Vanilloid Receptor 1 Agonists, Capsaicin and Resiniferatoxin, Enhance MHC Class I-restricted Viral Antigen Presentation in Virus-infected Dendritic Cells

Cell lines, reagents, and mice

A T cell hybridoma, B3Z86/90.14 (B3Z), was provided by Dr. Nilabh Shastri (University of California, Berkeley, CA, USA) (17). The T cell hybridomas, CD8 OVA1.3 and DOBW, were provided by Dr. Clifford V. Harding (Case Western Reserve University, Cleveland, OH, USA) (18, 19). Recombinant vaccinia virus encoding OVA (VV-OVA) was provided by Dr. Jonathan W. Yewdell (National Institutes of Health, Bethesda, MD, USA) (20). Both capsaicin (CP) and resiniferatoxin (RTX) were purchased from Sigma-Aldrich (St. Louis, MO, USA). The compounds were dissolved in absolute ethanol for in vitro experiments or in ethanol/tween-20/PBS (10/10/80) for in vivo experiments. Male, C57BL/6 and BALB/c mice (8~12-week-old) were purchased from Orient Co., Ltd. (Seoul, Korea). Mice were studied according to the protocols approved by the Animal Care Committee of Chungbuk National University.

Generation of bone marrow-derived DCs (BM-DCs)

BM-DCs were generated as described previously (21). Briefly, total BM cells obtained from mouse femurs were cultured in 6-well plates (5×10⁶/well) in culture medium containing 40 ng/ml GM-CSF and 20 ng/ml IL-4 (both from CreaGene, Seongnam, Korea). After 3 and 4 days, non-adherent cells were removed by gentle shaking and replacing the medium. Immature BM-DCs were harvested by gentle pipetting on day 6.

In vitro viral antigen-presentation assay

Immature BM-DCs (1×10⁷/ml) were infected with VV-OVA (multiplicity of infection [MOI]=5) for 20 min at 37℃. Virus-infected DCs were diluted to 1×10⁶/ml with DMEM containing 10% FBS and 1 µM cytosine β-D-arabinofuranoside (Sigma-Aldrich), and distributed to a 96-well microtiter plate (100 µl/well). After adding the indicated quantities of VR1 agonists, the plate was incubated for 6 h at 37℃. After fixing the cells with ice-cold, 1.0% paraformaldehyde for 5 min at room temperature, class I MHC-complexed OVA peptide quantities were assessed using CD8 OVA 1.3 cells (2×10⁵/well), which recognize OVA[257-264]-K^b complexes and secrete IL-2 (22).

VR1 agonist treatment and VV-OVA infection

C57BL/6 (H-2^b) mice were orally administrated CP (10 mg/kg) or RTX (0.1 mg/kg) for 3 days, and then infected with VV-OVA (5×10⁴ CFUs/mouse, i.v. administration). After infection, the mice were orally administered the same doses of CP or RTX for 5 consecutive days. On the final day of final VR1 agonist administration, functional assays were performed, such as the in vivo antigen-presentation assay, in vivo CTL assay, and OT-I T cell-adaptive transfer assay were performed.

In vivo viral antigen-presentation assay

DCs were isolated from the lymph nodes (popliteal, inguinal, mesenteric, and axillary) and spleens were collected and pooled (≥10 mice per treatment group), using a negative-selection method, as described previously (23). Class I-restricted T cell activation assays were then performed as described previously (17) using B3Z cells, which express β-galactosidase when activated with OVA[257-264]-K^b complexes. The B3Z cell responses were determined by X-gal (5-bromo-4-chloro-3-indoyl-β- D-galactopyranoside) staining (23).

Phenotype analysis

Cells were stained with mAbs recognizing murine cell surface markers as described previously (22), and flow cytometric analysis was performed on a FACS Canto II (BD Biosciences, San Jose, CA, USA). mAbs (anti-H2-K^b, anti-I-A^b, anti-CD80, anti-CD86, and an isotype-matched control) were purchased from BD Biosciences. The mAbs recognizing OVA[257-264]-K^b complexes, Alexa 488-conjugated 25-D1.16, was kindly provided by Dr. Jonathan W. Yewdell (National Institutes of Health, Bethesda, MD) (20).

In vivo CTL assay

In vivo evaluation of the cytotoxic activities was performed using 5,6-carboxyfluorescein diacetate succinimidyl ester (CFSE, Molecular Probes)-labeled target cells prepared from syngeneic spleen and lymph node cell suspensions, as described previously (23). Specific in vivo cytotoxicity was determined for the lymph node and spleen cells isolated from the recipient mice 18 h after the i.v. injection of target cells. The ratio between the percentage of uncoated vs. OVA[257-264]-coated (CFSE^low/CFSE^high) cells was calculated to obtain a numerical value of the cytotoxicity.

