Fig 1.
RT-PCR analysis of MmuPV1 and Cd8a transcripts in vaginal samples of wildtype, Tmc6-/-, Tmc8-/- and nude mice after MmuPV1 challenge.
Female Tmc6-/-, Tmc8-/- and wildtype FVB mice (n = 5/group, aged 2–3.5 months) or age-matched nude mice (as a positive control for each challenge site) were challenged intravaginally with 20μL crude MmuPV1 wart extract. An additional 3 mice per group of male and female Tmc6-/-, Tmc8-/- mice and wildtype mice that did not receive any challenge were included as a naïve negative control. To confirm that the vaginal challenge was successful, a vaginal brushing was harvested at day 16 post challenge (A,D), and a second at week 6 (B,E). RNA was purified from the vaginal brushings and levels of MmuPV1 (A-C), and Cd8a (D-F) transcripts assessed by qRT-PCR. At month 2, all animals were euthanized, and the vaginal tissue was removed and split in two. RNA was extracted from half for analysis for levels of MmuPV1 (C), and Cd8a (F) transcripts by qRT-PCR, and the remainder was fixed and processed for in situ hybridization (S1 and S2 Figs). Samples where no signal was detected at 40 cycles of qRT-PCR were assigned a value of 40 (indicated with a red dot). All qPCR data was presented as -ΔCq (-[MmuPV1 Cq–Capzb Cq]) or -ΔCq (-[CD8a Cq–Capzb Cq]) with standard error. Note, a higher, more positive number corresponds to more transcript detected. (G) Tmc6-/- (n = 10), Tmc8-/- (n = 10) and wildtype (n = 5) FVB mice (males, aged 1–2.5 months) were challenged on the ear with 2.8x1010 vge MmuPV1. At 3 months post-challenge the mice were sacrificed and the presence of MmuPV1 transcript in RNA extracted from the challenged ear was determined by qRT-PCR. MmuPV1 transcript was detected in 10/10 Tmc6-/- mice, 9/10 Tmc8-/- mice and 1/5 wildtype FVB mice. (H, I) Tmc6-/- (n = 10), Tmc8-/- (n = 10) and wildtype (n = 10) FVB mice (females, aged 1–2.5 months), as well as nude mice (n = 3) as a positive control, were challenged on the ear with 9.3x109 vge MmuPV1 and in the vagina with 1x108 vge MmuPV1. The mice were sacrificed at 3 months post-challenge and the presence of MmuPV1 transcript in RNA extracted from the challenged ear (H) and vagina (I) was determined by qRT-PCR.
Fig 2.
Leukocyte composition in the spleen of wildtype, Tmc6-/-, and Tmc8-/- mice.
Splenocytes were isolated from naïve wildtype (n = 5), Tmc6-/- (Tmc6 ko, n = 5), and Tmc8-/- mice (Tmc8 ko, n = 5) of the FVB strain (7–8 week old males). Individual immune subsets were stained for flow cytometry analysis. (A) Representative flow cytometry plots illustrating the gating strategy used to identify the immune subsets in the spleen. The following parameters were examined: frequencies of (B) CD11c+ dendritic cells, (C) F4/80+ macrophages, (D) CD19+ B cells, (E) CD3+ T cells, (F) CD8+ T cells, and (G) CD4+ T cells in CD45+ leukocytes, and (H) frequencies of CD25+FoxP3+ regulatory T cells within the CD4+ T cell population.
Fig 3.
Leukocyte composition in the spleen of wildtype, Tmc6-/-, and Tmc8-/- mice at 3 weeks after MmuPV1 challenge.
Naïve wildtype, Tmc6-/-, and Tmc8-/- mice (7–10 week old female) were challenged intravaginally with 108 vge MmuPV1, and sacrificed at 3 weeks post infection (n = 5/group). The levels of viral transcript were measured in vaginal tissues and confirmed successful infection (S1A Fig). Splenocytes were isolated and individual immune cell populations were stained for flow cytometry analysis. The following parameters were examined: frequencies of (A) CD11c+ dendritic cells, (B) F4/80+ macrophages, (C) CD19+ B cells, (D) CD8+ T cells, and (E) CD4+ T cells in CD45+ leukocytes. (F) frequencies of CD25+FoxP3+ regulatory T cells within the CD4+ T cell population.
Fig 4.
