Fig 1.
Design and Function of OVA and MC.OVA Vaccine Vectors.
(A) Linear diagrams showing the functional features of the OVA (upper) and MC.OVA (lower) vaccine constructs. (B, C) 293 cells were transfected with 1 μg of the indicated plasmid. Lysates were probed for OVA expression by Western blot and expression was measured relative to β-actin. (D) 293 cells transfected with NF-κB SEAP reporter and either GFP, OVA, or MC.OVA expression plasmids. Cells were plated with increasing concentrations of rim.
Fig 2.
MC improves DNA EP vaccine immunogenicity compared to Ag alone.
C57BL/6 mice were vaccinated with 25 μg of the indicated plasmids on days 0 and 21. One day following each vaccination, 1.25 mg/kg of rim was administered IP in the MC.OVA + rim treatment group. (A) On day 28 splenocytes were analyzed for numbers Ag-specific CD8+ T cells using H2-Kb SIINFEKL tetramers. Representative data from two independent experiments. (B) Ag-specific T cell function was assayed by IFN-γ ELISpot. Data from two independent experiments. (C) Representative images of ELISpot membranes. (D-E) Naïve mice were vaccinated on day 0 and again 20 days later with 25 μg of plasmid DNA followed by electroporation. MC.OVA + rim-treated mice were injected with 1.25 mg/kg IP the day following each vaccination. On day 27 splenocytes were isolated from the mice and incubated with SIINFEKL peptide ON and supernatants were collected and analyzed for 25 different cytokines/chemokines by multiplex (representative graphs shown). n = 5–10, *p<0.05, One-way ANOVA, Tukey correction for multiple comparisons, nd = not detectable.
Fig 3.
MC.OVA reduces tumor growth in B16-OVA bearing mice more effectively than vaccination with OVA alone.
C57BL/6 mice were injected with 2 x 105 B16-OVA cells subQ. Tumors were allowed to establish for 6 days before mice were randomized into treatment groups and vaccinated with 25 μg of either GFP, OVA, or MC.OVA plasmids on days 6, 12, and 19 post-tumor injection. Tumor volumes were determined by caliper measurements. (A) Tumor growth curves (error bars = SEM) for each treatment group. (B) Comparison of each group at day 22 post-tumor injection. Each point represents a single mouse (error bars SD). (C) Survival curves of tumor bearing mice. n = 5, *p<0.05, Two-way ANOVA, Holms-Šidák correction for multiple comparisons. Survival curve analysis by Log Rank (Mantel-Cox) test.
Fig 4.
MC adjuvant improves TAg-specific CTL response in tumor-bearing mice.
C57BL/6 mice were injected subQ with 5 x 105 E.G7 tumor cells. Mice were vaccinated with 25 μg pDNA encoding either GFP, OVA, or MC.OVA on days 5 and 11 post-tumor injection. One day following vaccination, 1.25 mg/kg rim was administered IP in MC.OVA + rim-treated mice. (A) Tumor volumes were determined by caliper measurements. (B) The experiment was terminated on day 19 post-tumor injection and IFN-γ ELISpot was performed to quantify SIINFEKL-specific T cells. (C) Right panel: Correlation between day 19 tumor volume and IFN-γ SFC. Left Panel: correlation between change in tumor volume from day 13–19 vs IFN-γ SFC. R2 values determined by non-linear regression using a one phase decay curve-fit. n = 4–5, *p<0.05, One-way ANOVA, Tukey correction for multiple comparisons.
Fig 5.
Cytokine secretion in keratinocytes and fibroblasts mediated by MC-activation.
(A-B) B6-MPEK (MPEK), a keratinocyte cell line, and NIH3T3, a fibroblast cell line, were transduced with lentivirus encoding the GFP backbone alone (control cells), OVA, or MC.OVA. Polyclonal lines stably expressing these proteins were puromycin-selected to >90% transgene+ purity. 2 x 105 cells were cultured with or without 10 nM rim for 48 hours. As a positive control, control cells were stimulated with 1 ng/ml LPS. Culture supernatants were analyzed for cytokine secretion by a 25-plex cytokine panel. (A-B, Upper panels) Fold induction of cytokines relative to control cell supernatants with 10 nM rimiducid. (A-B, Bottom panels) Representative graphs for each cell line showing the absolute quantitation of cytokines secreted in pg/ml. n = 3, *p<0.05, One-way ANOVA with Tukey correction for multiple comparisons, nd = not detectable.
