DCs induce tolerance of tumor-infiltrating effector T cells in the prostate tumor tissue. A, CD11c⁺ cells were isolated from the spleens and prostates of B6, TRAMP, and TRP-SIY mice by cell sorting and were then stained for PDCA-1, Gr-1, or B220. Dot plots shown are from five pooled mice in each group and are representative of two similar experiments. Experimental protocol (B) and effect of DC depletion on 2C T cell function (C). Cells from the spleens and prostates of BMT mice were stained for 2C TCR, Thy1.1, and intracellular IFN-γ. Density plots show Thy1.1 versus IFN-γ profiles of 2C TCR⁺ Thy1.1⁺ cells. Numbers indicate the average percentage of positive cells ± 1 SD. Data are representative of two to three mice per group. *p < 0.05 when compared with control.

Intraprostatic injection of Ag-loaded DCs 7 dpi delays tolerization of tumor-infiltrating effector T cells. Experimental protocol (A) and effect of intraprostatic injection of SIY-loaded mature DCs on IFN-γ expression by tumor-infiltrating 2C cells (B). Cells from prostates were stained for 2C TCR, Thy1.1 and intracellular IFN-γ. Density plots are as described in Fig. 1C. Numbers inside the plots indicate the percentage or the average percentage of positive cells ± 1 SD. Numbers outside the plots indicate total number of 2C cells ± 1 SD in the prostate. Data are from four independent experiments with two to three mice per group. C, SIY peptide and plain DC controls. Experimental protocol is the same as in A, except SIY peptide or plain DCs were injected, and prostates were analyzed 11 and 13 dpi. Representative data from two similar experiments are shown with two to three mice per group. D, Persistence of injected DCs. DCs were labeled with CFSE, loaded with SIY peptide, and injected into TRAMP prostates. Three and 8 d later, the presence of CFSE⁺ DCs in the indicated tissues was determined by flow cytometry. Dot plots show CD11c versus CFSE profiles of live cells. Numbers indicate percentage of CFSE⁺ cells. Representative data are shown from two to three mice per group. *p < 0.05 when compared with control.

Intraprostatic injection of Ag-loaded DCs reactivates persisting tolerized T cells in the prostate tumor tissue. Experimental protocol (A) and effect of intraprostatic injection of SIY-loaded mature DCs on reactivation of persisting tolerized 2C cells (B). Cells from prostates were stained for 2C TCR, Thy1.1, and intracellular IFN-γ. Dot plots show Thy1.1 versus 2C TCR profiles of live cells, and density plots are as described in Fig. 1C. Numbers inside the plots indicate the percentage or the average percentage of positive cells ± 1 SD in the gated region. Numbers outside the plots indicate total number of 2C cells ± 1 SD. Data are from three independent experiments with two to three mice per group. C, In vivo CTL assay. A 1:1 mixture of SIY-pulsed, CFSE-labeled, activated T cells (Thy1.1⁺) and unlabeled, unpulsed, activated T cells (Thy1.1⁺) were retro-orbitally injected into TRP-SIY mice (Thy1.2⁺) on day 34. The following day, the proportions of CFSE⁺ versus CFSE⁻ target cells were determined by flow cytometry. CFSE histograms are shown for live Thy1.1⁺ cells. Numbers indicate percentage of CFSE⁺ cells. Representative data from two similar experiments are shown. D, H&E stains of prostate tissues from TRP-SIY mice injected intraprostatically with either PBS or SIY-loaded DCs 5 d earlier. Arrow indicates leukocyte infiltration in the prostate. Scale bar, 100 μm. E, Effect of s.c. injection of SIY-loaded DCs. The experiment was performed as in A and B, except SIY-loaded mature DCs were injected s.c., and prostates were analyzed 35 dpi. *p < 0.05 when compared with control.