Blood Dendritic Cells Research Articles

P-type pilus PapG protein elicits toll-like receptor 2-mediated immune activation during cancer immunotherapy

The immune activation ability of FimH, an adhesion protein in pili of Escherichia coli (E. coli), has been recently reported. However, studies on the immune activity of PapG, another major pili terminal protein, have not been well explored. In this study, the immune stimulatory effect of purified recombinant PapG was evaluated. PapG treatment promoted dramatic changes in dendritic morphology of the bone marrow-derived dendritic cells (BMDCs) and induced upregulation of co-stimulatory molecule levels, major histocompatibility complex (MHC) I and II expression, and pro-inflammatory cytokine production in BMDCs. To identify the stimulatory receptor of PapG, an in silico study was performed. PapG exhibited strong binding affinity with murine toll-like receptor 2 (TLR2). In addition, PapG-induced activation of splenic DC and its subsets was unsuccessful in TLR2-knock out mice. Combination of PapG and ovalbumin (OVA) elicited OVA-specific T cell proliferation and cytokine production and cytotoxicity that consequently promoted anti-cancer immune responses against OVA-expressing B16 melanoma. Furthermore, PapG treatment induced activation of peripheral blood DCs and its subsets in humans in a TLR2 dependent manner. PapG-stimulated human conventional DC2 promoted syngeneic T cell proliferation and activation. The findings of this study demonstrated that PapG could be a useful immune stimulator for immunotherapy against cancer.

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.

Unlocking the secrets of NPSLE: the role of dendritic cell-secreted CCL2 in blood-brain barrier disruption.

This study employed RNA-seq technology and meta-analysis to unveil the molecular mechanisms of neuropsychiatric systemic lupus erythematosus (NPSLE) within the central nervous system. Downloaded transcriptomic data on systemic lupus erythematosus (SLE) from the Gene Expression Omnibus (GEO) and analyzed differential genes in peripheral blood samples of NPSLE patients and healthy individuals. Employed WGCNA to identify key genes related to cognitive impairment and validated findings via RNA-seq. Conducted GO, KEGG, and GSEA analyses, and integrated PPI networks to explore gene regulatory mechanisms. Assessed gene impacts on dendritic cells and blood-brain barrier using RT-qPCR, ELISA, and in vitro models. Public databases and RNA-seq data have revealed a significant upregulation of CCL2 (C-C motif chemokine ligand 2) in the peripheral blood of both SLE and NPSLE patients, primarily secreted by mature dendritic cells. Furthermore, the secretion of CCL2 by mature dendritic cells may act through the RSAD2-ISG15 axis and is associated with the activation of the NLRs (Nod Like Receptor Signaling Pathway) signaling pathway in vascular endothelial cells. Subsequent in vitro cell experiments confirmed the high expression of CCL2 in peripheral blood dendritic cells of NPSLE patients, with its secretion being regulated by the RSAD2-ISG15 axis and inducing vascular endothelial cell pyroptosis through the activation of the NLRs signaling pathway. Clinical trial results ultimately confirmed that NPSLE patients exhibiting elevated CCL2 expression also experienced cognitive decline. The secretion of CCL2 by dendritic cells induces pyroptosis in vascular endothelial cells, thereby promoting blood-brain barrier damage and triggering cognitive impairment in patients with systemic lupus erythematosus.

Profound deficiencies in mature blood and bone marrow progenitor dendritic cells in Chronic Lymphocyticcytic Leukemia patients.

