Peripheral Blood Dendritic Cells Subsets Research Articles

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.

Role of monocytes and dendritic cells in cardiac reverse remodelling after cardiac resynchronization therapy

Background and aimsMonocytes and dendritic cells (DC) are both key inflammatory cells, with recognized effects on cardiac repair. However, there are distinct subsets of monocytes with potential for beneficial or detrimental effects on heart failure (HF) pathogenesis. The connection between reverse cardiac remodelling, the potential anti-inflammatory effect of cardiac resynchronization therapy (CRT) and monocytes and DC homeostasis in HF is far from being understood. We hypothesized that monocytes and DC play an important role in cardiac reverse remodelling and CRT response. Therefore, we aimed to assess the potential role of baseline peripheral levels of blood monocytes and DC subsets and their phenotypic and functional activity for CRT response, in HF patients. As a secondary objective, we aimed to evaluate the impact of CRT on peripheral blood monocytes and DC subsets, by comparing baseline and post CRT circulating levels and phenotypic and functional activity.MethodsForty-one patients with advanced HF scheduled for CRT were included in this study. The quantification and phenotypic determination of classical (cMo), intermediate (iMo) and non-classical monocytes (ncMo), as well as of myeloid (mDC) and plasmacytoid DC (pDC) were performed by flow cytometry in a FACSCanto™II (BD) flow cytometer. The functional characterization of total monocytes and mDC was performed by flow cytometry in a FACSCalibur flow cytometer, after in vitro stimulation with lipopolysaccharide from Escherichia coli plus interferon (IFN)-γ, in the presence of Brefeldina A.Comparisons between the control and the patient group, and between responders and non-responders to CRT were performed.ResultsCompared to the control group, HF population presented a significantly lower frequency of pDC at baseline and a higher proportion of monocytes and mDC producing IL-6 and IL-1β, both before and 6-months after CRT (T6). There was a remarkable decrease of cMo and an increase of iMo after CRT, only in responders. The responder group also presented higher ncMo values at T6 compared to the non-responder group. Both responders and non-responders presented a decrease in the expression of CD86 in all monocyte and DC populations after CRT. Moreover, in non-responders, the increased frequency of IL-6-producing DC persisted after CRT.ConclusionOur study provides new knowledge about the possible contribution of pDC and monocytes subsets to cardiac reverse remodelling and response to CRT. Additionally, CRT is associated with a reduction on CD86 expression by monocytes and DC subsets and in their potential to produce pro-inflammatory cytokines, contributing, at least in part, for the well described anti-inflammatory effects of CRT in HF patients.

Ascaris suum excretory/secretory products differentially modulate porcine dendritic cell subsets.

Helminths produce excretory/secretory products (E/S) which can modulate the immune responses of their hosts. Dendritic cells (DC) are essential for initiating the host T cell response and are thus potential targets for modulation by helminth E/S. Here we study immunomodulation of porcine peripheral blood DC subsets following ex vivo stimulation with E/S from Ascaris suum, a common helminth of pigs with considerable public health and economic importance. Our data showed that the relative frequencies of DC subsets in porcine blood differ, with plasmacytoid DC (pDC) being the most prominent in healthy 6-month-old pigs. pDC are an important cytokine source, and we found that A. suum E/S suppressed production of the type 1 cytokines IL-12p40 and TNF-α by this subset following toll-like receptor (TLR) ligation. In contrast, conventional DC (cDC) are more efficient antigen presenters, and the expression of CD80/86, costimulatory molecules essential for efficient antigen presentation, were modulated differentially by A. suum E/S between cDC subsets. CD80/86 expression by type 1 cDC (cDC1) following TLR ligation was greatly suppressed by the addition of A. suum E/S, while CD80/86 expression by type 2 cDC (cDC2) was upregulated by A. suum E/S. Further, we found that IFN-γ production by natural killer (NK) cells following IL-12 and IL-18 stimulation was suppressed by A. suum E/S. Finally, in the presence of E/S, IFN-γ production by CD4+ T cells co-cultured with autologous blood-derived DC was significantly impaired. Together, these data provide a coherent picture regarding the regulation of type 1 responses by A. suum E/S. Responsiveness of pDC and cDC1 to microbial ligands is reduced in the presence of E/S, effector functions of Th1 cells are impaired, and cytokine-driven IFN-γ release by NK cells is limited.

