Myeloid Dendritic Cell Subsets Research Articles

Relationship between Phenotypic Changes of Dendritic Cell Subsets and the Onset of Plateau Phase during Intermittent Interferon Therapy in Patients with CHB*

ObjectiveThis study aimed to evaluate whether the onset of the plateau phase of slow hepatitis B surface antigen decline in patients with chronic hepatitis B treated with intermittent interferon therapy is related to the frequency of dendritic cell subsets and expression of the costimulatory molecules CD40, CD80, CD83, and CD86. MethodThis was a cross-sectional study in which patients were divided into a natural history group (namely NH group), a long-term oral nucleoside analogs treatment group (namely NA group), and a plateau-arriving group (namely P group). The percentage of plasmacytoid dendritic cell and myeloid dendritic cell subsets in peripheral blood lymphocytes and monocytes and the mean fluorescence intensity of their surface costimulatory molecules were detected using a flow cytometer. ResultsIn total, 143 patients were enrolled (NH group, n = 49; NA group, n = 47; P group, n = 47). The results demonstrated that CD141/CD1c double negative myeloid dendritic cell (DNmDC)/lymphocytes and monocytes (%) in P group (0.041 [0.024, 0.069]) was significantly lower than that in NH group (0.270 [0.135, 0.407]) and NA group (0.273 [0.150, 0.443]), and CD86 mean fluorescence intensity of DNmDCs in P group (1832.0 [1484.0, 2793.0]) was significantly lower than that in NH group (4316.0 [2958.0, 5169.0]) and NA group (3299.0 [2534.0, 4371.0]), Adjusted P all < 0.001. ConclusionReduced DNmDCs and impaired maturation may be associated with the onset of the plateau phase during intermittent interferon therapy in patients with chronic hepatitis B.

Mass Cytometry Reveals the Imbalanced Immune State in the Peripheral Blood of Patients with Essential Hypertension.

Mounting evidence has confirmed that essential hypertension (EH) is closely related to low-grade inflammation, but there is still a lack of in-depth understanding of the state of immune cells in the circulating blood of patients with EH. We analyzed whether hypertensive peripheral blood immune cell balance was destroyed. The peripheral blood mononuclear cells (PBMCs) of all subjects were analyzed using time-of-flight cytometry (CyTOF) based on 42 kinds of metal-binding antibodies. CD45+ cells were categorized into 32 kinds of subsets. Compared with the health control (HC) group, the percentage of total dendritic cells, two kinds of myeloid dendritic cell subsets, one intermediate/nonclassical monocyte subset and one CD4+ central memory T cell subset in the EH group, was significantly higher; the percentage of low-density neutrophils, four kinds of classical monocyte subsets, one CD14lowCD16- monocyte subset, one naive CD4+ and one naive CD8+ T cell subsets, one CD4+ effector and one CD4+ central memory T cell subsets, one CD8+ effector memory T cell subset, and one terminally differentiated γδ T cell subset, decreased significantly in EH. What is more, the expression of many important antigens was enhanced in CD45+ immune cells, granulocytes, and B cells in patients with EH. In conclusion, the altered number and antigen expression of immune cells reflect the imbalanced immune state of the peripheral blood in patients with EH.

Machine Learning-Based Single Cell and Integrative Analysis Reveals That Baseline mDC Predisposition Correlates With Hepatitis B Vaccine Antibody Response.

Vaccination to prevent infectious disease is one of the most successful public health interventions ever developed. And yet, variability in individual vaccine effectiveness suggests that a better mechanistic understanding of vaccine-induced immune responses could improve vaccine design and efficacy. We have previously shown that protective antibody levels could be elicited in a subset of recipients with only a single dose of the hepatitis B virus (HBV) vaccine and that a wide range of antibody levels were elicited after three doses. The immune mechanisms responsible for this vaccine response variability is unclear. Using single cell RNA sequencing of sorted innate immune cell subsets, we identified two distinct myeloid dendritic cell subsets (NDRG1-expressing mDC2 and CDKN1C-expressing mDC4), the ratio of which at baseline (pre-vaccination) correlated with the immune response to a single dose of HBV vaccine. Our results suggest that the participants in our vaccine study were in one of two different dendritic cell dispositional states at baseline – an NDRG2-mDC2 state in which the vaccine elicited an antibody response after a single immunization or a CDKN1C-mDC4 state in which the vaccine required two or three doses for induction of antibody responses. To explore this correlation further, genes expressed in these mDC subsets were used for feature selection prior to the construction of predictive models using supervised canonical correlation machine learning. The resulting models showed an improved correlation with serum antibody titers in response to full vaccination. Taken together, these results suggest that the propensity of circulating dendritic cells toward either activation or suppression, their “dispositional endotype” at pre-vaccination baseline, could dictate response to vaccination.

