Tumor-resident DCs are transformed from immunostimulatory to immunosuppressive during tumor progression. (A) Left, CD3⁺ T cells were obtained from spleens of advanced tumor-bearing p53/K-ras animals, and then added to cultures containing BMDCs (10:1) which were previously pulsed with lysed tumor cells. 5 d later, sorted (nonpulsed) CD45⁺CD11c⁺ DCs from the DLNs or tumors (tumor-infiltrating DCs; TIDCs) were co-cultured with the tumor-specific CD3⁺ T cells, after CFSE labeling (10:1). Right, percentage of proliferated T cells which were cultured with sorted DCs from the DLN of mice with advanced tumors or animals with primordial tumors (early; received adenovirus 7 d before) or DCs from advanced tumors (B). (C) ELISPOT analysis of the responses induced by CD45⁺CD11c⁺ DCs, sorted from the spleens of early and advanced tumor-bearing mice, and directly incubated with tumor-reactive T cells, obtained as in A. (D) Tumor-specific CD3⁺ T cells were obtained as in A, and then added to culture wells, which contained tumor pulsed BMDCs (1:1). Either sorted CD45⁺CD11c⁺CD11b^+/− DCs or unpulsed BMDCs were added to these cultures making a 1:1:1 ratio (T cell/pulsed BMDC/sorted DC/BMDC). (E) Left, proliferation indices of T cells in response to the addition of tumor infiltrating DCs (TIDCs) or BMDCs. Right, representative histogram of CFSE dye dilution. (F) Left, proliferation indices of T cells in response to the addition of BMDCs or DLN DCs sorted from mice with early or advanced tumors. Right, representative histogram of CFSE dilution. n = 4 mice/group. BMDC cultures were done in quad- or quintuplicate. Even stronger differences were obtained using all tumor-pulsed BMDCs (not depicted). Error bars, SEM. Data points on scatter plots represent experimental replicates for pooled DLN or tumors. Horizontal bars, SEM. *, P < 0.05. Representative of at least two independent experiments in all panels (n = 3–4 mice/group).