Erythrocyte lysing solutions have a detrimental effect in flow cytometric dendritic cell detection.

Flow cytometry (FCM) enumeration of peripheral blood dendritic cells (PBDCs) is a minimally invasive procedure extremely useful for immunological studies. Numbers of PBDCs vary depending on age, lifestyle, or in pathologies like cancer, leukemia or immunodeficiencies. Conventional methods for PBDC identification by FCM involve red blood cell lysis using either formaldehyde or ammonium chloride-based solutions. This specific procedure has been widely reported to cause a detrimental effect as well as an artifactual detection of target populations. Alternatively, minimal sample perturbation assays that avoid the use of erythrolytic solutions with centrifugation steps and preserve the native cellular state are simpler and more robust than conventional methods. In this study, we aimed to evaluate how conventional FCM assays can alter dendritic cell (DC) counting when compared with minimal sample perturbation protocols, in terms of absolute cell counting, percentage and stain index (SI) of PBDC subsets. We evaluated the use of three different erythrolytic solutions (CyLyse, OptiLyse C, and Pharm Lyse) on a series of n= 20 peripheral blood specimens for conventional and plasmacytoid DCs detection as well as for leukocyte and basophil detection. Our results showed a significant reduction of leukocytes and specifically, of DCs and basophils in terms of absolute number when using erythrolytic solutions. In conclusion, our study shows that PBDC counting is heavily affected when lysing solutions are used, indicating that these stellate-shaped populations appear to be more labile.

Reduced peripheral blood dendritic cell and monocyte subsets in MDS patients with systemic inflammatory or dysimmune diseases.

Systemic inflammatory and autoimmune diseases (SIADs) occur in 10-20% of patients with myelodysplastic syndrome (MDS). Recently identified VEXAS (Vacuoles, E1 enzyme, X-linked, Autoinflammatory, Somatic) syndrome, associated with somatic mutations in UBA1 (Ubiquitin-like modifier-activating enzyme 1), encompasses a range of severe inflammatory conditions along with hematological abnormalities, including MDS. The pathophysiological mechanisms underlying the association between MDS and SIADs remain largely unknown, especially the roles of different myeloid immune cell subsets. The aim of this study was to quantitatively evaluate peripheral blood myeloid immune cells (dendritic cells (DC) and monocytes) by flow cytometry in MDS patients with associated SIAD (n = 14, most often including relapsing polychondritis or neutrophilic dermatoses) and to compare their distribution in MDS patients without SIAD (n = 23) and healthy controls (n = 7). Most MDS and MDS/SIAD patients had low-risk MDS. Eight of 14 (57%) MDS/SIAD patients carried UBA1 somatic mutations, defining VEXAS syndrome.Compared with MDS patients, most DC and monocyte subsets were significantly decreased in MDS/SIAD patients, especially in MDS patients with VEXAS syndrome. Our study provides the first overview of the peripheral blood immune myeloid cell distribution in MDS patients with associated SIADs and raises several hypotheses: possible redistribution to inflammation sites, increased apoptosis, or impaired development in the bone marrow.

Early Immune Initiation by Porcine Cells following Toxoplasma gondii Infection versus TLR Ligation.

Containment of acute Toxoplasma gondii infection is dependent on an efficient interferon gamma response. However, the earliest steps of immune response initiation immediately following exposure to the parasite have not been previously characterized in pigs. Murine and human myeloid cells produce large quantities of interleukin (IL)-12 during early T. gondii infection. We therefore examined IL-12 expression by porcine peripheral blood monocytes and dendritic cell (DC) subsets following toll-like receptor (TLR) ligation and controlled T. gondii tachyzoite infection. We detected IL-12p40 expression by porcine plasmacytoid DC, but not conventional or monocyte-derived DC following TLR ligation. Unexpectedly, we also observed considerable IL-12p40 production by porcine CD3– NKp46+ cells—a classical natural killer cell phenotype—following TLR ligation. However, in response to T. gondii exposure, no IL-12 production was observed by either DC or CD3– NKp46+ cells. Despite this, IL-18 production by DC-enriched peripheral blood mononuclear cells was detected following live T. gondii tachyzoite exposure. Only combined stimulation of porcine peripheral blood mononuclear cells with recombinant IL-12p70 and IL-18 induced innate interferon gamma production by natural killer cells, while T cells and myeloid cells did not respond. Therefore, porcine CD3– NKp46+ cells serve as important IL-12 producers following TLR ligation, while IL-18 likely plays a prominent role in early immune response initiation in the pig following T. gondii infection.

