Blood Dendritic Cell Antigen Research Articles

Interactions that define the arrangement of sugar-binding sites in BDCA-2 and dectin-2 dimers.

The sugar-binding receptors dectin-2 and blood dendritic cell antigen 2 (BDCA-2) bind oligosaccharide ligands through extracellular carbohydrate-recognition domains (CRDs) and initiate intracellular signaling through Fc receptor γ adapters (FcRγ). Dectin-2 stimulates macrophages in response to pathogen binding while BDCA-2 modulates cytokine production in plasmacytoid dendritic cells. The oligomeric states of these receptors and the orientations of their CRDs have been investigated by analysis of a naturally occurring disulfide-bonded variant of BDCA-2 and by replacement of transmembrane domains with N-terminal dimerization domains to create extracellular domain dimers of both dectin-2 and BDCA-2. Analysis of these constructs, as well as previously described crystal structures of the CRDs from these proteins and a novel structure of an extended version of the extracellular domain of dectin-2, showed that there is only limited interaction of the CRDs in the dimers, but interactions can be stabilized by the presence of the neck region. The resulting orientation of sugar-binding sites in the dimers would favor crosslinking of multiple dimers by oligosaccharide ligands, causing clustering of FcRγ to initiate signaling.

A type I interferon regulatory network for human plasmacytoid dendritic cells based on heparin, membrane-bound and soluble BDCA-2

Plasmacytoid dendritic cells (pDCs) produce type I interferons (IFNs) after sensing viral/bacterial RNA or DNA by toll-like receptor (TLR) 7 or TLR9, respectively. However, aberrant pDCs activation can cause adverse effects on the host and contributes to the pathogenesis of type I IFN-related autoimmune diseases. Here, we show that heparin interacts with the human pDCs-specific blood dendritic cell antigen 2 (BDCA-2) but not with related lectins such as DCIR or dectin-2. Importantly, BDCA-2-heparin interaction depends on heparin sulfation and receptor glycosylation and results in inhibition of TLR9-driven type I IFN production in primary human pDCs and the pDC-like cell line CAL-1. This inhibition is mediated by unfractionated and low-molecular-weight heparin, as well as endogenous heparin from plasma, suggesting that the local blood environment controls the production of IFN-α in pDCs. Additionally, we identified an activation-dependent soluble form of BDCA-2 (solBDCA-2) in human plasma that functions as heparin antagonist and thereby increases TLR9-driven IFN-α production in pDCs. Of importance, solBDCA-2 levels in the serum were increased in patients with scrub typhus (an acute infectious disease caused by Orientia tsutsugamushi) compared to healthy control subjects and correlated with anti-dsDNA antibodies titers. In contrast, solBDCA-2 levels in plasma from patients with bullous pemphigoid or psoriasis were reduced. In summary, this work identifies a regulatory network consisting of heparin, membrane-bound and solBDCA-2 modulating TLR9-driven IFN-α production in pDCs. This insight into pDCs function and regulation may have implications for the treatment of pDCs-related autoimmune diseases.

An immunomodulatory antibody-drug conjugate targeting BDCA2 strongly suppresses plasmacytoid dendritic cell function and glucocorticoid responsive genes.

Blood dendritic cell antigen 2 (BDCA2) is exclusively expressed on plasmacytoid dendritic cells (pDCs) whose uncontrolled production of type I IFN (IFN-I) is crucial in pathogenesis of SLE and other autoimmune diseases. Although anti-BDCA2 antibody therapy reduced disease activity in SLE patients, its clinical efficacy needs further improvement. We developed a novel glucocorticoid receptor agonist and used it as a payload to conjugate with an anti-BDCA2 antibody to form an BDCA2 antibody-drug conjugate (BDCA2-ADC). The activation of BDCA2-ADC was evaluated in vitro. Inhibitory activity of BDCA2-ADC was evaluated in peripheral blood mononuclear cells or in purified pDCs under ex vivo toll-like receptor agonistic stimulation. The global gene regulation in purified pDCs was analysed by RNA-seq. The antigen-dependent payload delivery was measured by reporter assay. The BDCA2-ADC molecule causes total suppression of IFNα production and broader inhibition of inflammatory cytokine production compared with the parental antibody in human pDCs. Global gene expression analysis confirmed that the payload and antibody acted synergistically to regulate both type I IFN signature genes and glucocorticoid responsive genes in pDCs. Taken together, these data suggest dual mechanisms of BDCA2-ADC on pDCs and the potential for BDCA2-ADC to be the first ADC treatment for SLE in the world and a better treatment option than anti-BDCA2 antibody for SLE patients.

