 × 106 MLL/AF9-HPC-OVA cells in the third transplant (n = 5). Recipient mice were not irradiated. Results from the first and second transplants are shown in S1 Fig. (E) May–Giemsa staining of MLL/AF9-OVA leukemia cells that developed in non-irradiated recipients (Magnification: 400×). (F) Flow-cytometry analysis of GFP+ BM cells from mice with leukemia. Blue lines represent MLL/AF9-HPC cells.
Fig 2.
Spontaneous regression of leukemia was observed in the presence, but not in the absence, of adaptive immunity.
(A) Flow-cytometry analyses of BM cells of non-irradiated wild-type recipients 7 days after transplantation with different numbers (3 × 103, 3 × 104, or 3 × 105) of MLL/AF9-OVA leukemia cells. (B) FACS analysis of BM from non-irradiated wild-type or Rag2-/- mice transplanted with 3 × 104 MLL/AF9-OVA leukemia cells. Mice were analyzed 3 weeks after transplant. (C) Kaplan–Meier curves for overall survival of non-irradiated wild-type (n = 7) or Rag2-/- (n = 3) recipients transplanted with 3 × 104 MLL/AF9-OVA leukemia cells. (D) Percentages of GFP+ leukemia cells in BM after transplantation into non-irradiated wild-type mice were examined every week. Each dot and line corresponds to a recipient mouse. The results of four mice in which leukemia spontaneously regressed (Exp. 3 in Table 1) are shown. (E) FACS analysis of BM from non-irradiated wild-type or Rag2-/- mice transplanted with 3 × 104 of MLL/AF9 leukemia cells (OVA-). Mice were analyzed 3 weeks after transplant.
Table 1.
Transplant experiments performed in this study.
Fig 3.
Functional CTLs specific for the antigen expressed in leukemia cells were highly expanded in mice that did not develop leukemia.
Analysis of BM and spleen cells from mice 4 weeks (A–C, n = 5) or 6 weeks (D–F, n = 3) after transplant with 3 × 104 MLL/AF9-OVA leukemia cells. (A, D) Flow-cytometry analysis of the frequencies of GFP+ leukemia cells in the 7AAD- whole BM or spleen cells and those of H-2Kb/OVA tetramer-positive cells in CD8+ T cells. (B, E) Flow-cytometry analysis of cytokine production by CD8+ BM and spleen T cells, with or without SIINFEKL peptide stimulation. (C, F) Percentages of IFN-γ- and/or TNF-α-producing cells in BM or spleen CD8+ T cells, with or without SIINFEKL peptide stimulation. *: p < 0.05.
Fig 4.
In mice with advanced leukemia, CTLs specific for the antigen expressed in leukemia cells were also expanded, but could not suppress disease progression.
(A-C) Analysis of BM and spleen cells from mice with advanced MLL/AF9-OVA leukemia (A) Flow-cytometry analysis of the frequencies of GFP+ leukemia cells among the whole BM or spleen cells and the frequencies of H-2Kb/OVA tetramer-positive cells among CD8+ T cells. (B) Flow-cytometry analysis of cytokine production by CD8+ T cells in BM and spleen with or without SIINFEKL peptide stimulation. (C) Percentages of IFN-γ- and/or TNF-α-producing cells among CD8+ BM or spleen T cells, with or without SIINFEKL peptide stimulation (n = 3). *: p < 0.05 (D) Analysis of the expression of T-cell exhaustion–associated markers in H-2Kb/OVA tetramer-positive CD8+ T cells. BM cells from non-leukemic mice and mice with advanced leukemia were analyzed (n = 3 for each). Representative flow-cytometry analysis and bar graphs for mean fluorescence intensities (MFI) are shown. Dotted lines represent isotype controls.*: p < 0.05, N.S.: not statistically significant. (E) Analysis of the expression of H-2Kb, GFP, and the presentation of SIINFEKL peptide in leukemia cells that developed in wild-type or Rag2-/- recipients. Representative flow-cytometry analysis and bar graphs for MFI are shown. Dotted lines represent isotype controls.