Percentage of Myeloid Dendritic Cells in Peripheral Venous Blood Is Negatively Related to Incidence of Graves’ Orbitopathy

The aim of the study was to evaluate the distribution of blood dendritic cells (DCs) in patients with Graves' orbitopathy (GO) and to assess the influence of methylprednisolone therapy on subsets of peripheral blood mononuclear cells (PBMCs). Peripheral blood DC subsets were analyzed by flow cytometry in patients with active GO (n = 17), inactive GO (n = 8), and Graves' disease (GD) without GO (n = 8) and controls (n = 15); additionally, in patients with active GO (n = 17), analyses were done at three time points, i.e., before methylprednisolone treatment and after 6 weeks and after 12 weeks of the treatment. Percentage of myeloid DCs (mDCs) in PBMC fraction was significantly lower in patients with both active and inactive GO, compared to patients with GD without GO and controls (p < 0.05). In addition, mDCs were also documented to be an independent factor negatively associated with GO, however without essential differences between active and inactive phases. On the other hand, we did not observe any changes in the percentage of DCs after methylprednisolone therapy (p > 0.05). In the present study, we have succeeded to firstly demonstrate—according to our knowledge—that blood mDCs are negatively related to GO incidence.

Dendritic cell responses to Plasmodium falciparum in a malaria-endemic setting

BackgroundPlasmodium falciparum causes the majority of malaria cases worldwide and children in sub-Saharan Africa are the most vulnerable group affected. Non-sterile clinical immunity that protects from symptoms develops slowly and is relatively short-lived. Moreover, current malaria vaccine candidates fail to induce durable high-level protection in endemic settings, possibly due to the immunomodulatory effects of the malaria parasite itself. Because dendritic cells play a crucial role in initiating immune responses, the aim of this study was to better understand the impact of cumulative malaria exposure as well as concurrent P. falciparum infection on dendritic cell phenotype and function.MethodsIn this cross-sectional study, the phenotype and function of dendritic cells freshly isolated from peripheral blood samples of Malian adults with a lifelong history of malaria exposure who were either uninfected (n = 27) or asymptomatically infected with P. falciparum (n = 8) was assessed. Additionally, plasma cytokine and chemokine levels were measured in these adults and in Malian children (n = 19) with acute symptomatic malaria.ResultsWith the exception of lower plasmacytoid dendritic cell frequencies in asymptomatically infected Malian adults, peripheral blood dendritic cell subset frequencies and HLA-DR surface expression did not differ by infection status. Peripheral blood myeloid dendritic cells of uninfected Malian adults responded to in vitro stimulation with P. falciparum blood-stage parasites by up-regulating the costimulatory molecules HLA-DR, CD80, CD86 and CD40 and secreting IL-10, CXCL9 and CXCL10. In contrast, myeloid dendritic cells of asymptomatically infected Malian adults exhibited no significant responses above the uninfected red blood cell control. IL-10 and CXCL9 plasma levels were elevated in both asymptomatic adults and children with acute malaria.ConclusionsThe findings of this study indicate that myeloid dendritic cells of uninfected adults with a lifelong history of malaria exposure are able to up-regulate co-stimulatory molecules and produce cytokines. Whether mDCs of malaria-exposed individuals are efficient antigen-presenting cells capable of mounting an appropriate immune response remains to be determined. The data also highlights IL-10 and CXCL9 as important factors in both asymptomatic and acute malaria and add to the understanding of asymptomatic P. falciparum infections in malaria-endemic areas.

Alterations in peripheral blood monocyte and dendritic cell subset homeostasis in relapsing-remitting multiple sclerosis patients

Antigen-presenting cells participate and are implicated in the pathogenesis of multiple sclerosis. In our study we assessed the frequency of plasmacytoid (pDC) and myeloid (mDC) dendritic cells and the classical, intermediate and non-classical monocytes subsets, as well as their phenotypic and functional profile. We evaluated peripheral blood from relapsing-remitting patients treated with IFN-β in remission and relapse phases and from healthy subjects. In remission, we observed a decrease of mDC/pDC ratio and a return to normal values in relapse. In both phases the frequency of non-classical monocytes decreases. Concerning the phenotypic characterization, an increased HLA-DR expression was observed in remission and a decrease in relapse, revealing alterations in monocytes and dendritic cells homeostasis.