Chronic lymphocytic leukemia (CLL) patients are immunocompromised and highly vulnerable to serious recurrent infections. Conventional dendritic cells (cDCs) and plasmacytoid DCs (pDCs) are principal sensors of infection and are essential in orchestrating innate and adaptive immune responses to resolve infection. This study identified significant deficiencies in six functionally distinct DC subsets in blood of untreated CLL (UT-CLL) patients and selective normalization of pDCs in response to acalabrutinib (a Bruton tyrosine kinase inhibitor) therapy. DCs are continuously replenished from hematopoiesis in bone marrow (BM). Four BM developmental intermediates that give rise to cDCs and pDCs were examined and significant reductions of these were identified in UT-CLL patients supporting a precursor/progeny relationship. The deficiencies in blood DCs and BM DC progenitors were significantly associated with alterations in the Flt3/FL signaling pathway critical to DC development and function. Regarding clinical parameter, cDC subset deficiencies are associated with adverse prognostic indicators of disease progression, including IGHV mutation, CD49d, CD38, and ZAP-70 status. Importantly, UT-CLL patients with shared DC subset deficiencies had shorter time-to-first treatment (TTFT), uncovering a profound alteration in innate immunity with the potential to instruct therapeutic decision-making.

The Immune Response in Two Models of Traumatic Injury of the Immature Brain.

Traumatic brain injury (TBI) can cause major disability and increases the risk of neurodegeneration. Post-TBI, there is infiltration of peripheral myeloid and lymphoid cells; there is limited information on the peripheral immune response post-TBI in the immature brain-where injury may interfere with neurodevelopment. We carried out two injury types in juvenile mice: invasive TBI with a controlled cortical impact (CCI) and repetitive mild TBI (rmTBI) using weight drop injury and analysed the response at 5- and 35-days post-injury. In the two models, we detected the brain infiltration of immune cells (e.g., neutrophils, monocytes, dendritic cells, CD4+ T cells, and NK cells). There were increases in macrophages, neutrophils, and dendritic cells in the spleen, increases in dendritic cells in blood, and increases in CD8+ T cells and B cells in lymph nodes. These results indicate a complex peripheral immune response post-TBI in the immature brain, with differences between an invasive injury and a repetitive mild injury.

Endothelial biomarkers (Von willebrand factor, BDCA3, urokinase) as predictors of mortality in COVID-19 patients: cohort study

BackgroundSARS-CoV-2 is a systemic disease that affects endothelial function and leads to coagulation disorders, increasing the risk of mortality. Blood levels of endothelial biomarkers such as Von Willebrand Factor (VWF), Thrombomodulin or Blood Dendritic Cell Antigen-3 (BDCA3), and uUokinase (uPA) increase in patients with severe disease and can be prognostic indicators for mortality. Therefore, the aim of this study was to determine the effect of VWF, BDCA3, and uPA levels on mortality.MethodsFrom May 2020 to January 2021, we studied a prospective cohort of hospitalized adult patients with polymerase chain reaction (PCR)-confirmed COVID-19 with a SaO2 ≤ 93% and a PaO2/FiO2 ratio < 300. In-hospital survival was evaluated from admission to death or to a maximum of 60 days of follow-up with Kaplan-Meier survival curves and Cox proportional hazard models as independent predictor measures of endothelial dysfunction.ResultsWe recruited a total of 165 subjects (73% men) with a median age of 57.3 ± 12.9 years. The most common comorbidities were obesity (39.7%), hypertension (35.4%) and diabetes (30.3%). Endothelial biomarkers were increased in non-survivors compared to survivors. According to the multivariate Cox proportional hazard model, those with an elevated VWF concentration ≥ 4870 pg/ml had a hazard ratio (HR) of 4.06 (95% CI: 1.32–12.5) compared to those with a lower VWF concentration adjusted for age, cerebrovascular events, enoxaparin dose, lactate dehydrogenase (LDH) level, and bilirubin level. uPA and BDCA3 also increased mortality in patients with levels ≥ 460 pg/ml and ≥ 3600 pg/ml, respectively.ConclusionThe risk of mortality in those with elevated levels of endothelial biomarkers was observable in this study.

A Minimal PBPK/PD Model with Expansion-Enhanced Target-Mediated Drug Disposition to Support a First-in-Human Clinical Study Design for a FLT3L-Fc Molecule.

