Immunoprevention of chemical carcinogenesis through early recognition of oncogene mutations

Abstract

Prevention of tumors induced by environmental carcinogens has not been achieved. Skin tumors produced by polyaromatic hydrocarbons, such as 7,12-dimethylbenz(a)anthracene (DMBA), often harbor an H-ras point mutation, suggesting that it is a poor target for early immunosurveillance. The application of pyrosequencing and allele-specific PCR techniques established that mutations in the genome and expression of the Mut H-ras gene could be detected as early as 1 d after DMBA application. Further, DMBA sensitization raised Mut H-ras epitope-specific CTLs capable of eliminating Mut H-ras(+) preneoplastic skin cells, demonstrating that immunosurveillance is normally induced but may be ineffective owing to insufficient effector pool size and/or immunosuppression. To test whether selective pre-expansion of CD8 T cells with specificity for the single Mut H-ras epitope was sufficient for tumor prevention, MHC class I epitope-focused lentivector-infected dendritic cell- and DNA-based vaccines were designed to bias toward CTL rather than regulatory T cell induction. Mut H-ras, but not wild-type H-ras, epitope-focused vaccination generated specific CTLs and inhibited DMBA-induced tumor initiation, growth, and progression in preventative and therapeutic settings. Transferred Mut H-ras-specific effectors induced rapid tumor regression, overcoming established tumor suppression in tumor-bearing mice. These studies support further evaluation of oncogenic mutations for their potential to act as early tumor-specific, immunogenic epitopes in expanding relevant immunosurveillance effectors to block tumor formation, rather than treating established tumors.

FIGURE 1.

Early detection of H-ras mutations and oncogene expression in DMBA-treated skin. (A) Identification of DMBA-induced H-ras codon 61 mutations by pyrosequencing. Quantification of Q61L, Q61R, and G12E mutant H-ras alleles in genomic DNA from samples of skin treated with 0, 0.1 (400 nmol), or 1% (4 μmol) DMBA after 1 or 3 d (n = 2 mice per treatment; mean ± range is shown). Insets: pyrosequence tracing peaks for missense codon 61 mutation A → T. Pyrosequence quantification validation: the CH72 cell line, heterozygous for the H-ras Q61L allele, indicates 50% of its H-ras alleles are mutated. (B and C) Relative expression of Mut H-ras mRNA in (B) 1% or (C) 0.1% DMBA-treated skin samples. Skin samples from two mice per condition were examined. Densitometry of amplicons generated by H-ras Q61L-specific ACB-PCR and GAPDH-specific PCR of cDNA made from individual skin tissues. CH72 mRNA serves as a positive control. Bar graphs display Mut-H-ras gene expression levels when normalized to expression levels of a housekeeping gene GAPDH, as shown. The corresponding gel used to provide densitometry readings is shown below each bar graph (mean ± range of duplicate densitometry readings for each sample is shown). The data are from one of two separate experiments.

FIGURE 2.

Specificity of DMBA and Mut H-ras T cell responses. (A) DMBA-sensitized T cells respond to Mut H-ras peptide elicitation. (A) DTH responses. Ear-swelling responses elicited by the indicated peptide after DMBA sensitization are shown (n = 5 per group). (B) Cytokine production by DMBA-sensitized T cells stimulated by Mut H-ras peptide–pulsed BMDCs. T cells from the LNs of DMBA-sensitized mice were cocultured with DMBA-pulsed or unpulsed BMDCs from naive mice at a 1:10 ratio for 48 h. The mean and range concentration values of duplicate wells for IL-17 and IFN-γ ELISAs are shown. (C–F) Mut H-ras vaccines induce responses that can be elicited by DMBA. (C) Specificity of DC-based vaccine. Mice were vaccinated, then challenged by i.d. injection of the ear with WT or Mut H-ras peptide. Specific ear-swelling responses on day 3 are shown (n = 3 per group, repeated twice). (D) Engineered DC vaccination induces responses that are elicited by DMBA challenge. DC-GFP serves as a specificity control. Note that day 3 responses induced by Mut H-ras–engineered DC and peptide-pulsed DC-GFP to DMBA elicitation are equivalent. DC-GFP serves as a specificity control. (E) Specificity of DNA-based vaccine in A/J and C3H/HeN mice. A/J (left) or C3H/HeN (right) mice receiving DNA-based vaccines generate comparable responses to Mut H-ras peptide. Day 3 response is shown. (F) Mut H-ras DNA vaccination responses can be elicited by Mut H-ras peptide or DMBA. Day 3 response is shown (n = 5 per group, more than two experiments each). (G–I) DC–Mut H-ras vaccines eradicate Mut-H-ras mRNA expression in DMBA-treated skin. DC-vaccinated mice were challenged by either (G) i.d. injection of Mut H-ras peptide (100 μg) or (H and I) painting the dorsal skin with 100 μL 0.1% DMBA (400 nmol) (n = 5 mice per group). (G) Specific ear-swelling responses on day 3 and (H and I) Mut H-ras mRNA expression levels in skin, assessed by ACB-PCR on day 7. (H) gel (bottom). GAPDH expression is a loading control (bottom row). (H, top) Relative H-ras (Q61L) mRNA expression: the ratio of Mut H-ras over the GAPDH densitometry values is shown for tumors per mouse (3 tumors pooled per mouse, n = 5 mice per group). (I) Scatter plot of data in (H). Significant differences (by Student t test) between Mut H-ras–immunized samples from controls are shown. *p < 0.05, **p < 0.01, ***p < 0.001. n.s., not significant.