OT-I T cell transfer experiment

CD8⁺ T cells were isolated from the pooled spleens and lymph nodes of OT-I mice using a CD8⁺ T Cell Isolation kit (Miltenyi Biotec Inc., Auburn, CA, USA). The cells were labelled with 50 µM CFSE, and injected into the tail vein of mice (1×10⁷ cells/mouse). After 4 days, proliferation of CFSE-labeled OT-I CD8⁺ T cells was determined for the lymph node and spleen cells isolated from the recipient mice by flow cytometry.

Intracellular IFN-γ staining

Intracellular IFN-γ staining was performed as described previously (24) using a FITC-labeled anti-CD8 mAb and a PE-conjugated anti-IFN-γ mAb (BD Biosciences) (24). Cells were then analyzed using a FACSCanto II flow cytometer (BD Biosciences), and the number of CD8⁺ cells expressing IFN-γ was enumerated using FlowJo software (TreeStar, Ashland, OR, USA).

Preparation of microspheres containing OVA (OVA-microspheres)

OVA-microspheres were prepared using a homogenization/ solvent evaporation method, as described previously (21), with 400 µl of OVA-containing water (50 mg OVA/ ml) and 2 ml of ethyl acetate containing poly(lactic-coglycolic acid) (100 mg/ml, Sigma-Aldrich). Fluorescent microspheres were prepared by adding FITC (5 µg/ml) to the ethyl acetate phase.

Exogenous antigen-presentation assays

BM-DCs, generated from the BM cells of C57BL/6 or BALB/c mice, were seeded in 96-well microtiter plates (1×10⁵/well) in the presence of different concentrations of CP or RTX, incubated for 4 h at 37℃, and then OVA-microspheres (50 µg/ml as OVA) were added. After 2 h incubation at 37℃, the cells were fixed with 1.0% paraformaldehyde for 5 min at room temperature. Class I MHC-complexed OVA peptide quantities were assessed using CD8 OVA 1.3 cells (2×10⁵/well), which recognize OVA[257-264]-K^b complexes and secrete IL-2 (22). Class II MHC-complexed OVA peptide quantities were assessed using DOBW cells (1×10⁵/well), which recognize OVA[323-339]-I-A^d complexes and secrete IL-2 (19).

Statistical analysis

The statistical significance of the difference between the control and treatment groups was assessed using a one-way ANOVA followed by a Student's t-test.

VR1 agonists increase MHC class I-restricted OVA presentation in BM-DCs infected with VV-OVA

To examine the effects of VR1 agonists on MHC class I-restricted endogenous antigen presentation, BM-DCs were infected with VV-OVA (MOI=5), and then cultured in the presence of different concentrations of CP or RTX. After 6 h, the cells were fixed with paraformaldehyde, washed, and then the amounts of MHC class I (K^b)-OVA peptide complexes on the cell surface were assessed using CD8 OVA1.3 cells. Both CP and RTX increased MHC class I-restricted presentation of virus-encoded OVA (Fig. 1A). The ability of CP and RTX to increase MHC class I-restricted endogenous antigen presentation reached near maximum levels at 32 nM and 6.4 nM, respectively, showing that RTX is much more potent than CP in increasing MHC class I-restricted endogenous antigen presentation in DCs.

Figure 1
VR1 agonists increase MHC class I-restricted OVA presentation in DCs infected with VV-OVA. (A) BM-DCs (1×10⁵/well) generated from C57BL/6 (H-2^b) mice were infected with VV-OVA (MOI=5), and cultured for 6 h in the presence of the indicated amounts of CP or RTX. The amounts of H-2K^b-OVA peptide complexes were assessed using CD8 OVA1.3 cells. (B, C) VR1 agonists increased MHC class I-restricted DC presentation of OVA peptides in mice infected with VV-OVA. C57BL/6 mice were orally administered CP (10 mg/kg) or RTX (0.1 mg/kg) was for 3 days, and then then were infected with VV-OVA (5×10⁴ CFUs/mouse, i.v.). After virus infection, the mice were orally administrated the same doses of CP or RTX for consecutive 5 days. On the final day of VR1 agonist administration, DCs were isolated from the lymph nodes and spleens of the mice, and the amounts of K^b-OVA peptide complexes on the cell surface were evaluated using OVA-specific CD8 T hybridoma cells (B3Z cells). ^**p<0.01.

