Suppression of antigen-specific CD4+ T cell activation by SRA/CD204 through reducing the immunostimulatory capability of antigen-presenting cell

Abstract

Pattern recognition scavenger receptor SRA/CD204, primarily expressed on specialized antigen-presenting cells (APCs), including dendritic cells (DCs) and macrophages, has been implicated in multiple physiological and pathological processes, including atherosclerosis, Alzheimer's disease, endotoxic shock, host defense, and cancer development. SRA/CD204 was also recently shown to function as an attenuator of vaccine response and antitumor immunity. Here, we, for the first time, report that SRA/CD204 knockout (SRA(-/-)) mice developed a more robust CD4(+) T cell response than wild-type mice after ovalbumin immunization. Splenic DCs from the immunized SRA(-/-) mice were much more efficient than those from WT mice in stimulating naïve OT-II cells, indicating that the suppressive activity of SRA/CD204 is mediated by DCs. Strikingly, antigen-exposed SRA(-/-) DCs with or without lipopolysaccharide treatment exhibited increased T-cell-stimulating activity in vitro, which was independent of the classical endocytic property of the SRA/CD204. Additionally, absence of SRA/CD204 resulted in significantly elevated IL12p35 expression in DCs upon CD40 ligation plus interferon gamma (IFN-γ) stimulation. Molecular studies reveal that SRA/CD204 inhibited the activation of STAT1, mitogen activated protein kinase p38, and nuclear factor-kappa B signaling activation in DCs treated with anti-CD40 antibodies and IFN-γ. Furthermore, splenocytes from the generated SRA(-/-) OT-II mice showed heightened proliferation upon stimulation with OVA protein or MHC-II-restricted OVA(323-339) peptide compared with cells from the SRA(+/+) OT-II mice. These results not only establish a new role of SRA/CD204 in limiting the intrinsic immunogenicity of APCs and CD4(+) T cell activation but also provide additional insights into the molecular mechanisms involved in the immune suppression by this molecule.

Figure 1. Lack of SRA/CD204 potentiates an OVA-specific CD4⁺ T cell response by enhancing T cell-stimulating functions of DCs

A and B. SRA/CD204 suppresses OVA-MPL induced expansion of adoptively transferred OT-II cells. Mice (n=5) received CFSE-labeled OT-II cells, followed by OVA-MPL immunization. Draining lymph nodes (A) and spleen (B) were analyzed using FACS by gating on CD4⁺CSFE⁺ cells. Representative histograms from two separate experiments with similar results are shown (*p<0.01). C. Increased IFN-γ production by OVA-specific CD4⁺ T cells from SRA^−/− mice. Mice (n=5) were immunized with OVA-MPL or left untreated. Splenocytes were stimulated with OVA_323-339 and assayed for IFN-γ secretion using ELISPOT (*p<0.01). D. Increased T cell-stimulating activity of DCs from immunized SRA^−/− mice. Splenic CD11c⁺ DCs were magnetically isolated from the immunized WT or SRA^−/− mice and used to stimulate OT-II cells. Cell proliferation was measured using ³H-TdR uptake assays (*p<0.01). Data are means ± SE. Results representative of three independent experiments are shown.

Figure 2. OVA-specific CD4⁺ T cells from OVA-MPL immunized SRA^−/− mice produced more Th1 cytokine IFN-γ

A and B. WT and SRA^−/− mice (n=5) were immunized with OVA-MPL twice. Splenocytes were stimulated with OVA_323-339 and subjected to intracellular cytokine staining assays. Representative histograms from three independent experiments are shown. B. The effect of SRA/CD204 on the percentage of OVA-specific CD4⁺IFN-γ⁺ T cells (left) and CD4⁺IL-4⁺ T cells (right) before and after vaccination are also shown (**p<0.005, NS, not significant). C. Supernatants from OVA_323-339-stimulated cells were collected and assayed for the levels of IFN-γ (left) and IL-4 (right) by ELISA (**p<0.005). Data are means ± SE and represent three independent experiments.

Figure 3. SRA/CD204 deficiency does not affect Tregs and their suppressive activity

A and B. Following OVA-MPL immunization (n=3), Tregs in the spleen were stained with CD4 and Foxp3 antibodies. Representative histograms from two experiments with similar results are shown (A). The percentage of CD4⁺Foxp3⁺ and CD4⁺Foxp3⁻ cells in total splenocytes, as well as the percentage of CD4⁺Foxp3⁺ in total CD4⁺ cells are also analyzed (B). C. CD4⁺CD25⁺ Treg cells were isolated from control or OVA-MPL immunized mice, and co-cultured with OT-II cells. Proliferation of OT-II cells in response to OVA_323-339 stimulation was measured using ³H-TdR uptake assays. D. CD4⁺CD25⁻ cells and CD4⁺CD25⁺ Tregs were isolated from OVA-MPL immunized mice, co-cultured at different ratios as indicated. Proliferation of CD4⁺CD25⁻ effector T cells upon stimulation of CD3/CD28 mAb was examined using ³H-TdR uptake assays (top). IFN-γ (middle) and IL-2 (bottom) levels in the culture supernatants were measured by ELISA. Results representative of two independent experiments with similar results are shown.

