Inbred FVB/N and C57BL/6 mice were purchased from National Laboratory Animal Center (Taipei, Taiwan) at the age of 6–8 weeks, and housed in the Animal Care Facility at Chang Gung Memorial Hospital (CGMH) under the standard guidelines from ‘Guide for the Care and Use of Laboratory Animals’ and with the approval of the CGMH Committee on Animal Research.

FVB/N or C57BL/6 females were caged with males of the same strain in the afternoon and checked for vaginal plugs the following morning. The day when the plug was observed was designated as day 0 of the pregnancy. Under anesthesia with ketamine (100 mg/kg) and xylazine (10 mg/kg), the uteri of GD₁₄ pregnant mice were exposed through a vertical laparotomy. A 60 µm glass micropipette with beveled tip was used to inject 0.125, 0.25 and 0.5 µg endotoxin-free HPV E7 peptides (full-length sequence of 98 amino acids: MHGDTPTLHE YMLDLQPETT DLYCYEQLND SSEEEDEIDG PAGQAEPDRA HYNIVTFCCK CDSTLRLCVQ STHVDIRTLE DLLMGTLGIV CPICSQKP, MyBioSource, San Diego, California, USA) in 5 µL saline into peritoneal cavities of all fetuses at a litter via transuterine approach. Then, the abdomen of the pregnant mice was closed in two layers of 5–0 silk suture. After the operation, all mice were housed in an undisturbed room without bedding changes until the pups were 1 week old. Pups were weaned at 3 weeks of age. Control mice received saline injection at the same gestation age.

Cells from GD₁₄ fetal liver or peritoneum9 were pulsed with HPV E7 peptides (25 µg/2 ×10⁷ cells) overnight. After vigorous washes with phosphate buffered saline (PBS), cells were fixed with cold methanol and permeabilized with 0.5% Tween-20 for 30 min. Then, they were subjected to the treatment of mouse anti-E7 monoclonal IgG₁ (ED17, Santa Cruz Biotechnology), followed by fluorophore-conjugated anti-mouse IgG antibody and 4’,6-diamidino-2-phenylindole (DAPI) nuclear counterstain. Finally, cells were analyzed by flow-cytometry and cytospun on slides for confocal microscopic examinations.

Cells from GD₁₄ fetal liver or peritoneum were adjusted at a concentration of 4 ×10⁶ cells/mL RPMI 1640 medium and incubated with foreign antigen of 20 µg Alexa Fluor 647-labeled OVA (OVA-647, Molecular Probes) overnight. After being washed vigorously with PBS, cells were then stained with fluorophore-conjugated anti-CD45, F4/80, CD11b and Gr1 monoclonal antibodies (eBioscience). Samples were acquired by BD FACSCanto II flow cytometry system. To the exclusion of dead cells using propidium iodide staining, fetal MPs defined as OVA-laden cells were analyzed by BD FACSDiva software.

Pooled GD₁₄ fetal cells, collected from peritoneum by saline lavage and dissociated livers by passage through 70 µm cell strainers,9 were subjected to Ficoll density gradient centrifugation for the enrichment of fetal MPs. Enriched MPs (>80%, 5×10⁶ cells/mL) were pulsed with HPV E7 peptides (25 µg/2×10⁷ cells) overnight in RPMI 1640 medium and washed vigorously with PBS. Cells were intraperitoneally injected into adult FVB/N or C57BL/6 mice (8–12 weeks old) at a dose of 2×10⁷ cells/mouse in 200 µL saline. Control mice were injected with fetal MPs maintained overnight in HPV E7-free medium. Four to 6 weeks later, recipients were examined for serum anti-E7 IgG₁ levels, T-cell polarization and immune response to HPV E7. Additionally, C57BL/6 recipients were examined for HPV E7-specific cytotoxic CD8⁺ T-cells in peripheral blood and spleen 1 week after intraperitoneal challenge of 10⁵ TC-1 cells. Leukocytes enriched by Ficoll density gradient centrifugation were stained using biotin-labeled Pro5^® MHC Class I pentamers of H-2D^b/RAHYNIVTF (HPV16 E7 49–57, Peptide code: 502H, ProImmune) in combination with fluorophore-conjugated streptavidin and anti-CD8α monoclonal antibodies (eBioscience). Cells were acquired and analyzed by flow cytometry.

