Distinct mechanisms govern populations of myeloid-derived suppressor cells in chronic viral infection and cancer.

Myeloid-derived suppressor cells (MDSCs) are major negative regulators of immune responses in cancer and chronic infections. It remains unclear if regulation of MDSC activity in different conditions is controlled by similar mechanisms. We compared MDSCs in mice with cancer and lymphocytic choriomeningitis virus (LCMV) infection. Chronic LCMV infection caused the development of monocytic MDSCs (M-MDSCs) but did not induce polymorphonuclear MDSCs (PMN-MDSCs). In contrast, both MDSC populations were present in cancer models. An acquisition of immune-suppressive activity by PMN-MDSCs in cancer was controlled by IRE1α and ATF6 pathways of the endoplasmic reticulum (ER) stress response. Abrogation of PMN-MDSC activity by blockade of the ER stress response resulted in an increase in tumor-specific immune response and reduced tumor progression. In contrast, the ER stress response was dispensable for suppressive activity of M-MDSCs in cancer and LCMV infection. Acquisition of immune-suppressive activity by M-MDSCs in spleens was mediated by IFN-γ signaling. However, it was dispensable for suppressive activity of M-MDSCs in tumor tissues. Suppressive activity of M-MDSCs in tumors was retained due to the effect of IL-6 present at high concentrations in the tumor site. These results demonstrate disease- and population-specific mechanisms of MDSC accumulation and the need for targeting different pathways to achieve inactivation of these cells.

Central Roles of OX40L-OX40 Interaction in the Induction and Progression of Human T Cell-Driven Acute Graft-versus-Host Disease.

Graft-versus-host disease (GVHD) is one of the major obstacles for the success of allogeneic hematopoietic stem cell transplantation. Here, we report that the interaction between OX40L and OX40 is of critical importance for both induction and progression of acute GVHD (aGVHD) driven by human T cells. Anti-human OX40L monoclonal antibody (hOX40L) treatment could thus effectively reduce the disease severity in a xenogeneic-aGVHD (x-aGVHD) model in both preventative and therapeutic modes. Mechanistically, blocking OX40L-OX40 interaction with an anti-hOX40L antibody reduces infiltration of human T cells in target organs, including liver, gut, lung, and skin. It also decreases IL-21- and TNF-producing T cell responses, while promoting regulatory T cell (Treg) responses without compromising the cytolytic activity of CD8+ T cells. Single blockade of hOX40L was thus more effective than dual blockade of IL-21 and TNF in reducing the severity of aGVHD as well as mortality. Data from this study indicate that OX40L-OX40 interactions play a central role in the pathogenesis of aGVHD induced by human T cells. Therapeutic strategies that can efficiently interrupt OX40L-OX40 interaction in patients might have potential to provide patients with an improved clinical benefit.

Galectin-9 inhibits TLR7-mediated autoimmunity in murine lupus models.

Uncontrolled secretion of type I IFN, as the result of endosomal TLR (i.e., TLR7 and TLR9) signaling in plasmacytoid DCs (pDCs), and abnormal production of autoantibodies by B cells are critical for systemic lupus erythematosus (SLE) pathogenesis. The importance of galectin-9 (Gal-9) in regulating various autoimmune diseases, including lupus, has been demonstrated. However, the precise mechanism by which Gal-9 mediates this effect remains unclear. Here, using spontaneous murine models of lupus (i.e., BXSB/MpJ and NZB/W F1 mice), we demonstrate that administration of Gal-9 results in reduced TLR7-mediated autoimmune manifestations. While investigating the mechanism underlying this phenomenon, we observed that Gal-9 inhibits the phenotypic maturation of pDCs and B cells and abrogates their ability to mount cytokine responses to TLR7/TLR9 ligands. Importantly, immunocomplex-mediated (IC-mediated) and neutrophil extracellular trap-mediated (NET-mediated) pDC activation was inhibited by Gal-9. Additionally, the mTOR/p70S6K pathway, which is recruited by both pDCs and B cells for TLR-mediated IFN secretion and autoantibody generation, respectively, was attenuated. Gal-9 was found to exert its inhibitory effect on both the cells by interacting with CD44.

