CP mediates maturation of VR1^+/+ DCs but not VR1^–/– DCs. (A) Schematic representation of VR1 protein structure in VR1^+/+ and VR1^–/– mice including the position of primers used to detect VR1 mRNA in DCs by RT-PCR. (B) RNA isolated from VR1^+/+ and VR1^–/– bone marrow culture (day 6) was subjected to RT-PCR for mouse VR1. PCR product was separated on 1% agarose gel and visualized with ethidium bromide. Lanes 1 and 2, PCR product from VR1^+/+ and VR1^–/– DCs by using forward primer 297 and reverse primer 1,562; lanes 3 and 4, PCR product by using forward primer 1,561 and reverse primer 1,562 (in A). (Because the VR1^–/– mice have been created by insertional inactivation rather than deletion of the VR1 gene, they still express the product of the gene, which is still recognizable by the Ab against VR1; therefore, the lack of functional VR1 cannot be shown by using Ab-based methods.) (C) Day 6 BMCs from VR1^+/+ (Left) and VR1^–/– mice were cultured with different doses of CP with or without CP as indicated or with LPS (1 μg/ml) for 20 h at 37°C or heat shocked at 41°C for 6 h and allowed to recover for 16 h at 37°C. Cells were analyzed for expression of IA^b by using FACS. Increase in mean fluorescence intensity values as compared with vehicle-treated controls are plotted for IA^b.

Adjuvanticity of CP. (A) Experimental design. (B) Expansion of OT1 cells in the draining lymph node of VR1^+/+ mice after 3 days of injection of OVA and CP was determined by flow cytometry by using K^b-SIINFEKL tetramer staining. (C) Proportion of CD8⁺ T cells that are K^b-SIINFEKL⁺ in the spleen and draining lymph node of VR1^+/+ mice after 3 days of injection. (D) Clonal expansion of OVA-specific CD8⁺ T cells in the draining lymph node of VR1^+/+ mice 3 days after injection by using CD45.1 as a clonotypic marker. (E) In vivo effector function of OT1 cells was determined by CTL assay in vivo. C57BL/6 splenocytes from naive mice were pulsed with OVA 8 mer. The peptide-pulsed cells were labeled with CFSE^high, and unpulsed cells were labeled with CFSE^low. Both cell types were transferred i.v. to day 3 OVA/CP-injected mice. Specific cytotoxic effector function of OT1 cells was determined by monitoring the elimination of peptide-pulsed targets. The ratios of percentages of unpulsed vs. pulsed targets is shown.

Expression of VR1 on mouse bone marrow-derived DCs. (A) RNA isolated from mouse BMC (day 6) was subjected to RT-PCR for mouse VR1. PCR product was separated on 1% agarose gel and visualized with ethidium bromide. Lane 1, 100-bp DNA ladder; lane 2, VR1 PCR product. (B) VR1 protein expression was determined by loading ≈0.1 × 10⁶ BMC (day 6) lysate per lane and separating on a 10% SDS/PAGE. VR1 immunoreactivity was determined by using anti-VR1 Ab alone (lane 1), anti-VR1 Ab with irrelevant peptide (IP) (lane 2), or anti-VR1 Ab with neutralizing peptide (NP) (lane 3). (C and D) Intracellular staining of VR1 protein. (C) CD11c⁺ cells were purified from day 6 BMCs (≈98%), as described in Materials and Methods.(D) The purified cells were permeabilized and incubated with anti-VR1 Ab or isotype control rabbit IgG at different dilutions (Left) or anti-VR1 Ab (1:2,500) with or without neutralizing peptide or irrelevant peptide (Right). After incubation with primary Ab cells were washed and stained with FITC-conjugated goat anti-rabbit Ab and analyzed by FACs.

Injection of CP leads to maturation and migration of skin DCs in a VR1-dependent manner. (A) Experimental design. (B) Lymph node cells from vehicle- and CP-injected mice (see A) were stained for CD11c and IA^b and analyzed by flow cytometry. Increase in MFI values as compared with vehicle-treated controls for surface marker IA^b on CD11c⁺ cells is plotted. (C) Analysis of CD11c⁺ cells (R1) in the draining lymph nodes of vehicle- and CP-injected VR1^+/+ mice with respect to size, surface expression of IA^b (high, R2; and low, R3), CD86, CD11b, and CD205 by flow cytometry.