Polysaccharides from Astragalus membranaceus elicit T cell immunity by activation of human peripheral blood dendritic cells

Astragalus membranaceus is a widely used herbal medicine in Asia. It has been recognized as possessing various biological properties, however, studies on the activity of the A. membranaceus polysaccharide (AMP), a major component of A. membranaceus, on human peripheral blood dendritic cells (PBDCs) have not been thoroughly investigated. In this study, we found that AMP induced changes in dendritic morphology and the upregulation of activation marker expression and inflammatory cytokine production in human blood monocyte-derived dendritic cells (MDDCs). The AMP promoted the activation of both blood dendritic cell antigen 1+ (BDCA1+) and BDCA3+ PBDCs. AMP-induced secretion of cytokines in the peripheral blood mononuclear cells (PBMCs) was mainly due to PBDCs. Finally, activated BDCA1+ and BDCA3+ PBDCs by AMP elicited proliferation and activation of autologous T cells, respectively. Hence, these data demonstrated that AMPs could activate dendritic and T cells in human blood, and may provide a new direction for the application of AMPs in the regulation of human immunity.

Research in Brief

Litifilimab, a humanised monoclonal antibody against blood dendritic cell antigen 2 (BDCA2), might be an effective treatment for systemic lupus erythematosus (SLE). In a phase 2 trial, Richard Furie and colleagues randomly assigned 132 participants with SLE to receive litifilimab 50 mg (n=6), 150 mg (n=6), or 450 mg (n=64), or placebo (n=56) subcutaneously at weeks 0, 2, 4, 8, 12, 16, and 20. Among the 102 participants who received 450 mg litifilimab or placebo and who had at least four tender joints and at least four swollen joints at baseline, the least-squares mean change in the total number of active joints from baseline to week 24 (the primary outcome) was –15·0 (SD 1·2) on litifilimab versus –11·6 (1·3) on placebo (mean difference –3·4; 95% CI –6·7 to –0·2; p=0·04).

Imaging Mass Cytometry Reveals Predominant Innate Immune Signature and Endothelial-Immune Cell Interaction in Juvenile Myositis Compared to Lupus Skin.

Cutaneous inflammation can signal disease in juvenile dermatomyositis (DM) and childhood-onset systemic lupus erythematosus (cSLE), but we do not fully understand cellular mechanisms of cutaneous inflammation. In this study, we used imaging mass cytometry to characterize cutaneous inflammatory cell populations and cell-cell interactions in juvenile DM as compared to cSLE. We performed imaging mass cytometry analysis on skin biopsy samples from juvenile DM patients (n=6) and cSLE patients (n=4). Tissue slides were processed and incubated with metal-tagged antibodies for CD14, CD15, CD16, CD56, CD68, CD11c, HLA-DR, blood dendritic cell antigen 2, CD20, CD27, CD138, CD4, CD8, E-cadherin, CD31, pan-keratin, and type I collagen. Stained tissue was ablated, and raw data were acquired using the Hyperion imaging system. We utilized the Phenograph unsupervised clustering algorithm to determine cell marker expression and permutation test by histoCAT to perform neighborhood analysis. We identified 14 cell populations in juvenile DM and cSLE skin, including CD14+ and CD68+ macrophages, myeloid and plasmacytoid dendritic cells (pDCs), CD4+ and CD8+ T cells, and B cells. Overall, cSLE skin had a higher inflammatory cell infiltrate, with increased CD14+ macrophages, pDCs, and CD8+ T cells and immune cell-immune cell interactions. Juvenile DM skin displayed a stronger innate immune signature, with a higher overall percentage of CD14+ macrophages and prominent endothelial cell-immune cell interaction. Our findings identify immune cell population differences, including CD14+ macrophages, pDCs, and CD8+ T cells, in juvenile DM skin compared to cSLE skin, and highlight a predominant innate immune signature and endothelial cell-immune cell interaction in juvenile DM, providing insight into candidate cell populations and interactions to better understand disease-specific pathophysiology.