Potent Immune Modulation by MEDI6383, an Engineered Human OX40 Ligand IgG4P Fc Fusion Protein.

Ligation of OX40 (CD134, TNFRSF4) on activated T cells by its natural ligand (OX40L, CD252, TNFSF4) enhances cellular survival, proliferation, and effector functions such as cytokine release and cellular cytotoxicity. We engineered a recombinant human OX40L IgG4P Fc fusion protein termed MEDI6383 that assembles into a hexameric structure and exerts potent agonist activity following engagement of OX40. MEDI6383 displayed solution-phase agonist activity that was enhanced when the fusion protein was clustered by Fc gamma receptors (FcγRs) on the surface of adjacent cells. The resulting costimulation of OX40 on T cells induced NFκB promoter activity in OX40-expressing T cells and induced Th1-type cytokine production, proliferation, and resistance to regulatory T cell (Treg)-mediated suppression. MEDI6383 enhanced the cytolytic activity of tumor-reactive T cells and reduced tumor growth in the context of an alloreactive human T cell:tumor cell admix model in immunocompromised mice. Consistent with the role of OX40 costimulation in the expansion of memory T cells, MEDI6383 administered to healthy nonhuman primates elicited peripheral blood CD4 and CD8 central and effector memory T-cell proliferation as well as B-cell proliferation. Together, these results suggest that OX40 agonism has the potential to enhance antitumor immunity in human malignancies. Mol Cancer Ther; 17(5); 1024-38. ©2018 AACR.

NFATC3 promotes IRF7 transcriptional activity in plasmacy--toid dendritic cells.

Plasmacytoid dendritic cells (pDCs) rapidly produce large amounts of type 1 interferon (IFN) after Toll-like receptor 7 and 9 engagements. This specialized function of type 1 IFN production is directly linked to the constitutive expression of IRF7, the master transcription factor for type 1 IFN production. However, the IRF7 regulatory network in pDCs remains largely unknown. In this study, we identify that the transcription factor NFATC3 specifically binds to IRF7 and enhances IRF7-mediated IFN production. Furthermore, knockout of NFATC3 greatly reduced the CpG DNA-induced nuclear translocation of IRF7, which resulted in impaired type 1 IFN production in vitro and in vivo. In addition, we found that NFATC3 and IRF7 both bound to type 1 IFN promoters and that the NFAT binding site in IFN promoters was required for IRF7-mediated IFN expression. Collectively, our study shows that the transcription factor NFATC3 binds to IRF7 and functions synergistically to enhance IRF7-mediated IFN expression in pDCs.

Human NLRP3 inflammasome senses multiple types of bacterial RNAs.

Inflammasomes are multiprotein platforms that activate caspase-1, which leads to the processing and secretion of the proinflammatory cytokines IL-1ß and IL-18. Previous studies demonstrated that bacterial RNAs activate the nucleotide-binding domain, leucine-rich-repeat-containing family, pyrin domain-containing 3 (NLRP3) inflammasome in both human and murine macrophages. Interestingly, only mRNA, but neither tRNA nor rRNAs, derived from bacteria could activate the murine Nlrp3 inflammasome. Here, we report that all three types of bacterially derived RNA (mRNA, tRNA, and rRNAs) were capable of activating the NLRP3 inflammasome in human macrophages. Bacterial RNA's 5'-end triphosphate moieties, secondary structure, and double-stranded structure were dispensable; small fragments of bacterial RNA were sufficient to activate the inflammasome. In addition, we also found that 20-guanosine ssRNA can activate the NLRP3 inflammasome in human macrophages but not in murine macrophages. Therefore, human and murine macrophages may have evolved to recognize bacterial cytosolic RNA differently during bacterial infections.

Helicase proteins DHX29 and RIG-I cosense cytosolic nucleic acids in the human airway system.

