Conventional DCs Research Articles

DC-Derived Exosomes for Cancer Immunotherapy

Simple SummaryDendritic cells (DCs)-based cancer vaccines have not succeeded in generating significant clinical responses despite their capacity to induce host anti-tumor CD8 T cell immunity, and one major hurdle is tumor-mediated immunosuppression. Exosomes are nano-sized inert membrane vesicles derived from the endocytic pathway that play a critical role in intercellular communication. DC-derived exosomes (DCexos) additionally carried MHC class I/II (MHCI/II) often complexed with antigens and co-stimulatory molecules, thus capable of priming antigen-specific CD4 and CD8 T cells. Indeed, vaccines with DCexos have been shown to exhibit better anti-tumor efficacy in eradicating tumors compared to DC vaccines in pre-clinical models. Coupled with their resistance to tumor immunosuppression, DCexo-based cancer vaccines have been heralded as the superior alternative cell-free therapeutic vaccines over DC vaccines, and have now been tested in multiple clinical trials. In this review, current studies of DCexo cancer vaccines as well as potential future directions will be discussed.As the initiators of adaptive immune responses, DCs play a central role in regulating the balance between CD8 T cell immunity versus tolerance to tumor antigens. Exploiting their function to potentiate host anti-tumor immunity, DC-based vaccines have been one of most promising and widely used cancer immunotherapies. However, DC-based cancer vaccines have not achieved the promised success in clinical trials, with one of the major obstacles being tumor-mediated immunosuppression. A recent discovery on the critical role of type 1 conventional DCs (cDC1s) play in cross-priming tumor-specific CD8 T cells and determining the anti-tumor efficacy of cancer immunotherapies, however, has highlighted the need to further develop and refine DC-based vaccines either as monotherapies or in combination with other therapies. DC-derived exosomes (DCexos) have been heralded as a promising alternative to DC-based vaccines, as DCexos are more resistance to tumor-mediated suppression and DCexo vaccines have exhibited better anti-tumor efficacy in pre-clinical animal models. However, DCexo vaccines have only achieved limited clinical efficacy and failed to induce tumor-specific T cell responses in clinical trials. The lack of clinical efficacy might be partly due to the fact that all current clinical trials used peptide-loaded DCexos from monocyte-derived DCs. In this review, we will focus on the perspective of expanding current DCexo research to move DCexo cancer vaccines forward clinically to realize their potential in cancer immunotherapy.

Suppression of plasmacytoid dendritic cell migration to colonic isolated lymphoid follicles abrogates the development of colitis

BackgroundDendritic cells (DCs) play a pivotal role in maintaining immunological homeostasis by orchestrating innate and adaptive immune responses via migration to inflamed sites and the lymph nodes (LNs). Plasmacytoid DCs (pDCs) have been reported to accumulate in the colon of inflammatory bowel disease (IBD) patients and dextran sulfate sodium (DSS)-induced colitis mice. However, the role of pDCs in the progression of colonic inflammation remains unclear. Methods80 compounds in natural medicines were searched for inhibitors of pDC migration using bone marrow-derived pDCs (BMpDCs) and conventional DCs (BMcDCs). BALB/c mice were given 3% DSS in the drinking water to induce acute colitis. Compounds, which specifically inhibited pDC migration, were administrated into DSS-induced colitis mice. FindingsAstragaloside IV (As-IV) and oxymatrine (Oxy) suppressed BMpDC migration but not BMcDC migration. In DSS-induced colitis mice, the number of pDCs was markedly increased in the colonic lamina propria (LP), and the expression of CCL21 was obviously observed in colonic isolated lymphoid follicles (ILFs). As-IV and Oxy reduced symptoms of colitis and the accumulation of pDCs in colonic ILFs but not in the colonic LP. Moreover, in a BMpDC adoptive transfer model, BMpDC migration to colonic ILFs was significantly decreased by treatment with As-IV or Oxy. InterpretationpDCs accumulated in the colon of colitis mice, and As-IV and Oxy ameliorated colitis by suppressing pDC migration to colonic ILFs. Accordingly, the selective inhibition of pDC migration may be a potential therapeutic approach for treating colonic inflammatory diseases.

Circulating Plasmacytoid and Conventional Dendritic Cells Are Numerically and Functionally Deficient in Patients With Scrub Typhus.