Pattern recognition receptor expression and maturation profile of dendritic cell subtypes in human tonsils and lymph nodes

Dendritic cells (DCs) with capacity of antigen cross-presentation are of key interest for immunotherapy against cancer as they can induce antigen-specific cytotoxic T lymphocyte (CTL) responses. This study describes frequencies of DC subtypes in human tonsils and lymph nodes, and phenotypic aspects that may be targeted by adjuvant measures.From human tonsils and neck lymph nodes, DCs were identified through flow cytometry, and subsets of plasmacytoid DCs (pDCs) and myeloid DCs (mDCs) were investigated. Maturity status was assessed and surface receptors with CTL-promoting potentials were studied.CD123+ pDCs as well as CD1c+, CD141+, and CD1c-CD141- mDCs were detected in tonsils and lymph nodes. Both sites featured a similar presence of DC subsets, with CD123+ pDC being dominant and CD141+ mDCs least frequent. Based on CD80/CD86 expression, all DC subtypes featured a low degree of maturation. Expression of pattern recognition receptors (PRRs) CD206, CD207, DC-SIGN, TLR2, and TLR4, as well as the chemokine receptor XCR1, indicated DC subset-specific receptor profiles.We conclude that tonsils and lymph nodes share common features in terms of DC subset frequency and maturation as well as PRR and XCR1 expression pattern. Our work suggests that both sites may be considered for vaccine deposition in DC-mediated immunotherapy.

Comprehensive analysis utilizing flow cytometry and immunohistochemistry reveals inflammatory changes in local endometrial and systemic dendritic cell populations in endometriosis.

What are the detailed endometrial tissue specific and systemic dendritic cell (DC) subset disturbances in endometriosis? This study confirms myeloid DC (mDC) and plasmacytoid DC subsets are readily identified in endometrial tissue and shows both endometrial and circulating differences in DC populations in women with endometriosis, with disease stage-specific relationships evident locally in the endometrium. Immune factors in the uterus, the peritoneal environment and systemically are implicated in the pathogenesis and progression of both endometriosis and infertility. While there is some evidence that endometrial DC populations are altered in endometriosis, DC subset involvement in both the endometrium and peripheral blood have not been comprehensively investigated so the functional consequences have been unknown. This prospective cross-sectional cohort study compares circulating and endometrial DC populations in women of reproductive age with and without endometriosis (n = 55 and 30, respectively), wherein each participant donated samples at a single time point. Study participants were surveyed for menstrual cycle phase, American Society for Reproductive Medicine (ASRM) endometriosis disease stage and fertility status (where possible). Peripheral blood samples were processed into mononuclear cells for analysis by flow cytometry, and endometrial samples were analysed by immunohistochemistry and dissociated into single-cell suspension for flow cytometry. In the endometrium of women with endometriosis, IRF-8+ cells were increased during the proliferative phase (P = 0.014), total DC proportions increased in the secretory phase (P = 0.038) and normal menstrual cyclical fluctuations in CD1c+ and IRF-8+ cells blunted; indicative of a consistently inflammatory tissue environment. The inflammatory changes in CD141+ and IRF-8+ populations in the endometrium of women with endometriosis were particularly evident in more advanced ASRM stages of the disease (respective P-values 0.032 and 0.045). There was also evidence of systemic inflammation in women with endometriosis, with increased circulating CD141+ mDC proportions (overall P = 0.040, secretory phase P = 0.021). N/A. As is common in this type of study, one of the main limitations was small sample numbers, particularly during the menstrual phase of the cycle. Further phenotyping of local and circulating immune cell subtypes is critical to improving understanding of endometriosis pathogenesis and immune contributions to infertility associated with the disease. This research was financially supported by a Sydney Medical School and Balnaves Foundation Kick Start Grant and the Department of Obstetrics, Gynaecology and Neonatology at The University of Sydney. The authors have no conflicts of interest to declare.

Absence of HIF1A Leads to Glycogen Accumulation and an Inflammatory Response That Enables Pancreatic Tumor Growth.

Cancer cells respond to hypoxia by upregulating the hypoxia-inducible factor 1α (HIF1A) transcription factor, which drives survival mechanisms that include metabolic adaptation and induction of angiogenesis by VEGF. Pancreatic tumors are poorly vascularized and severely hypoxic. To study the angiogenic role of HIF1A, and specifically probe whether tumors are able to use alternative pathways in its absence, we created a xenograft mouse tumor model of pancreatic cancer lacking HIF1A. After an initial delay of about 30 days, the HIF1A-deficient tumors grew as rapidly as the wild-type tumors and had similar vascularization. These changes were maintained in subsequent passages of tumor xenografts in vivo and in cell lines ex vivo. There were many cancer cells with a "clear-cell" phenotype in the HIF1A-deficient tumors; this was the result of accumulation of glycogen. Single-cell RNA sequencing (scRNA-seq) of the tumors identified hypoxic cancer cells with inhibited glycogen breakdown, which promoted glycogen accumulation and the secretion of inflammatory cytokines, including interleukins 1β (IL1B) and 8 (IL8). scRNA-seq of the mouse tumor stroma showed enrichment of two subsets of myeloid dendritic cells (cDC), cDC1 and cDC2, that secreted proangiogenic cytokines. These results suggest that glycogen accumulation associated with a clear-cell phenotype in hypoxic cancer cells lacking HIF1A can initiate an alternate pathway of cytokine and DC-driven angiogenesis. Inhibiting glycogen accumulation may provide a treatment for cancers with the clear-cell phenotype. SIGNIFICANCE: These findings establish a novel mechanism by which tumors support angiogenesis in an HIF1α-independent manner.