The recognition of cytoplasmic nucleic acid is critical for innate immune responses against microbial infection and is responsible for autoimmunity induced by dead cells. Here, we report the identification of a unique cytosolic nucleic acid cosensor in human airway epithelial cells and fibroblasts: DEAH (Asp-Glu-Ala-His) box polypeptide 29 (DHX29), a member of the DExD/H (Asp-Glu-x-Asp/His)-box helicase family. Knocking down DHX29 by siRNA attenuated the ability of cells to mount type I IFN and IL-6 in response to cytosolic nucleic acids and various viruses by blocking the activation of interferon regulatory factor 3 and NF-κB-p65. The cytosolic nucleic acid sensing by DHX29 in human epithelial cells and fibroblasts is independent of stimulator of interferon genes but is dependent on retinoic acid-inducible gene 1 (RIG-I) and mitochondrial antiviral signaling protein (MAVS). DHX29 binds directly to nucleic acids and interacts with RIG-I and MAVS through its helicase 1 domain, activating the RIG-I-MAVS-dependent cytosolic nucleic acid response. These results suggest that DHX29 is a cytosolic nucleic acid cosensor that triggers RIG-I/MAVS-dependent signaling pathways. This study will have important implications in drug and vaccine design for control of viral infections and viral-induced pathology in the airway.

Proatherogenic conditions promote autoimmune T helper 17 cell responses in vivo.

Patients with systemic autoimmune diseases show increased incidence of atherosclerosis. However, the contribution of proatherogenic factors to autoimmunity remains unclear. We found that atherogenic mice (herein referred to as LDb mice) exhibited increased serum interleukin-17, which was associated with increased numbers of T helper 17 (Th17) cells in secondary lymphoid organs. The environment within LDb mice was substantially favorable for Th17 cell polarization of autoreactive T cells during homeostatic proliferation, which was considerably inhibited by antibodies directed against oxidized low-density lipoprotein (oxLDL). Moreover, the uptake of oxLDL induced dendritic-cell-mediated Th17 cell polarization by triggering IL-6 production in a process dependent on TLR4, CD36, and MyD88. Furthermore, self-reactive CD4(+) T cells that expanded in the presence of oxLDL induced more profound experimental autoimmune encephalomyelitis. These findings demonstrate that proatherogenic factors promote the polarization and inflammatory function of autoimmune Th17 cells, which could be critical for the pathogenesis of atherosclerosis and other related autoimmune diseases.

The DHX33 RNA helicase senses cytosolic RNA and activates the NLRP3 inflammasome.

The NLRP3 inflammasome plays a major role in innate immune responses by activating caspase-1, resulting in secretion of interleukin-18 (IL-18) and IL-1ß. Although cytosolic double-stranded RNA (dsRNA) and bacterial RNA are known to activate the NLRP3 inflammasome, the upstream sensor is unknown. We investigated the potential function of DExD/H-box RNA helicase family members (previously shown to sense cytosolic DNA and RNA to induce type 1 interferon responses) in RNA-induced NLRP3 inflammasome activation. Among the helicase family members tested, we found that targeting of DHX33 expression by short hairpin RNA efficiently blocked the activation of caspase-1 and secretion of IL-18 and IL-1ß in human macrophages that were activated by cytosolic poly I:C, reoviral RNA, or bacterial RNA. DHX33 bound dsRNA via the helicase C domain. DHX33 interacted with NLRP3 and formed the inflammasome complex following stimulation with RNA. We therefore identified DHX33 as a cytosolic RNA sensor that activates the NLRP3 inflammasome.

Plasmacytoid dendritic cells promote immunosuppression in ovarian cancer via ICOS costimulation of Foxp3(+) T-regulatory cells.

Epithelial ovarian cancer (EOC) is the fifth most common cause of cancer death among women. Despite its immunogenicity, effective antitumor responses are limited, due, in part, to the presence of forkhead box protein 3-positive (Foxp3(+)) T regulatory (Treg) cells in the tumor microenvironment. However, the mechanisms that regulate the accumulation and the suppressive function of these Foxp3(+) Treg cells are poorly understood. Here, we found that the majority of Foxp3(+) Treg cells accumulating in the tumor microenvironment of EOCs belong to the subset of Foxp3(+) Treg cells expressing inducible costimulator (ICOS). The expansion and the suppressive function of these cells were strictly dependent on ICOS-L costimulation provided by tumor plasmacytoid dendritic cells (pDC). Accordingly, ICOS(+) Foxp3(+) Treg cells were found to localize in close vicinity of tumor pDCs, and their number directly correlated with the numbers of pDCs in the tumors. Furthermore, pDCs and ICOS(+) Foxp3(+) Treg cells were found to be strong predictors for disease progression in patients with ovarian cancer, with ICOS(+) Treg cell subset being a stronger predictor than total Foxp3(+) Treg cells. These findings suggest an essential role for pDCs and ICOS-L in immunosuppression mediated by ICOS(+) Foxp3(+) Treg cells, leading to tumor progression in ovarian cancer.