BackgroundDendritic cells (DCs) are specialized antigen-presenting cells known to bridge innate and adaptive immune reactions. However, the relationship between circulating DCs and Orientia tsutsugamushi infection is unclear. Therefore, this study aimed to examine the level and function of plasmacytoid DCs (pDCs) and conventional DCs (cDCs), two subsets of circulating DCs, in scrub typhus patients.MethodsThe study included 35 scrub typhus patients and 35 healthy controls (HCs). pDC and cDC levels, CD86 and CD274 expression, and cytokine levels were measured using flow cytometry.ResultsCirculating pDC and cDC levels were found to be significantly reduced in scrub typhus patients, which were correlated with disease severity. The patients displayed increased percentages of CD86+ pDCs, CD274+ pDCs, and CD274+ cDCs in the peripheral blood. The alterations in the levels and surface phenotypes of pDCs and cDCs were recovered in the remission state. In addition, the production of interferon (IFN)-α and tumor necrosis factor (TNF)-α by circulating pDCs, and interleukin (IL)-12 and TNF-α by circulating cDCs was reduced in scrub typhus patients. Interestingly, our in vitro experiments showed that the percentages of CD86+ pDCs, CD274+ pDCs, and CD274+ cDCs were increased in cultures treated with cytokines including IFN-γ, IL-12, and TNF-α.ConclusionsThis study demonstrates that circulating pDCs and cDCs are numerically deficient and functionally impaired in scrub typhus patients. In addition, alterations in the expression levels of surface phenotypes of pDCs and cDCs could be affected by pro-inflammatory cytokines.

Lenalidomide Acts As a Positive Immunomodulator through Modulating the Functions of Human Plasmacytoid Dendritic Cells

Background: Lenalidomide (LEN), is a core anti-multiple myeloma (MM) drug (so-called Immunomodulatory Drugs; IMiD). LEN has been reported to enhance function and proliferation of effector immune cells such as T cells and NK cells. In addition, LEN treatment leads to relatively low rate onset of herpes zoster viral infection compared to treatment of bortezomib as an alternative therapeutic tool for MM. However, cellular and molecular mechanisms underlying its immunomodulatory effects still remain largely unclear. Although there is evidence indicating the immunomodulatory effects of LEN on mouse conventional DCs, there are no reports showing its effects on human DC subsets. Human plasmacytoid DCs (pDCs) dedicate a large proportion of their transcription machinery to making type I interferons (IFNs) and are the major source of IFN-α in the blood (Ito T. Blood 2006; 107: 2423), which have the potential to enhance immune effectors such as T cells, B cells, monocytes, and NK cells. Thus, pDCs play a central role in antiviral immune response in innate host defense. Therefore, we focused on the effects of LEN on the functions of human pDCs.Methods: Using cell sorting, flow cytometry, microarray, Bio-Plex Multiplex assay, and ELISA methods, functions were analyzed in blood human BDCA4+ pDCs from healthy adult volunteers. This study was approved by the Institutional Review Board of Kansai Medical University.Results: We found that 3 to 300 nM of bortezomib killed pDCs but 0.1 to 3 µM of LEN (covering clinical in vivo plasma concentration range for both agents) did not affect pDC survival and their CD86 expression in response to CpG2216. Bortezomib inhibited dose-dependently pDC-derived IFN-α but 0.1 to 3 µM of LEN sustained IFN-α level in the pDC culture when stimulated with optimal concentration of CpG2216 (3 µM). Of note, in stimulation with low concentrated CpG2216 (0.1 µM), 1 µM of LEN enhanced pDC-derived IFN-α production (184 % of control culture; p=0.027). These results indicate that the clinical concentration of LEN at least sustained and potentially enhanced IFN-α production from pDCs in response to weak stimulation. To seek the chemokine production by pDCs enhanced by the LEN treatment, we used gene microarray analysis and found transcript expressions of CXCL2, CXCL8, CXCL9, CXCL10, CXCL11 and CCL19 were upregulated in LEN-treated pDCs. CXCL2 potentially attracts CTLs and NK cells, CXCL8, CXCL9, CXCL10, and CXCL11 do effector memory T cells, and CCL19 does naïve T and B cells (Piqueras B. Blood 2006; 107: 2613). By Bio-Plex Multiplex assay in the supernatants of pDCs in response to CpG in the presence or absence of 1 µM LEN, we confirmed at protein levels that these chemokines were upregulated (CXCL2; 167 % of control culture, CXCL8; 140 % of cont., CXCL9; 181 % of cont., CXCL10; 160 % of cont., CXCL11; 265 % of cont., and CCL19; 279 % of cont.). In addition, we found LEN upregulated expression of GITR-ligand on pDCs, which has been reported to promote NK cell activity in pDC-NK cell interaction.Conclusion: LEN enhances production of the pDC-derived chemokines, which allows attraction of several types of immune effectors, and in turn the sustained pDC-derived endogenous type I IFNs and the upregulated GITR-ligand expression may potentially contribute to efficient activation of the attracted immune cells. These data suggest LEN functions as a positive immunomodulator through elaborately modulating pDC functions. Thus, our findings provide a plausible explanation for an advantage of its use for anti-viral defense against herpes and unveiled a part mechanism of IMiD function of LEN in the treatment for MM. DisclosuresIto:bristol myers squibb: Honoraria; Celgene Corporation: Honoraria. Nomura:Celgene K.K.: Honoraria, Research Funding; Jansen Pharmaceutical K.K.: Honoraria; Takeda Pharmaceutical Co.,LTD.: Honoraria, Research Funding; Asahi Kasei Pharma K.K.: Honoraria; Ono Pharmaceutical Co.,LTD.: Honoraria; Fujimoto Pharmaceutical Co.: Research Funding; Bristol-Meyer Squibb K.K.: Honoraria.