The immune landscape of vaginal brushings from wildtype, Tmc6-/-, and Tmc8-/- mice, either with or without MmuPV1 challenge.
Vaginal brushings were collected 17 days after MmuPV1 challenge from all groups of mice from the experiment described in Fig 3 (n = 5/group). Vaginal cell suspensions were stained with selected immune subsets for flow cytometry analysis. The following parameters were examined: frequencies of (A) CD45+ leukocytes, (B) CD3+ T cells, (C) CD8+ T cells, and (D) CD4+ T cells in total cells.
Fig 5.
MHC complex expression on CD11c+ dendritic cells and interferon-γ production by CD8+ T cells of naive wildtype, Tmc6-/-, and Tmc8-/- mice.
Flow cytometric analysis of the surface expression of MHC class I and II molecules and CD11c on splenocytes of naïve wildtype, Tmc6-/-, and Tmc8-/- mice (n = 5/group, 7–8 week old males). (A) The gMFI values for MHC class I on CD11c+ cells. (B) The gMFI values for MHC class II on CD11c+ cells. (C-D) Wildtype, Tmc6-/-, and Tmc8-/- mice (10–14 week old females) were challenged with 3.7x1010 vge MmuPV1(n = 5/group) or naïve (n = 2 wildtype and 3 Tmc6 -/-). Keratinocytes were collected 2 weeks after immunization through samplings using vaginal swabs and stained with antibodies against CD49f, MHC class I, and MHC class II. FACS analysis of the expression of MHC class I and II molecules on CD49f+ keratinocytes of FVB, Tmc6-/-, and Tmc8-/- mice. (C) The gMFI values for MHC class I on CD49f+ cells. (D) The gMFI values for MHC class II on CD49f+ cells. (E-F) Splenocytes were isolated from wildtype, Tmc6-/-, and Tmc8-/- mice (n = 5/group, 7–10 week old female) at 3 weeks post challenge intravaginally with 108 vge MmuPV1 or mock challenge (see Fig 2). The splenocytes were stimulated with Phorbol 12-myristate 13-acetate and Ionomycin (P+I) in (E) or anti-CD3 plus anti-CD28 antibody (F) for 24 h. Stimulated cells were analyzed for IFNγ expression in CD8+ T cells by surface and intracellular staining and flow cytometry. Statistical analysis utilized the Student’s T-test. Representative gating is shown in S4 and S5 Figs.
Fig 6.
Activation, proliferation, and intracellular zinc levels of CD8 T cells of wildtype, Tmc6-/-, and Tmc8-/- mice.
Naïve CD8 T cells were magnetically isolated from the spleens of wildtype, Tmc6-/-, and Tmc8-/- mice (n = 5/group, 9–13 week old female), and labeled with CellTrace Far Red. The CD8+ T cells were activated through either PMA/Ionomycin or anti-CD3/anti-CD28 for 24 h. Statistical analysis utilized the Student’s T-test. (A) Representative gating strategy to analyze the expression of CD3, CD8, CD44, and CD62L on T cells before enrichment. (B) The flow cytometric plot shows the expression of CD62L and CD44 before and after enrichment. (C) The percentage of CD25+CD8+ T cells in total CD8 T cells. (D) The percentage of dividing CD8+ T cells. (E) The concentration of free zinc was measured using FluoZin-3. Representative gating is shown in S4 and S5 Figs.
Fig 7.
Control of vaccinia virus expressing luciferase after percutaneous challenge of Tmc6-/-, Tmc8-/- or wildtype FVB mice.
Infectivity in wildtype, Tmc6-/- and Tmc8-/- FVB mice of recombinant vaccinia virus expressing luciferase (VV-luc) was assessed in 6–8 week old Tmc6-/-, Tmc8-/- and wildtype FVB male mice (n = 10/group). Briefly, mice were anesthetized and 5x105 pfu (5μL) of recombinant vaccinia virus expressing luciferase (VV-luc) was applied to tail skin 1 cm from the base of the tail on day 0. The skin area was then gently scratched 15 times with a bifurcated needle. Mice were imaged by IVIS Spectrum in vivo imaging system series 2000 (PerkinElmer) at days 2, 6, 9 and 12. Total photon counts were quantified in the tumor site by using Living Image 2.50 software (PerkinElmer). Luminescence imaging from each group is presented, quantification of luminescence signal in the region of interest (ROI) shown graphically.
Fig 8.