CD2AP/SHIP1 complex positively regulates plasmacytoid dendritic cell receptor signaling by inhibiting the E3 ubiquitin ligase Cbl.

The human plasmacytoid dendritic cell (pDC) receptor BDCA2 forms a complex with the adaptor FcεR1γ to activate an ITAM-signaling cascade. BDCA2 receptor signaling negatively regulates the TLR7/9-mediated type 1 IFN responses in pDCs, which may play a key role in controlling self-DNA/RNA-induced autoimmunity. We report in this article that CD2-associated adaptor protein (CD2AP), which is highly expressed in human pDCs, positively regulates BDCA2/FcεR1γ receptor signaling. By immunoprecipitation and mass spectrometry analyses, we found that CD2AP bound to SHIP1. Knockdown of CD2AP or SHIP1 reduced the BDCA2/FcεR1γ-mediated ITAM signaling and blocked its inhibition of TLR9-mediated type 1 IFN production. Knockdown of CD2AP or SHIP1 also enhanced the ubiquitination and degradation of Syk and FcεR1γ that was mediated by the E3 ubiquitin ligase Cbl. This led us to discover that, upon BDCA2 cross-linking, the CD2AP/SHIP1 complex associated with Cbl and inhibited its E3 ubiquitin ligase activity. In human primary pDCs, cross-linking of the BDCA2/FcεR1γ complex induced the recruitment of the CD2AP/SHIP1/Cbl complex to the plasma membrane of pDCs, where it colocalized with the BDCA2/FcεR1γ complex. Therefore, CD2AP positively regulates BDCA2/FcεR1γ signaling by forming a complex with SHIP1 to inhibit the E3 ubiquitin ligase Cbl.

Phospholipid scramblase 1 regulates Toll-like receptor 9-mediated type I interferon production in plasmacytoid dendritic cells.

Toll-like receptor 9 (TLR9) senses microbial DNA in the endosomes of plasmacytoid dendritic cells (pDCs) and triggers MyD88-dependent type I interferon (IFN) responses. To better understand TLR9 biology in pDCs, we established a yeast two-hybrid library for the identification of TLR9-interacting proteins. Here, we report that an IFN-inducible protein, phospholipid scramblase 1 (PLSCR1), interacts with TLR9 in pDCs. Knockdown of PLSCR1 expression by siRNA in human pDC cell line led to a 60-70% reduction of IFN-α responses following CpG-ODN (oligodeoxynucleotide) stimulation. Primary pDCs from PLSCR1-deficient mice produced lower amount of type 1 IFN than pDCs from the wild-type mice in response to CpG-ODN, herpes simplex virus and influenza A virus. Following CpG-A stimulation, there were much lower amounts of TLR9 in the early endosomes together with CpG-A in pDCs from PLSCR1-deficient mice. Our study demonstrates that PLSCR1 is a TLR9-interacting protein that plays an important role in pDC's type 1 IFN responses by regulating TLR9 trafficking to the endosomal compartment.

TSLP and immune homeostasis.

In an immune system, dendritic cells (DCs) are professional antigen-presenting cells (APCs) as well as powerful sensors of danger signals. When DCs receive signals from infection and tissue stress, they immediately activate and instruct the initiation of appropriate immune responses to T cells. However, it has remained unclear how the tissue microenvironment in a steady state shapes the function of DCs. Recent many works on thymic stromal lymphopoietin (TSLP), an epithelial cell-derived cytokine that has the strong ability to activate DCs, provide evidence that TSLP mediates crosstalk between epithelial cells and DCs, involving in DC-mediated immune homeostasis. Here, we review recent progress made on how TSLP expressed within the thymus and peripheral lymphoid and non-lymphoid tissues regulates DC-mediated T-cell development in the thymus and T-cell homeostasis in the periphery.