Commitment to dendritic cells and monocytes.

Dendritic cells (DCs) and monocytes are widely conserved immune cells in vertebrates that arise from hematopoietic stem cells via intermediate progenitors. The progenitors that strictly give rise to DCs or monocytes have been recently identified both in humans and in mice, thereby revealing their differentiation pathways. Advances in analysis technologies have further deepened our understanding of the development of DCs and monocytes from progenitor population-based to individual progenitor cell-based commitment. Since DC-committed progenitors, common DC progenitors (CDPs) and precursor conventional DCs (pre-cDCs) do not differentiate into monocytes, DCs are a distinct lineage from monocytes, although monocytes can acquire DC functions upon activation at tissues where they arrive.

Dysregulation of Rnf 213 gene contributes to T cell response via antigen uptake, processing, and presentation.

Growing evidence suggest the association between Moyamoya disease (MMD) and immune systems, such as antigen presenting cells in particular. Rnf213 gene, a susceptibility gene for MMD, is highly expressed in immune tissues, however, its function remains unclear. In addition, the physiological role of RNF213 gene polymorphism c.14576G > A (rs112735431), susceptibility variant for MMD, is also poorly understood. By studying Rnf213-knockout (Rnf213-KO) mice with deletion of largest exon32 and Rnf213-knockin (Rnf213-KI) mice with insertion of single-nucleotide polymorphism corresponding to c.14576G > A mutation in MMD patients, we aimed to investigate the role of RNF213 in dendritic cell development, and antigen processing and presentation. First, we found a high level of Rnf213 gene expression in conventional DCs and monocytes. Second, flow cytometric and confocal microscopic analysis revealed ovalbumin protein-pulsed Rnf213-KO and Rnf213-KI DCs showed impaired antigen uptake, proteolysis and reduced numbers of endosomes and lysosomes, and thereby failed to activate and proliferate antigen-specific T cells efficiently. In addition, Rnf213-KI DCs showed a similar phenotype to that of Rnf213-KO BMDCs. In conclusion, our findings suggest the critical role of RNF213 in antigen uptake, processing and presentation.

Conventional type 2 lung dendritic cells are potent inducers of follicular helper T cells in the asthmatic lung