Impact of adoptive transfer of splenotypes from naïve or vaccinated wildtype FVB mice on MmuPV1 transcript levels in Tmc6-/- or Tmc8-/- FVB mice.
Tmc6-/- (n = 18), Tmc8-/- (n = 9) FVB mice (females, aged 3 months) were challenged intravaginally with 5.1x1011 vge MmuPV1, and their vagina was first sampled at 5 weeks post-challenge (A-D). Two weeks later, 9/18 of the challenged Tmc6-/- mice (B) and 3/9 of the challenged Tmc8-/- mice (D), as well as 5 of 10 additional wildtype FVB mice (female, 3 months old), were then administered 15 μg of hCRT-mE6mE7mL2 DNA intramuscularly three times at 2 week intervals with electroporation. The remaining 7/14 Tmc6-/- mice (A), 6/9 of Tmc8-/- mice (C) and 5/10 wildtype FVB mice were not vaccinated. Two weeks after the vaccination was completed, serum was collected and analyzed in Fig 9A. A second vaginal sampling from the challenged mice was taken 4 weeks after the final vaccination (A-D), and the levels of MmuPV1 transcript present in the paired samples was compared by qRT-PCR. There was no significant difference by paired T test in the level of MmuPV1 transcript present before versus after vaccination in the vaccinated (n = 9, p = 0.24, B) or unvaccinated Tmc6-/- mice (n = 9, p = 0.24, A), or in the vaccinated (n = 3, p = 0.46, D) or unvaccinated Tmc8-/- mice (n = 6, p = 0.11, C). Splenocytes (106) from 5 naïve wildtype FVB mice were transferred into 3 of the Tmc6-/- (E) and 2 of the Tmc8-/- mice (G) from the above experiment. Likewise, splenocytes (106) from 5 wildtype FVB mice vaccinated with hCRT-mE6mE7mL2 DNA were transferred into 3 of the Tmc6-/- (F) and 2 of the Tmc8-/- mice (H) from the above experiment. One month after adoptive transfer, the vaginal tracts were sampled to measure MmuPV1 transcript levels of all ten mice (E-H). No significant change in the levels of MmuPV1 transcript from before versus a month after adoptive transfer were observed, either in the Tmc6-/- or the Tmc8-/- mice, or either those that received splenocytes from wildtype FVB mice previously vaccinated with hCRT-mE6mE7mL2 DNA (F,H) or not (E,G).
Fig 9.
L2-specific Antibody and E6/E7-specific CD8+ T cell responses of FVB mice vaccinated with hCRT-mE6mE7mL2 DNA.
Tmc6-/-, Tmc8-/—and wildtype female FVB mice were injected intramuscularly with 15μg of hCRT-mE6mE7mL2 DNA vaccine followed by electroporation on days 1, 15 and 29. One month after the final vaccination, sera were collected. Sera were collected were also collected from unvaccinated wildtype female FVB mice as a negative control. Sera (n = 3/group) were analyzed by ELISA for reactivity against MmuPV1 L2-6His (A). Briefly, 1μg/ml of MmuPV1 L2 protein in PBS was coated on BRANDplates microplates overnight at 4°C. After 16 h, the plates were washed, blocked with eBioscience ELISA/ELISPOT Diluent, and added serial 2-fold dilution of serum for two hours at room temperature. Goat anti-mouse IgG-HRP secondary antibody was added at 1:5000 dilution for 1 hour, followed by TMB substrate. The OD at 450nm was determined by 800 TS Absorbance Reader (BioTek Instruments, Inc). Characterization of MmuPV1 E6 (B,D) and E7 epitopes (C,E) recognized by CD8+ T cells of C57BL/6 (C,D) or FVB mice. Mice (n = 3) were injected intramuscularly with 15μg of hCRT-mE6mE7mL2 DNA vaccine followed by electroporation on days 1, 8 and 15. One week after the final vaccination, splenocytes were collected. To determine the epitopes, a panel of 20mer peptides, each overlapping by 15 amino acids were incubated with splenocytes in the presence of Golgi plug for 16 hours. Splenocytes stimulated with eBioscience Cell Stimulation Cocktail was used as a positive control. The cells were stained for interferon-γ and CD8, and analyzed by flow cytometry using a CytoFLEX S (Beckman) and data were analyzed by FlowJo software. Percentage of interferon-γ producing CD8+ T cells is presented.