FLT3L-Fc is a half-life extended, effectorless Fc-fusion of the native human FLT3-ligand. In cynomolgus monkeys, treatment with FLT3L-Fc leads to a complex pharmacokinetic/pharmacodynamic (PK/PD) relationship, with observed nonlinear PK and expansion of different immune cell types across different dose levels. A minimal physiologically based PK/PD model with expansion-enhanced target-mediated drug disposition (TMDD) was developed to integrate the molecule's mechanism of action, as well as the complex preclinical and clinical PK/PD data, to support the preclinical-to-clinical translation of FLT3L-Fc. In addition to the preclinical PK data of FLT3L-Fc in cynomolgus monkeys, clinical PK and PD data from other FLT3-agonist molecules (GS-3583 and CDX-301) were used to inform the model and project the expansion profiles of conventional DC1s (cDC1s) and total DCs in peripheral blood. This work constitutes an essential part of our model-informed drug development (MIDD) strategy for clinical development of FLT3L-Fc by projecting PK/PD in healthy volunteers, determining the first-in-human (FIH) dose, and informing the efficacious dose in clinical settings. Model-generated results were incorporated in regulatory filings to support the rationale for the FIH dose selection.

Novel highly immunogenic DNA vaccine regimen potently activates lymph nodes in macaques.

Abstract Background We evaluated a novel DNA vaccine delivery by LION™ nanoparticles for its ability to induce innate and adaptive immune responses in Rhesus macaques. Methods CoV-2 Spike or HIV Env DNA were formulated with a cationic nanocarrier (Lipid InOrganic Nanoparticle; LION™) administered via the IM route. Chemokine/cytokine and vaccine-specific responses were monitored in blood and lymph nodes (LN) by proteomics, flow, antibody assays. Results DNA/LION™ induced strong cellular and higher humoral responses than DNA/Electroporation and induced rapid (4-24hr) transient innate responses. Flow analysis showed activation of T, B, myeloid incl. dendritic cells in blood and their trafficking to LN with a strong increase of Tfh, Germinal Center Tfh CD4 and CD8 cells. Cell subsets were activated by cytokine networks incl. CXCL11, IL-7 and IL-15. GC-B cell activation directly correlated with increase of FLT3L and CXCL12 and importantly with antibody levels. Conclusion DNA/LION™ led to robust induction of cellular and humoral responses against different immunogens, serving as promising platform of effective nucleic acid vaccine delivery. Transient cytokine responses, incl. IL-15, resulted in strong activation of draining lymph nodes, and contributed to shaping adaptive (cellular and humoral) immunity. Our data showed rapid and coordinated cytokine responses to the vaccine and highlight the important role of innate responses to vaccination in modulating adaptive immunity.

Potency of Peripheral Blood- and Umbilical Cord Blood-derived Dendritic Cells and Their Secretomes as Vaccines for Cancer

Dendritic cell (DC) vaccines, as immunotherapy agents, can gather up and transport cancer-related antigens to T lymphocytes, activating anti-tumor effector responses. After being activated by DC, cytotoxic T lymphocyte cells (CTL) will secrete the cytolytic granzyme B that can effectively induce rapid apoptosis of target cells. On the other hand, DC also secrete several cytokines and a large number of exosomes, which together operate as a whole antigen-presenting entity. The efficacy of the vaccine’s treatment may be affected by the sources used for DC vaccines. Umbilical cord blood (UCB) from healthy donors can be employed when autologous cancer patient’s peripheral blood (PB) cannot be used as a source for isolating DC due to genetic abnormalities. Comparing UCB to other sources, there is a painless method of collecting sources as opposed to PB, which necessitates a venipuncture or leukapheresis procedure to isolate the blood. Many studies related to the use of PB-DC have been carried out, but research on potential comparisons between PB-DC and UCB-DC is still very limited. In this review, the potential of PB- and UCB-derived DC and their secretomes for cancer will be discussed.Keywords: dendritic cells, vaccines, umbilical cord blood, peripheral blood

Generation and characterization of mAb 61H9 against junctional adhesion molecule-a with potent antitumor activity.