In vivo role of pDCs in regulating adaptive immunity.

Plasmacytoid dendritic cells (pDCs) have potential influence on innate and adaptive immune responses. In this issue of Immunity, Takagi et al. (2011) established mice lacking pDCs and explored the impact of pDC ablation in vivo.

Aspartate-glutamate-alanine-histidine box motif (DEAH)/RNA helicase A helicases sense microbial DNA in human plasmacytoid dendritic cells.

Toll-like receptor 9 (TLR9) senses microbial DNA and triggers type I IFN responses in plasmacytoid dendritic cells (pDCs). Previous studies suggest the presence of myeloid differentiation primary response gene 88 (MyD88)-dependent DNA sensors other than TLR9 in pDCs. Using MS, we investigated C-phosphate-G (CpG)-binding proteins from human pDCs, pDC-cell lines, and interferon regulatory factor 7 (IRF7)-expressing B-cell lines. CpG-A selectively bound the aspartate-glutamate-any amino acid-aspartate/histidine (DExD/H)-box helicase 36 (DHX36), whereas CpG-B selectively bound DExD/H-box helicase 9 (DHX9). Although the aspartate-glutamate-alanine-histidine box motif (DEAH) domain of DHX36 was essential for CpG-A binding, the domain of unknown function 1605 (DUF1605 domain) of DHX9 was required for CpG-B binding. DHX36 is associated with IFN-alpha production and IRF7 nuclear translocation in response to CpG-A, but DHX9 is important for TNF-alpha and IL-6 production and NF-kappaB activation in response to CpG-B. Knocking down DHX9 or DHX36 significantly reduced the cytokine responses of pDCs to a DNA virus but had no effect on the cytokine responses to an RNA virus. We further showed that both DHX9 and DHX36 are localized within the cytosol and are directly bound to the Toll-interleukin receptor domain of MyD88 via their helicase-associated domain 2 and DUF domains. This study demonstrates that DHX9/DHX36 represent the MyD88-dependent DNA sensors in the cytosol of pDCs and suggests a much broader role for DHX helicases in viral sensing.

Thymic stromal lymphopoietin-activated plasmacytoid dendritic cells induce the generation of FOXP3+ regulatory T cells in human thymus.

Human thymus contains major dendritic cell (DC) subsets, myeloid DCs (mDCs), and plasmacytoid DCs (pDCs). We previously showed that mDCs, educated by thymic stromal lymphopoietin (TSLP) produced by the epithelial cells of the Hassall's corpuscles, induced differentiation of CD4(+)CD25(-) thymocytes into Forkhead Box P3(+) (FOXP3(+)) regulatory T cells (T(R)) within the medulla of human thymus. In this study, we show that pDCs expressed the TSLP receptor and IL-7 receptor alpha complexes upon activation and became responsive to TSLP. TSLP-activated human pDCs secrete macrophage-derived chemokine CCL-22 and thymus- and activation-regulated chemokine CCL-17 but not Th1- or Th2-polarizing cytokines. TSLP-activated pDCs induced the generation of FOXP3(+) T(R) from CD4(+)CD8(-)CD25(-) thymocytes, which could be strongly inhibited by Th1-polarizing cytokine IL-12 or Th2-polarizing cytokine IL-4. Interestingly, the FOXP3(+) T(R) induced by the TSLP-pDCs expressed more IL-10 but less TGF-beta than that induced by the TSLP-mDCs. These data suggest that TSLP expressed by thymic epithelial cells can activate mDCs and pDCs to positively select the FOXP3(+) T(R) with different cytokine production potential in human thymus. The inability of TSLP to induce DC maturation without producing Th1- or Th2-polarizing cytokines may provide a thymic niche for T(R) development.

Distinct signal codes generate dendritic cell functional plasticity.