Trial of Anti-BDCA2 Antibody Litifilimab for Systemic Lupus Erythematosus

BackgroundAntibody-binding of blood dendritic cell antigen 2 (BDCA2), which is expressed exclusively on plasmacytoid dendritic cells, suppresses the production of type I interferon that is involved in the pathogenesis of systemic lupus erythematosus (SLE). The safety and efficacy of subcutaneous litifilimab, a humanized monoclonal antibody that binds to BDCA2, in patients with SLE have not been extensively studied.MethodsWe conducted a phase 2 trial of litifilimab involving participants with SLE. The initial trial design called for randomly assigning participants to receive litifilimab (at a dose of 50, 150, or 450 mg) or placebo administered subcutaneously at weeks 0, 2, 4, 8, 12, 16, and 20, with the primary end point of evaluating cutaneous lupus activity. The trial design was subsequently modified; adults with SLE, arthritis, and active skin disease were randomly assigned to receive either litifilimab at a dose of 450 mg or placebo. The revised primary end point was the change from baseline in the total number of active joints (defined as the sum of the swollen joints and the tender joints) at week 24. Secondary end points were changes in cutaneous and global disease activity. Safety was also assessed.ResultsA total of 334 adults were assessed for eligibility, and 132 underwent randomization (64 were assigned to receive 450-mg litifilimab, 6 to receive 150-mg litifilimab, 6 to receive 50-mg litifilimab, and 56 to receive placebo). The primary analysis was conducted in the 102 participants who had received 450-mg litifilimab or placebo and had at least four tender and at least four swollen joints. The mean (±SD) baseline number of active joints was 19.0±8.4 in the litifilimab group and 21.6±8.5 in the placebo group. The least-squares mean (±SE) change from baseline to week 24 in the total number of active joints was –15.0±1.2 with litifilimab and –11.6±1.3 with placebo (mean difference, –3.4; 95% confidence interval, –6.7 to –0.2; P=0.04). Most of the secondary end points did not support the results of the analysis of the primary end point. Receipt of litifilimab was associated with adverse events, including two cases of herpes zoster and one case of herpes keratitis.ConclusionsIn a phase 2 trial involving participants with SLE, litifilimab was associated with a greater reduction from baseline in the number of swollen and tender joints than placebo over a period of 24 weeks. Longer and larger trials are required to determine the safety and efficacy of litifilimab for the treatment of SLE. (Funded by Biogen; LILAC ClinicalTrials.gov number, NCT02847598.)

Research in Brief

Litifilimab, a humanised monoclonal antibody against blood dendritic cell antigen 2 (BDCA2), might be an effective treatment for cutaneous lupus erythematosus, according to a phase 2 trial by Victoria Werth and colleagues. 132 participants with cutaneous lupus erythematosus, with or without systemic manifestations, were randomly assigned, stratified by Cutaneous Lupus Erythematosus Disease Area and Severity Index–Activity (CLASI-A) score and presence of discoid lupus erythematosus, to receive 50 mg (n=26), 150 mg (n=25), or 450 mg (n=48) subcutaneous litifilimab or placebo (n=33) at weeks 0, 2, 4, 8, and 12. At week 16, the least-squares mean differences in change in CLASI-A score from baseline between litifilimab and placebo were −24·3% (95% CI −43·7 to −4·9) for 50 mg litifilimab, −33·4% (−52·7 to −14·1) for 150 mg, and −28·0% (−44·6 to −11·4) for 450 mg. A best-fitting dose–response model across the three drug doses showed a significant effect of litifilimab compared with placebo. Three cases of hypersensitivity, three oral herpes infections, and one herpes zoster infection occurred in patients taking litifilimab.

