Blood Plasmacytoid Dendritic Cells Research Articles

Assessing the Innate Sensing of HIV-1 Infected CD4<sup>+</sup> T Cells by Plasmacytoid Dendritic Cells Using an <em>Ex vivo</em> Co-culture System.

HIV-1 innate sensing requires direct contact of infected CD4+ T cells with plasmacytoid dendritic cells (pDCs). In order to study this process, the protocols described here use freshly isolated human peripheral blood mononuclear cells (PBMCs) or plasmacytoid dendritic cells (pDCs) to sense infections in either T cell line (MT4) or heterologous primary CD4+ T cells. In order to ensure proper sensing, it is essential that PBMC are isolated immediately after blood collection and that optimal percentage of infected T cells are used. Furthermore, multi-parametric flow cytometric staining can be used to confirm that PBMC samples contain the different cell lineages at physiological ratios. A number of controls can also be included to evaluate viability and functionality of pDCs. These include, the presence of specific surface markers, assessing cellular responses to known agonist of Toll-Like Receptors (TLR) pathways, and confirming a lack of spontaneous type-I interferon (IFN) production. In this system, freshly isolated PBMCs or pDCs are co-cultured with HIV-1 infected cells in 96 well plates for 18-22 hr. Supernatants from these co-cultures are then used to determine the levels of bioactive type-I IFNs by monitoring the activation of the ISGF3 pathway in HEK-Blue IFN-α/β cells. Prior and during co-culture conditions, target cells can be subjected to flow cytometric analysis to determine a number of parameters, including the percentage of infected cells, levels of specific surface markers, and differential killing of infected cells. Although, these protocols were initially developed to follow type-I IFN production, they could potentially be used to study other imuno-modulatory molecules released from pDCs and to gain further insight into the molecular mechanisms governing HIV-1 innate sensing.

Assessing the Innate Sensing of HIV-1 Infected CD4+ T Cells by Plasmacytoid Dendritic Cells Using an Ex vivo Co-culture System.

HIV-1 innate sensing requires direct contact of infected CD4+ T cells with plasmacytoid dendritic cells (pDCs). In order to study this process, the protocols described here use freshly isolated human peripheral blood mononuclear cells (PBMCs) or plasmacytoid dendritic cells (pDCs) to sense infections in either T cell line (MT4) or heterologous primary CD4+ T cells. In order to ensure proper sensing, it is essential that PBMC are isolated immediately after blood collection and that optimal percentage of infected T cells are used. Furthermore, multi-parametric flow cytometric staining can be used to confirm that PBMC samples contain the different cell lineages at physiological ratios. A number of controls can also be included to evaluate viability and functionality of pDCs. These include, the presence of specific surface markers, assessing cellular responses to known agonist of Toll-Like Receptors (TLR) pathways, and confirming a lack of spontaneous type-I interferon (IFN) production. In this system, freshly isolated PBMCs or pDCs are co-cultured with HIV-1 infected cells in 96 well plates for 18-22 hr. Supernatants from these co-cultures are then used to determine the levels of bioactive type-I IFNs by monitoring the activation of the ISGF3 pathway in HEK-Blue IFN-α/β cells. Prior and during co-culture conditions, target cells can be subjected to flow cytometric analysis to determine a number of parameters, including the percentage of infected cells, levels of specific surface markers, and differential killing of infected cells. Although, these protocols were initially developed to follow type-I IFN production, they could potentially be used to study other imuno-modulatory molecules released from pDCs and to gain further insight into the molecular mechanisms governing HIV-1 innate sensing.

PTPN22 Variant R620W Is Associated With Reduced Toll-like Receptor 7-Induced Type I Interferon in Systemic Lupus Erythematosus.