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VR1 agonists increase MHC class I-restricted DC presentation of OVA peptides in mice infected with VV-OVA

To examine whether VR1 agonists also increase MHC class I-restricted endogenous antigen presentation in vivo , CP (10 mg/kg) or RTX (0.1 mg/kg) was orally administered to C57BL/6 mice for 3 days, followed by infection with VV-OVA (5×10⁴ CFUs/mouse, i.v.). After virus infection, the mice were orally administrated with the same doses of CP or RTX for consecutive 5 days. On the final day of VR1 agonist administration, DCs were isolated from the lymph nodes and spleens of the mice, and the amounts of K^b-OVA peptide complexes on the cell surface were evaluated using OVA-specific CD8 T hybridoma cells (B3Z cells). Both CP and RTX significantly increased MHC class I-restricted presentation of virus-encoded OVA in both lymph node DCs and spleen DCs (Fig. 1B, C).

The amounts of K^b-OVA peptide complexes on the cell surface of lymph node and spleen DCs were directly determined using the F(ab')2 fragment of a SIINFEKL-H-2K^b-specific mAb (25-D1.16). Oral administration of CP and RTX (Fig. 2A) profoundly increased the cell surface expression of SIINFEKL-H-2K^b complexes in the spleen DCs of VV-OVA-infected mice. We then examined whether CP and RTX increased the expression of MHC class I molecules on DCs. Both CP and RTX increased the expression levels of H-2K^b molecules, but not the expression levels of I-A^b molecules, in the spleen DCs of VV-OVA-infected mice (Fig. 2B).

Figure 2
VR1 agonists increased the expression of SIINFEKL-H-2K^b complexes in mice infected with VV-OVA. Mice were orally administered CP or RTX for 3 days, infected with VV-OVA, and then treated with the same doses of CP or RTX for consecutive 5 days, as described in Fig. 1. DCs were isolated from the lymph nodes and spleens. (A) The amounts of K^b-OVA peptide complexes on the cell surface were directly determined using the F(ab')2 fragment of a SIINFEKL-H-2K^b-specific mAb (25-D1.16). (B) Expression levels of H-2K^b and I-A^b molecules. Blue dotted line, isotype control; green shaded area, DCs from mice not treated with a VR1 agonist (CP or RTX); red line, DCs from VR1 agonist-treated mouse.

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Oral administration of VR1 agonists increased OVA-specific CTL activity in mice infected with VV-OVA

The effects of VR1 agonists on the induction of CTL activity were examined in mice orally administered CP or RTX for 3 days, infected with VV-OVA, and then treated with the same doses of CP or RTX for consecutive 5 days, as described in Fig. 1. On the final day of VR1 agonist administration, in vivo CTL assays were performed using CFSE-labeled syngeneic target cells. Representative histograms are shown in Fig. 3. Oral administration of CP (Fig. 3A) or RTX (Fig. 3B) significantly increased OVA-specific CTL activities in the lymph nodes and spleens.

Figure 3
VR1 agonists increased OVA-specific CTL activity in mice injected with VV-OVA. Mice were orally administered CP or RTX for 3 days, infected with VV-OVA, and then treated with the same doses of CP or RTX for consecutive 5 days, as described in Fig. 1. On the final day of VR1 agonist administration, and in vivo CTL assays were performed using CFSE-labeled syngeneic target cells. (A) Representative histograms of lymph node and spleen cells, and the percentage of specific killing of OVA[257-264] peptide-pulsed target cells in the lymph nodes and spleens are shown (n=5 for each group). Each data point represents the mean±SD of values obtained from 2 individual experiments. ^*p<0.05; ^**p<0.01.

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Oral CP administration increased the proliferation and activation of adaptively transferred OT-I cells

To confirm that CP increases MHC class I-restricted OVA presentation in DCs of VV-OVA infected mice, an adaptive transfer experiment was performed, in which OVA-specific CD8⁺ T cells (OT-I cells) were transferred to VV-OVA-infected mice. Mice were orally administered CP, infected with VV-OVA, and then treated with the same doses of CP for consecutive 5 days, as described in Fig. 1. On the final day of CP administration, CFSE-labeled OT-I cells were injected through the tail vein. After 4 days, proliferation of OT-I cells in the spleens and lymph nodes was determined by the progressive dilution of CFSE intensity, as determined by flow cytometry. Representative histograms are shown in Fig. 4. OT-1 cells in the lymph node and spleen expanded significantly in CP-treated mice, compared with vehicle-administered control mice.