Human Miscarriage Is Associated With Dysregulations in Peripheral Blood-Derived Myeloid Dendritic Cell Subsets.

Dendritic cells (DC) are critically involved in decisions related to the acceptance or rejection of the foreign fetal antigens by the maternal immune system. However, particularly for human peripheral blood DCs (PBDC), available literature is rather inconsistent and the factors regulating these cells are ill-defined. Here, we investigated the phenotype and functionality of different human PBDC subsets during normal and pathologic pregnancies and studied an involvement of human chorionic gonadotropin (hCG) in PBDC regulation. Peripheral blood samples were obtained from normal pregnant women in all three trimesters, from first trimester miscarriage patients and from healthy non-pregnant women. Samples were analyzed for plasma hCG levels, for regulatory T (Treg) cell numbers, for frequencies of total and mature plasmacytoid (PDC) and myeloid (MDC1 and MDC2) PBDC subsets and for their cytokine secretion. In vitro assays, culturing PDC, MDC1 or MDC2 in the presence of two trophoblast cell lines, placenta explant supernatants or two hCG preparations were performed. The Treg-inducing capability of hCG- or non-hCG-treated stimulated MDC1 was assessed. Total and mature MDC1 and MDC2 frequencies increased during the first and second trimester of normal pregnancy, respectively. Miscarriage was associated with a reduced MDC1 and an increased MDC2 activation profile. PDC were not altered neither during normal pregnancy progression nor during miscarriage. In vitro, the culture of isolated PBDC subsets in the presence of placenta-derived factors impaired the maturation of MDC1 and differentially affected PDC maturation. An inhibitory effect on MDC1 and PDC maturation was also proven for the urine-derived hCG preparation. Finally, we observed a Treg cell elevation during early normal pregnancy that was not present in miscarriages. Stimulated MDC1 induced Treg cells in vitro, however, hCG was not involved in this process. Our findings suggest that during normal pregnancy PBDC subsets are differentially regulated dependent on gestational age. Miscarriage seems to be associated with dysregulations in the myeloid PBDC subsets and with disturbances in Treg cell frequencies. Moreover, our results propose an interdependency between MDC1 and Treg cells during early pregnancy. hCG, although shown to impair MDC1 maturation, does not seem to be a key regulator of PBDC alterations during pregnancy.

Introduction of Human Flt3-L and GM-CSF into Humanized Mice Enhances the Reconstitution and Maturation of Myeloid Dendritic Cells and the Development of Foxp3+CD4+ T Cells.

Two cytokines, fms-related tyrosine kinase 3 ligand (Flt3-L) and granulocyte-macrophage colony-stimulating factor (GM-CSF) are considered to be the essential regulators of dendritic cell (DC) development in vivo. However, the combined effect of Flt3-L and GM-CSF on human DCs has not been evaluated in vivo. In this study, we, therefore, aimed at evaluating this using a humanized mouse model. Humanized non-obese diabetic/SCID/Jak3null (hNOJ) mice were constructed by transplanting hematopoietic stem cells from human umbilical cord blood into newborn NOJ mice, and in vivo transfection (IVT) was performed by hydrodynamic injection-mediated gene delivery using plasmids encoding human Flt3-L and GM-CSF. Following IVT, Flt3-L and GM-CSF were successfully induced in hNOJ mice. At 10 days post-IVT, we found, in the spleen, that treatment with both Flt3-L and GM-CSF enhanced the reconstitution of two myeloid DC subsets, CD14−CD1c+ conventional DCs (cDCs) and CD14−CD141+ cDCs, in addition to CD14+ monocyte-like cells expressing CD1c and/or CD141. GM-CSF alone had less effect on the reconstitution of these myeloid cell populations. By contrast, none of the cytokine treatments enhanced CD123+ plasmacytoid DC (pDC) reconstitution. Regardless of the reconstitution levels, three cell populations (CD1c+ myeloid cells, CD141+ myeloid cells, and pDCs) could be matured by treatment with cytokines, in terms of upregulation of CD40, CD80, CD86, and CD184/CXCR4 and downregulation of CD195/CCR5. In particular, GM-CSF contributed to upregulation of CD80 in all these cell populations. Interestingly, we further observed that Foxp3+ cells within splenic CD4+ T cells were significantly increased in the presence of GM-CSF. Foxp3+ T cells could be subdivided into two subpopulations, CD45RA−Foxp3hi and CD45RA−Foxp3lo T cells. Whereas CD45RA−Foxp3hi T cells were increased only after treatment with GM-CSF alone, CD45RA−Foxp3lo T cells were increased only after treatment with both Flt3-L and GM-CSF. Treatment with Flt3-L alone had no effect on the number of Foxp3+ T cells. The correlation analysis demonstrated that the development of these Foxp3+ subpopulations was associated with the maturation status of DC(-like) cells. Taken together, this study provides a platform for studying the in vivo effect of Flt3-L and GM-CSF on human DCs and regulatory T cells.