Protein tyrosine phosphatase nonreceptor type 22 (PTPN22) is associated with an increased risk of systemic lupus erythematosus (SLE). PTPN22 encodes Lyp, and a disease-associated coding variant bears an R620W substitution (LypW). LypW carriage is associated with impaired production of type I interferon (IFN) by myeloid cells following Toll-like receptor (TLR) engagement. The aim of this study was to investigate the effects of LypW carriage on TLR signaling in patients with SLE. Plasma IFNα concentrations and whole-blood IFN gene scores were compared in SLE patients who were LypW carriers and those who were noncarriers. TLR-7 agonist R848-stimulated IFNα and tumor necrosis factor levels, IFN-dependent gene expression, and STAT-1 activation were determined in peripheral blood mononuclear cells (PBMCs) and/or plasmacytoid dendritic cells (PDCs) obtained from these patients. The effect of LypW expression on the systemic type I IFN response to R848 stimulation in vivo was assessed in transgenic mice. Plasma IFNα levels and whole-blood IFN gene signatures were comparable in SLE patients who were LypW carriers and those who were noncarriers. However, PBMCs from LypW carriers produced less IFNα and showed reduced IFN-dependent gene up-regulation and STAT-1 activation after R848 stimulation. The frequency of PDCs producing IFNα2 and the per-cell IFNα2 levels were significantly reduced in LypW carriers. LypW-transgenic mice displayed reduced TLR-7-induced circulating type I IFN responses. PDCs from SLE patients carrying the disease-associated PTPN22 variant LypW showed a reduced capacity for TLR-7 agonist-induced type I IFN production, even though LypW carriers displayed systemic type I IFN activation comparable with that observed in noncarriers. LypW carriage identifies SLE patients who may harbor defects in TLR- and PDC-dependent host defense or antiinflammatory functions.

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.

A novel CD141+ human blood plasmacytoid dendritic cell subtype produces high levels of IFN-α and acquires virus-associated cellular material from live tumor cells (VIR9P.1136)

Abstract Dendritic cells (DCs) are a heterogeneous population important in regulating both innate and adaptive immunity. Human blood DCs are broadly characterized as pDC(BDCA2+), mDC1(BDCA1+), and mDC2(BDCA3+(CD141)). While much work has focused on the functional role of CD141hi mDCs, we investigated the maturation potential and functional significance of a novel subtype of classically defined plasmacytoid DCs (BDCA2+) co-expressing intermediate levels of BDCA3. Using flow cytometry, we identified four human blood DC subsets: BDCA2+/3-, BDCA2+/3int, BDCA2-/3int, and BDCA2-/3hi. Upon stimulation with TLR9 agonists CpG-A or HSV-1, the BDCA2+/3int DCs had the highest frequency of IFN-α+ and TNF-α+ cells among these DC subtypes. The BDCA2+/3int compared to BDCA2+/3- DCs also become more activated and mature with relatively higher expression of co-stimulatory and migratory surface receptors in response to HSV-1. IRF7, the master regulator of IFN production in pDCs, was constitutively expressed by BDCA2+/3- and BDCA2+/3int ­DCs but not by BDCA2- DCs. Both BDCA2+/3int and to a lesser extent, BDCA2+/3- DC preferentially acquired cellular material from HSV-infected Raji cells. These data indicate that BDCA2+/3int DCs exhibit major functions associated with classical pDCs and are phenotypically and functionally distinct from BDCA2-/3int and BDCA2-/3hi DCs. Together these results describe a novel CD141+ pDC subset that is very potent and virus responsive as compared to the CD141hi mDCs.

Peripheral Blood Plasmacytoid Dendritic Cells at Day 100 Can Predict Outcome after Allogeneic Stem Cell Transplantation

The rapidly increasing use of allogeneic stem cell transplantation (allo-SCT) emphasizes the need for identifying variables predictive of its outcome. Plasmacytoid dendritic cells (pDCs) play a major role in establishing immune competence and in several autoimmune diseases. Thus, we investigated whether pDCs might influence the outcome of patients after allo-SCT in 79 consecutive patients who underwent this procedure. pDCs were identified in the blood of patients at day 100 after allo-SCT by staining peripheral blood mononuclear cells for surface markers and intracellular cytokines and analyzing them on a flow cytometer. We found the pDC level at day 100 was not influenced by patient or graft characteristics, and only the absence of previous grades II to IV acute graft-versus-host disease was significantly associated with higher levels of blood pDCs after allo-SCT (OR, .67; 95% CI, .54 to .83; P = .0004). Using the median value of pDCs at day 100 to divide the patients into 2 distinct groups, we observed that a low pDC level was correlated with a worse overall survival (55% versus 86%, P = .007). In a multivariate analysis, only low pDC level (OR, 3.41; 95% CI, 1.19 to 9.79; P = .02) and older patient age (OR, 5.16; 95% CI, 1.15 to 23.14; P = .03) were significantly predictive of increased risk of death. We conclude that monitoring of pDC may be useful for patient management and may have a significant impact on the probability of a favorable outcome of allo-SCT.