Figure 4
CP increased the proliferation of adaptively transferred OTI cells. Mice were orally administered CP, infected with VV-OVA, and then treated with the same doses of CP for consecutive 5 days, as described in Fig. 1. On the final day of CP administration, CFSE-labeled OT-I cells were injected through the tail vein. After 4 days, proliferation of OT-I cells in the spleens and lymph nodes was determined by flow cytometry.

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To examine the effects of CP on IFN-γ production in CD8 T cells, total spleen cells and lymph node cells were isolated from mice treated with CP and adaptively transferred with OT-1 cells, and were re-stimulated with OVA_[257-264]-SIINFEKL peptide-pulsed normal BM-DCs in the presence of monensin for 6 h. The cells were stained with an anti-CD8 mAb, permeabilized, and stained with an anti-IFN-γ mAb. Representative histograms are shown in Fig. 5A. Oral administration of CP significantly increased the frequency of IFN-γ-producing CD8 T cells among total CD8 T cells (Fig. 5B).

Figure 5
CP increased IFN-γ producing CD8 T cells in mice infected with rVV-OVA. Mice were orally administered CP, infected with VV-OVA, and then treated with the same doses of CP for consecutive 5 days, as described in Fig. 1. On the final day of CP administration, CFSE-labeled OT-I cells were injected through the tail vein. After 4 days, the lymph node and spleen cells were isolated and restimulated for 6 h with OVA[257-264] peptide-pulsed DCs. The cells were stained with an anti-CD8 mAb, then permeabilized and stained with an anti-IFN-γ mAb. (A) Representative histograms are shown. (B) The percentages of IFN-γ⁺ cells among total CD8⁺ T cells are shown graphically (n=5 for each group). ^**p<0.01.

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VR1 agonists increase class I, but not class II, MHC-restricted processing of exogenous antigen in BM-DCs

The effects of VR1 agonists on the MHC-restricted exogenous antigen presentation were examined using BM-DCs. For MHC class I-restricted exogenous antigen presentation assays, BM-DCs were generated from BM cells of C57BL/6 mice, treated with the indicated concentrations of CP or RTX for 4 h, and then allowed to phagocytose OVA-microspheres for 2 h. The BM-DCs were washed and fixed with paraformaldehyde. Class I MHC-complexed OVA peptide quantities were assessed using CD8 OVA 1.3 hybriboma cells. The results showed that CP and RTX efficiently enhanced class I MHC-restricted exogenous OVA presentation (Fig. 6A). For MHC class II-restricted exogenous antigen presentation assays, BM-DCs were generated from the BM cells of BALB/c mice, treated with the indicated concentrations of CP or RTX for 4 h, and then allowed to phagocytose OVA-microspheres for 2 h. The BM-DCs were washed and fixed with paraformaldehyde. Class II MHC-complexed OVA peptide quantities were assessed using DOBW cells. The results showed that CP and RTX did not affect class II MHC-restricted exogenous OVA presentation in BM-DCs (Fig. 6B).

Figure 6
VR1 agonists increases class I, but not class II, MHC-restricted presentation of exogenous antigen in BM-DCs. (A) BM-DCs generated from BM cells of C57BL/6 mice were treated with the indicated concentrations of CP or RTX for 4 h, and then allowed to phagocytose OVA-microspheres for 2 h. After fixing, the amounts of H-2K^b-OVA peptide complexes were assessed using CD8 OVA 1.3 cells. (B) BM-DCs generated from BM cells of BALB/c mice were treated with the indicated concentrations of CP or RTX for 4 h, and then allowed to phagocytose OVA-microspheres for 2 h. After fixing, the amounts of I-A^d-OVA peptide complexes were assessed using DOBW cells. (C, D) Effects of CP and RTX on phagocytic activity. BM-DCs generated from BM cells of C57BL/6 mice were incubated with FITC-labeled OVA-microspheres, washed, harvested, and analyzed by flow cytometry. A representative set of histograms from 3 individual experiments is shown. Shaded histograms, phagocytic activity of DCs in the absence of CP or RTX; thick line histograms, phagocytic activity of DCs in the presence of CP or RTX.

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To test whether the class I MHC-restricted antigen presentation-enhancing activity of CP and RTX was due to the enhancement of phagocytic activity, BM-DCs generated from BM cells of C57BL/6 mice were incubated with FITC-labeled OVA-microspheres, washed, and then harvested by gentle pipetting after cooling on ice. Flow cytometric analysis of the harvested cells showed that CP and RTX did not affect the phagocytic activity of BM-DCs (Fig. 6C, D).