Transcriptional Profiling of Age-Associated Gene Expression Changes in Human Circulatory CD1c+ Myeloid Dendritic Cell Subset.

Immune dysfunction is a hallmark of aging and is thought to be responsible for the age-associated diseases. Dendritic cells (DCs) of the immune system function as initiators and regulators of the immune responses. Recent studies have highlighted the division of labor between various DC subsets. CD1c+ DC subset has emerged as a major inducer of CD4 T cell response. There is a scarcity of information regarding the age-associated changes in the functions of DC subsets in the elderly. Here, we investigated the changes in transcriptional profile of CD1c+ DC subset from healthy aged and young individuals using RNA sequencing. Our results suggest that majority of the genes in DCs are upregulated with age. Glucose transport, GPCR, and potassium channel genes are all upregulated in DCs from aged as compared to young indicating an enhanced activation state of DCs from aged individuals. The expression of histones, small nucleolar RNA H/ACA box (SNORA) and small nucleolar RNA C/D/box (SNORD), and long non-coding RNA (lncRNA) is also substantially upregulated in the DCs from aged. In contrast, the antigen-presenting and energy generating pathways are downregulated. In summary, DCs from aged subjects display an activated state coupled with reduced antigen presentation which may be responsible for age-associate immune dysfunction.

Low-density lipoprotein receptor–related protein 1 attenuates house dust mite–induced eosinophilic airway inflammation by suppressing dendritic cell–mediated adaptive immune responses

Low-density lipoprotein receptor-related protein 1 (LRP-1) is a scavenger receptor that regulates adaptive immunity and inflammation. LRP-1 is not known to modulate the pathogenesis of allergic asthma. We sought to assess whether LRP-1 expression by dendritic cells (DCs) modulates adaptive immune responses in patients with house dust mite (HDM)-induced airways disease. LRP-1 expression on peripheral blood DCs was quantified by using flow cytometry. The role of LRP-1 in modulating HDM-induced airways disease was assessed in mice with deletion of LRP-1 in CD11c+ cells (Lrp1fl/fl; CD11c-Cre) and by adoptive transfer of HDM-pulsed CD11b+ DCs from Lrp1fl/fl; CD11c-Cre mice to wild-type (WT) mice. Human peripheral blood myeloid DC subsets from patients with eosinophilic asthma have lower LRP-1 expression than cells from healthy nonasthmatic subjects. Similarly, LRP-1 expression by CD11b+ lung DCs was significantly reduced in HDM-challenged WT mice. HDM-challenged Lrp1fl/fl; CD11c-Cre mice have a phenotype of increased eosinophilic airway inflammation, allergic sensitization, TH2 cytokine production, and mucous cell metaplasia. The adoptive transfer of HDM-pulsed LRP-1-deficient CD11b+ DCs into WT mice generated a similar phenotype of enhanced eosinophilic inflammation and allergic sensitization. Furthermore, CD11b+ DCs in the lungs of Lrp1fl/fl; CD11c-Cre mice have an increased ability to take up HDM antigen, whereas bone marrow-derived DCs display enhanced antigen presentation capabilities. This identifies a novel role for LRP-1 as a negative regulator of DC-mediated adaptive immune responses in the setting of HDM-induced eosinophilic airway inflammation. Furthermore, the reduced LRP-1 expression by circulating myeloid DCs in patients with eosinophilic asthma suggests a possible role for LRP-1 in modulating type 2-high asthma.

Early Immune Regulatory Changes in a Primary Controlled Human Plasmodium vivax Infection: CD1c+ Myeloid Dendritic Cell Maturation Arrest, Induction of the Kynurenine Pathway, and Regulatory T Cell Activation.