Dendritic cell integrin expression patterns regulate inflammation in the rheumatoid arthritis joint.

ObjectivesImmune dysregulation contributes to the development of RA. Altered surface expression patterns of integrin adhesion receptors by immune cells is one mechanism by which this may occur. We investigated the role of β2 integrin subunits CD11a and CD11b in dendritic cell (DC) subsets of RA patients.MethodsTotal β2 integrin subunit expression and its conformation (‘active’ vs ‘inactive’ state) were quantified in DC subsets from peripheral blood (PB) and SF of RA patients as well as PB from healthy controls. Ex vivo stimulation of PB DC subsets and in vitro-generated mature and tolerogenic monocyte-derived DCs (moDCs) were utilized to model the clinical findings. Integrin subunit contribution to DC function was tested by analysing clustering and adhesion, and in co-cultures to assess T cell activation.ResultsA significant reduction in total and active CD11a expression in DCs in RA SF compared with PB and, conversely, a significant increase in CD11b expression was found. These findings were modelled in vitro using moDCs: tolerogenic moDCs showed higher expression of active CD11a and reduced levels of active CD11b compared with mature moDCs. Finally, blockade of CD11b impaired T cell activation in DC–T cell co-cultures.ConclusionFor the first time in RA, we show opposing expression of CD11a and CD11b in DCs in environments of inflammation (CD11alow/CD11bhigh) and steady state/tolerance (CD11ahigh/CD11blow), as well as a T cell stimulatory role for CD11b. These findings highlight DC integrins as potential novel targets for intervention in RA.

Impaired Phagocytosis in Dendritic Cells From Pediatric Patients With Type 1 Diabetes Does Not Hamper Their Tolerogenic Potential.

Type 1 diabetes (T1D) is prompted by defective immunological tolerance, an event in which dendritic cells (DCs) are crucial as immune response orchestrators. In fact, they contribute to maintaining tolerance to self-antigens, but they can also prompt an immunogenic response against them, leading to autoimmunity. Countless factors can potentially impact on the proper functionality of the DCs, which range from altered subset distribution, impaired phagocytic function to abnormal gene expression. Moreover, in T1D, metabolic dysregulation could impair DC functions as well. Indeed, since T1D clinical course is likely to be more aggressive in children and adolescents and entails severe dysglycemia, the aim of this study was to analyze circulating DCs subpopulations in pediatric T1D at different stages, as well as to characterize their phagocytosis ability and tolerance induction potential. Thus, pediatric patients newly diagnosed with T1D, with established disease and control subjects were recruited. Firstly, DCs subsets from peripheral blood were found quantitatively altered during the first year of disease, but recovered in the second year of progression. Secondly, to study the tolerogenic functionality of DCs, liposomes with phosphatidylserine (PS) were designed to mimic apoptotic beta cells, which are able to induce tolerance, as previously demonstrated by our group in DCs from adult patients with T1D. In this study, monocyte-derived DCs from pediatric patients with T1D and control subjects were assessed in terms of PS-liposomes capture kinetics, and transcriptional and phenotypic changes. DCs from pediatric patients with T1D were found to phagocyte PS-liposomes more slowly and less efficiently than DCs from control subjects, inversely correlating with disease evolution. Nonetheless, the transcription of PS receptors and immunoregulatory genes, cytokine profile, and membrane expression of immunological markers in DCs was consistent with tolerogenic potential after PS-liposomes phagocytosis. In conclusion, T1D progression in childhood entails altered peripheral blood DCs subsets, as well as impaired DCs phagocytosis, although tolerance induction could still function optimally. Therefore, this study provides useful data for patient follow-up and stratification in immunotherapy clinical trials.

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.

Severe depletion of peripheral blood dendritic cell subsets in obstructive sleep apnea patients: A new link with cancer?