BackgroundFollicular helper T (Tfh) cells represent a unique subset of helper CD4+ T cells in lymphoid follicles. Recently, Tfh cells were shown to play an important role in asthma through B cell differentiation. Conventional lung DCs are classified into two major subsets: conventional type 1 (cDC1) and type 2 (cDC2). Although the two subsets are different in driving particular T cell responses, the subset that induces Tfh cells in the asthmatic lung primarily has yet to be fully elucidated. MethodsWe evaluated Tfh cells, defined by the expression of CD4 and CXCR5, in HDM-challenged mice. Next, we characterized cDC1 and cDC2 purified from antigen-primed lung and examined their Tfh cell-inducing capacity. Additionally, the ability of lung DC-induced Tfh cells to cause germinal center B (GCB) cells to produce antigen-specific IgE was assessed. ResultsIn HDM-challenged mice, Bcl-6-expressing Tfh cells were significantly increased in the mediastinal lymph nodes. Lung cDC2, but not lung cDC1, increased after HDM priming, and cDC2 secreted larger amounts of IL-6 with higher ICOS-L expression than cDC1. In the co-cultures with OVA-specific naïve CD4+ T cells, cDC2 from OVA-primed lung induced Bcl-6-expressing Tfh cells more efficiently, together with larger amounts of IL-6 and IL-21, than cDC1. Blockage of IL-6 or ICOS-L significantly reduced Tfh cell induction. Finally, cDC2-induced Tfh cells enabled GCB cells to produce OVA-specific IgE. ConclusionsIn asthmatic lung, cDC2 is the primary DC subset responsible for Tfh cell differentiation and plays an important role in humoral immunity in asthma by inducing Tfh cells.

MHC Class I on murine hematopoietic APC selects Type A IEL precursors in the thymus.

TCRαβ+ CD8α+ CD8β- intestinal intraepithelial lymphocytes (CD8αα IEL) are gut T cells that maintain barrier surface homeostasis. Most CD8αα IEL are derived from thymic precursors (IELp) through a mechanism referred to as clonal diversion. In this model, self-reactive thymocytes undergo deletion in the presence of CD28 costimulation, but in its absence undergo diversion to the IEL fate. While previous reports showed that IELp were largely β2m dependent, the APC that drive the development of these cells are poorly defined. We found that both CD80 and CD86 restrain IELp development, and conventional DCs play a prominent role. We sought to define a CD80/86 negative, MHCI positive APC that supports the development to the IEL lineage. Chimera studies showed that MHCI needs to be expressed on hematopoietic APC for selection. As thymic hematopoietic APC are heterogeneous in their expression of MHCI and costimulatory molecules, we identified four thymic APC types that were CD80/86neg/low and MHCI+ . However, selective depletion of β2m in individual APC suggested functional redundancy. Thus, while hematopoietic APC play a critical role in clonal diversion, no single APC subset is specialized to promote the CD8αα IEL fate.

CXCR4 signaling controls dendritic cell location and activation at steady state and in inflammation

AbstractDendritic cells (DCs) encompass several cell subsets that collaborate to initiate and regulate immune responses. Proper DC localization determines their function and requires the tightly controlled action of chemokine receptors. All DC subsets express CXCR4, but the genuine contribution of this receptor to their biology has been overlooked. We addressed this question using natural CXCR4 mutants resistant to CXCL12-induced desensitization and harboring a gain of function that cause the warts, hypogammaglobulinemia, infections, and myelokathexis (WHIM) syndrome (WS), a rare immunodeficiency associated with high susceptibility to the pathogenesis of human papillomavirus (HPV). We report a reduction in the number of circulating plasmacytoid DCs (pDCs) in WHIM patients, whereas that of conventional DCs is preserved. This pattern was reproduced in an original mouse model of WS, enabling us to show that the circulating pDC defect can be corrected upon CXCR4 blockade and that pDC differentiation and function are preserved, despite CXCR4 dysfunction. We further identified proper CXCR4 signaling as a critical checkpoint for Langerhans cell and DC migration from the skin to lymph nodes, with corollary alterations of their activation state and tissue inflammation in a model of HPV-induced dysplasia. Beyond providing new hypotheses to explain the susceptibility of WHIM patients to HPV pathogenesis, this study shows that proper CXCR4 signaling establishes a migration threshold that controls DC egress from CXCL12-containing environments and highlights the critical and subset-specific contribution of CXCR4 signal termination to DC biology.

CSF1R defines the mononuclear phagocyte system lineage in human blood in health and COVID-19

SummaryMononuclear phagocytes defend tissues, present antigens, and mediate recovery and healing. To date, we lack a marker to unify mononuclear phagocytes in humans or that informs us about their origin. Here, we reassess mononuclear phagocyte ontogeny in human blood through the lineage receptor CSF1R, in the steady state and in COVID-19. We define CSF1R as the first sensitive and reproducible pan-phagocyte lineage marker, to identify and enumerate all conventional monocytes, and the myeloid dendritic cells. In the steady state, CSF1R is sufficient for sorting and immuno-magnetic isolation. In pathology, changes in CSF1R are more sensitive than CD14 and CD16. In COVID-19, a significant drop in membrane CSF1R is useful for stratifying patients, beyond the power of cell categories published thus far, which fail to capture COVID-19 specific events. Importantly, CSF1R defines cells which are neither conventional monocytes nor DCs, which are missed in published analysis. CSF1R decrease can be linked ex vivo to high CSF1 levels. Blood assessment of CSF1R+ cells opens a developmental window to the Mononuclear Phagocyte System in transit from bone marrow to tissues, supports isolation and phenotypic characterisation, identifies novel cell types, and singles out CSF1R inhibition as therapeutic target in COVID-19 and other diseases.