Abstract 174: Role of Cardiomyocyte-Localized Nuclear Factor (NF)-κB in Diet-Induced Diabetic Cardiomyopathy

Role of Inflammatory signaling in the development of obesity- and type 2 diabetes (T2D)-associated cardiomyopathy remains unclear. We hypothesized that cardiomyocyte-specific inflammatory activation, independent of systemic inflammation, is sufficient for the genesis of diabetic cardiomyopathy. Wild-type (WT) and transgenic (Tg) C57BL/6 mice with myocyte-restricted overexpression of phosphorylation-resistant IκBα (IκBαS32A,S36A) were fed a high fat (HFD, 45% kcal fat) or control diet (CD, 10% kcal fat) for 28 weeks to induce obesity and T2D. These Tg mice exhibit attenuated NF-kB activation, a central regulator of inflammation. After 28 weeks of feeding, WT HFD mice exhibited increased body weight, hyperglycemia, hyperinsulinemia, insulin resistance, and glucose intolerance. In contrast, although HFD-fed Tg mice exhibited significantly (p < 0.05) increased body weight, the mice still maintained normal fasting blood glucose, insulin levels, and insulin sensitivity compared to CD Tg mice, indicating absence of overt T2D. Echocardiography revealed that both HFD-fed WT and Tg mice developed concentric left ventricular (LV) hypertrophy with increased septal and posterior wall thickness, decreased LV end-diastolic diameter and had unchanged LV ejection fraction suggesting development of LV hypertrophy in both strains regardless of the severity of the metabolic disorder. Analysis of mononuclear phagocyte cell profiles revealed similar trends in WT and IκB Tg mice. HFD fed mice had higher levels of pro-inflammatory (CD11b+Ly6chi) and anti-inflammatory (CD11b+Ly6clo) monocytes in the blood, heart, spleen and adipose tissue compared to CD fed mice. Analysis of antigen presenting cells indicated that HFD fed mice in both genotypes had significantly increased levels of both classical (CD11c+CD45R–) and plasmacytoid (CD11cintCD45R+) dendritic cells in blood, heart, spleen and adipose tissues compared to respective CD fed mice. Thus, we conclude that cardiomyocyte inflammation is central to the development of metabolic abnormalities related to obesity, whereas neither cardiomyocyte-specific nor systemic insulin resistance is required for the development of the hypertrophic cardiomyopathic phenotype.

EFFECT OF TEMOZOMIDE ON HUMAN DENDRITIC CELLS

BACKGROUND: We are undertaking dendritic cell (DC) therapy against glioma stem-like cells (GSCs) with concomitant maintenance chemotherapy using temozolomide (TMZ). In general, anti-cancer agents are known to suppress immune response in patients. TMZ also suppresses immunity by inducing lymphopenia and myelosuppression in malignant glioma patients. However, there has been no study about the effect of TMZ on DC function. In this study, we evaluate whether concomitant TMZ affects DC function in vitro. METHODS: We isolated human blood plasmacytoid DCs (pDCs) and myelocytoid DCs (mDCs) from healthy volunteers by flow cytometry. These DC subsets were cultured in flat-bottomed 96-well plates at 5 × 104 cells in the final 200 µl of medium per well for 24 h. In the first set of experiment, we assessed cytotoxic activity of TMZ on DCs at clinically relevant concentration. After 24 h, viable cells were measured by a trypan-blue exclusion test. We next measured the Toll-like receptor ligand (TLRL) -induced cytokine production by DCs in the presence of TMZ. DC subsets were stained with FITC-labeled anti-CD83 and PE-labeled anti-CD86 antibody and then analyzed by FACS calibur. We also measured secreted IL-12p40 and IFN-alpha concentration in the supernatant of the cultures by ELISA. RESULTS: Analysis of trypan-blue exclusion of the dead cells showed that over 300 microM of TMZ started to kill both DC subsets and there was no cytocidal effect at 100 microM, a clinically relevant concentration. Therefore, we thereafter used 10 microM to 300 microM for the following assays. We found that TMZ did not affect IFN-alpha production by pDCs and IL-12 production by mDCs in response to various TLRLs. In addition, up to 100 microM of concentration, TMZ did not influence the CD83 and CD86 expression on both DC subsets. CONCLUSIONS: Temozolomide did not significantly affect on DC function, suggesting that DC therapy and concomitant chemotherapy using TMZ may be possible, although further assessment in the clinical setting is necessary. In the presentation, we also would like to present preliminary data on interaction between DCs and two types of GSCs (proneural and mesenchymal). SECONDARY CATEGORY: n/a.