Plasmodium vivax malaria remains a major public health problem. The requirements for acquisition of protective immunity to the species are not clear. Dendritic cells (DC) are essential for immune cell priming but also perform immune regulatory functions, along with regulatory T cells (Treg). An important function of DC involves activation of the kynurenine pathway via indoleamine 2,3-dioxygenase (IDO). Using a controlled human experimental infection study with blood-stage P. vivax, we characterized plasmacytoid DC (pDC) and myeloid DC (mDC) subset maturation, CD4+ CD25+ CD127lo Treg activation, and IDO activity. Blood samples were collected from six healthy adults preinoculation, at peak parasitemia (day 14; ∼31,400 parasites/ml), and 24 and 48 h after antimalarial treatment. CD1c+ and CD141+ mDC and pDC numbers markedly declined at peak parasitemia, while CD16+ mDC numbers appeared less affected. HLA-DR expression was selectively reduced on CD1c+ mDC, increased on CD16+ mDC, and was unaltered on pDC. Plasma IFN-γ increased significantly and was correlated with an increased kynurenine/tryptophan (KT) ratio, a measure of IDO activity. At peak parasitemia, Treg presented an activated CD4+ CD25+ CD127lo CD45RA- phenotype and upregulated TNFR2 expression. In a mixed-effects model, the KT ratio was positively associated with an increase in activated Treg. Our data demonstrate that a primary P. vivax infection exerts immune modulatory effects by impairing HLA-DR expression on CD1c+ mDC while activating CD16+ mDC. Induction of the kynurenine pathway and increased Treg activation, together with skewed mDC maturation, suggest P. vivax promotes an immunosuppressive environment, likely impairing the development of a protective host immune response.

Exposure to cryopreserved platelets mediates suppression of myeloid dendritic cell subset immune responses

Exposure to cryopreserved platelets mediates suppression of myeloid dendritic cell subset immune responses

Evidence of regulatory myeloid dendritic cells and circulating inflammatory epidermal dendritic cells-like modulated by Toll-like receptors 2 and 7/8 in adults with atopic dermatitis.

Atopic dermatitis (AD) is a chronic, inflammatory skin disease characterized by intense pruritus and xerosis. Dendritic cells (DC) play an essential role in tissue inflammation in atopic dermatitis (AD) skin, especially the inflammatory epidermal dendritic cells (IDEC), a particular subset of myeloid dendritic cells (mDC). The aim of the present study was to assess the phenotype and function of mDC and circulating IDEC-like in peripheral blood mononuclear cells (PBMC) of adults with AD. We selected 21 AD patients and 21 non-AD controls, age and gender matched. Expressions of FcεRI, CD36, TNF, IFN- γ, and IL-10 in mDC were analyzed by flow cytometry under various stimuli, such as staphylococcal enterotoxin B (SEB), TLR2 (Pam3CSK4), TLR4 (LPS), and TLR7/8 (CL097) agonists. The most prominent findings in AD patients were: (i) enhanced frequency of IL-10 under TLR4 (LPS), and decreased frequency of IFN-γ and TNF under TLR2 (Pam3CSK4) and 7/8 (CL097) stimulation in classic mDC; (ii) elevation of circulating IDEC-like frequency with TLR2 (Pam3CSK4) stimuli, augmented frequency of IFN-γ in nonstimulated condition, and of IL-10 under TLR7/8 (CL097) stimuli in IDEC-like population. In AD individuals, classic mDC showed an immunomodulatory profile, favoring tolerance in a combined action with IDEC-like, and inducing Th1 polarization. Our findings indicate a potential role of IDEC-like in the maintenance of inflammation in atopic dermatitis patients; moreover, IDEC-like may exert a regulatory impact on T cells of AD individuals through IL-10, often induced by agonist mimicking single stranded RNA virus.

CLEC12A-Mediated Antigen Uptake and Cross-Presentation by Human Dendritic Cell Subsets Efficiently Boost Tumor-Reactive T Cell Responses.

Potent immunotherapies are urgently needed to boost antitumor immunity and control disease in cancer patients. As dendritic cells (DCs) are the most powerful APCs, they are an attractive means to reinvigorate T cell responses. An appealing strategy to use the effective Ag processing and presentation machinery, T cell stimulation and cross-talk capacity of natural DC subsets is in vivo tumor Ag delivery. In this context, endocytic C-type lectin receptors are attractive targeting molecules. In this study, we investigated whether CLEC12A efficiently delivers tumor Ags into human DC subsets, facilitating effective induction of CD4(+) and CD8(+) T cell responses. We confirmed that CLEC12A is selectively expressed by myeloid cells, including the myeloid DC subset (mDCs) and the plasmacytoid DC subset (pDCs). Moreover, we demonstrated that these DC subsets efficiently internalize CLEC12A, whereupon it quickly translocates to the early endosomes and subsequently routes to the lysosomes. Notably, CLEC12A Ab targeting did not negatively affect DC maturation or function. Furthermore, CLEC12A-mediated delivery of keyhole limpet hemocyanin resulted in enhanced proliferation and cytokine secretion by keyhole limpet hemocyanin-experienced CD4(+) T cells. Most importantly, CLEC12A-targeted delivery of HA-1 long peptide resulted in efficient Ag cross-presentation by mDCs and pDCs, leading to strong ex vivo activation of HA-1-specific CD8(+) T cells of patients after allogeneic stem cell transplantation. Collectively, these data indicate that CLEC12A is an effective new candidate with great potential for in vivo Ag delivery into mDCs and pDCs, thereby using the specialized functions and cross-talk capacity of these DC subsets to boost tumor-reactive T cell immunity in cancer patients.

