Figure 3. High levels of functional regulatory T cells infiltrate murine pancreatic adenocarcinoma.

(A) Prevalence of Treg in various compartments (tumor, tumor-draining lymph nodes, non-draining lymph nodes and spleen) in the tumor-bearing mouse at 4 weeks post tumor implantation. Compared to other compartments, there were higher numbers of Treg found in the tumor. Histogram shows percentage of CD4⁺ cells that are Foxp3⁺. Results represent mean ± SEM, * P < 0.005, from 5 independent experiments, each comprising 2–4 mice. (B) Tumor-derived Foxp3⁺ cells are suppressive in vivo. To examine the suppressive capacity of tumor-infiltrating Treg in Foxp3^gfp mice, CD4⁺ cells were isolated from tumors 4–5 weeks after inoculation with Pan02, then sorted to collect the Foxp3⁺ Treg subset. CFSE-labeled thy1.1⁺ CD4⁺ CD25⁻ T effector cells were subsequently co-cultured for 72 hours either alone (filled histogram) or with the sorted thy1.2⁺CD4⁺Foxp3⁺ cells in a 1:1 ratio (open histogram). For flow cytometric analysis, CFSE dilution was evaluated in thy1.1⁺ cells. Figure is representative of 2 independent experiments, each comprising 4–5 mice. (C) Tumor-derived CD4⁺CD25⁺ cells have equivalent suppressive ability to CD4⁺CD25⁺ cells from lymph nodes and spleen. To examine the suppressive capacity of tumor-infiltrating Treg, CD4⁺CD25⁺ cells were isolated from the spleen, tumor-draining lymph nodes and tumor of C57BL/6 (thy1.2⁺) mice 5 weeks after inoculation with Pan02. CFSE-labeled thy1.1⁺CD4⁺ CD25⁻ T effector cells were subsequently co-cultured for 72 hours either alone (filled histograms) or with CD4⁺CD25⁺ cells (at a 1:1 ratio) derived from tumor; tumor-draining lymph nodes; or spleen (open histograms). Figure is representative of 2 independent experiments, each comprising 3 mice.