SRA/CD204 attenuates immunostimulatory activity of TLR4-engaged DCs in vivo. (A) Increased IFN-γ production by OVA-specific CTLs from SRA^−/− DC-immunized mice. WT mice (n = 3) were immunized with OVA-pulsed DCs activated with or without LPS. Splenocytes were harvested and restimulated with OVA_257-264 and subjected to an ELISPOT assay (*P < .001, LPS-treated SRA^−/− DC vs LPS-treated WT DC). (B) Improved OVA-specific cytolytic activity in SRA^−/− DC-immunized mice. After vaccinations with WT or SRA^−/− DCs, mice were inoculated intravenously with 5 × 10⁶ OVA_257-264-pulsed, CFSE^high splenocytes and nonpulsed, CFSE^low population. Lymph node cells were analyzed by FACS after 12 hours. Data represent an example of the FACS data from 3 mice per experimental group. (C) Increased tumor-lytic activity of T-effector cell from SRA^−/− DC-immunized WT mice. Splenocytes from WT or SRA^−/− DC-immunized mice were restimulated with OVA_257-264, and subjected to chromium release assays using OVA_257-264-pulsed B16 cells as targets. Data shown as mean plus or minus SD are representative of 2 experiments.

Hematopoietic cells are involved in SRA/CD204 deficiency-enhanced antitumor immunity after OVA-MPL vaccination. (A) Enhanced antitumor efficacy in immunized SRA^−/− mice. Age-matched WT and SRA^−/− C57BL/6 mice (n = 5) were immunized twice at a weekly interval with 50 μg of OVA protein mixed with 10 μg of MPL. One week after booster immunization, mice were challenged with 3 × 10⁵ B16-OVA tumor cells (*P < .001, SRA^−/− vs WT mice, ANOVA test). Experiments were performed twice, and a representative experiment is shown. (B) Hematopoietic cells required for the enhanced vaccine activities in SRA^−/− mice. Recipient SRA^−/− mice (n = 5) were whole body irradiated and received 10⁷ total BM cells from donor WT or SRA^−/− mice. Eight weeks later, recipient mice were immunized with OVA-MPL and challenged with 2 × 10⁵ B16-OVA cells (P < .001, SRA^−/− BM vs WT BM, ANOVA test). Results are representative of 2 separate experiments.

SRA/CD204 silencing enhances immunostimulatory capability of TLR4-activated DCs. (A) Lentivirus-mediated RNA interference efficiently down-regulates SRA/CD204 expression in DCs. BM-DCs (10⁵ cells/well) were transfected with LV-scramble shRNA, LV-SRA shRNA at a multiplicity of infection of 50, or left untreated. Cells were harvested 2 days later and analyzed by immunoblotting using anti-SRA/CD204 antibodies. (B) Silencing of SRA/CD204 in DCs by lentivirus encoding SRA shRNA, as indicated by FACs analysis. (C) SRA/CD204 silencing enhances ability of DCs to prime naive OT-I cells. BM-DC cells were transfected with LV-SRA shRNA, LV-scramble shRNA, or left untreated. DCs were harvested 2 days after infection and pulsed with OVA protein. After LPS stimulation and extensive washes, DCs were cocultured with OT-I cells at different ratios. OT-I cell proliferation was measured by ³H-thymidine incorporation assays. (D) Increased IL-2 production by OT-I cells stimulated with SRA/CD204-silenced DCs. After incubation, the levels of IL-2 in the supernatant were determined using an ELISA (*P < .005, SRA shRNA vs scramble control). (E) SRA-silenced DCs were stimulated with or without LPS, followed by coculture with OT-I cells at a ratio of 1:10. T-cell proliferation was assayed by ³H-thymidine uptake (*P < .005, SRA shRNA vs control). Representative results from 2 independent experiments are shown. Error bars indicate SE.

Enhanced proliferation of OVA-specific CD8⁺ T cells in SRA/CD204 KO mice. (A) Enhanced expansion of adoptively transferred OT-I cells in SRA^−/− mice. Mice received CFSE-labeled OT-I cells on day −1, and were immunized with OVA-MPL on day 0. Spleens were harvested at different time points and subjected to FACS analysis by gating on CD8⁺ cells. OT-I cells were also transferred to mice without immunization and used as controls. Data are representative of 2 separate experiments in which at least 3 mice in each group were analyzed. (B) Increased frequency of endogenous OVA-specific CD8⁺ T cells in SRA^−/− mice. Representative FACS dot plots of tetramer staining. WT and SRA^−/− mice (n = 3) were immunized with OVA-MPL. PBLs were collected on day 5 and stained with anti-CD8 antibodies and OVA tetramer. (C) Kinetic analysis of the frequency of OVA-specific CD8⁺ T cells. After immunization, CD8⁺OVA tetramer⁺ T cells in PBLs were assessed on days 3, 5, 7, and 14. Percentage of tetramer-positive cells gated on CD8⁺ T cells. Data shown as mean plus or minus SD are representative of 2 experiments (P < .001, SRA^−/− vs WT mice, ANOVA test). (D) Stronger recall response against OVA in SRA^−/− mice. Two months after vaccination, mice were immunized with OVA, and analyzed for CD8⁺OVA tetramer⁺ T cells in PBLs.

