Figure 6.
Prazosin Enhances Cross-Presentation and Cross-Priming
(A) Antigen cross-presentation assay with B3Z hybridoma in the presence of increasing concentrations of prazosin or tamoxifen. The cells were pulsed with sOVA for 45 min, followed by 3.5 h incubation in the presence of the indicated compounds. Representative of three independent experiments.
(B) The effect of prazosin on cross-presentation of cell-derived antigens. 3T3 cells expressing cytosolic OVA were used as antigen source and co-cultured with MutuDCs in for 5 h in the presence or absence of prazosin. Mean from three independent experiments ±SE.
(C) Phagocytosis efficiency in the presence and absence of prazosin. 3T3s were labeled with PKH26, and acquisition of the dye by MutuDCs was analyzed after 2 h of co-culture. Mean from three independent experiments ± SE.
(D) MutuDCs were incubated with sOVA or sOVA EF for 45 min followed by 3.5 h incubation with prazosin. B3Z assay was used to monitor cross-presentation efficiency (representative plot from three independent experiments, error bars indicate SEM from technical duplicates).
(E) For the analysis of DC activation, MutuDCs were incubated with sOVA/EF sOVA in the presence and in the absence of prazosin for 5 h, washed, further incubated for 16 h at 37°C, and stained with anti-CD86 (gated for live cells only).
(F) MutuDCs were incubated with the MHC I peptide in the presence or absence of prazosin for 5 h, washed, fixed, and incubated with B3Z hybridoma for 16 h. Mean from three independent experiments ±SE.
(G) MutuDCs were incubated with sOVA EF in the presence of indicated compounds for 5 h, and antigen cross-presentation was detected with B3Z hybridomas. Mean from three independent experiments ±SE.
(H) The effect of prazosin on antigen presentation to OT-I and OT-II cells. MutuDC were incubated with sOVA EF, OVA-expressing 3T3s, or MHC class I or II peptides in the presence or absence of prazosin or Poly(I:C).
(I) Tumor growth. Mice were injected subcutaneously (SC) with the MC38-OVA tumor cell. When tumors became detectable, the animals were treated systemically (intraperitoneally, i.p.) with 0.5 mg prazosin or vehicle control, 3× week. Mice pooled from two independent experiments. The numbers indicate number of mice with tumors smaller than 250 mm3 at the end of the experiment. Lower panel represents best-fit curves for control and prazosin-treated groups, where means and SD were calculated using loess regression, the statistical significance was calculated using ANOVA with the Tukey test and false discovery rate (FDR) Benjamini-Hochberg correction. ∗∗∗p < 0.001 ∗∗∗∗p < 0.0001.
(J) Tumor growth curves for mice injected s.c. with the B16-OVA tumor cells. From the day when tumors became detectable, mice were treated three times per week with 0.5 mg prazosin, 150 μg anti-PD-1, or the combination of both. Mice pooled from three independent experiments. The last panel represents best-fit curves for all groups, where means and SD were calculated using loess regression. The statistical significance was calculated using ANOVA with the Dunnett’s test and FDR Benjamini-Hochberg correction. ∗∗p < 0.01, ∗∗∗∗p < 0.0001.
See also Figure S6.