Monophosphoryl lipid A-induced activation of plasmacytoid dendritic cells enhances the anti-cancer effects of anti-PD-L1 antibodies.

Monophosphoryl lipid A (MPLA) is a toll-like receptor 4 ligand that promotes immune activation in mice and humans, without undesired inflammation. Immunotherapy by the combining immune checkpoint blockade and MPLA has shown promising anti-cancer effects in both mice and humans. In this study, we explored how MPLA enhanced the anti-cancer effects of anti-PD-L1 antibodies (Abs). Anti-cancer immunity induced by the combination of anti-PD-L1 Abs and MPLA failed in CD4 and CD8 cell-depleted mice. Moreover, the combination treatment of anti-PD-L1 Abs and MPLA synergistically enhanced the activation of plasmacytoid dendritic cells (pDCs) in the mouse in vivo, while conventional DCs were not. In addition, mice treated with anti-PD-L1 Abs and MPLA were not protected from B16 melanoma by blockade of interferon-alpha receptor (IFNAR). The combination of anti-PD-L1 Abs and MPLA also promoted human peripheral blood pDC activation and induced IFN-α-dependent T cell activation. Therefore, these results demonstrate that MPLA enhances anti-PD-L1 Ab-mediated anti-cancer immunity through the activation and IFN-α production of pDCs.

Plasmacytoid dendritic cells cross-prime naive CD8 T cells by transferring antigen to conventional dendritic cells through exosomes

Although plasmacytoid dendritic cells (pDCs) have been shown to play a critical role in generating viral immunity and promoting tolerance to suppress antitumor immunity, whether and how pDCs cross-prime CD8 T cells in vivo remain controversial. Using a pDC-targeted vaccine model to deliver antigens specifically to pDCs, we have demonstrated that pDC-targeted vaccination led to strong cross-priming and durable CD8 T cell immunity. Surprisingly, cross-presenting pDCs required conventional DCs (cDCs) to achieve cross-priming in vivo by transferring antigens to cDCs. Taking advantage of an in vitro system where only pDCs had access to antigens, we further demonstrated that cross-presenting pDCs were unable to efficiently prime CD8 T cells by themselves, but conferred antigen-naive cDCs the capability of cross-priming CD8 T cells by transferring antigens to cDCs. Although both cDC1s and cDC2s exhibited similar efficiency in acquiring antigens from pDCs, cDC1s but not cDC2s were required for cross-priming upon pDC-targeted vaccination, suggesting that cDC1s played a critical role in pDC-mediated cross-priming independent of their function in antigen presentation. Antigen transfer from pDCs to cDCs was mediated by previously unreported pDC-derived exosomes (pDCexos), that were also produced by pDCs under various conditions. Importantly, all these pDCexos primed naive antigen-specific CD8 T cells only in the presence of bystander cDCs, similarly to cross-presenting pDCs, thus identifying pDCexo-mediated antigen transfer to cDCs as a mechanism for pDCs to achieve cross-priming. In summary, our data suggest that pDCs employ a unique mechanism of pDCexo-mediated antigen transfer to cDCs for cross-priming.