Junctional adhesion molecule-A (JAM-A) is an adhesion molecule that exists on the surface of certain types of cells, including white blood cells, endothelial cells, and dendritic cells. In this study, the cDNA sequences of JAM-A-Fc were chemically synthesized with optimization for mammalian expression. Afterward, we analyzed JAM-A protein expression through transient transfection in HEK293 cell lines. Mice were immunized with JAM-A-Fc protein, and hybridoma was prepared by fusing myeloma cells and mouse spleen cells. Antibodies were purified from the hybridoma supernatant and four monoclonal strains were obtained and numbered 61H9, 70E5, 71A8, and 74H3 via enzyme-linked immunosorbent assay screening. Immunofluorescence staining assay showed 61H9 was the most suitable cell line for mAb production due to its fluorescence signal being the strongest. Flow cytometric analysis proved that 61H9 possessed high affinity. Moreover, antagonism of JAM-A mAb could attenuate the proliferative, migrative, and invasive abilities of ESCC cells and significantly inhibit tumor growth in mice. By examining hematoxylin-eosin staining mice tumor tissues, we found inflammatory cells infiltrated lightly in the anti-JAM-A group. The expression of BCL-2 and IκBα in the anti-JAM-A group were decreased in mice tumor tissues compared to the control group. Ultimately, a method for preparing high-yield JAM-A-Fc protein was created and a high affinity mAb against JAM-A with an antitumor effect was prepared.

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.

Exploiting Leishmania-Primed Dendritic Cells as Potential Immunomodulators of Canine Immune Response.

Dendritic cells (DCs) capture pathogens and process antigens, playing a crucial role in activating naïve T cells, bridging the gap between innate and acquired immunity. However, little is known about DC activation when facing Leishmania parasites. Thus, this study investigates in vitro activity of canine peripheral blood-derived DCs (moDCs) exposed to L. infantum and L. amazonensis parasites and their extracellular vesicles (EVs). L. infantum increased toll-like receptor 4 gene expression in synergy with nuclear factor κB activation and the generation of pro-inflammatory cytokines. This parasite also induced the expression of class II molecules of major histocompatibility complex (MHC) and upregulated co-stimulatory molecule CD86, which, together with the release of chemokine CXCL16, can attract and help in T lymphocyte activation. In contrast, L. amazonensis induced moDCs to generate a mix of pro- and anti-inflammatory cytokines, indicating that this parasite can establish a different immune relationship with DCs. EVs promoted moDCs to express class I MHC associated with the upregulation of co-stimulatory molecules and the release of CXCL16, suggesting that EVs can modulate moDCs to attract cytotoxic CD8+ T cells. Thus, these parasites and their EVs can shape DC activation. A detailed understanding of DC activation may open new avenues for the development of advanced leishmaniasis control strategies.

The CSF-1R inhibitor pexidartinib affects FLT3-dependent DC differentiation and may antagonize durvalumab effect in patients with advanced cancers.

Tumor-associated macrophages (TAMs) are a critical determinant of resistance to PD-1/PD-L1 blockade. This phase 1 study (MEDIPLEX, NCT02777710) investigated the safety and efficacy of pexidartinib, a CSF-1R-directed tyrosine kinase inhibitor (TKI), and durvalumab (anti-PD-L1) in patients with advanced colorectal and pancreatic carcinoma with the aim to enhance responses to PD-L1 blockade by eliminating CSF-1-dependent suppressive TAM. Forty-seven patients were enrolled. No unexpected toxicities were observed, one (2%) high microsatellite instability CRC patient had a partial response, and seven (15%) patients experienced stable disease as their best response. Increase of CSF-1 concentrations and decrease of CD14lowCD16high monocytes in peripheral blood mononuclear cells (PBMCs) confirmed CSF-1R engagement. Treatment decreased blood dendritic cell (DC) subsets and impaired IFN-λ/IL-29 production by type 1 conventional DCs in ex vivo TLR3-stimulated PBMCs. Pexidartinib also targets c-KIT and FLT3, both key growth factor receptors of DC development and maturation. In patients, FLT3-L concentrations increased with pexidartinib treatment, and AKT phosphorylation induced by FLT3-L ex vivo stimulation was abrogated by pexidartinib in human blood DC subsets. In addition, pexidartinib impaired the FLT3-L- but not GM-CSF-dependent generation of DC subsets from murine bone marrow (BM) progenitors in vitro and decreased DC frequency in BM and tumor-draining lymph node in vivo. Our results demonstrate that pexidartinib, through the inhibition of FLT3 signaling, has a deleterious effect on DC differentiation, which may explain the limited antitumor clinical activity observed in this study. This work suggests that inhibition of FLT3 should be considered when combining TKIs with immune checkpoint inhibitors.