Recent evidence suggests that alterations of the immune responses are associated with the inflammatory nature of obstructive sleep apnea (OSA) and of its related co-morbidities. In this scenario, we asked whether circulating dendritic cell (DC) subsets may be possible players as their role has not yet been detailed. The frequency distribution of peripheral blood myeloid (mDC1 and mDC2) and plasmacytoid (p) DCs was investigated by mean of multi-parametric flow cytometry in 45 OSA patients (mean age: 53 yrs; M = 29) at the time of the first diagnosis and compared to 30 age- and sex-matched healthy controls. Oxidative burst (OB) and serum levels of tumor necrosis factor (TNF)-α, (interleukin) (IL)-6, interferon (INF)-γ, IL-2, IL-4, IL-10 and vascular endothelial growth factor (VEGF) were also analyzed. All subsets of circulating DCs were significantly depleted in OSA patients as compared to healthy subjects (p < 0.01, in all instances), with mDC2 and pDC subtypes being more severely compromised. These findings were co-existing with higher levels of OB along with an increased expression of IL-6, IL-10, TNF-α, IFN-γ, and VEGF (p < 0.005 in all instances). In particular, IL6 levels were significantly higher (p = 0.013) in severe OSA patients (apnea/hypopnea index >30) and were inversely correlated with both mDC2 (r = −0.802, p < 0.007) and pDC (r = −0.317, p = 0.04) subsets. We first provide evidence for a constitutive reduction of all circulating DC subsets in OSA patients. Perturbation of DCs coexists with an inflammatory milieu and is negatively correlated with the expression of IL-6, which is actually recognized as a pivotal inhibitor of DC maturation. Future studies exploring the contribution of DCs in the pathogenesis of OSA and of its complications should be encouraged.

Myeloid Dendritic Cells Are Enriched in Lymph Node Tissue of Early Rheumatoid Arthritis Patients but not in At Risk Individuals.

Lymph nodes (LNs) are highly organized structures where specific immune responses are initiated by dendritic cells (DCs). We investigated the frequency and distribution of human myeloid (mDCs) and plasmacytoid (pDCs) in LNs and blood during the earliest phases of rheumatoid arthritis (RA). We included 22 RA-risk individuals positive for IgM rheumatoid factor and/or anti-citrullinated protein antibodies, 16 biological-naïve RA patients and 8 healthy controls (HCs). DC subsets (CD1c+ mDCs and CD304+ pDCs) in LN tissue and paired peripheral blood were analyzed using flow cytometry and confocal microscopy. In blood of RA patients a significant decreased frequency of pDCs was found, with a similar trend for mDCs. In contrast, mDC frequencies were higher in RA compared with HCs and RA-risk individuals, especially in LN. Frequency of mDCs seemed higher in LNs compared to paired blood samples in all donors, while pDCs were higher in LNs only in RA patients. As expected, both mDCs and pDCs localized mainly in T-cell areas of LN tissue. In conclusion, compared with RA-risk individuals, mDCs and pDCs were enriched in the LN tissue of early-RA patients, while their frequency in RA-risk individuals was comparable to HCs. This may suggest that other antigen-presenting cells are responsible for initial breaks of tolerance, while mDCs and pDCs are involved in sustaining inflammation.

The prevalence of regulatory T and dendritic cells is altered in peripheral blood of women with pre-eclampsia.

Regulatory T cells (Tregs) and dendritic cell (DC) subsets play an essential role in the development of pregnancy immune tolerance. We aimed to investigate the proportion of CD4+CD25+FOXP3+ Tregs and DC subsets in peripheral blood of patients with pre-eclampsia compared to normal pregnant women. Peripheral blood samples were collected from 25 women with pre-eclampsia and 30 women with normal, healthy pregnancies. The ratio of CD4+CD25+ T cells in all CD4+ T cells, expression of FOXP3, and the ratio of myeloid DC (mDC) versus plasmacytoid DC (pDC) were assessed by flow cytometry. Levels of IL-10 and TGF-β in culture supernatants of purified CD4+CD25+ T cells were analyzed by enzyme-linked immunosorbent assay. The mean ratio of CD4+CD25+ T cells in CD4+ T lymphocytes in peripheral blood was lower in pre-eclampsia women than in normal pregnancies. The expression of Tregs marker FOXP3 in the pre-eclampsia group was significantly lower than the control group. The levels of IL-10 and TGF-β in supernatants of CD4+CD25+ T cells were significantly decreased in the pre-eclampsia group compared with the control group. The ratio of mDC/pDC was significantly higher in the pre-eclampsia group when compared with the control group. A decreased proportion and secretion of related inhibitory cytokines of CD4+CD25+FOXP3+ Tregs was found in the peripheral blood of pre-eclampsia, while the ratio of mDC/pDC increased. It is speculated that Tregs and DCs may play a role in the decreased immunosuppressive function of pre-eclampsia patients.

