Figure 5. Direct antigen presentation by leukemia cells is required for in vivo antigen recognition by TCR_Tg101.

(A) TCR_2C or TCR_Tg101 (1 × 10⁵) were CTV-labeled and cultured in vitro with C1498.SIY cells at various ratios for 72 h, at which point TCR_2C and TCR_Tg101 proliferation, as measured by CTV dilution, was assessed by flow cytometry. TCR_2C and TCR_Tg101 stimulated with anti-CD3 and anti-CD28 antibodies or left unstimulated served as positive and negative controls for proliferation, respectively.

(B) CTV dilution profiles of TCR_2C or TCR_Tg101 cultured for 72 h with CD8α⁺ DCs, CD8 α⁻ DCs, or macrophages isolated from spleens of naive C57BL/6 mice or C57BL/6 mice challenged 3 h earlier with C1498.SIY cells i.v.

(C–F) CTV-labeled TCR_Tg101 (CD45.1.2) were adoptively transferred into CD11c-DTR bone marrow chimeric mice (CD45.2) that received DT or PBS prior to and following i.v. C1498 cell challenge.

(C) Representative FACS plot showing frequencies of CD11c⁺ cells in spleens of CD11c-DTR bone marrow chimeras after DT or PBS treatment. Gating was performed on live CD3⁻CD19⁻CD11c⁺ cells.

(D) Representative FACS plots showing CTV dilution of TCR_Tg101 (TCRb⁺CD8⁺CD45.1.2 cells) in livers of naive or C1498 cell-challenged CD11c-DTR bone marrow chimeric mice (treated with DT or PBS) at day 12.

(E and F) Quantitative data from (D) are shown.

(G) CTV dilution profiles of TCR_2C and TCR_Tg101 cultured with BMDCs pulsed with C1498.SIY or C1498 cell lysates, respectively. CTV dilution profile of TCR_Tg101 cultured with live C1498 cells was included as a positive control (lower right). Representative FACS plots are shown.

(H and I) 2 × 10⁶ CTV-labeled TCR_Tg101 (CD45.2) were transferred i.v. into B6.SJL (CD45.1) mice followed by an i.v. challenge with 10⁶ C1498 or C1498.H2-Kb^−/− cells the following day. On day 18, TCR_Tg101 proliferation was assessed.

(H) Representative FACS plots showing CTV dilution and CD44 expression on TCR_Tg101 in livers of mice previously challenged with C1498 or C1498.H2-Kb^−/− cells.

(I) Quantitative data from (H) are shown.

(J–L) 2 × 10⁶ CTV-labeled TCR_Tg101 (CD45.1.2) were transferred i.v. into B6.SJL (CD45.1) mice followed by a s.c. challenge with 10⁶ C1498 or C1498.H2-Kb^−/− cells the following day. 14 days later, TCR_Tg101 frequencies and numbers were assessed in tdLNs and tumors.

(J) Representative FACS plots showing TCR_Tg101 frequencies in tdLNs of mice with s.c. C1498 or Kb^−/− C1498 tumors.

(K and L) Quantitative data showing frequencies (K) and numbers (L) of TCR_Tg101 in tdLNs of mice with s.c C1498 or Kb^−/− C1498 tumors.

(M) Representative FACS plots showing TCR_Tg101 frequencies among total CD8⁺ T cell populations in s.c. C1498 or Kb^−/− C1498 tumors.

(N and O) Quantitative data showing TCR_Tg101 frequencies (N) and numbers/mg of tumor tissue (O).

Data are representative (I) of or pooled (E–F, K–L, and M–O) from 2 independent experiments with 3 mice/group and presented as mean ± SD. *p < 0.05; **p < 0.01; ***p < 0.001; n.s., not significant.