The Aryl Hydrocarbon Receptor Antagonist StemRegenin 1 Promotes Human Plasmacytoid and Myeloid Dendritic Cell Development from CD34+ Hematopoietic Progenitor Cells

The superiority of dendritic cells (DCs) as antigen-presenting cells has been exploited in numerous clinical trials, where generally monocyte-derived DCs (Mo-DCs) are injected to induce immunity in patients with cancer or infectious diseases. Despite promising expansion of antigen-specific T cells, the clinical responses following vaccination have been limited, indicating that further improvements of DC vaccine potency are necessary. Pre-clinical studies suggest that vaccination with combination of primary DC subsets, such as myeloid and plasmacytoid blood DCs (mDCs and pDCs, respectively), may result in stronger clinical responses. However, it is a challenge to obtain high enough numbers of primary DCs for immunotherapy, since their frequency in blood is very low. We therefore explored the possibility to generate them from hematopoietic progenitor cells (HPCs). Here, we show that by inhibiting the aryl hydrocarbon receptor with its antagonist StemRegenin 1 (SR1), clinical-scale numbers of functional BDCA2(+)BDCA4(+) pDCs, BDCA1(+) mDCs, and BDCA3(+)DNGR1(+) mDCs can be efficiently generated from human CD34(+) HPCs. The ex vivo-generated DCs were phenotypically and functionally comparable to peripheral blood DCs. They secreted high levels of pro-inflammatory cytokines such as interferon (IFN)-α, interleukin (IL)-12, and tumor necrosis factor (TNF)-α and upregulated co-stimulatory molecules and maturation markers following stimulation with Toll-like receptor (TLR) ligands. Further, they induced potent allogeneic T-cell responses and activated antigen-experienced T cells. These findings demonstrate that SR1 can be exploited to generate high numbers of functional pDCs and mDCs from CD34(+) HPCs, providing an alternative option to Mo-DCs for immunotherapy of patients with cancer or infections.

Hepatitis C virus alternate reading frame protein decreases interferon-α secretion in peripheral blood mononuclear cells

The hepatitis C virus (HCV) alternate reading frame protein (ARFP or F protein) of the HCV 1b genotype is a double-frameshift product of the HCV core protein (Core). The discovery of HCV F protein challenges various biological functions attributed to Core. However, the specific characteristics of the host cellular immune response to F protein during HCV infection have yet to be fully elucidated. Therefore, the present study investigated the cytokine response to HCV Core or F protein in peripheral blood mononuclear cells (PBMCs) and plasmacytoid dendritic cells (PDCs) from patients with chronic HCV and healthy donors in vitro. The results demonstrated that the levels of interferon (IFN)-α, analyzed by an enzyme-linked immunosorbent assay, secreted by PBMCs in patients positive for the anti-F protein antibody, were lower than those of patients negative for the anti-F protein antibody. Moreover, the frequency of PDCs in patients negative for the anti-F protein antibody, were higher than in the group positive for the anti-F protein antibody. Furthermore, HCV F protein and Core not only inhibited specific unmethylated CpG oligonucleotide sequences of type A (CpG‑A)-induced IFN-α production by PBMCs and PDCs, but also upregulated the production of interleukin (IL)-10 by PBMCs in patients with chronic HCV and healthy controls. Notably, following neutralization of IL-10 in the media and in vitro Core or F protein stimulation, levels of IFN-α were increased. Moreover, the results revealed that the roles of F protein and Core were similar with regard to the induction of apoptosis of PDCs in patients with chronic HCV. These findings suggest that F protein may inhibit PBMC IFN-α secretion by regulating the production of IL-10, and may contribute to an increase in the rates of apoptosis in PDCs. In conclusion, the results have revealed a potential involvement of F protein in the mechanisms of chronic hepatitis C.

