Huanfa Yi

Mouse CD11b+Gr-1+ Myeloid Cells Can Promote Th17 Cell Differentiation and Experimental Autoimmune Encephalomyelitis

Myeloid-derived suppressor cells (MDSCs) have been a focus of recent study on tumor-mediated immune suppression. However, its role in Th17 cell differentiation and the pathogenesis of autoimmune diseases (e.g., multiple sclerosis) has not been determined. We show in this study that development of experimental autoimmune encephalomyelitis (EAE) in mice is associated with a profound expansion of CD11b+Gr-1+ MDSCs, which display efficient T cell inhibitory functions in vitro. Unexpectedly, these MDSCs enhance the differentiation of naive CD4+ T cell precursors into Th17 cells in a highly efficient manner under Th17-polarizing conditions, as indicated by significantly increased number of Th17 cells, elevation of IL-17A production, and upregulation of the orphan nuclear receptor RORA and RORC. Mechanistic studies show that IL-1β represents a major mediator of MDSC-facilitated Th17 differentiation, which depends on the IL-1 receptor on CD4+ T cells but not MDSCs. Selective depletion of MDSCs using gemcitabine results in a marked reduction in the severity of EAE (e.g., decreased clinical scores and myelin injury), which correlates with reduced Th17 cells and inflammatory cytokines (IL-17A and IL-1β) in the lymphoid tissues and spinal cord. Adoptive transfer of MDSCs after gemcitabine treatment restores EAE disease progression. Together, we demonstrate for the first time, to our knowledge, that excessive and prolonged presence of MDSCs can drive a Th17 response and consequently contributes to the pathogenesis of EAE. These new findings provide unique insights into the pleiotropic functions of MDSCs and may help explain the failure of immunosuppressive MDSCs to control Th17/IL-17–dependent autoimmune disorders.

Pattern Recognition Scavenger Receptor CD204 Attenuates Toll-like Receptor 4-induced NF-κB Activation by Directly Inhibiting Ubiquitination of Tumor Necrosis Factor (TNF) Receptor-associated Factor 6

The collaboration and cross-talk between different classes of innate pattern recognition receptors are crucial for a well coordinated inflammatory response and host defense. Here we report a previously unrecognized role of scavenger receptor A (SRA; also known as CD204) as a signaling regulator in the context of Toll-like receptor 4 (TLR4) activation. We show that SRA/CD204 deficiency leads to greater sensitivity to LPS-induced endotoxic shock. SRA/CD204 down-regulates inflammatory gene expression in dendritic cells by suppressing TLR4-induced activation of the transcription factor NF-κB. For the first time, we demonstrate that SRA/CD204 executes its regulatory functions by directly interacting with the TRAF-C domain of TNF receptor-associated factor 6 (TRAF6), resulting in inhibition of TRAF6 dimerization and ubiquitination. The attenuation of NF-κB activity by SRA/CD204 is independent of its ligand-binding domain, indicating that the signaling-regulatory feature of SRA/CD204 can be uncoupled from its conventional endocytic functions. Collectively, we have identified the molecular linkage between SRA/CD204 and the TLR4 signaling pathways, and our results reveal a novel mechanism by which a non-TLR pattern recognition receptor restricts TLR4 activation and consequent inflammatory response.

Pattern recognition scavenger receptor SRA/CD204 down-regulates Toll-like receptor 4 signaling–dependent CD8 T-cell activation

Class A scavenger receptor (SRA), also known as CD204, has been shown to participate in the pathogenesis of atherosclerosis and the pattern recognition of pathogen infection. However, its role in adaptive immune responses has not been well defined. In this study, we report that the lack of SRA/CD204 promotes Toll-like receptor (TLR)4 agonist-augmented tumor-protective immunity, which is associated with enhanced activation of CD8(+) effector T cell and improved inhibition of tumor growth. Dendritic cells (DCs) deficient in SRA/CD204 display more effective immunostimulatory activities upon TLR4 engagement than those from wild-type counterparts. Silencing of SRA/CD204 by RNA interference improves the ability of DCs to prime antigen-specific CD8(+) T cells, suggesting that antigen-presenting cells, for example, DCs, play a major role in SRA/CD204-mediated immune modulation. Our findings reveal a previously unrecognized role for SRA/CD204, a non-TLR pattern recognition receptor, as a physiologic negative regulator of TLR4-mediated immune consequences, which has important clinical implications for development of TLR-targeted immunotherapeutic intervention.