Blastic Plasmacytoid Dendritic Cell Neoplasm: Progress in Cell Origin, Molecular Biology, Diagnostic Criteria and Therapeutic Approaches

SummaryBlastic plasmacytoid dendritic cell neoplasm (BPDCN) is a rare hematological malignancy characterized by recurrent skin nodules, an aggressive clinical course with rapid involvement of hematological organs, and a poor prognosis with poor overall survival. BPDCN is derived from plasmacytoid dendritic cells (pDCs) and its pathogenesis is unclear. The tumor cells show aberrant expression of CD4, CD56, interleukin-3 receptor alpha chain (CD123), blood dendritic cell antigen 2 (BDCA 2/CD303), blood dendritic cell antigen 4 (BDCA4) and transcription factor (E protein) E2-2 (TCF4). The best treatment drugs are based on experience by adopting those used for either leukemia or lymphoma. Relapse with drug resistance generally occurs quickly. Stem cell transplantation after the first complete remission is recommended and tagraxofusp is the first targeted therapy. In this review, we summarize the differentiation of BPDCN from its cell origin, its connection with normal pDCs, clinical characteristics, genetic mutations and advances in treatment of BPDCN. This review provides insights into the mechanisms of and new therapeutic approaches for BPDCN.

POS0698 BIIB059 DEMONSTRATED A CONSISTENT THERAPEUTIC EFFECT ON SRI-4 RESPONSE ACROSS SUBGROUPS OF PARTICIPANTS WITH SYSTEMIC LUPUS ERYTHEMATOSUS IN THE LILAC PHASE 2 STUDY

Background:Type I interferons and other inflammatory mediators derived from plasmacytoid dendritic cells (pDCs) are implicated in systemic lupus erythematous (SLE) pathology. BIIB059 is a humanized monoclonal antibody that targets blood dendritic cell antigen 2 (BDCA2), a pDC-specific receptor. The binding of BIIB059 to BDCA2 leads to rapid internalization of BDCA2 from the surface of pDCs and subsequent inhibition of interferon, cytokine, and chemokine production. In Part A of the 2-part, phase 2 LILAC study (NCT02847598), BIIB059 significantly reduced SLE activity, as evidenced by reduced total active joint count (primary endpoint) and higher SLE Responder Index (SRI-4)1 response (a secondary endpoint) versus placebo.2Objectives:To evaluate SRI-4 response for BIIB059 versus placebo at Week 24 in SLE participant subgroups.Methods:Enrollment in LILAC Part A was open to adults fulfilling ≥ 4 of 11 revised 1997 ACR criteria for classification of SLE, with ≥ 4 tender and ≥ 4 swollen joints, active skin disease, and positive lupus antibodies. Participants were randomized to receive either BIIB059 450 mg or placebo subcutaneously every 4 weeks for 20 weeks (with an additional dose at Week 2). SRI-4 response at Week 24 was analyzed in subgroups, though analyses were limited by small sample sizes and were not powered for statistical testing.Results:In LILAC Part A, 64 and 56 participants were dosed with BIIB059 450 mg and placebo, respectively. At week 24, SRI-4 response rate was observed in favor of BIIB059 regardless of the baseline disease activity, such as SLEDAI-2K <10 versus ≥10, presence of BILAG-2004 grade A or B arthritis, oral corticosteroid usage, positivity for anti-ds DNA autoantibody and/or complement status, with point estimates for least-squares mean differences as well as corresponding 95% CIs consistently favoring BIIB059 (Figure 1). The incidence of adverse events in the overall study population was similar between the placebo and BIIB059 groups.2Conclusion:BIIB059 treatment was associated with greater SRI-4 response rate, consistent among different subgroups of baseline disease activity as measured by SLEDAI-2K and BILAG-2004, glucocorticoid dosage, and serology. These findings provide additional evidence of the potential benefit of BIIB059 for the treatment of patients with active SLE.

Human Peripheral Blood Dendritic Cell and T Cell Activation by Codium fragile Polysaccharide.