Longitudinal Analysis of Distribution and Function of Plasmacytoid Dendritic Cells in Peripheral Blood and Gut Mucosa of HIV Infected Patients

Aberrant activation of plasmacytoid dendritic cells (pDCs) with excessive production of interferon alpha (IFNα) represents one of the hallmarks of immune activation during chronic phase of human immunodeficiency virus (HIV) infection. A number of studies have shown that disruption of mucosal integrity in the gut is a cause of persistent immune activation. However, little is known about the role that pDCs play in this process, and our current understanding comes from the simian immunodeficiency virus macaque model. Thus, in the present study we sought to investigate the frequency and function of pDCs in peripheral blood and gut samples from HIV-infected individuals before and 6 months after initiation of antiretroviral therapy (ART). We show that circulating pDCs were depleted in ART-naive HIV+ patients, and upregulated the gut-homing receptor CD103 compared with uninfected controls. By converse, pDCs accumulated in the terminal ileum of ART-naive HIV individuals compared with controls. Baseline levels of IFNα production and markers of immune activation in gut samples of ART-naive HIV subjects were elevated. All these parameters declined after 6 months of ART. Our results suggest that in chronic HIV infection, pDCs migrate from peripheral blood to the gut-associated lymphatic tissue, where they may contribute to immune activation.

Whole blood stimulation with Toll-like receptor (TLR)-7/8 and TLR-9 agonists induces interleukin-12p40 expression in plasmacytoid dendritic cells in rhesus macaques but not in humans

Macaques provide important animal models in biomedical research into infectious and chronic inflammatory disease. Therefore, a proper understanding of the similarities and differences in immune function between macaques and humans is needed for adequate interpretation of the data and translation to the human situation. Dendritic cells are important as key regulators of innate and adaptive immune responses. Using a new whole blood assay we investigated functional characteristics of blood plasmacytoid dendritic cells (pDC), myeloid dendritic cells (mDC) and monocytes in rhesus macaques by studying induction of activation markers and cytokine expression upon Toll-like receptor (TLR) stimulation. In a head-to-head comparison we observed that rhesus macaque venous blood contained relatively lower numbers of pDC than human venous blood, while mDC and monocytes were present at similar percentages. In contrast to humans, pDC in rhesus macaques expressed the interleukin (IL)-12p40 subunit in response to TLR-7/8 as well as TLR-9 stimulation. Expression of IL-12p40 was confirmed by using different monoclonal antibodies and by reverse transcription-polymerase chain reaction (RT-PCR). Both in humans and rhesus macaques, TLR-4 stimulation induced IL-12p40 expression in mDC and monocytes, but not in pDC. The data show that, in contrast to humans, pDC in macaques are able to express IL-12p40, which could have consequences for evaluation of human vaccine candidates and viral infection.

A Proof-of-Concept, Randomized, Controlled Trial of Omalizumab in Patients With Severe, Difficult-to-Control, Nonatopic Asthma

While up to 50% of patients with severe asthma have no evidence of allergy, IgE has been linked to asthma, irrespective of atopic status. Omalizumab, an anti-IgE monoclonal antibody, is reported to significantly benefit a subset of patients with severe, persistent, allergic asthma. Therefore, we investigated whether omalizumab has biologic and clinical effects in patients with refractory nonatopic asthma. Forty-one adult patients who, despite daily treatment with or without maintenance oral corticosteroids, had severe, nonatopic, refractory asthma according to GINA (Global Initiative for Asthma) step 4, were randomized to receive omalizumab or placebo in a 1:1 ratio. The primary end point was the change in expression of high-affinity IgE receptor (FcεRI) on blood basophils and plasmacytoid dendritic cells (pDC2) after 16 weeks. The impact of omalizumab on lung function and clinical variables was also examined. Compared with placebo, omalizumab resulted in a statistically significant reduction in FcεRI expression on basophils and pDC2 (P < .001). The omalizumab group also showed an overall increase in FEV1 compared with baseline (+250 mL, P = .032; +9.9%, P = .029). A trend toward improvement in global evaluation of treatment effectiveness and asthma exacerbation rate was also observed. Omalizumab negatively regulates FcεRI expression in patients with severe nonatopic asthma, as it does in severe atopic asthma. Omalizumab may have a therapeutic role in severe nonatopic asthma. Nonetheless, our preliminary findings support further investigation to better assess the clinical efficacy of omalizumab. ClinicalTrials.gov; No.: NCT01007149; URL: www.clinicaltrials.gov and European Clinical Trials Database, EudraCT; No.: 2009-010937-38; URL: https://www.clinicaltrialsregister.eu.