Abstract 4537: SBT6050, a HER2-directed TLR8 ImmunoTAC™therapeutic, is a potent human myeloid cell agonist that provides opportunity for single agent clinical activity

Abstract SBT6050, a novel therapeutic comprised of a potent toll-like receptor (TLR) 8 agonist conjugated to a HER2-directed monoclonal antibody that binds an epitope distinct from trastuzumab, is designed for systemic delivery and tumor-localized activation of human myeloid cells in the presence of moderate and high HER2-expressing tumor cells. Many solid tumors, including those expressing HER2, are refractory to immunotherapy due to immune-suppressive mechanisms, loss of HLA, low neoantigen availability, and/or minimal T cell infiltrates. These tumors frequently contain abundant populations of tumor-associated myeloid cells. Activation of these cells through TLR agonism has emerged as a promising approach in overcoming resistance mechanisms to current cancer immunotherapies. Unlike other endosomal TLRs such as TLR7 and TLR9, TLR8 is highly expressed in human myeloid cells known to be prevalent in human tumors such as conventional DCs and macrophages. TLR8's restricted myeloid cell expression removes the risk of inducing T cell death or tumor cell proliferation as described for other innate immune activators such as STING. Agonism of TLR8 in human myeloid cells activates a broad spectrum of anti-tumor immune mechanisms, including proinflammatory cytokine production, repolarization of suppressive myeloid cells and the priming of CTL responses. These functions cannot be replicated by a potent TLR7 agonist or with clinical agents such as resiquimod that agonize TLR7 and only weakly engage TLR8. Here we present data demonstrating the superiority of SBT6050 at activating human myeloid cells compared to HER2 antibody conjugates that use either a selective TLR7 agonist or resiquimod. In vitro studies with human immune cells show that SBT6050 potently induces multiple anti-tumor immune activities, including proinflammatory cytokine and chemokine production, inflammasome activation, direct activation of DCs and indirect T and NK cell cytolytic activity. Our data indicate the favorable profile of SBT6050 is likely due to efficient engagement of TLR8 in conjugate form and TLR8's unique expression profile, and not due to differences in the potency of the small molecule payloads. Using an SBT6050 mouse surrogate we show in vivo evidence for intratumoral TLR agonist activation of several myeloid driven anti-tumor pathways leading to curative single agent efficacy in both a T cell-excluded syngeneic tumor model and a xenograft model lacking T, B and NK cells. Collectively, these data demonstrate that SBT6050 displays an attractive functional profile unachievable with agonist-based antibody conjugates directed against other innate immune receptors. These data also highlight the potential for SBT6050 to be clinically active as a monotherapy. SBT6050 is projected to enter the clinic in 2020 for patients with moderate or high HER2-expressing tumors. Citation Format: Michael R. Comeau, Ty Brender, Monica Childs, Jamie Brevik, Damion Winship, Heather Metz, Jenny Chang, Jeffrey Adamo, Ben Setter, Hengyu Xu, Li-Qun Fan, Brenda Stevens, Sean W. Smith, Phil Tan, Robert DuBose, Yvette Latchman, Peter Baum, Valerie Odegard. SBT6050, a HER2-directed TLR8 ImmunoTAC™therapeutic, is a potent human myeloid cell agonist that provides opportunity for single agent clinical activity [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4537.

Quantitative and qualitative impairments in dendritic cell subsets of patients with ovarian or prostate cancer

BackgroundDendritic cells (DCs) are the most efficient antigen-presenting cells, hence initiating a potent and cancer-specific immune response. This ability (mainly using monocyte-derived DCs) has been exploited in vaccination strategies for decades with limited clinical efficacy. Another alternative would be the use of conventional DCs (cDCs) of which at least three subsets circulate in human blood: cDC1s (CD141bright), cDC2s (CD1c+) and plasmacytoid DCs. Despite their paucity, technical advances may allow for their selection and clinical use. However, many assumptions concerning the DC subset biology depend on observations from mouse models, hindering their translational potential. In this study, we characterise human DCs in patients with ovarian cancer (OvC) or prostate cancer (PrC). Patients and methodsWhole blood samples from patients with OvC or PrC and healthy donors (HDs) were evaluated by flow cytometry for the phenotypic and functional characterisation of DC subsets. ResultsIn both patient groups, the frequency of total CD141+ DCs was lower than that in HDs, but the cDC1 subset was only reduced in patients with OvC. CD141+ DCs showed a reduced response to the TLR3 agonist poly (I:C) in both groups of patients. An inverse correlation between the frequency of cDC1s and CA125, the OvC tumour burden marker, was observed. Consistently, high expression of CLEC9A in OvC tissue (The Cancer Genome Atlas data set) indicated a better overall survival. ConclusionscDC1s are reduced in patients with OvC, and CD141+ DCs are quantitatively and qualitatively impaired in patients with OvC or PrC. CD141+ DC activation may predict functional impairment. The loss of cDC1s may be a bad prognostic factor for patients with OvC.

