A titration of the RAHYNIVTF-loaded tetramer demonstrates that the TILS induced in the C57BL/6 mice have a higher avidity than those in the E6/E7 transgenic mice. 1 x 10⁵ TC-1 tumor cells in Matrigel were implanted s.c. in both six- to eight-week-old C57BL/6 and six-to eight-week old E6/E7 transgenic mice. Seven days later, when palpable tumors about 5 mm in size form, the mice were immunized i.p. with 1 x 10⁸ LM-LLO-E7, 2.5 x 10⁸ LM-ActA-E7, or left untreated. Mice were boosted on day 14 with the same vaccine dose. On day 21, mice were sacrificed, and each group had tumors pooled together, digested with collagenase, crushed, filtered, and made into a single cell suspension as described in Materials and Methods. Cells were stained for CD62L, CD8α, CD11b, and the H-2D^b tetramer loaded with the RAHYNIVTF peptide at dilutions of 1:50, 1:300, and 1:3000. 7AAD was used as a viability dye. Cells shown in (a) were positive for CD8α and the E7 tetramer, were negative for 7AAD and CD11b, and were low for CD62L. Histograms shown in (b) were gated from the data in (a) and overlayed. (a) Naive mice fail to accumulate any TILs. Mice immunized with LM-LLO-E7 or LM-ActA-E7 have an increased frequency of TILs at any given concentration of tetramer in the WT mice as opposed to those in the transgenic mice. (b) The MFI was also higher for the overall CD8+ effector T cells found in the tumor for the C57BL/6 mice (thick line) as opposed to the transgenic mice (thin line). Data shown are representative of one of two experiments.