Figure 2.

Enhanced numbers of activated tumor-specific CD8⁺ T cells in TDLNs of mice following induction of acute local inflammation. BALB/c mice bearing Colo26-HA (A–C) or Colo26 (D) tumors were subjected to PDT treatments, causing different levels of acute inflammation, or injected with the photosensitizer and not exposed to laser treatment (Control). Twenty-four hours later, TDLN were harvested, single-cell suspensions generated, and the phenotype of CD8⁺ T cells specific for the HA antigen (tet⁺) was determined. A, flow cytometry depicting staining for tet-HA, CD8, CD44, and CD69. The boxed area in the first column identifies the tet-HA⁺CD8⁺ T cells. The second column depicts CD44 staining and is gated on the tet-HA⁺CD8⁺ T-cell population. The third column is gated on the tet-HA⁺CD8⁺CD44^hi population and shows staining for CD69. The numbers indicate percentages of cells in the respective gates. Data from one representative experiment is shown. B, left, average percentage of CD44^hi cells among the tet-HA⁺CD8⁺ population from three individual experiments. Right, the corresponding average number of live cells, as determined with trypan blue, recovered from the TDLNs. C, absolute number of tumor-specific CD8⁺ T-cell effector cells that expressed CD69 (left) and CD25 (right) following treatment. Experiments depicted in B and C were repeated twice with five mice per group; bars, SE. *, P < 0.03, compared with low-inflammation PDT. D, TDLNs were harvested at 24 h posttreatment and single-cell suspensions were generated. The number of cells able to specifically recognize tumor antigen AH1 and secrete IFN-γ as a consequence was determined with ELISPOT assays. Nonspecific staining with an irrelevant peptide (PA1) was subtracted from each group. Columns, average number of IFN-γ⁺ cells (each group contains at least six mice per group); bars, SE. *, P < 0.01, compared with low-inflammation PDT.