FIGURE 3.

Vaccine-induced CTL activity and CD8 T cell expansion. (A) In vivo CTL assay. C3H/HeN mice were immunized and boosted with DNA-based vaccines or PBS (Control), then injected with CFSE-labeled splenocytes pulsed with WT H-ras (CFSE^lo) or Mut H-ras (CFSE^hi) peptides. The CFSE-stained cells remaining in spleen were analyzed the following day. The % specific cytotoxicity is indicated. (B and C) DC-based immunization raises DMBA-responsive Mut H-ras–specific effector T cells. A/J mice were immunized with DC vaccines once (Prime only) or twice (Prime/Boost), as indicated. At 10 d later, mice were challenged with either Mut H-ras peptide or DMBA. At 3 d later, LN cells were prepared and cultured with PMA + ionophore (B and C) or the indicated stimulus (D) and then stained for intracellular cytokines and phenotypic markers. (B and C) IFN-γ⁺ CD8 T cells expand in a vaccine dose–dependent manner. (B) IFN-γ⁺ CD8⁺ T cells. Mut ras peptide or DMBA elicits IFN-γ⁺ CD8 cells. The % of IFN-γ⁺ gated CD8 cells is shown. (C) Selective expansion of CD8 over CD4 T cells. The absolute number of IFN-γ⁺ CD8 T cells per million LN cells expanded in vivo by either Mut ras peptide or DMBA challenge (top row). CD8/CD4 effector cell ratio = the number of IFN-γ⁺ CD8 T cells over the number of IL-4⁺ CD4 T cells per million LN cells (mean ± SEM of triplicate mice). (D) Ag specificity of CTL. Bar graphs display the % of CD11c^hi target cells within the CD11c⁺ gate for each set of stimuli per immunized group. LN cells from immunized mice were harvested 3 d after DMBA Ag elicitation and then cultured for 6 h with PMA + ionophore or overnight with peptides, as indicated. The percentage of CD11c^hi target cells in the CD11c DC gate was determined by flow cytometric analysis. The presence of WT H-ras peptide or PMA + ionophore did not affect DC profiles; however, Mut H-ras peptide cultures lost 87% of CD11c^hi cells. (n = 3 per group). Student t test: *p < 0.05, ***p < 0.001 (one of more than three similar experiments).

FIGURE 4.

Immunoprevention against chemical carcinogenesis in two mouse strains and evidence for immunoediting. (A–C) Engineered DC-based vaccination of A/J mice. Mice were immunized twice with the indicated DC vaccine, then subjected to DMBA/TPA carcinogenesis over 25 wk (n = 20 per group). The individual number of tumors per mouse (A) and the average tumor volume per mouse (B) per group are shown (mean ± SEM). Tumor incidence and growth rates were inhibited by 70% and 90%, respectively (the Tukey significance test, p < 0.01). (A and B) Two-way ANOVA and the Tukey posttest calculated significance are indicated: *p < 0.05, **p < 0.01. (C) Photodocumentation of representative mice per vaccination group is shown. (D–G) DNA-based epicutaneous vaccination of C3H/HeN mice. Mice received three vaccinations over 15 d, then were subjected to DMBA/TPA carcinogenesis over 24 wk (n = 20 per group). (D) The average tumor numbers per mouse in each group. (E) The average tumor volume per mouse in each group. The Mut H-ras DNA–vaccinated group developed 50% fewer tumors, and tumor growth was inhibited by >85% (p < 0.01, p < 0.001, respectively). (F) Representative photographs of tumors that develop in DNA-vaccinated mice. (G) Tumor-free plot. Tumor penetrance was inhibited in C3H/HeN mice immunized with Mut H-ras DNA–based vaccine. Tumor-free mice are defined as having no tumors or only small tumors < 3 mm³. (H and I) Immunoediting in tumors from Mut H-ras–vaccinated mice. (H) Relative quantification of Mut H-ras mRNA expression levels in individual tumors. The densitometry ratio for Mut H-ras ACB-PCR and matched GAPDH amplicon products of each tumor sample, as shown for six tumors in (I), as individual data points. The number of tumors screened was as follows: PBS, n = 30; DC–WT H-ras, n = 25; DC–Mut H-ras, n = 20. Expression was negligible in 45% of tumors from Mut H-ras–vaccinated mice and in 15–22% of tumors from the control groups. (G and H) Two-way ANOVA statistical significance is indicated: **p < 0.01, ***p < 0.001. (I) Loss of H-ras Q61L mutation expression in tumors from DC–Mut H-ras–vaccinated mice. The Q61L H-ras allele (upper band, red arrow) detected by ACB-PCR in mRNA from individual tumors harvested from A/J mice after 25 wk of carcinogenesis. GAPDH serves as an internal gene expression control.

