E7-specific CD8+ T cells are detected at different frequencies in the spleens of tumor-free mice.

Mean fluorescent intensity (MFI) of CD8 and CD3 staining of tetramer positive TILs from normal and transgenic mice.

Mean fluorescent intensity (MFI) of tetramer staining of CD8+ TILs from normal and transgenic mice vaccinated with LM-LLO-E7 or LM-ActA-E7.

(a) Gross photograph of an 18 month old E6/E7 transgenic mouse with an enlarged thyroid visible externally. (b and c) Photomicrograph of a thyroid gland from a 6 month old WT mouse at 5x (b) and 20x (c) magnification. (d and e) Photomicrograph of a thyroid gland from a 6 month old transgenic mouse at 5x (d) and 20x (e) magnification. The thyroid follicles are engorged with colloid and are irregular in shape. Also found are solid masses of cells with little or no follicular organization. A papillary carcinoma is evident in (e).

(a) Tissue-specific expression of the E7 transgene is detected in the thyroid only but not in the liver, spleen or whole thymus. Organs were isolated and RT-PCR was performed as described in Materials and Methods. Lanes: 1, liver; 2, spleen; 3, thyroid; 4, whole thymus. (b) Medullary thymic epithelial cells (mTECs) express E7. mTECs were isolated by cell sorting into an RNA protection solution as detailed in Materials and Methods. RT-PCR results are shown for equivalent amounts of cDNA loaded for 40 cycles. Lanes: 5, cathepsin S; 6, E7; 7, actin; 8, negative control (water).

1 x 10⁵ TC-1 tumor cells were implanted s.c. at day 0 in six to eight week old mice. When palpable tumors formed seven days later, mice were i.p. immunized with 1 x 10⁸ LM-LLO-E7, 2.5 x 10⁸ LM-ActA-E7, left naive, or treated with LM-NP as a control. Mice were boosted with the same vaccine on day 14. Every seven days, tumors were measured using a caliper in two dimensions, and the average of those values was plotted against days post tumor implantations for each mouse. Values shown are only for surviving mice. Depicted is one of 3 experiments.

1 x 10⁵ TC-1 tumor cells were implanted s.c. at day 0 in six- to eight-week-old E6/E7 transgenic mice. For experimental groups, mice were depleted of regulatory T cells using i.p. injections of 0.5 mg of PC61 anti-CD25 mAb on days 4 and 6, which was then followed either by i.p. immunization with 1 x 10⁸ LM-LLO-E7 or the mice were left naive. In control groups, when palpable tumors formed seven days later, mice were i.p. immunized with 1 x 10⁸ LM-LLO-E7 or left naive. Mice were boosted with the same vaccine on day 14. Tumors were measured in two dimensions using a caliper, and the average of those values was plotted against days post tumor implantation for each mouse. Depicted is one of 2 experiments.

Either six- to eight-week-old WT or transgenic mice were immunized with 1 x 10⁸ LM-LLO-E7 or 2.5 x 10⁸ LM-ActA-E7 on days zero and seven. On day 14, splenocytes were removed and cultured with irradiated TC-1 cells for 4 days. Following this culture step, splenocytes were used in a four-hour ⁵¹Cr release assay. (a and b) EL4 cells alone. (c and d) EL4 cells pulsed with 1 µM of the RAHYNIVTF peptide with differing concentrations of effector cells to target cells. (e and f) EL4 cells plus differing concentrations of peptide with an effector to target cell ratio of 50:1. Assays were preformed in triplicate with the mean number plotted and standard deviation present as error bars. Depicted is one of 2 experiments.

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.