ER stress and its regulator X-box binding protein-1 enhance polyIC induced innate immune response in dendritic cells

Multiple physiological and pathological conditions interfere with the function of the endoplasmic reticulum (ER). However, much remains unknown regarding the impact of ER stress on inflammatory responses in dentritic cells (DCs) upon the recognition of pathogen molecules. We show that ER stress greatly potentiates the expression of inflammatory cytokines and IFN‐β in murine DCs stimulated by polyIC, a synthetic mimic of virus dsRNA. Both toll‐like receptor 3 and melanoma differentiation‐associated gene‐5 are involved in the enhanced IFN‐β production, which is associated with increased activation of NF‐κB and IRF3 signaling as well as the splicing of X‐box‐binding protein‐1 (XBP‐1), an important regulator involved in ER stress response. Surprisingly, silencing of XBP‐1 reduces polyIC‐stimulated IFN‐β expression in the presence or absence of ER stress, indicating that XBP‐1 may be essential for polyIC signaling and ER stress‐amplified IFN‐β production. Overexpression of a spliced form of XBP‐1 (XBP‐1s) synergistically augments polyIC‐induced inflammatory response. For the first time, we show that XBP‐1s overexpression‐enhanced IFN‐β production in DCs markedly suppresses vesicular stomatitis virus infection, revealing a previously unrecognized role for XBP‐1 in an antiviral response. Our findings suggest that evolutionarily conserved ER stress response and XBP‐1 may function collaboratively with innate immunity to maintain cellular homeostasis.

Myeloid-derived suppressor cells have a proinflammatory role in the pathogenesis of autoimmune arthritis

Objectives Although myeloid-derived suppressor cells (MDSCs) have been linked to T cell tolerance, their role in autoimmune rheumatoid arthritis (RA) remains elusive. Here we investigate the potential association of MDSCs with the disease pathogenesis using a preclinical model of RA and specimen collected from patients with RA. Methods The frequency of MDSCs in blood, lymphoid tissues, inflamed paws or synovial fluid and their association with disease severity, tissue inflammation and the levels of pathogenic T helper (Th) 17 cells were examined in arthritic mice or in patients with RA (n=35) and osteoarthritis (n=15). The MDSCs in arthritic mice were also characterised for their phenotype, inflammation status, T cell suppressive activity and their capacity of pro-Th17 cell differentiation. The involvement of MDSCs in the disease pathology and a Th17 response was examined by adoptive transfer or antibody depletion of MDSCs in arthritic mice or by coculturing mouse or human MDSCs with naïve CD4+ T cells under Th17-polarising conditions. Results MDSCs significantly expanded in arthritic mice and in patients with RA, which correlated positively with disease severity and an inflammatory Th17 response. While displaying T cell suppressive activity, MDSCs from arthritic mice produced high levels of inflammatory cytokines (eg, interleukin (IL)-1β, TNF-α). Mouse and human MDSCs promoted Th17 cell polarisation ex vivo. Transfer of MDSCs facilitated disease progression, whereas their elimination in arthritic mice ameliorated disease symptoms concomitant with reduction of IL-17A/Th17 cells. Conclusions Our studies suggest that proinflammatory MDSCs with their capacity to drive Th17 cell differentiation may be a critical pathogenic factor in autoimmune arthritis.