FIGURE 5.

Phenotypic and functional characterization of tumor-bearing vaccinated mice: (A) Flow cytometry multivariate plots. (B–I) Subset quantification in LNs (duplicate mice; mean ± range): (B) IL-17⁺ CD4 T cells; (C) CD69⁺ CD4 T cells; (D) IL-17⁺ CD8 T cells; (E) CD69⁺ CD8 T cells; (F) IL-12⁺ MHC^hi cells; (G) IFN-γ⁺ IL-17⁻ CD8 T cells; (H) Gr1⁺ CD11c⁺ cells. Control: mice are vaccinated, DMBA treated, but not TPA treated. Carcinogenesis: mice are vaccinated, DMBA and TPA treated for 20 wk. (I) Gr-1^hi, CD11b⁺ cells. Fold change from control (dotted line). Student t test: *p < 0.05, **p < 0.01. (J and K) In vivo CTL activity detected in immunized control and tumor-bearing mice. (J). Histogram display of CFSE^lo and CFSE^hi target cells in the spleen, 1 d after target cell transfer. Gated CFSE-labeled target cells: CFSE^lo control (white) and the % of CFSE^hi peptide–pulsed population (black) per sample (n = 3). (K) The % of Mut-H-ras–specific cytotoxicity for each group (mean ± SEM).

FIGURE 6.

Therapeutic regression of established tumors by transfer of cells from Mut H-ras–immunized, tumor-bearing mice. LN and splenocytes (1:4 cells) from carcinogenesis-treated vaccinated mice were harvested at 28 wk and pooled for infusion into control GFP-vaccinated tumor-bearing mice (n = 2 per group). Phenotypic analysis indicated there were no significant differences in T cells contained in each pool: 3 million CD4 (range: 2.8–3.3) and 1 million CD8 (range: 1.4–1.6) T cells were transferred. (A) Effect of cell transfer on established tumors. Photos of tumors in recipient mice over 3 wk; arrows highlight tumor progression (white) and regression (red). Magnified view (original magnification ×3.6): tumor regression in Mut H-ras recipient mice at 3 wk. (B) Kinetics of tumor growth and regression. Tumor change from week 0: average tumor number per mouse (left), average tumor size per tumor (right). (C) Immunoediting of tumor H-ras expression. Tumor biopsy specimens obtained at 30 wk were analyzed for Mut H-ras mRNA expression by ACB-PCR (n = 5 per group). GAPDH gene expression of each sample is a loading control (upper panels). (D) Skin tumor infiltrating CD4 T cells. Cryosections of tumors stained with conjugated Abs: mouse CD4 (FITC⁺, green), Foxp3⁺ (PE), and DAPI (blue nuclei). Foxp3⁺ cells have pink nuclear staining (pink arrows). Foxp3⁻ CD4⁺ T cells are indicated by green arrows (upper panels). CD8⁺ T cell infiltration in and around cutaneous tumors. Mouse CD8⁺ T cells [PE, red arrows (lower panels)]. Scale bar, 50 μm. (E) Enumeration of T cell subsets in tumor sections. Tumors infiltrating CD8, CD4, and Foxp3⁺ T cells were counted in at least five non-overlapping views per section. (n = 6–8 tumors per group). The % of CD4 (green) T cells stained with Foxp3 (pink)⁺ nuclei was determined in the same view, as described. Bar graph depicts the ratio of CD8:CD4 average values per group. Text indicates the fold increase in the CD8:CD4 ratio compared with mock (PBS)–vaccinated mice. *p < 0.05, **p < 0.01, ***p < 0.001. n.s., not significant.