Our adaptive immune system induces distinct responses to different pathogens because of the functional plasticity of dendritic cells (DCs); however, how DCs program unique responses remains unclear. Here, we found that the cytokine thymic stromal lymphopoietin (TSLP) potently transduced a unique T helper type 2 (T(H)2)-inducing compound signal in DCs. Whereas activation of nuclear factor kappaB (predominantly p50) drove DCs to produce OX40L to induce T(H)2 differentiation, the activation of signal transducer and activator of transcription 6 (STAT6) triggered DCs to secrete chemokines necessary for the recruitment of T(H)2 cells. In addition, TSLP signaling limited the activation of STAT4 and interferon regulatory factor 8 (IRF-8), which are essential factors for the production of the T(H)1-polarizing cytokine interleukin-12 (IL-12). By contrast, Toll-like receptor ligands and CD40 ligand did not activate STAT6 in myeloid DCs, but instead increased the abundance of STAT4 and IRF-8 to induce T(H)1 responses through the production of IL-12. Therefore, we propose that the functional plasticity of DCs relies on elaborate signal codes that are generated by different stimuli.

TSLP production by epithelial cells exposed to immunodeficiency virus triggers DC-mediated mucosal infection of CD4+ T cells.

Mucosal dendritic cells have been implicated in the capture, storage, and transmission of HIV to CD4(+) T cells as well as in the promotion of HIV replication in activated CD4(+) T cells during the cognate T-cell and DC interaction. We report that HIV induces human genital mucosal epithelial cells to produce thymic stromal lymphopoietin (TSLP) via activation of the NFkappaB signaling pathway. The TSLP secreted by HIV exposed epithelial cells activated DC, which promoted proliferation and HIV-1 replication of co-cultured autologous CD4(+) T cells. In rhesus macaques, we observed dramatic increases in TSLP expression concurrent with an increase in viral replication in the vaginal tissues within the first 2 weeks after vaginal SIV exposure. These data suggest that HIV-mediated TSLP production by mucosal epithelial cells is a critical trigger for DC-mediated amplification of HIV-infection in activated CD4(+) T cells. The cross talk between mucosal epithelial cells and DC, mediated by HIV-induced TSLP, may be an important mechanism for the high rate of HIV infection in women through the vaginal mucosa.

TSLP and IL-7 use two different mechanisms to regulate human CD4+ T cell homeostasis.

Whether thymic stromal lymphopoietin (TSLP) directly induces potent human CD4(+) T cell proliferation and Th2 differentiation is unknown. We report that resting and activated CD4(+) T cells expressed high levels of IL-7 receptor a chain but very low levels of TSLP receptor (TSLPR) when compared with levels expressed in myeloid dendritic cells (mDCs). This was confirmed by immunohistology and flow cytometry analyses showing that only a subset of mDCs, with more activated phenotypes, expressed TSLPR in human tonsils in vivo. IL-7 induced strong STAT1, -3, and -5 activation and promoted the proliferation of naive CD4(+) T cells in the presence of anti-CD3 and anti-CD28 monoclonal antibodies, whereas TSLP induced weak STAT5 activation, associated with marginally improved cell survival and proliferation, but failed to induce cell expansion and Th2 differentiation. The effect of TSLP on enhancing strong human T cell proliferation was observed only when sorted naive CD4(+) T cells were cultured with mDCs at levels as low as 0.5%. TSLP could only induce naive CD4(+) T cells to differentiate into Th2 cells in the presence of allogeneic mDCs. These results demonstrate that IL-7 and TSLP use different mechanisms to regulate human CD4(+) T cell homeostasis.

Regulation of TLR7/9 responses in plasmacytoid dendritic cells by BST2 and ILT7 receptor interaction.

Plasmacytoid dendritic cells (pDCs) produce copious type I interferon (IFN) upon sensing nucleic acids through Toll-like receptor (TLR) 7 and TLR9. Uncontrolled pDC activation and IFN production are implicated in lymphopenia and autoimmune diseases; therefore, a mechanism controlling pDC IFN production is essential. Human pDCs specifically express an orphan receptor, immunoglobulin-like transcript 7 (ILT7). Here, we discovered an ILT7 ligand expressed by human cell lines and identified it as bone marrow stromal cell antigen 2 (BST2; CD317). BST2 directly binds to purified ILT7 protein, initiates signaling via the ILT7-FcepsilonRIgamma complex, and strongly inhibits production of IFN and proinflammatory cytokines by pDCs. Readily induced by IFN and other proinflammatory cytokines, BST2 may modulate the human pDC's IFN responses through ILT7 in a negative feedback fashion.