Natural polysaccharides exhibit an immunostimulatory effect with low toxicity in humans and animals. It has shown that polysaccharide extracted from Codium fragile (CFP) induces anti-cancer immunity by dendritic cell (DC) activation, while the effect of CFP has not examined in the human immune cells. In this study, we found that CFP promoted the upregulation of CD80, CD83 and CD86 and major histocompatibility complex (MHC) class I and II in human monocyte-derived dendritic cells (MDDCs). In addition, CFP induced the production of proinflammatory cytokines in MDDCs. Moreover, CFP directly induced the activation of Blood Dendritic Cell Antigen (BDCA)1+ and BDCA3+ subsets of human peripheral blood DCs (PBDCs). The CFP-stimulated BDCA1+ PBDCs further promoted activation and proliferation of syngeneic CD4 T cells. The CFP-activated BDCA3+ PBDCs activated syngeneic CD8 T cells, which produced cytotoxic mediators, namely, cytotoxic T lymphocytes. These results suggest that CFP may be a candidate molecule for enhancing immune activation in humans.

Identification of the immunological profile in rejection-free heart transplantation

BackgroundTolerance induction following organ transplantation can be achieved by adoptive cell transfer of regulatory T-cells (Tregs) or dendritic cells (DCs). However, the target immunological profile is unknown. The present study aimed to identify an immunological profile connected to tolerance induction following heart transplantation (HTx). MethodsBlood samples of long-term rejection-free HTx patients (LT-HTx, n = 20) and patients on the HTx waiting list (pre-HTx, n = 20) were compared. Flow cytometric and multiplex analyses of DCs, Tregs, subsets of both cell types and serum cytokines were performed. Furthermore, principle component and cluster analysis was used to identify a target immunological profile using a multiparametric dataset. ResultsPlasmacytoid DCs expressing blood DC antigen (BDCA) 2 and BDCA4 were significantly increased in LT-HTx patients (BDCA2+: 29.4 ± 10.1%, p = .022; BDCA4+: 26.4 ± 9.3%, p = .008) compared to pre-HTx patients (BDCA2+: 22.8 ± 7.2%; BDCA4+: 18.9 ± 7.4%). The percentage of total Tregs and of their CD62L+ subset was reduced in LT-HTx patients (%Tregs: 9.1 ± 3.7%, p = .026; %CD62L+: 85.1 ± 11.9%, p = .009) compared to pre-HTx patients (%Tregs: 11.8 ± 3.6%; %CD62L+: 93.3 ± 4.5%). LT-HTx patients showed different cytokine levels than pre-HTx patients. Principle component and cluster analysis revealed that the total DCs, BDCA2+ and BDCA4+ DCs and CD147+ Tregs had the strongest influence to distinguish among long-term rejection-free and pre-HTx patients. ConclusionIn conclusion, we defined the immune status of pre-HTx patients and the target immunological profile of LT-HTx patients. These data may help to establish a monitoring tool that is based on a multiparametric dataset.

Predictive value of plasmacytoid dendritic cells and Toll-like receptor-9 regarding the treatment efficacy of interferon-α in HBeAg-positive chronic hepatitis B patients.

Hepatitis B virus (HBV) infection represents a public health threat and a challenge for the medical community. Untimely treatment may lead to liver cirrhosis and even liver cancer. At present, the major treatment for hepatitis B e antigen (HBeAg)-positive chronic hepatitis B patients includes administration of interferon-α (IFN-α), which has anti-viral and immunomodulatory effects. Plasmacytoid dendritic cells (pDCs) and Toll-like receptor-9 (TLR-9) have important roles in anti-viral therapy. However, their predictive value regarding the efficacy of IFN-α treatment of HBeAg-positive chronic hepatitis B (CHB) patients has remained elusive. A total of 178 patients with CHB and HBeAg-positive status, who had not received any previous anti-HBV treatment, were enrolled in the present study. All patients were treated with IFN-α. HBV DNA load, hepatitis B surface antigen and serum alanine aminotransferase were measured prior to and following 48 weeks of treatment. According to HBV levels, the patients were divided into a response group and non-responders group. To determine the amount of pDCs, blood dendritic cell antigen 2 (BDCA-2)- and immunoglobulin-like transcript 7 (ILT7)-expressing cells in liver biopsies were detected using immunohistochemistry. TLR-9 expression in peripheral blood mononuclear cells was determined by reverse transcription-quantitative PCR. There was no significant difference in the proportion of pDCs (BDCA-2; ILT7) and TLR-9 mRNA expression between the response group and the non-responders group prior to IFN-α treatment. After IFN-α treatment, BDCA-2, ILT7 and TLR-9 mRNA expression was obviously increased in the response group compared with that in the non-responders group (P<0.05). Increased expression of BDCA-2, ILT7 and TLR-9 mRNA was negatively correlated with HBV DNA (P<0.05). Increased levels of pDCs and TLR-9 were negatively correlated with HBV DNA, and were thus capable of predicting the IFN-α treatment response in patients with CHB and HBeAg-positive status.