Absence of scavenger receptor A promotes dendritic cell-mediated cross-presentation of cell-associated antigen and antitumor immune response

Given the primary expression of scavenger receptor A (SRA) or CD204 on antigen‐presenting cells, we investigate the immunoregulatory activities of SRA/CD204 in the context of cross‐presentation of cell‐associated antigen and the immunogenicity of dying tumor cells. Immunization with dying prostate cancer cells results in profoundly increased control of subsequently inoculated tumors in SRA/CD204 knockout mice. Using OVA‐expressing RM1 prostate tumor line (RM1‐OVA), we show for the first time that SRA absence greatly enhances dendritic cells (DCs)‐mediated cross‐presentation of OVA antigen derived from dying RM1 cells. While the phagocytic ability of DCs is not significantly impacted by the lack of SRA/CD204, DCs deficient in SRA/CD204 display increased expression of inflammatory cytokines and chemokines, as well as co‐stimulatory molecules upon interaction with dying RM1 cells, implicating a suppressive regulation of the functional activation of DCs by SRA/CD204. Further, SRA/CD204‐deficient DCs pulsed with dying RM1‐OVA cells are more effective than wild‐type counterparts in priming antigen‐specific T‐cell responses, resulting in improved control of RM1 tumor growth in both prophylactic and therapeutic settings. Our findings suggest that the increased immunogenicity of dying tumor cells in SRA/CD204 knockout mice is attributed to the altered functions of DCs in the absence of SRA/CD204, which underscores the important role of SRA/CD204 in host immune homeostasis. Selective downregulation or blockade of this immunoregulatory molecule may lead to enhanced potency of DC‐based vaccines capable of breaking immune tolerance against cancer.

CD204 Suppresses Large Heat Shock Protein-Facilitated Priming of Tumor Antigen gp100-Specific T Cells and Chaperone Vaccine Activity against Mouse Melanoma

We previously reported that scavenger receptor A (SRA/CD204), a binding structure on dendritic cells (DCs) for large stress/heat shock proteins (HSPs; e.g., hsp110 and grp170), attenuated an antitumor response elicited by large HSP-based vaccines. In this study, we show that SRA/CD204 interacts directly with exogenous hsp110, and lack of SRA/CD204 results in a reduction in the hsp110 binding and internalization by DCs. However, SRA−/− DCs pulsed with hsp110 or grp170-reconstituted gp100 chaperone complexes exhibit a profoundly increased capability of stimulating melanoma Ag gp100-specific naive T cells compared with wild-type (WT) DCs. Similar results were obtained when SRA/CD204 was silenced in DCs using short hairpin RNA-encoding lentiviruses. In addition, hsp110-stimulated SRA−/− DCs produced more inflammatory cytokines associated with increased NF-κB activation, implicating an immunosuppressive role for SRA/CD204. Immunization with the hsp110-gp100 vaccine resulted in a more robust gp100-specific CD8+ T cell response in SRA−/− mice than in WT mice. Lastly, SRA/CD204 absence markedly improved the therapeutic efficacy of the hsp110-gp100 vaccine in mice established with B16 melanoma, which was accompanied by enhanced activation and tumor infiltration of CD8+ T cells. Given the presence of multiple HSP-binding scavenger receptors on APCs, we propose that selective scavenger receptor interactions with HSPs may lead to highly distinct immunological consequences. Our findings provide new insights into the immune regulatory functions of SRA/CD204 and have important implications in the rational design of protein Ag-targeted recombinant chaperone vaccines for the treatment of cancer.

Targeting the immunoregulator SRA/CD204 potentiates specific dendritic cell vaccine-induced T cell response and antitumor immunity

Although dendritic cell (DC) vaccines offer promise as cancer immunotherapy, further improvements are needed to amplify their clinical therapeutic efficacy. The pattern recognition scavenger receptor SRA/CD204 attenuates the ability of DCs to activate CD8(+) T-cell responses. Therefore, we examined the impact of SRA/CD204 on antitumor responses generated by DC vaccines and we also evaluated the feasibility of enhancing DC vaccine potency by SRA/CD204 blockade. DCs from SRA/CD204-deficient mice were more immunogenic in generating antitumor responses to B16 melanoma, compared with DCs from wild-type mice. Similarly, siRNA-mediated knockdown of SRA/CD204 by lentiviral vectors improved the ability of wild-type DCs to stimulate the expansion and activation of CD8(+) T cells specific for idealized or established melanoma antigens in mice. Using SRA/CD204-silenced DCs to generate antigen-targeted vaccines, we documented a marked increase in the level of antitumor immunity achieved against established B16 tumors and metastases. This increase was associated with enhanced activation of antigen specific CTLs, greater tumor infiltration by CD8(+) T cells and NK cells, and increased intratumoral ratios of both CD4(+) and CD8(+) T-effector cells to CD4(+)CD25(+) T-regulatory cells. Our studies establish that downregulating SRA/CD204 strongly enhances DC-mediated antitumor immunity. In addition, they provide a rationale to enhance DC vaccine potency through SRA/CD204-targeting approaches that can improve clinical outcomes in cancer treatment.