Fig 6.
MC-mediated immune stimulatory properties of fibroblasts and keratinocytes.
(A) NIH3T3 or (B) MPEK stable cells lines expressing either GFP alone (neg. control), OVA or MC.OVA were incubated for 60 hours. MC.OVA +rim had 10 nM rim present in the culture. After 60 hours, cells were harvested and MHC-I expression was analyzed by flow cytometry. (C-E) MPEK stable cells surface expression of CD80, CD86, and CD40. MFI values of live cells are reported relative to the mean MFI of negative control cells, transduced with an empty-vector, in each respective sample. (F) CD3+CD8+ OT-1 or WT T cells were purified by MACs and labeled with CellTrace violet (CTV) dye. Then 5 x 104 CD3+CD8+ CTV dye-labeled OT-1 or WT T cells were incubated with 5 x 104 B6-MPEK cells for 60 hours. By flow cytometry, live CTV+ cells were analyzed for CTV dye dilution as a measure of cell proliferation. (G) Representative histograms of live CTV+ cells. Percentages represent the percent of cells proliferating. n = 3, *p<0.05, One-way ANOVA, Tukey correction for multiple comparisons.
Fig 7.
Expression of MC and Ag in cutaneous atypical APCs contribute to adjuvant-enhanced EP vaccine-mediate Ag-specific CD8+ T cell priming and is partly CD8α+/CD103+ DC-independent.
C57BL/6 mice were vaccinated on days 0 and 21 with 25 μg pDNA by EP, rim was administered 1.25 mg/kg IP the day following each vaccination in MC.OVA.miR142T + rim-treated mice. (A) Splenocytes were extracted 7 days after the final vaccination (day 28) and restimulated with SIINFEKL peptide overnight. IFN-γ SFCs were quantitated by ELISpot. n = 5, *p<0.05, One-way ANOVA, Tukey correction for multiple comparisons. (B) C57BL/6 mice were injected in alternating flanks 21 days apart with 1 x 105 MPEK cells stables (neg. control, OVA, MC.OVA). Rimiducid was administered at 1.25 mg/kg IP 24 hours after each injection of cells. 7 days following the second injection, splenocytes were analyzed for SIINFEKL-specific IFN-γ-secreting CD8+ T cell by ELISpot. Error bars represent 95% confidence interval. *p<0.05 when compared to MPEK-OVA, One-way ANOVA, Dunnett’s correction for multiple comparisons. (C) Either C57BL/6 or Batf3-/- mice were vaccinated in the same manner as described for panel A. SIINFEKL-specific IFN-γ-secreting splenocytes were quantitated by ELISpot. n = 4–5, *p<0.05, One-way ANOVA, Tukey correction for multiple comparisons within mouse strain.
Fig 8.
Anti-tumor and cytotoxic CD8+ T cell responses are increased by cutaneous atypical APCs expressing MC and T-Ag.
(A-B) In vivo CTL assay. Splenocytes from naïve, syngeneic C57BL/6 mice were isolated and labeled with either 0.5 μM (Lo) or 5 μM (Hi) CTV dye, then pulsed with 10 ng of either irrelevant H2-Kb ICPMYARV (β-gal) peptide (Lo), or target H2-Kb SIINFEKL (OVA) peptide (Hi). Ag-pulsed and dye-labeled splenocytes were mixed 1:1 (Hi:Lo) and a total of 1e7 cells per mouse was injected IV into vaccinated mice 7 days after the last vaccination. After 7 hours, splenocytes were extracted and analyzed for the presence of CTV dye-labeled cells. The Hi:Lo CTV+ cell ratio was proportional to the levels of target-specific killing. (B) Representative histograms of live, CTV+ splenocytes. Values are the mean ± SD of % specific lysis of target cells. n = 4–5, *p<0.05, One-way ANOVA, Tukey correction for multiple comparisons. (C) C57BL/6 mice were injected subQ with 1e6 E.G7 tumor cells on day 0. Tumors were allowed to establish for 2 days. On days 2 and 9, mice were vaccinated with 25 μg pDNA by EP. Tumor volumes were determined by caliper measurements. n = 10, *p<0.05 when compared to OVA.miR142T, Two-way ANOVA with repeated measures, Holm-Šidák correction for multiple comparisons, error bars represent SEM.