Within 5–7 weeks after in utero HPV E7 injection or 4 weeks after adoptive transfer of HPV E7 loaded-fetal MPs, the recipients were subjected to blood sampling for measuring serum anti-E7 IgG₁ levels. ELISA microtiter plates were coated with 100 ng/mL HPV E7 peptides overnight, blocked with 3% bovine serum albumin (BSA, Sigma) in PBS and incubated with 100 µL of diluted samples at room temperature for 60 min. After washing, biotinylated anti-mouse IgG₁ (RMG1-1, BioLegend) was added in each well, followed by streptavidin-horseradish peroxidase (Sigma). The reaction was developed by adding 100 µL NeA-blue tetramethylbenzidine substrate (Clinical Science Products) and stopped with 2 M H₂SO₄. The optical density at 450 nm was measured in an automatic ELISA reader. Serum anti-E7 IgG₁ levels were determined by the standard curve of mouse anti-E7 IgG₁ mAb (ED17, Santa Cruz Biotechnology).

Splenic lymphocytes were enriched by density gradient centrifugation and then cultured in triplicate each with 2×10⁵ cells in 200 µL RPMI 1640 medium containing 10% fetal calf serum in 96-well plates. Responder lymphocytes were stimulated with HPV E7 (100 ng/mL), BSA (100 ng/mL) or Con-A (1 µg/mL). IL2 secretion marks a critical landmark in the process of T-cell activation by antigen stimulation and a T-cell commitment to further proliferation and differentiation programs.14 On day 3, T-cell activation was quantified by monitoring the release of IL2 within culture supernatants using ELISA.15 16 For the measurement of lymphocyte proliferation, day 5 cells were first subjected to 16 hours incubation with tritiated thymidine (ICN Biomedicals) at a final concentration of 1 µCi per well and then harvested for counting incorporated tritium in a liquid scintillation counter (1450 Microbeta Plus counter). Lymphocyte proliferation was determined by the readout of incorporated tritium as counts per minute.

Enriched splenic lymphocytes were cultured in triplicate at a density of 5×10⁶ cells/mL in 500 µL RPMI 1640 with 10% fetal calf serum under the stimulation of HPV E7 (100 ng/mL). Cytokines of IL-4, IL-5 and IFNγ in supernatants of 5-day cell cultures were quantified using ELISA assay kits according to the manufacturer’s protocols (BioLegend, San Diego, California, USA).

Splenic lymphocytes of C57BL/6 recipients were harvested by density gradient centrifugation 4 weeks after adoptive transfer of C57BL/6 fetal MPs with or without HPV E7 loading. The lymphocytes were restimulated with HPV E7 (1 µg/2×10⁷ cells) for 24 hours in RPMI 1640. Then, 2×10⁵ lymphocytes were transferred to 96-well culture plates containing 4×10⁴ viable TC-1 cells in 100 µL phenol-red free RPMI medium per well. After overnight incubation, cell-mediated cytotoxicity to TC-1 cells was examined under a fluorescence microscope following the treatment of DAPI viability dye and then further quantified by the supernatant lactate dehydrogenase (LDH) released from lysed cells using the CytoTox 96^® Non-Radioactive Cytotoxicity Assay kit (Promega). The percentage of specific release of LDH was determined by the following equation of (experimental release – spontaneous T-cell release – spontaneous TC-1 cell release)/(maximal TC-1 cell release – spontaneous TC-1 cell release) × 100%.