Methods for generating the CD137L-DC-EBV-VAX anti-cancer vaccine.

Dendritic cells (DC) are professional antigen presenting cells (APCs) that can efficiently present captured antigens to cytotoxic T cells and initiate powerful antigen-specific responses. Therefore, DC have been explored for cancer immunotherapy. However, due to the scarcity of DCs in the peripheral blood, ex-vivo expansion is required to generate sufficient DCs before use. The majority of DC-based tumor vaccines utilize monocyte-derived DC (mo-DC) that are generated with GM-CSF and IL-4. Here, we describe the generation of a novel type of DC, CD137L-DC, which are generated from monocytes by stimulation with a CD137 ligand agonist, and that proved to be more potent than classical mo-DC in inducing cytotoxic responses against tumor associated viruses, such as EBV and HBV in vitro. In a phase I clinical trial on patients with locally recurrent or metastatic NPC, a CD137L-DC-EBV vaccine showed good tolerability and prolonged patient survival, providing a basis for further development of CD137L-DC vaccines for immunotherapy.

Immunoregulatory molecule expression on extracellular microvesicles in people living with HIV.

People living with HIV (PLWH) now benefit from combined antiviral treatments that durably control viral replication. These antiretroviral treatments decrease mortality and improve quality of life in PLWH, but do not completely control the excessive non-specific activation of the immune system in PLWH. This chronic immune activation is a key element of HIV immunopathology that contributes to the pathophysiology of inflammatory comorbid conditions, such as cardiovascular disorders, cancer and autoimmune diseases. Circulating non-exosomal extracellular vesicles, also known as microparticles (MPs) are detected in these diseases and have been linked to immune activation. The objective of this study was to characterize the MPs present in PLWH and to assess their association with chronic immune activation. We performed flow cytometry for the complete phenotypic characterization of MPs from fresh plasma from PLWH and from people without HIV as the control group. The absolute number, size and cellular origin of MPs were evaluated. The immunoregulatory profile was determined by cell origin, for MPs derived from platelets (PMPs), monocytes (MMPs) and T lymphocytes (LMPs). PLWH had significantly more circulating MPs than controls, for MPs of all sizes originating from T lymphocytes, red blood cells, neutrophils, dendritic cells, B lymphocytes and endothelial cells. PMPs and MMPs were not more numerous in PLWH, but the immunoregulatory phenotypes of these MPs differed between PLWH and controls. These differences in immunoregulatory molecule expression profile were also observed for LMPs. PDL1, ICOSL, CCR5, TGFβ1, MHC classes I and II, TRAIL, CXCR4, OX40, DC-SIGN, CTLA4 and PDL2 were more strongly expressed on the surface of MPs from PLWH than on those from controls. MPs are an important element in intercellular communication, making it possible to transfer phenotypes and functions to immune cells. The significantly higher numbers of MPs expressing diverse immunomodulatory molecules in PLWH may make a major contribution to the maintenance and/or the development of immune-cell activation in these individuals.

MicroRNA-150-5P regulates Th1/Th2 cytokines expression levels by targeting EGR2 in allergic rhinitis.

MiR-150-5p is one of the miRNAs in the expression profile of miRNAs, and in many previous studies, it has been shown that miR-150-5p may play an important role in peripheral blood dendritic cells (DCs) of allergic rhinitis (AR) patients. We sought to investigate the role and mechanism of miR-150-5p in regulating DC function by modulating EGR2 and influencing T cell derivation to promote AR development. The expression of miR-150-5p and EGR2 in AR patients was examined by real-time quantitative polymerase chain reaction (qRT-PCR), the expression of IL-4 cytokines in the supernatant of AR patients was tested by enzyme-linked immunosorbent assay (ELISA), and the expression of eosinophils in the supernatant of AR patients was measured by HE staining. The expression of EGR2 was detected by immunohistochemistry and fluorescent m-immunohistochemistry. MiR-150-5p expression was up-regulated and EGR2 expression was down-regulated in peripheral blood DCs from AR patients. miR-150-5p upregulated DCs, which promoted T-cell differentiation. miR-150-5p further regulated EGR2, which suppressed DCs and caused alteration of T-cell differentiation, in turn triggering the occurrence of AR. MiR-150-5p and its target gene EGR2 are involved in the development of AR, and DCs foster T-cell differentiation in peripheral blood of AR patients.