350 Histone demethylase LSD1 is required for LC embryonic development but dispensable for LC maintenance and repopulation

Langerhans cells (LCs), the sole dendritic cell subpopulation in the epidermis, are potent regulators of immune surveillance and tolerance. Unlike conventional DCs, LCs follow unique patterns of development and maintenance under steady and inflamed states. Lysine-specific demethylase 1 (LSD1), known to mediate the demethylation of lysine amino acids on histone proteins, plays a key role in the maintenance and differentiation of hematopoietic stem cells. However, the role of LSD1 in LC ontogeny and homeostasis remains unknown. To address this, we generated Csf1rCreLSD1fl/fl conditional knockout (Csf1r.LSD1-KO) mice in which LSD1 was deficient in macrophage/monocytes and LC precursors from embryonic stage. We found a robust reduction of LC precursors in Csf1r.LSD1-KO mice at embryonic day 18.5 and postnatal day 0, suggesting that LSD1 is required for LC ontogeny at late embryonic stage. To investigate whether LSD1 is involved in LC maintenance and repopulation, we created CD11cCreLSD1fl/fl conditional knockout (CD11c.LSD1-KO) mice in which LSD1 was deficient in all DC populations including epidermal LCs after birth. Flow cytometry and immunofluorescent staining of skin showed that the frequency and number of LCs were unaltered in 2-week-old and adult CD11c.LSD1-KO mice compared to wild-type mice. Using an ultraviolet C (UVC)-induced skin damage model, we found that long-term LCs were able to repopulate to the inflamed epidermis of CD11c.LSD1-KO mice 2 weeks after UVC treatment. Overall, our data suggests that LSD1 is critical for LC embryonic development, but not required for the LC maintenance at steady state and repopulation under inflammatory conditions.

360 CBFβ2 is required for LC hemostasis and repopulation but not required for its embryonic development

Langerhans cells (LCs), established through the differentiation of embryonic LC precursors, are the sole dendritic cell subpopulation in the epidermis and potent regulators of immune surveillance and tolerance. Unlike conventional DCs, LCs follow unique patterns of development and maintenance under steady and inflamed states. Several transcription factors are essential for LC differentiation, including Runx3, which functions by forming heterodimers with the non-DNA binding β-subunit, CBFβ (Core-Binding Factor Subunit Beta) 1 or 2.

OX40+ plasmacytoid dendritic cells in the tumor microenvironment promote antitumor immunity.

Plasmacytoid DCs (pDCs), the major producers of type I interferon, are principally recognized as key mediators of antiviral immunity. However, their role in tumor immunity is less clear. Depending on the context, pDCs can promote or suppress antitumor immune responses. In this study, we identified a naturally occurring pDC subset expressing high levels of OX40 (OX40+ pDC) enriched in the tumor microenvironment (TME) of head and neck squamous cell carcinoma. OX40+ pDCs were distinguished by a distinct immunostimulatory phenotype, cytolytic function, and ability to synergize with conventional DCs (cDCs) in generating potent tumor antigen-specific CD8+ T cell responses. Transcriptomically, we found that they selectively utilized EIF2 signaling and oxidative phosphorylation pathways. Moreover, depletion of pDCs in the murine OX40+ pDC-rich tumor model accelerated tumor growth. Collectively, we present evidence of a pDC subset in the TME that favors antitumor immunity.

IFNAR signaling augments T cell and plasmacytoid dendritic cell activation during costimulation blockade resistant rejection

Abstract Costimulation blockade (CoB) is a promising new transplant immunosuppression strategy offering improved long-term allograft survival without the nephrotoxicity of calcineurin inhibitors. However, increased risks of T cell mediated acute rejection have impeded the widespread adoption of CoB. Type I interferon (IFN) produced predominantly by plasmacytoid dendritic cells (pDCs) in response to innate immune signals, induce systemic inflammation that may prime the adaptive immune system for acute rejection. Here we investigate how signaling through the type I interferon receptor (IFNAR) contributes to CoB-resistant allograft rejection. We performed fully MHC-mismatched skin grafts from Balb/cJ donors to C57BL/6 recipients. Mice were treated with CoB alone (250 μg CTLA4-Ig + 250 μg anti-CD154 given by IP injection day 0, 2, 4, 6) or in conjunction with anti-IFNAR (250 μg given day 0, 2, 4, 6). CoB treatment improved graft survival but resulted in CoB-resistant rejection, while CoB+anti-IFNAR significantly improved graft survival. We found that CoB+anti-IFNAR led to a reduction in the activation and effector function of CD8+ T cells compared to CoB alone, demonstrated by reduced frequencies of CD44+CD8+ T cells and an inhibition of IFN-gamma and TNF-alpha production by CD8+ T cells. In addition, we observed reduced CD80 and MHC I expression on conventional DCs (cDCs) and pDCs treated with CoB+anti-IFNAR compared to treatment with CoB alone. These data suggest that signaling through IFNAR augments cDC and pDC activation and is associated with CD8+ T cell activation and effector functions in order to promote CoB-resistant allograft rejection.