Abstract # 1701 Anti-CD40 prevents the corticosterone-induced loss of splenic dendritic cells in C57BL/6 mice

Dendritic cells (DCs) are important immune cells linking the innate and adaptive immune response. DCs capture antigen to present to and activate naive T cells. Subsets of DCs vary based on protein expression. This research focused on total DCs (CD11c+), and the myeloid and cross-priming DC subsets. Corticosterone (CORT), a glucocorticoid hormone released in response to stress, greatly reduces DC numbers and alters DC function and subsequent T cell responses. The current study quantifies DC recovery after CORT exposure and demonstrates that the loss of DCs can be prevented. C57BL/6 mice were exposed to exogenous CORT in the drinking water for 24 h. The splenic DC populations were then quantified by flow cytometry. CORT exposure significantly decreased the numbers of all splenic DCs. Mice were then provided varying recovery times (3, 5, or 10 days) post CORT exposure. DCs quickly returned to normal levels, and even exceeded the percentages in control untreated mice. Injection of mice with anti-CD40 antibody triggers DC maturation. Thus, it was hypothesized that injection of anti-CD40 may prevent the CORT-induced loss of DCs. Mice received two injections of anti-CD40 antibody and were immediately supplied with CORT following the second injection. When provided anti-CD40 prior to CORT exposure, the loss of DCs was prevented. These studies contribute to the ongoing exploration of the mechanisms underlying the immunological effects of stress-induced glucocorticoids.

Abstract LB-081: In situ vaccination of low-grade lymphoma with intratumoral Flt3L and poly-ICLC with low-dose radiotherapy

Abstract Background: Lymphomas are the 5th most common cancer in the United States, 40% of these are cases are indolent non-Hodgkin's lymphoma (iNHL) and are incurable with standard therapy. A previous trial of in situ vaccination in iNHLs, in which intratumoral CpG, the TLR9 agonist, was combined with low dose radiation to induce a systemic immune response against tumor. In this trial induction of tumor-specific CD8 T cell responses and durable clinical remissions of patients’ untreated sites of disease was seen in some patients. One limitation in this previous trial may have been the scarcity of intratumoral dendritic cells (DC) and the suppressive tumor microenvironment. DC are uniquely able to endocytose dying (e.g. irradiated) tumor cells for cross-presentation to anti-tumor CD8 T cells. In this new iteration of in situ vaccine, Flt3L is added as a priming step to increase the presence of intratumoral DCs ahead of vaccination. Fms-like tyrosin kinase 3 ligand (Flt3L) induces tumor leukocyte infiltration and regression in lymphoma tumors in pre-clinical trials, and CDX-301- a formulation of Flt3L - was shown to mobilize BDCA-1 and BDCA-3 myeloid DC subsets in an early phase trial. These DC subsets respond to several TLR agonists and cross-present antigens more effectively than plasmacytoid DCs. While pDCs are high expressors of TLR9, responsive to CpG, myeloid dendritic cells express a wider array of TLRs, including high levels of TLR3. Methods: This Phase I/II trial tests the hypothesis that this novel in situ vaccination will induce clinical remissions at distant (untreated) tumor sites in two cohorts of patients with either previously untreated or relapsed/refractory iNHL (n = 15 per group). Intratumoral CDX-301 25ug/kg is injected into a palpable lymph node for 9 days, followed 2Gy local radiotherapy on day 9 and 10 to the target lymph node. On day 10, following radiation therapy, intratumoral poly-ICLC 2mg is injected to activate local DCs. Poly-ICLC 2mg is then injected on day 14, day 17 (1 week after initial dose) and weekly thereafter for a total of 9 treatments over 8 weeks. Response is assessed with CT scans every three months as per the Revised Response Criteria for Malignant Lymphoma, also known as the Cheson criteria, and leukemic phase of lymphoma monitored by peripheral blood flow cytometry. The trial started accrual in January 2014 and is ongoing, 11 have been enrolled including 8 untreated patients and 3 with refractory disease. To perform correlative studies during the clinical trial we perform excisional biopsy of a distal lymph node before starting trial to assess baseline immunomodulatory or immunosuppressive characteristics of a patients tumor microenvironment. Fine needle aspirate (FNA) of the target tumor (site of Flt3L, Radiation and poly-ICLC administration) treatment at weeks 0, 2, 4 and 6, after which target tumor is significantly smaller and biopsy not typically possible. Blood is collected for immune monitoring at weeks 0, 2, 4, 6, 8, 12 to assess for transient enrichment of DC populations, and monitoring for tumor-specific effector T cell populations. Citation Format: Thomas U. Marron, Nina Bhardwaj, Linda Hammerich, Seunghee Kim-Schulze, Tibor Keler, Tom Davis, Elizabeth Crowley, Andres M. Salazar, Joshua Brody. In situ vaccination of low-grade lymphoma with intratumoral Flt3L and poly-ICLC with low-dose radiotherapy. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-081.