Correlation analysis between peripheral blood dendritic cell subsets and PD-1 in patients with peritoneal adenocarcinoma.

The aim of this study was to explore the association between differential percentages of dendritic cell (DC) subsets in peripheral blood and malignancy (grade and lymph node metastasis) of peritoneal adenocarcinoma patients and the frequencies of dendritic cell subsets in the normal controls. The peripheral blood of 30 patients with peritoneal adenocarcinoma and 12 healthy controls were collected for multicolor flow cytometry analysis. Peritoneal adenocarcinoma patients were grouped according to the malignant degree (grade and lymph node metastasis). Percentages of myeloid DCs (mDCs) and its subsets MDC1 and MDC2 in DCs were lower in peripheral blood of patients with peritoneal adenocarcinoma than in normal controls. The percentages of plasmacytoid dendritic cells (pDCs) and CD16+mDCs in DCs were higher than in normal controls. Compared with poor differentiation grade, patients with well/moderate differentiation grade had an increased percentage of CD16+mDCs. Contrary to CD16+mDCs, the percentage of MDC1 was lower in the well/moderate differentiation grade group. In patients with no lymph node metastasis, pDCs and CD16+mDCs levels were higher compared with patients with lymph node metastasis. mDCs and MDC1 levels had opposite results. pDCs were positively correlated with CD16+mDCs in peripheral blood of peritoneal patients, as was mDCs and MDC1. CD16+mDCs were negatively correlated with MDC1. The percentages of pDCs and CD16+mDCs in DCs were positively correlated with CD3+CD8+T cells, and pDCs also positively correlated with CD8+PD-1+T cells. Our results revealed that DCs subsets correlated with peritoneal adenocarcinoma malignancy. Dendritic cells play an independent role in the immune function of peritoneal adenocarcinoma.

Fully closed and automated enrichment of primary blood dendritic cells for cancer immunotherapy.

Dendritic cell (DC) vaccination is a promising approach to induce tumor-specific immune responses in cancer patients. Until recently, most DC vaccines were based on in vitro-differentiated monocyte-derived DCs. However, through development of efficient isolation techniques, the use of primary blood dendritic cell subsets has come within reach. Manufacturing of blood-derived DCs has multiple advances over monocytes-derived DCs, including more standardized isolation and culture protocols and shorter production processes. In peripheral blood, multiple DC subsets can be distinguished based on their phenotype and function. Plasmacytoid DC (pDC) and myeloid/conventional DCs (cDC) are the two main DC populations, moreover cDC can be further subdivided into CD141/BDCA3+ DC (cDC1) and CD1c/BDCA1+ DC (cDC2). In three separate clinical DC vaccination studies in melanoma and prostate cancer patients, we manufactured DC vaccines consisting of pDCs only, cDC2s only, or a combination of pDC and cDC2s, which we called natural DCs (nDC). Here, we describe a fully closed and automated GMP-compliant method to enrich naturally circulating DCs and present the results of enrichment of primary blood DCs from aphaeresis products of 8 healthy donors, 21 castrate-resistant prostate cancer patients, and 112 stage III melanoma patients. Although primary blood DCs are relatively scarce in aphaeresis material, our results show that it is feasible to isolate highly pure pDC, cDC2, or nDC with sufficient yield to manufacture DC vaccines for natural DC-based immunotherapy.