Specific Immune Tolerance Response Triggered by Extracorporal Photopheresis after Heart Transplantation

Purpose Extracorporeal photopheresis (ECP) treatment following heart transplantation (HTx) may induce transplant tolerance. A monitoring tool for successful ECP treatment would allow determination of patient-specific duration and frequency of ECP therapy. We evaluated the immunological effects of ECP long-term therapy. Methods ECP therapy started 3 months after HTx (n=19). ECP therapy included a total of 5 cycles, each on 2 consecutive days every 4-8 wks over a mean period of 8.4±0.4 months. Blood samples were drawn prior to ECP (baseline), before each cycle and 4 months after the last cycle. Flow cytometry analysis of all samples specific subsets of both dendritic cells (DCs) and regulatory T cells (Tregs), which are involved in the immune tolerance response. Results Compared to baseline, ECP reduced CD4+ T cells (baseline: 23.2±9.2%, cycle 5: 23.6±3.4%, after ECP: 16.3±7.4%, p 0.01). DC subsets expressing blood dendritic cell antigen (BDCA) 1, 2, 3 or 4 increased during ECP but returned to baseline levels 4 months after ECP (BDCA1prior ECP: 42.1±13.4%, BDCA1cycle 5: 53.4±3.7%, BDCA1after ECP: 43.6±8.7%, BDCA2prior ECP: 20.2±9.1%, BDCA2cycle 5: 32.6±3.2%, BDCA2after ECP: 24.0±6.0%, BDCA3prior ECP: 73.2±17.8%, BDCA3cycle 5: 90.4±5.0%, BDCA3after ECP: 75.4±12.4%, BDCA4prior ECP: 21.6±10.0%, BDCA4cycle 5: 31.7±3.9%, BDCA4after ECP: 21.4±5.9%, all p≤0.01). Conclusion Our results suggest that a combined increase of BDCA1+, 2+, 3+ and 4+ DCs, total Tregs and the highly suppressive CD39+ Treg subset could be useful to monitor ECP therapy after HTx, and, to establish an individual ECP treatment schedule. Future studies need to explore ECP effects on immune cells in the context with clinical events like rejection or infection.

A promising approach to targeting type 1 IFN in systemic lupus erythematosus.

Despite advances in understanding systemic lupus erythematosus (SLE) pathogenesis, most clinical trials of new targeted therapies have been met with disappointment. The type I IFN pathway is believed to play an important role in SLE, and the proposed involvement of this pathway helps explain the frustration behind the failure at targeting either IFN-α or the type 1 IFN receptor itself. In this issue of the JCI, Furie et al. report on an intriguing phase 1b study that demonstrates an approach for inhibiting this pathway in the skin using an mAB (BIIB059) that targets the blood DC antigen 2 (BDCA-2) receptor on plasmacytoid DCs (pDCs). BIIB059 decreased IFN expression and improved cutaneous lupus disease activity, with a favorable safety profile. Whether or not this strategy will be effective in managing SLE in other organs remains unanswered. However, these results suggest that closing the door on targeting the type 1 IFN pathway in SLE may be premature and highlight the emerging question of whether an organ-specific approach toward lupus trials and treatment should be the wave of the future.

A pre-clinical quantitative model predicts the pharmacokinetics/pharmacodynamics of an anti-BDCA2 monoclonal antibody in humans.