Dendritic cells in renal biopsies of patients with acute tubulointerstitial nephritis

Dendritic cells (DCs) play a critical role in the regulation of the adaptive immune response and can be separated into 2 major subsets: myeloid (mDC) and plasmacytoid (pDC) DCs. Acute tubulointerstitial nephritis (ATIN) is a common cause of injury to renal tubules and interstitium resulting from the interplay of tubular cells and inflammatory cells and their products. However, the involvement of DCs in ATIN is still unknown. In this study, the participation and localization of myeloid (CD1c) and plasmacytoid (CD303) DC subsets in the biopsies from patients with ATIN (n=20), lupus nephritis (n=17, positive control), or minimal change disease (n=14, negative control) were investigated. DCs were identified morphologically within the tubulointerstitium in the renal biopsies by transmission electron microscopy interacting with surrounding tubules and inflammatory cells. Direct immunofluorescence showed that both CD1c+DCs and CD303+ DCs exist in all the renal biopsies. As compared with minimal change disease, biopsies with ATIN had significantly increased CD1c+DCs (P<.001) and CD303+ DCs (P<.001). The number of CD1c+DCs in ATIN was significantly higher than that in lupus nephritis (P<.02), whereas the number of CD303+ DCs in ATIN was slightly but not significantly higher than that in lupus nephritis (P=.2). DCs in the biopsies with ATIN were restricted to the tubulointerstitium forming dense networks, and most of them maintained immature state. All these findings suggest that DC subsets may be differentially involved in the pathogenesis of ATIN. Their potential role in intrarenal regulation of immune responses in ATIN is proposed.

Dendritic Cell Subsets in Bone Marrow and Peripheral Blood of Patients with Myelodysplastic Syndromes Display Numeric and Functional Defects

Abstract Introduction Immune response plays an important role in the pathogenesis and progression of myelodysplastic syndromes (MDS). It has been shown that a consistent immunological signature in MDS comprises an increase in Th17 cells in low risk MDS and an expansion of Tregs in high risk subtypes. The presence of high Treg numbers in lower risk stages is an independent prognostic value and is associated with poor survival. Whereas the immune response in low risk MDS is pro-inflammatory, high risk MDS is characterized by immunosuppression which could contribute into the expansion of the dysplastic clone and malignant transformation. The exact role of dendritic cells (DC) in inducing this immune dysfunction is yet to be understood. The aim of this comparative study was therefore to investigate the frequency and function of different DC subsets in the bone marrow (BM) and peripheral blood (PB) of MDS patients compared to healthy donors (HD). Patients and methods The enumeration of DC subsets was performed in 110 MDS and in 19 HD bone marrow samples. In peripheral blood 34 MDS and 18 HD samples were investigated. DC were identified by the expression of HLA-DR and the lack of the lineage markers CD14 and CD19. They were further subdivided into CD303+ plasmacytoid DC (pDC), and the CD11c+ myeloid subsets CD1c+ myeloid DC 1 (mDC1), CD141hi myeloid DC 2 (mDC2) and CD16+ 6-Sulfo LacNac (Slan) DC. Furthermore, their ability to upregulate co-stimulatory molecules was assessed by flow cytometry and the secretion of cytokines in response to TLR ligands was analyzed using Cytometric Bead Array (CBA). Their capacity to induce allogeneic T cell proliferation was evaluated in a mixed leukocyte reaction (MLR). Results The frequencies of pDC, mDC1 and mDC2 but not of SlanDC were significantly lower in patients' BM compared to HDs' BM (pDC 0.33% vs 0.72%, p=0.01; mDC1 0.30% vs 0.73%, p&lt;0.001; mDC2 0.01% vs 0.05%, p&lt;0.0001; SlanDC 0.10% vs 0.13%, p=0.20). In PB all DC subsets were significantly lower in patients' samples compared to HDs' samples (pDC 0.15% vs 0.28%, p=0.02; mDC1 0.12% vs 0.61%, p&lt;0.0001; mDC2 0.003% vs 0.045%, p&lt;0.0001; SlanDC 0.19% vs 0.46%, p=0.04). A positive correlation was found between the frequencies of DC subsets in a paired assessment of PB and BM of 30 MDS patients (pDC r=0.77, mDC1 r=0.57, mDC2 r=0.34, SlanDC r=0.60). Of note, in a more advanced disease state DC subsets showed a progressive decline in frequency. To investigate DC function they were isolated or FACS sorted from HD BM (n=3) or patient BM (n=4). Based on their TLR expression profile a combination of LPS and R848 has been used to stimulate DC overnight. In HD upregulation of the co-stimulatory molecules CD80 and CD86 was observed after this stimulation. However, MDS DC stimulated with the same combination of TLR ligands showed a reduced upregulation of CD80 and CD86 (median fluorescence intensity (MFI) CD80: SlanDC 2323 vs 6254, MFI CD86: mDC1 2785 vs 4608; SlanDC 2344 vs 5087). They also produced lower levels of pro-inflammatory cytokines compared to HD DC as measured in culture supernatants (mDC1 TNF [pg/ml] 997 vs 2980, IL-6 [pg/ml] 843 vs 2806, IL-8 [pg/ml] 966 vs 5549 and IL-12p70 [pg/ml] 105 vs 1115). Additionally, after 5 days co-culture of CFSE labeled T cells and DC, mDC1 and SlanDC derived from MDS BM displayed a defective induction of allogeneic CD4+ and CD8+ T cell proliferation compared to HD (mDC1 3.08% vs 10.49% CD4+ T cell proliferation and 4.54% vs 15.07% CD8+ T cell proliferation; SlanDC 1.28% vs 5.23% CD4+ T cell proliferation and 1.50% vs 8.67% CD8+ T cell proliferation). Conclusion Our data clearly point to both a numeric and functional impairment of myeloid DC subsets in BM as well as in PB of patients with MDS. A further decline of DC frequencies in high risk MDS may support the therapeutic targeting of DC in the BM microenvironment of these patients to redress immune dysfunction and possibly prevent further progression to acute myeloid leukemia. Extensive research is needed to further delineate the differences in DC function and immune composition in MDS risk categories. Disclosures Mufti: Celgene Corporation: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