Evidence for effect modification by serum clusterin on dendritic cells in association with dementia

AbstractBackgroundAlzheimer’s disease (AD) and AD related dementias (ADRD) are complex heterogeneous diseases with multiple pathogenic mechanisms. Previous experimental studies showed that serum clusterin has a significant role in the maturation of dendritic cells (DCs). Studies showed that myeloid DCs (mDCs) in blood were dysregulated in AD and higher levels of serum clusterin related to higher incidence of dementia. We evaluated the association of DCs in peripheral blood and serum clusterin and their interaction with AD/ADRD.MethodWe used data from 3072 participants in the Health and Retirement Study (HRS) who participated in the 2016 Venous Blood Study (VBS), and had flow cytometry, clusterin and cognition assessed. We used generalized survey logit models to evaluate the association between the DCs, clusterin, and dementia after adjusting for age, sex, race, education, comorbidity, body mass index (BMI) and Cytomegalovirus (CMV) seroprevalence. Utilizing the RNAseq data, we performed differential gene expression (DGE) analysis on DCs, clusterin and dementia to identify overlapping genes in the biological process.ResultWe found that after covariate adjustment, a higher percentage of dendritic cells was associated with lower odds of having dementia (OR = 0.61, 95% CI: [0.44, 0.86]; p = 0.005). We found that higher levels of serum clusterin was associated with higher odds of having dementia. This relationship was somewhat attenuated after covariate adjustment (OR = 1.07, 95% CI [0.99, 1.16], p = 0.09). We found a significant interaction between serum clusterin and DCs in the association with dementia (p = 0.02) with those having low DCs and high clusterin being at increased ADRD risk (OR = 1.25) and those with high DCs and low clusterin being at lower ADRD risk (OR = 0.48) as compared to those who have both low DCs and low clusterin. We identified three genes (TUBB4A, WASHC5 and GSTA4) that were dysregulated in the pathways of DCs and clusterin in association with dementia.ConclusionThis study has shown, for the first time, an interaction between serum clusterin and DCs in peripheral blood that is associated with increased risk of dementia in a large sample of older adults that needs to be validated in future studies.

Alteration of Immunoregulatory Patterns and Survival Advantage of Key Cell Types in Food Allergic Children.

All allergic responses to food indicate the failure of immunological tolerance, but it is unclear why cow's milk and egg (CME) allergies resolve more readily than reactivity to peanuts (PN). We sought to identify differences between PN and CME allergies through constitutive immune status and responses to cognate and non-cognate food antigens. Children with confirmed allergy to CME (n = 6) and PN (n = 18) and non-allergic (NA) (n = 8) controls were studied. Constitutive secretion of cytokines was tested in plasma and unstimulated mononuclear cell (PBMNC) cultures. Blood dendritic cell (DC) subsets were analyzed alongside changes in phenotypes and soluble molecules in allergen-stimulated MNC cultures with or without cytokine neutralization. We observed that in allergic children, constitutively high plasma levels IL-1β, IL-2, IL-4, IL-5 and IL-10 but less IL-12p70 than in non-allergic children was accompanied by the spontaneous secretion of sCD23, IL-1β, IL-2, IL-4, IL-5, IL-10, IL-12p70, IFN-γ and TNF-α in MNC cultures. Furthermore, blood DC subset counts differed in food allergy. Antigen-presenting cell phenotypic abnormalities were accompanied by higher B and T cell percentages with more Bcl-2 within CD69+ subsets. Cells were generally refractory to antigenic stimulation in vitro, but IL-4 neutralization led to CD152 downregulation by CD4+ T cells from PN allergic children responding to PN allergens. Canonical discriminant analyses segregated non-allergic and allergic children by their cytokine secretion patterns, revealing differences and areas of overlap between PN and CME allergies. Despite an absence of recent allergen exposure, indication of in vivo activation, in vitro responses independent of challenging antigen and the presence of unusual costimulatory molecules suggest dysregulated immunity in food allergy. Most importantly, higher Bcl-2 content within key effector cells implies survival advantage with the potential to mount abnormal responses that may give rise to the manifestations of allergy. Here, we put forward the hypothesis that the lack of apoptosis of key immune cell types might be central to the development of food allergic reactions.