C57BL/6 recipients were subjected to subcutaneous 10⁵ TC-1 cell inoculation on their backs or abdominal walls 6 weeks after birth (HPV E7 fetal recipients) or 4 weeks following cellular transfer of HPV E7-loaded fetal MPs. TC-1 tumor growth was monitored in two perpendicular directions of the long dimension (length) and the short dimension (width) using a vernier caliper once per 2–3 days for 1 month and then weekly till excessive tumor burden (>3 cm). Tumor volumes were calculated according to the formula: (length × width²)/2.17 The data were then used to plot a graph of tumor growth kinetics. As for survival analyses, the survival time of mice was defined by estimating the length of time from the date of TC-1 tumor cell inoculation to the date of recipients’ death. Cases were considered as censored if the animals remained alive at the end (day 63) of the survival studies or were ethically euthanized before day 63 due to excessive tumor burden (>3 cm). At sacrifice, recipients that were cutaneous tumor-free would undergo histological examinations of the lung, liver and spleen following H&E staining to rule out TC-1 metastatic spread. Cutaneous TC-1 tumors, if available in the recipients, were also harvested for histological examinations at the end of the study. Controls were mice subjected to in utero saline injection or the injection of fetal MPs without HPV E7 pulse.

Alternatively, C57BL/6 mice were first subcutaneously inoculated with 10⁵ TC-1 cells. Two or 4 days later, mice were subjected to the adoptive transfer of 2×10⁷ HPV E7-pulsed GD₁₄ C57BL/6 fetal MPs as the strategy of cellular therapy against pre-existing TC-1 tumor cells. Tumor growth and recipients’ survivals were monitored as described above.

Sections of paraffin-embedded tumor specimens were deparaffinized, rehydrated in xylene and a series of graded ethanol. Sections were subjected to antigen retrieval with citrate buffer (pH 6.0) and then incubated with fluorophore-conjugated anti-F4/80 (1 :25, RM8, eBioscience) and anti-CD11c (1:50, N418, eBioscience) monoclonal antibodies. Visualization of the nuclei was achieved by Hoechst 33342 staining (1: 20,000, Invitrogen). Sections were mounted with Dako fluorescence mounting medium. Images were taken using a confocal microscope.

The equality of means was examined by Student’s t-test between two independent or paired groups, or one-way analysis of variance among three or more groups with post hoc multiple comparisons by Fisher’s least significant difference. Plots of survival time were constructed by Kaplan-Meier method. The log-rank test was employed to compare survival curves. Differences were regarded as significant in all tests at p<0.05.

GD₁₄ FVB/N murine fetuses were subjected to intraperitoneal injection of 0.125, 0.25 or 0.5 µg endotoxin-free HPV E7. None of fetal recipients survived the dose of 0.5 µg HPV E7. Serum anti-E7 IgG₁ was first examined at the age of 4–6 weeks and significantly generated for the doses of 0.125 and 0.25 µg (figure 1A). However, its levels did not differ between the two doses. At follow-up of 4 months old, serum anti-E7 IgG₁ persisted and even notably increased (figure 1B). The results supported the induction of B-cell humoral immunity following in utero HPV E7 exposure.

In HPV E7 fetal recipients, their lymphocyte responses to HPV E7 were further evaluated in vitro at 6–8 weeks old. The activation and proliferation of recipient lymphocytes were quantified by IL2 secretion (figure 1C) and tritium incorporation (figure 1D) in cell culture systems under HPV E7 and third-party antigen stimulation. Recipient lymphocytes significantly secreted IL2 and proliferated specifically in response to HPV E7 stimulation as compared with wild type mice and saline controls. It supported the occurrence of T-cell immunization to HPV E7 antigen following in utero HPV E7 exposure.