Figure 4. SRA/CD204 suppresses the ability of DCs to stimulate OVA-specific CD4⁺ T cells in vitro in the presence or absence of LPS treatment

A and B. Immunogenicity of LPS-stimulated SRA^−/− DCs following OVA protein loading. BM-DCs were pulsed with OVA protein (10 μg/ml) and treated with LPS. DCs were then cultured with OT-II cells at different ratios for 72 h. Cell proliferation was measured using ³H-TdR uptake assays (A). Supernatants were collected after 48 h and assayed for IL-2 using ELISA (B). *p<0.01. Data are means ± SE. Results representative of three independent experiments are shown. C and D. Effects of SRA/CD204 on DC-mediated T cell stimulation by other TLR ligands. BM-DCs were pulsed with OVA protein and stimulated with PAM₃CSK₄, poly (I:C), LPS, ssRNA40 or CpG1826. DCs were then cultured with OT-II cells at a ratio of 1:20 for 72 h. Cell proliferation (C) and IL-2 production (D) were assayed. *p<0.01. Data are shown as means ± SE and represent the results from two independent experiments with similar results. E and F. Immunostimulatory activity of SRA^−/− DCs without LPS stimulation. DCs were pulsed with different concentrations of OVA protein for 3 h, and co-cultured with OT-II at 1:10 ratio for 72 h. Cell proliferation and IL-2 production were analyzed using ³H-TdR uptake assays (E) and ELISA (F), respectively. **p<0.005. Data are shown as means ± SE and represent the results from two independent experiments.

Figure 5. SRA/CD204 reduces the ability of DQ-OVA protein-pulsed DCs to activate CD4⁺ T cells

A and B. Modest reduction in binding and uptake of DQ-OVA in the absence of SRA/CD204. WT or SRA^−/− BM-DCs were incubated with DQ-OVA (20 μg/ml) at 4 °C for 30 min. Cells were washed and incubated at 37 °C for indicated times. Cells were examined for the levels of DQ-OVA by immunoblotting (A). B. Following incubation with DQ-OVA at 4 °C, cells were washed and culture at 37 °C for 1 h. FACS analysis was used to examine the fluorescence in WT and SRA^−/− DCs. Representative histograms of three separate experiments with similar results are shown. C, D and E. Increased T cell-stimulating capability of SRA^−/− DCs. BM-DCs were incubated with DQ-OVA at 37 °C for 3 h. After washing, cells were cultured with OT-II cells at a ratio of 1:5. T cell proliferation was examined based on ³H-TdR uptake (C). Supernatants were assayed for IFN-γ (D) and IL-2 (E) using ELISA. **p<0.005. Data are shown as means ± SE and represent the results from two independent experiments.

Figure 6. SRA/CD204 limits the ability of OVA_323-339 peptide-pulsed DCs to activate CD4⁺ T cells

A. WT and SRA^−/− BM-DCs were pulsed with different concentrations of OVA_323-339 peptide for 3 h. Cell proliferation was measured after culture with OT-II cells at a ratio of 1:10. *p<0.01. B. BM-DCs pulsed with OVA_323-339 peptide (0.1 μg/ml) were cultured with OT-II cells at different ratios, followed by cell proliferation assays. *p<0.01. C. Day 7 BM-DCs were treated with IFN-γ or IFN-γ plus anti-CD40 mAbs for 24 h. IL12p35 mRNA expression was analyzed using quantitative real-time RT-PCR. **p<0.005. Gene expression was normalized relative to the expression of β-actin. Data are shown as means ± SE and represent the results from two independent experiments with similar results. D. BM-DCs were treated with anti-CD40 mAbs or anti-CD40 Abs plus IFN-γ for indicated times. Phosphorylation of STAT1 was examined using immunoblotting. E. SRA/CD204 suppresses activation of STAT1, p38 and NF-κB. WT and SRA^−/− DCs were treated with anti-CD40 mAbs plus IFN-γ. Cell lysates were prepared and analyzed for phosphorylation of STAT1, p38 and NF-κB p65 using immunoblotting. β-actin serves as a loading control.

Figure 7. Splenocytes from the SRA^−/− OT-II mice display increased proliferation upon OVA stimulation

A. PCR genotyping analysis of the SRA^−/− OT-II transgenic mice. The band of 154bp indicates WT allele in WT SRA^+/+ mouse or heterozygous SRA^+/− mouse. The band of 202bp indicates the mutant allele. B. Expression of SRA/CD204 on splenocytes from SRA^+/+ OT-II (dotted line) and SRA^−/− OT-II (solid line) transgenic mice were examined using FITC-labeled anti-SRA/CD204 antibodies and FACS analysis. Shaded area represents the IgG isotype control. C. Immunoblotting analysis of SRA/CD204 protein expression using splenocytes from the SRA^+/+ OT-II and SRA^−/− OT-II mice. D. SRA/CD204 absence does not affect the development of OT-II cells. Cells prepared from lymph nodes and spleen of the SRA^+/+ OT-II and SRA^−/− OT-II mice were stained for TCR-Vα2 and CD4 expression and analyzed by FACS. The numbers in the gates represent the percentage of CD4⁺ OT-II T cells. E and F. 2×10⁵ splenocytes from the SRA^+/+ OT-II or SRA^−/− OT-II mice were incubated with different concentrations of OVA protein (E) or OVA_323-339 peptide (F) as indicated in the absence of LPS. Cell proliferation was examined using ³H-TdR uptake assays (*p<0.01). Data are presented as means ± SE. Results representative of two independent experiments are shown.