Profiling of primary peripheral blood- and monocyte-derived dendritic cells using monoclonal antibodies from the HLDA10 Workshop in Wollongong, Australia.

Dendritic cells (DCs) arise from hematopoietic stem cells and develop into a discrete cellular lineage distinct from other leucocytes. Mainly three phenotypically and functionally distinct DC subsets are described in the human peripheral blood (PB): plasmacytoid DCs (pDCs), which express the key marker CD303 (BDCA-2), and two myeloid DC subsets (CD1c+ DC (mDC1) and CD141+ DC (mDC2)), which express the key markers CD1c (BDCA-1) and CD141 (BDCA-3), respectively. In addition to these primary cell subsets, DCs can also be generated in vitro from either CD34+ stem/progenitor cells in the presence of Flt3 (Fms-related tyrosine kinase 3) ligand or from CD14+ monocytes (monocyte-derived DCs (mo-DCs)) in the presence of granulocyte–macrophage colony-stimulating factor+interleukin-4 (GM-CSF+IL-4). Here we compare the reactivity patterns of HLDA10 antibodies (monoclonal antibody (mAb)) with pDCs, CD1c+ DCs and CD141+ DCs, as well as with CD14+-derived mo-DCs cultured for 7 days in the presence of 100 ng/ml GM-CSF plus 20 ng/ml IL-4. A detailed profiling of these DC subsets based on immunophenotyping and multicolour flow cytometry analysis is presented. Using the panel of HLDA10 Workshop mAb, we could verify known targets selectively expressed on discrete DC subsets including CD370 as a selective marker for CD141+ DCs and CD366 as a marker for both myeloid subsets. In addition, vimentin and other markers are heterogeneously expressed on all three subsets, suggesting the existence of so far not identified DC subsets.

Preserved dendritic cell HLA-DR expression and reduced regulatory T cell activation in asymptomatic Plasmodium falciparum and P. vivax infection.

Clinical illness with Plasmodium falciparum or Plasmodium vivax compromises the function of dendritic cells (DC) and expands regulatory T (Treg) cells. Individuals with asymptomatic parasitemia have clinical immunity, restricting parasite expansion and preventing clinical disease. The role of DC and Treg cells during asymptomatic Plasmodium infection is unclear. During a cross-sectional household survey in Papua, Indonesia, we examined the number and activation of blood plasmacytoid DC (pDC), CD141(+), and CD1c(+) myeloid DC (mDC) subsets and Treg cells using flow cytometry in 168 afebrile children (of whom 15 had P. falciparum and 36 had P. vivax infections) and 162 afebrile adults (of whom 20 had P. falciparum and 20 had P. vivax infections), alongside samples from 16 patients hospitalized with uncomplicated malaria. Unlike DC from malaria patients, DC from children and adults with asymptomatic, microscopy-positive P. vivax or P. falciparum infection increased or retained HLA-DR expression. Treg cells in asymptomatic adults and children exhibited reduced activation, suggesting increased immune responsiveness. The pDC and mDC subsets varied according to clinical immunity (asymptomatic or symptomatic Plasmodium infection) and, in asymptomatic infection, according to host age and parasite species. In conclusion, active control of asymptomatic infection was associated with and likely contingent upon functional DC and reduced Treg cell activation.