Lymphocyte polarization in HPV E7 fetal recipients was assessed by IFNγ, IL4 and IL5 secretion in in vitro lymphocyte culture under HPV E7 stimulation at the age of 6–8 weeks. HPV E7 recipient lymphocytes significantly generated higher levels of IFNγ, as opposed to those of saline controls (figure 1E). However, it made no difference in IL4 and IL5 secretions between HPV E7 recipients and saline controls. These findings indicated a Th1-skewed cytokine profile, suggesting that in utero exposure to HPV E7 endowed the recipient mice with Th1-skewed lymphocytes.

GD₁₄ C57BL/6 murine fetuses were subjected to in utero exposure to HPV E7. Postnatally, they were given subcutaneous inoculation of TC-1 cells at the age of 6 weeks to simulate leftover transformed cancer cells egressing during embryogenesis. The tumor cells slowly expanded by day 14. From day 14 onwards, the TC-1 tumor burden significantly decreased in HPV E7 recipients, but rapidly increased in saline controls (figure 2A). Among six HPV E7 recipients, four were able to eliminate TC-1 tumors by day 28, and then afterwards stayed tumor-free locally as well as in the lung, liver and spleen on histopathological examinations at sacrifice. The other two showed slow tumor regrowth from day 21 onwards after initial shrinkage within days 14–21. Overall, HPV E7 recipients were not in a state of lower tumor burdens until day 28, as compared with their counterpart controls (figure 2B). Within 2-month follow-up, two saline control cases succumbed to tumor expansion on days 43 and 48, respectively, and the third was ethically euthanized on day 61 due to restricted mobility from an excessive tumor burden. In contrast, all the HPV E7 recipients survived TC-1 cell challenge, experiencing superior overall survivals to their controls (figure 2C).

In the control group with in utero saline injection, the tumor was composed of TC-1 cells with nuclear pleomorphism and focal areas of tumor necrosis (figure 3A). In HPV E7 recipients that failed to completely eliminate TC-1 tumors, the residual tumor mass developed massive central necrosis with capsular infiltration by tumor cells and mononuclear lymphocytes (figure 3B). It supported the immune-mediated cytotoxicity against TC-1 cells. Additionally, numerous F4/80⁺CD11c⁺ macrophages were found to reside within capsular TC-1 cell nests of the residual tumor (figure 3C), exhibiting the capacity of tumor cell phagocytosis. These antitumor activities might contribute to less tumor burden in HPV E7 recipients. Remarkably, TC-1 cells also invaded beyond the tumor capsule (figure 3B). This provided the pathological evidence of tumor invasiveness as the barrier to complete tumor eradication.

Cells harvested from GD₁₄ fetal peritoneum and liver were pulsed with OVA-647 and subjected to immunophenotyping for surface markers of CD45, F4/80, CD11b and Gr1. Fetal MPs, functionally defined by their phagocytosis of OVA-647, were CD45⁺ hematopoietic cells and accounted for about 60% and 75% of cells harvested from fetal peritoneum and liver, respectively (figure 4A). Conventional F4/80⁺ macrophages were estimated as <1% of fetal phagocytes in the peritoneum and <3% in the liver (figure 4A), presenting as F4/80⁺CD11b⁺Gr1⁺, F4/80⁺CD11b⁺Gr1⁻ and F4/80⁺CD11b⁻Gr1⁻ rather than F4/80⁺CD11b⁻Gr1⁺ at that time (figure 4B). F4/80⁻ cells comprised over 95% of functionally defined fetal MPs and were rarely present as F4/80⁻CD11b⁻Gr1⁺.

After cocultured with HPV E7 overnight, fetal MPs were demonstrated to take up HPV E7 (figure 5A,B). To assess their ability to elicit lymphocytic cytotoxicity, we first subjected C57BL/6 mice to the injection of C57BL/6 fetal MPs loaded with HPV E7. One month later, C57BL/6 recipients were found to generate significant levels of serum anti-E7 IgG₁ (figure 5C). The activation and proliferation of recipient lymphocytes in response to HPV E7 were also examined in in vitro cell culture systems. Recipient lymphocytes exhibited the activity of lymphocyte activation and proliferation specifically in response to HPV E7 stimulation as evidenced by significant IL2 secretion (figure 5D) and tritium incorporation (figure 5E). Further evaluation of lymphocyte polarization in recipients by ELISA disclosed a Th1-skewed phenotype (figure 5F).