Reduced BDCA3+ dendritic cells in nasal mucosa induce severe inflammation in patients with allergic rhinitis and chronic rhinosinusitis

Abstract Dendritic cells (DCs) are specialized antigen-presenting cells that play a crucial role in T helper (Th) cell differentiation. DCs of human airway comprise conventional DCs (cDCs) and plasmacytoid DCs (pDCs), of which the cDCs lineage consists of cDC type 1 (cDC1) and cDC type 2 (cDC2). These DCs subsets differ in ontogeny and functional properties in immune response. Thus, we aimed to evaluate the expression of different dendritic cell subsets in nasal mucosa of patients with allergic rhinitis (AR) and chronic rhinosinusitis (CRS). Nasal polyp tissues or ethmoid mucosa from patients undergoing endoscopic sinus surgery or septoplasty were collected. The expression of DCs was investigated by flow cytometry via surface markers including BDCA-1 (CD1c) for cDC1, BDCA-3 (CD141) for cDC2, and BDCA-2 (CD303) for pDC. In subjects with AR, BDCA-3+cDC levels significantly decreased in patients with accompanying CRS, more reduced in eosinophilic CRS (ECRS) than in non-eosinophilic CRS (NECRS), compared to non-CRS patients. In subjects with CRS, the expression of BDCA-3+cDC decreased in patients with accompanying AR; more reduced in multiple allergens sensitization. Consequently, the IF result showed CD4+ CD25+ Foxp3+ regulatory T cell expression was significantly decreased in CRS patients with AR compared to those without AR. Furthermore, the level of BDCA-3+cDC was also inversely related with preoperative CT scores, serum eosinophils and IgE levels. We suggest that BDCA-3+cDC may play an essential role in immunoregulation and induction of clinical tolerance. Reduced frequency of BDCA-3+cDC followed by impaired Treg expression in CRS patients with AR might reflect the mechanism of severe inflammation in patients of CRS combined with AR.

Long non-coding RNA lnc-DC in dendritic cells regulates trophoblast invasion via p-STAT3-mediated TIMP/MMP expression.

Dendritic cells are the primary antigen-presenting cells that contact trophoblasts at the beginning of pregnancy. Excessive DCs maturity is described in some pregnancy complications, such as pre-eclampsia and fetal growth restriction, which are characterized by impaired trophoblast invasion. However, the mechanism is unclear. The long non-coding RNA long non-coding RNA DC (lnc-DC) is expressed exclusively in conventional human DCs and induces DC differentiation and maturation by promoting signal transducer and activator of transcription 3 (STAT3) phosphorylation. Our previous investigation proved lnc-DC and p-STAT3 are elevated in pre-eclampsia. This research is to study the mechanism of lnc-DC and trophoblast invasion. We transfected DCs with lnc-DC shRNA or a lentivirus for lnc-DC overexpression and cocultured these treated DCs with trophoblast under different conditions. Transwell assay and wound healing assay were used to detect the trophoblast invasion ability. We also tested the matured DCs and Th1 cells as well as the p-STAT3. We found that lnc-DC promoted DC maturation and inhibited trophoblast invasion without the involvement of CD4+ T cells. And the p-STAT3 agonist could reverse the lnc-DC function. Mature DCs may be involved in altering trophoblast invasion through the overexpression of lnc-DC, which increases p-STAT3 levels and the tissue inhibitor of metalloproteinase-1 (TIMP-1)/matrix metalloproteinase-9 (MMP-9) and tissue inhibitor of metalloproteinase-2 (TIMP-2)/matrix metalloproteinase-2 (MMP-2) ratios. Thus, lnc-DC is a promising novel target for regulating trophoblast invasion.