BIIB059 is a novel humanized monoclonal antibody (mAb) that is currently under development for the treatment of Systemic Lupus Erythematosus and Cutaneous Lupus Erythematosus. BIIB059 is targeted against the blood dendritic cell antigen 2 (BDCA2), a receptor exclusively expressed on the surface of plasmacytoid dendritic cells (pDCs). Herein, we utilized pre-clinical pharmacokinetic (PK) and pharmacodynamic (PD) data to develop a non-human primate (NHP) model and to address whether the NHP model can be successfully scaled to predict the human PK/PD. In particular, PK data from 17 cynomolgus monkeys were utilized for PK model development, wherein BIIB059 was administered intravenously (1 and 10mg/kg single-dosing and 5mg/kg multiple-dosing) or subcutaneously (0.2 and 7.5mg/kg single-dosing). Additionally, PD data (BDCA2 receptor density on pDCs) from 6 cynomolgus monkeys were used for the development of the PD model. The developed NHP two-compartment PK model, linked with an indirect response PD model, was subsequently scaled to humans by combining traditional allometric PK scaling with sensitivity-analysis-driven scaling of the PD. The scaled PK/PD model was then used to simulate the human PK/PD for different dose levels. When clinical data from the BIIB059 Phase I study became available, they were used to evaluate the predictability of the scaled PK/PD model and the model simulations were in agreement with the clinical data. Therefore, the presented approach is suggested to be employed in scaling pre-clinical mAb models to support the selection of safe first-in-human doses and, more broadly, the prediction of PK/PD in the clinic.

Identification of serum glycoprotein ligands for the immunomodulatory receptor blood dendritic cell antigen 2.

Blood dendritic cell antigen 2 (BDCA-2) is a C-type lectin found on the surface of plasmacytoid dendritic cells. It functions as a glycan-binding receptor that downregulates the production of type I interferons and thus plays a role in oligosaccharide-mediated immunomodulation. The carbohydrate recognition domain in BDCA-2 binds selectively to galactose-terminated bi-antennary glycans. Because the plasmacytoid dendritic cells function in a plasma environment rich in glycoproteins, experiments have been undertaken to identify endogenous ligands for blood dendritic cell antigen 2. A combination of blotting, affinity chromatography and proteomic analysis reveals that serum glycoprotein ligands for BDCA-2 include IgG, IgA and IgM. Compared to binding of IgG, which was previously described, IgA and IgM bind with higher affinity. The association constants for the different subclasses of immunoglobulins are below and roughly proportional to the serum concentrations of these glycoprotein ligands. Binding to the other main serum glycoprotein ligand, α2-macroglobulin, is independent of whether this protease inhibitor is activated. Binding to all of these glycoprotein ligands is mediated predominantly by bi-antennary glycans in which each branch bears a terminal galactose residue. The different affinities of the glycoprotein ligands reflect the different numbers of these galactose-terminated glycans and their degree of exposure on the native glycoproteins. The results suggest that normal serum levels of immunoglobulins could downmodulate interferon stimulation of further antibody production.

Packed Red Blood Cell Transfusion Modulates Myeloid Dendritic Cell Activation and Inflammatory Response In Vitro.

Transfusion of packed red blood cells (PRBCs) modulates patients' immune responses and clinical outcomes; however, the underpinning mechanism(s) remain unknown. The potential for PRBC to modulate myeloid dendritic cells (mDC) and blood DC antigen 3 was assessed using an in vitro transfusion model. In parallel, to model processes activated by viral or bacterial infection, toll-like receptor agonists polyinosinic:polycytidylic acid or lipopolysaccharide were added. Exposure to PRBC upregulated expression of CD83 and downregulated CD40 and CD80 on both DC subsets, and it suppressed production of interleukin (IL)-6, IL-8, IL-12, tumor necrosis factor-α, and interferon-gamma-inducible protein-10 by these cells. Similar effects were observed when modeling processes activated by concurrent infection. Furthermore, exposure to PRBC at date of expiry was associated with more pronounced effects in all assays. Our study suggests PRBC have an impact on recipient DC function, which may result in failure to establish an appropriate immune response, particularly in patients with underlying infection.