Following adoptive transfer of HPV E7-loaded fetal MPs, C57BL/6 recipients were examined for the generation of HPV E7-specific cytotoxic CD8⁺ T-cells using HPV E7 peptide/MHC class I (H-2D^b) pentamers. Circulating and splenic CD8⁺ T-cells specific to an HPV E7 epitope could be detected 1 week after intraperitoneal challenge of TC-1 tumor cells (figure 6A). The cell-mediated cytotoxicity of recipient lymphocytes against TC-1 cells was morphologically evaluated by DAPI viability dye, which is impermeant to live cells but penetrates the membrane and binds to nucleic acid in nonviable cells. After incubated with the recipient lymphocytes, TC-1 tumor cells displayed very bright fluorescence under a fluorescence microscope (figure 6B). The lymphocytic cytotoxicity was further quantified by LDH release from dead cells. It showed that TC-1 cytolysis significantly increased in recipients subjected to adoptive transfer of HPV E7-loaded fetal MPs (figure 6C), especially when the recipient lymphocytes were rechallenged in advance with HPV E7 before their incubation with TC-1 tumor cells.

To evaluate the protective effects of HPV E7-loaded fetal MPs on TC-1 tumorigenesis, we treated seven C57BL/6 mice with HPV E7-loaded fetal MPs 4 weeks prior to subcutaneous challenge of TC-1 tumor cells (figure 7A,B). After given a tumorigenic dose of TC-1 cells, five of the seven recipients were tumor-free 3 weeks after tumor cell inoculation and remained so afterwards. The other two exhibited sluggish tumor growth, in sharp contrast to rapid tumor growth in wild types as well as the controls receiving fetal MPs without HPV E7 loading. Thus, this preventive HPV E7-loaded fetal MP vaccination effectively rendered the recipients resistant to TC-1 tumorigenesis, in line with the results observed following in utero exposure to HPV E7.

To further evaluate the therapeutic effects of HPV E7-loaded fetal MPs on pre-existing or growing TC-1 tumor cells, we first subjected C57BL/6 mice to subcutaneous inoculation of TC-1 cells, followed 2 or 4 days later by the injection of HPV E7-loaded fetal MPs. Neither of the strategies could cure mice with the tumors derived from a tumorigenic dose of TC-1 cells, as evidenced by the observation that the recipients either lived with progressive enlargement of cutaneous TC-1 tumors or died prematurely during follow-up (figure 7C). However, both strategies significantly slowed down cutaneous TC-1 tumor growth, achieving less cutaneous tumor burdens in comparison with wild types and the controls receiving fetal MPs only (figure 7D). Thus, the therapeutic HPV E7-loaded fetal MP vaccination could only attenuated cutaneous TC-1 tumor growth but never achieved complete tumor clearance.

Given HPV E7-loaded fetal MPs as preventive or therapeutic cellular vaccines described above, the recipients were examined for survivals following TC-1 tumor challenge within a period of 2 months (figure 7E). It was worth noting that preventive HPV E7-loaded fetal MPs conferred an excellent protection against TC-1 tumor challenge, accounting for 71% (5/7) of animals immunized with HPV E7-loaded MPs to live free from cutaneous TC-1 tumors and metastatic lesions at 2-month follow-up, which was superior to wild types or their counterpart controls. However, the recipients receiving the therapeutic MP vaccines either died of tumor expansion or were ethically sacrificed due to excessive tumor burden at follow-up. Overall, the therapeutic MP vaccines provided no survival benefits to the recipients.