Costimulatory Molecules and Immune Checkpoints Are Differentially Expressed on Different Subsets of Dendritic Cells.

Dendritic cells (DCs) play a crucial role in initiating and shaping immune responses. The effects of DCs on adaptive immune responses depend partly on functional specialization of distinct DC subsets, and partly on the activation state of DCs, which is largely dictated by environmental signals. Fully activated immunostimulatory DCs express high levels of costimulatory molecules, produce pro-inflammatory cytokines, and stimulate T cell proliferation, whereas tolerogenic DCs express low levels of costimulatory molecules, produce immunomodulatory cytokines and impair T cell proliferation. Relevant to the increasing use of immune checkpoint blockade in cancer treatment, signals generated from inhibitory checkpoint molecules on DC surface may also contribute to the inhibitory properties of tolerogenic DCs. Yet, our knowledge on the expression of inhibitory molecules on human DC subsets is fragmentary. Therefore, in this study, we investigated the expression of three immune checkpoints on peripheral blood DC subsets, in basal conditions and upon exposure to pro-inflammatory and anti-inflammatory stimuli, by using a flow cytometric panel that allows a direct comparison of the activatory/inhibitory phenotype of DC-lineage and inflammatory DC subsets. We demonstrated that functionally distinct DC subsets are characterized by differential expression of activatory and inhibitory molecules, and that cDC1s in particular are endowed with a unique immune checkpoint repertoire characterized by high TIM-3 expression, scarce PD-L1 expression and lack of ILT2. Notably, this unique cDC1 repertoire was subverted in a group of patients with myelodysplastic syndromes included in the study. Applied to the characterization of DCs in the tumor microenvironment, this panel has the potential to provide valuable information to be used for investigating the role of DC subsets in cancer, guiding DC-targeting treatments, and possibly identifying predictive biomarkers for clinical response to cancer immunotherapy.

The effect of everolimus and low-dose cyclophosphamide on immune cell subsets in patients with metastatic renal cell carcinoma: results from a phase I clinical trial

For the treatment of metastatic renal cell cancer several strategies are used among which the mTOR inhibitor everolimus. As mTOR plays an important role in the immune system, e.g., by controlling the expression of the transcription factor FoxP3 thereby regulating regulatory T cells (Tregs), it plays a key role in the balance between tolerance and inflammation. Previous reports showed stimulatory effects of mTOR inhibition on the expansion of Tregs, an effect that can be considered detrimental in terms of cancer control. Since metronomic cyclophosphamide (CTX) was shown to selectively deplete Tregs, a phase 1 clinical trial was conducted to comprehensively investigate the immune-modulating effects of several dosages and schedules of CTX in combination with the standard dose of everolimus, with the explicit aim to achieve selective Treg depletion. Our data show that 50 mg of CTX once daily and continuously administered, in combination with the standard dose of 10 mg everolimus once daily, not only results in depletion of Tregs, but also leads to a reduction in MDSC, a sustained level of the CD8+ T-cell population accompanied by an increased effector to suppressor ratio, and reversal of negative effects on three peripheral blood DC subsets. These positive effects on the immune response may contribute to improved survival, and therefore this combination therapy is further evaluated in a phase II clinical trial.

Welcome message from the ISSHP President

Regulatory T cells (Tregs) and dendritic cell (DC) subsets play an essential role in the development of pregnancy immune tolerance. We aimed to investigate the proportion of CD4+CD25+FOXP3+ Tregs and DC subsets in peripheral blood of patients with pre-eclampsia compared to normal pregnant women.Peripheral blood samples were collected from 25 women with pre-eclampsia and 30 women with normal, healthy pregnancies. The ratio of CD4+CD25+ T cells in all CD4+ T cells, expression of FOXP3, and the ratio of myeloid DC (mDC) versus plasmacytoid DC (pDC) were assessed by flow cytometry. Levels of IL-10 and TGF-β in culture supernatants of purified CD4+CD25+ T cells were analyzed by enzyme-linked immunosorbent assay.The mean ratio of CD4+CD25+ T cells in CD4+ T lymphocytes in peripheral blood was lower in pre-eclampsia women than in normal pregnancies. The expression of Tregs marker FOXP3 in the pre-eclampsia group was significantly lower than the control group. The levels of IL-10 and TGF-β in supernatants of CD4+CD25+ T cells were significantly decreased in the pre-eclampsia group compared with the control group. The ratio of mDC/pDC was significantly higher in the pre-eclampsia group when compared with the control group.A decreased proportion and secretion of related inhibitory cytokines of CD4+CD25+FOXP3+ Tregs was found in the peripheral blood of pre-eclampsia, while the ratio of mDC/pDC increased. It is speculated that Tregs and DCs may play a role in the decreased immunosuppressive function of pre-eclampsia patients.