Scavenger receptor a restrains T‐cell activation and protects against concanavalin A‐induced hepatic injury

Negative feedback immune mechanisms are essential for maintenance of hepatic homeostasis and prevention of immune‐mediated liver injury. We show here that scavenger receptor A (SRA/CD204), a pattern recognition molecule, is highly up‐regulated in the livers of patients with autoimmune or viral hepatitis, and of mice during concanavalin A (Con A)‐induced hepatitis (CIH). Strikingly, genetic SRA ablation strongly sensitizes mice to Con A‐induced liver injury. SRA loss, increased mortality and liver pathology correlate with excessive production of IFN‐γ and heightened activation of T cells. Increased liver expression of SRA primarily occurs in mobilized hepatic myeloid cells during CIH, including CD11b+Gr‐1+ cells. Mechanistic studies establish that SRA on these cells functions as a negative regulator limiting T‐cell activity and cytokine production. SRA‐mediated protection from CIH is further validated by adoptive transfer of SRA+ hepatic mononuclear cells or administration of a lentivirus‐expressing SRA, which effectively ameliorates Con A‐induced hepatic injury. Also, CIH and clinical hepatitis are associated with increased levels of soluble SRA. This soluble SRA displays a direct T‐cell inhibitory effect and is capable of mitigating Con A‐induced liver pathology. Conclusion: Our findings demonstrate an unexpected role of SRA in attenuation of Con A‐induced, T‐cell‐mediated hepatic injury. We propose that SRA serves as an important negative feedback mechanism in liver immune homeostasis, and may be exploited for therapeutic treatment of inflammatory liver diseases. (HEPATOLOGY 2013)

In situ vaccination with CD204 gene-silenced dendritic cell, not unmodified dendritic cell, enhances radiation therapy of prostate cancer

Given the complexity of prostate cancer progression and metastasis, multimodalities that target different aspects of tumor biology, for example, radiotherapy in conjunction with immunotherapy, may provide the best opportunities for promoting clinical benefits in patients with high-risk localized prostate cancer. Here, we show that intratumoral administration of unmodified dendritic cells (DC) failed to synergize with fractionated radiotherapy. However, ionizing radiation combined with in situ vaccination with DCs, in which the immunosuppressive scavenger receptor A (SRA/CD204) has been downregulated by lentivirus-mediated gene silencing, profoundly suppressed the growth of two mouse prostate cancers (e.g., RM1 and TRAMP-C2) and prolonged the lifespan of tumor-bearing animals. Treatment of subcutaneous tumors with this novel combinatorial radioimmunotherapeutic regimen resulted in a significant reduction in distant experimental metastases. SRA/CD204-silenced DCs were highly efficient in generating antigen or tumor-specific T cells with increased effector functions (e.g., cytokine production and tumoricidal activity). SRA/CD204 silencing-enhanced tumor cell death was associated with elevated IFN-γ levels in tumor tissue and increased tumor-infiltrating CD8+ cells. IFN-γ neutralization or depletion of CD8+ cells abrogated the SRA/CD204 downregulation-promoted antitumor efficacy, indicating a critical role of IFN-γ–producing CD8+ T cells. Therefore, blocking SRA/CD204 activity significantly enhances the therapeutic potency of local radiotherapy combined with in situ DC vaccination by promoting a robust systemic antitumor immunity. Further studies are warranted to test this novel combinatorial approach for translating into improved clinical outcomes in patients with prostate cancer. Mol Cancer Ther; 11(11); 2331–41. ©2012 AACR.