The MEK1/2-ERK Pathway Inhibits Type I IFN Production in Plasmacytoid Dendritic Cells.

Recent studies have reported that the crosslinking of regulatory receptors (RRs), such as blood dendritic cell antigen 2 (BDCA-2) (CD303) or ILT7 (CD85g), of plasmacytoid dendritic cells (pDCs) efficiently suppresses the production of type I interferons (IFN-I, α/β/ω) and other cytokines in response to toll-like receptor 7 and 9 (TLR7/9) ligands. The exact mechanism of how this B cell receptor (BCR)-like signaling blocks TLR7/9-mediated IFN-I production is unknown. Here, we stimulated BCR-like signaling by ligation of RRs with BDCA-2 and ILT7 mAbs, hepatitis C virus particles, or BST2 expressing cells. We compared BCR-like signaling in proliferating pDC cell line GEN2.2 and in primary pDCs from healthy donors, and addressed the question of whether pharmacological targeting of BCR-like signaling can antagonize RR-induced pDC inhibition. To this end, we tested the TLR9-mediated production of IFN-I and proinflammatory cytokines in pDCs exposed to a panel of inhibitors of signaling molecules involved in BCR-like, MAPK, NF-ĸB, and calcium signaling pathways. We found that MEK1/2 inhibitors, PD0325901 and U0126 potentiated TLR9-mediated production of IFN-I in GEN2.2 cells. More importantly, MEK1/2 inhibitors significantly increased the TLR9-mediated IFN-I production blocked in both GEN2.2 cells and primary pDCs upon stimulation of BCR-like or phorbol 12-myristate 13-acetate-induced protein kinase C (PKC) signaling. Triggering of BCR-like and PKC signaling in pDCs resulted in an upregulation of the expression and phoshorylation of c-FOS, a downstream gene product of the MEK1/2-ERK pathway. We found that the total level of c-FOS was higher in proliferating GEN2.2 cells than in the resting primary pDCs. The PD0325901-facilitated restoration of the TLR9-mediated IFN-I production correlated with the abrogation of MEK1/2-ERK-c-FOS signaling. These results indicate that the MEK1/2-ERK pathway inhibits TLR9-mediated type I IFN production in pDCs and that pharmacological targeting of MEK1/2-ERK signaling could be a strategy to overcome immunotolerance of pDCs and re-establish their immunogenic activity.

Immunomodulatory effect of cryopreserved platelets: altered BDCA3+ dendritic cell maturation and activation in vitro.

Cryopreservation of platelets (PLTs) is useful in remote areas to overcome logistic problems associated with supply and can extend the shelf life to 2 years. During cryopreservation, properties of PLTs are modified. Whether changes in the cryopreserved PLT (CPP) product are associated with modulation of recipients' immune function is unknown. We aimed to characterize the immune profile of myeloid dendritic cells (mDCs) and the specialized blood DC antigen (BDCA)3+ subset after exposure to CPPs. Using an in vitro whole blood model of transfusion, the effect of CPPs on mDC and BDCA3+ DC surface antigen expression and inflammatory mediator production was examined using flow cytometry. In parallel, polyinosinic:polycytidylic acid (poly(I:C)) or lipopolysaccharide (LPS) was utilized to model processes activated in viral or bacterial infection, respectively. Cryopreserved PLTs had minimal impact on mDC responses but significantly modulated BDCA3+ DC responses in vitro. Exposure to CPPs alone up regulated BDCA3+ DC CD86 expression and suppressed interleukin (IL)-8, tumor necrosis factor (TNF)-α, and interferon-γ inducible protein (IP)-10 production. In both models of infection-related processes, exposure to CPPs down regulated BDCA3+ DC expression of CD40, CD80, and CD83 and suppressed BDCA3+ DC production of IL-8, IL-12, and TNF-α. CPPs suppressed CD86 expression in the presence of LPS and IP-10 and IL-6 production with poly(I:C). Cryopreserved PLTs may be immunosuppressive, and this effect is more evident when processes associated with infection are concurrently activated, especially for BDCA3+ DCs. This suggests that transfusion of CPPs in patients with infection may result in impaired BDCA3+ DC responses.