Phenotypic and Transcriptomic Analysis of Peripheral Blood Plasmacytoid and Conventional Dendritic Cells in Early Drug Naïve Rheumatoid Arthritis.

Dendritic cells (DCs) are key orchestrators of immune function. To date, rheumatoid arthritis (RA) researchers have predominantly focused on a potential pathogenic role for CD1c+ DCs. In contrast, CD141+ DCs and plasmacytoid DCs (pDCs) have not been systematically examined, at least in early RA. In established RA, the role of pDCs is ambiguous and, since disease duration and treatment both impact RA pathophysiology, we examined pDCs, and CD1c+ and CD141+ conventional DCs (cDCs), in early, drug-naïve RA (eRA) patients. We analyzed the frequency and phenotype of pDCs, CD1c+, and CD141+ DCs from eRA patients and compared findings with healthy controls. In parallel, we performed transcriptional analysis of >600 immunology-related genes (Nanostring) from peripheral blood pDCs, CD1c+ DCs, B cells, T cells, and monocytes. All DC subsets were reduced in eRA (n = 44) compared with healthy controls (n = 30) and, for pDCs, this was most marked in seropositive patients. CD141+ and CD1c+ DCs, but not pDCs, had a comparatively activated phenotype at baseline (increased CD86) and CD1c+ DC frequency inversely associated with disease activity. All DC frequencies remained static 12 months after initiation of immunomodulatory therapy despite a fall in activation markers (e.g., HLA-DR, CD40). There was no association between the whole blood interferon gene signature (IGS) and pDC or CD1c+ DC parameters but an inverse association between CD141+ DC frequency and IGS was noted. Furthermore, IFN-I and IFN-III mRNA transcripts were comparable between eRA pDC and other leukocyte subsets (B cells, CD4+, and CD8+ T cells and monocytes) with no obvious circulating cellular source of IFN-I or IFN-III. Transcriptomic analysis suggested increased pDC and CD1c+ DC proliferation in eRA; pDC differentially expressed genes also suggested enhanced tolerogenic function, whereas for CD1c+ DCs, pro-inflammatory transcripts were upregulated. This is the first detailed examination of DC subsets in eRA peripheral blood. Compared with CD1c+ DCs, pDCs are less activated and may be skewed toward tolerogenic functions. CD141+ DCs may be implicated in RA pathophysiology. Our findings justify further investigation of early RA DC biology.

Rehmannia glutinosa polysaccharide promoted activation of human dendritic cells

In our previous study, we showed that Rehmannia glutinosa polysaccharide (RGP) treatment induced maturation of dendritic cells (DCs) and that it had an anticancer effect in mice. The effect of RGP has not been studied in human DCs, including monocyte-derived DCs (MDDCs) and peripheral blood DCs (PBDCs). In this study, we examined DC activation by RGP in human cells. The dendritic morphology of RGP-treated MDDCs was substantially altered as compared with that of phosphate-buffered saline (PBS)-treated control cells. Moreover, RGP treatment markedly decreased phagocytic activity and increased expression levels of co-stimulatory molecules in MDDCs. In addition, RGP treatment elevated the production of proinflammatory cytokines. Furthermore, RGP-induced activation of MDDCs was dependent on the phosphorylation of extracellular signal-regulated kinase (ERK), p38, and c-Jun N-terminal kinase (JNK). RGP-treated MDDCs promoted upregulation of T-cell activation, including proliferation and interferon-gamma (IFN-γ) and tumor necrosis factor-alpha (TNF-α) production. Analysis of the effect of RGP in PBDC subsets revealed that it induced upregulation of co-stimulatory molecule expression and proinflammatory cytokine production. These data suggest that RGP may function as an immune stimulatory molecule in humans.