SRA/CD204 dampens TLR4 agonist-stimulated inflammatory response in DCs. (A) Increased SRA/CD204 expression during DC differentiation and activation. BM cell culture in the presence of GM-CSF was analyzed for SRA/CD204 expression by immunoblotting. Day 8 WT BM-DCs were treated with LPS overnight. (B) Increased SRA/CD204 expression in APCs in response to LPS stimulation in vivo. WT mice were challenged with 10 μg of LPS by tail vein injection. Splenic DCs and Mφ were isolated using magnetic beads 48 hours later, and stained for SRA/CD204 expression (broken line, isotype control; solid line, no LPS treatment; gray filled, LPS treatment). (C) Increased up-regulation of costimulatory molecules in SRA^−/− splenic DCs. After LPS challenge, splenic DCs were isolated and stained with antibodies for MHC class II and costimulatory molecules. Fold increase in mean fluorescence intensity (MFI) is shown. Data shown are representative of 3 experiments. (D) Representative histogram profiles showing higher levels of CD86 in SRA^−/− splenic DCs after LPS stimulation in vivo (open histogram, no LPS treatment; gray histogram, LPS treatment). (E) Enhanced LPS-induced gene transcription of inflammatory mediators in SRA^−/− DCs. BM-DCs were stimulated with LPS (100 ng/mL) for 4 hours or overnight. Total RNA was prepared and subjected to RT-PCR analysis using specific primers. β-Actin was used as an internal control.

SRA/CD204 suppresses immunostimulatory activity of TLR4-engaged DCs in vitro. (A) Increased proliferation of OT-I cells primed by LPS-activated SRA^−/− DCs. BM-DCs derived from WT or SRA^−/− mice were pulsed with OVA protein (10 μg/mL) for 3 hours and subsequently activated with or without LPS for 2 hours. Cells were cocultured with OT-I cells for 72 hours. OT-I cell proliferation was measured by ³H-thymidine incorporation assays (*P < .005, LPS-treated SRA^−/− DC vs LPS-treated WT DC). (B) OVA-pulsed, LPS-stimulated BM-DCs were cocultured with 5 μM CFSE-labeled OT-I cells. OT-I cell proliferation was assessed on day 3 by FACS based on the dilution of CFSE intensity. Representative histograms from 2 independent experiments are shown (P < .001, SRA^−/− vs WT). (C) Increased IL-2 production by SRA^−/− DC-stimulated OT-I cells. After incubation, the levels of IL-2 in the supernatant were determined by ELISA. (D) DCs were pulsed with OVA_257-264 (1 μg/mL) and activated with LPS. After coculturing with OT-I cells, IL-2 levels were determined using an ELISA. Data shown as mean plus or minus SD (*P < .005, SRA^−/− vs WT). (E) Enhanced immunostimulatory capability of DCs from immunized SRA/CD204 KO mice. Splenic DCs were isolated by magnetic selection from immunized WT or SRA^−/− mice, and cocultured with OT-I cells for 3 days. ³H-TdR incorporation assays were used to determine OT-I cell proliferation was assayed (*P < .01, SRA^−/− vs WT). (F) Increased Pmel cell proliferation by LPS-stimulated SRA^−/− DCs. DCs were pulsed with gp100 protein antigens (20 μg/mL), and treated with or without LPS. DCs were then cocultured with gp100-specific CD8⁺ T cells purified from Pmel transgenic mice, followed by ³H-TdR incorporation assays (*P < .005, LPS-treated SRA^−/− DC vs LPS-treated WT DC). Representative results from 2 independent experiments are shown.

Enhanced effector functions of OVA-specific CD8⁺ T cells in SRA/CD204 KO mice. (A) Increased IFN-γ production by OVA-specific CTLs from SRA^−/− mice. Animals (n = 5) were immunized with OVA alone, OVA-MPL, or left untreated. One week after booster immunization, splenocytes were restimulated with OVA_257-264 and subjected to an ELISPOT assay (*P < .01, SRA^−/− vs WT mice; error bars indicate SE). (B) Increased cytolytic activity of effector T cells from SRA^−/− mice. Splenocytes were restimulated with OVA_257-264 in the presence of IL-2 for 5 days, and chromium release assays were carried out using OVA-expressing EG7 cells as targets. OVA-negative EL4 cells were used as controls. Data shown as mean plus or minus SD are representative of 2 experiments. (C) Increased OVA-specific cytolytic activity in immunized SRA^−/− mice. Immunized WT and SRA^−/− mice (n = 3) were injected intravenously with 5 × 10⁶ OVA_257-264-pulsed, CFSE^high splenocytes mixed with nonpulsed, CFSE^low splenocytes at 1:1 ratio. Splenocytes and lymph node (LN) cells were analyzed 12 hours later by FACS. Representative histogram profiles from 2 experiments are shown. (D) Antibody response to OVA antigen not affected by SRA/CD204 deficiency. WT and SRA^−/− mice (n = 5) were immunized with OVA-MPL or left untreated. Sera were collected 7 days after booster immunization and analyzed for IgG levels against OVA by ELISAs (*P > .5, WT vs SRA^−/−). (E) Intrinsic T-cell function not altered by SRA/CD204 deficiency. Naive T cells (> 98% purity) were stimulated with 10 μg/mL anti-CD3, anti-CD28 mAb in precoated wells, or PHA (1 μg/mL) at 37°C for 72 hours. Cell proliferation was determined by measuring thymidine incorporation (counts per minute) using a scintillation counter (*P > .5, WT vs SRA^−/−). (F) Similar in vivo proliferation of T cells from WT and SRA^−/− mice. CFSE-labeled CD3⁺ T cells from WT or SRA^−/− mice were adoptively transferred to RAG-1 mice (n = 3). The recipient mice were then immunized with OVA-MPL. CFSE intensity was measured 5 days later using FACS by gating on CD3- and CFSE-positive cells (P > .5, WT vs SRA^−/−).