C‐Reactive Protein Inhibits Plasmacytoid Dendritic Cell Interferon Responses to Autoantibody Immune Complexes

C-reactive protein (CRP) is a serum pattern recognition molecule that binds to apoptotic cells and nucleoprotein autoantigens and Fcγ receptors (FcγR). In systemic lupus erythematosus (SLE), immune complexes (ICs) containing nucleoprotein autoantigens activate plasmacytoid dendritic cells (PDCs) to produce type I interferon (IFN), which contributes to disease pathogenesis. Autoantibody ICs are taken up by PDCs through FcγR type IIa into endosomes, where the nucleic acid components activate Toll-like receptor 7 (TLR-7) or TLR-9. The objective of this study was to investigate the effect of CRP on PDC and monocyte responses to nucleoprotein autoantigens and ICs. Peripheral blood mononuclear cells (PBMCs), purified monocytes, and PDCs were isolated from healthy volunteers and stimulated with autoantibody ICs containing apoptotic cells, small nuclear RNPs (snRNPs), or DNA, or directly with TLR-7 and TLR-9 agonists. Supernatants were analyzed for IFNα and cytokine levels by enzyme-linked immunosorbent assay and multiplex assay. Small nuclear RNPs were fluorescence-labeled, and the effect of CRP on binding, uptake, and intracellular localization of autoantibody snRNP complexes was measured by flow cytometry and confocal microscopy. CRP bound to autoantigen did not induce IFNα in PBMCs or PDCs, whereas complexes formed with autoantibody did. Significantly, CRP inhibited the IFNα response to both anti-U1 RNP-snRNP complexes and anti-DNA-DNA complexes, but not to other TLR-7 and TLR-9 agonists. CRP directly inhibited PDC IFNα release, promoted PDC differentiation, and increased late endosome localization of autoantigen in PDCs and monocytes. CRP is a regulator of the type I IFN response to SLE ICs. CRP increased the intracellular processing of ICs in late endosomes, which is associated with decreased synthesis of type I IFN after intracellular TLR activation.

AB0202 Changes and significance of dendritic cells in peripheral blood in patients with systemic lupus erythematosus

BackgroundSystemic lupus erythematosus (SLE) is an autoimmune disease characterized by immunological intolerance, hyperactivity of various immunocyte and production of a variety of autoantibodies. Although etiology of the disease remains unclear,...

Regulatory T cells, dendritic cells and neutrophils in patients with renal cell carcinoma

We evaluated dendritic cells (DC), regulatory T lymphocytes (Treg) and neutrophils in 37 patients with newly diagnosed renal cell carcinoma (RCC) in the tumor and peripheral blood (PB) and correlated these parameters with tumor staging (early-T1, 2, late-T3, 4 and metastatic disease). The number of myeloid and plasmacytoid DC in blood of RCC patients was higher than in healthy controls. The percentage of myeloid dendritic cells (mDC) from CD45+ cells in tumors was higher in comparison with peripheral blood irrespective of disease stage. Higher percentage of these cells expressed a maturation marker in the periphery in the early stage (CD83 expressing cells). The number of plasmacytoid dendritic cells (pDC) in PB was similar in both early and late stage groups, but the early group displayed a significantly higher percentage of pDC in tumor cell suspension. Neutrophil counts in the peripheral blood of RCC patients were higher than in healthy controls, but the counts in both tumor stage groups were similar. The proportion of neutrophils from CD45+ cells was higher in late stage tumors. Higher percentage of Treg from CD4+ cells was detected in renal carcinoma tissue in comparison to PB with no difference between stages of the disease. Our results reflect the complex interplay between various cells of the immune system and the tumor microenvironment. Activation of dendritic cell subpopulations at early stages of the disease course is counterbalanced by the early appearance of T regulatory cells both in the periphery and tumor tissue. Later stages are characterized by the accumulation of neutrophils in the tumor. Appropriate timing of anticancer strategies, especially immunotherapy, should take these dynamics of the immune response in RCC patients into account.

Peripheral Blood CD4 T-Cell and Plasmacytoid Dendritic Cell (pDC) Reactivity to Herpes Simplex Virus 2 and pDC Number Do Not Correlate with the Clinical or Virologic Severity of Recurrent Genital Herpes

Leukocytes participate in the immune control of herpes simplex virus (HSV). Data from HIV coinfections, germ line mutations, and case reports suggest involvement of CD4 T cells and plasmacytoid dendritic cells (pDC). We investigated the relationships between these cells and recurrent genital herpes disease severity in the general population. Circulating CD4 T-cell responses to HSV-2 were measured in specimens from 67 immunocompetent individuals with measured genital lesion and HSV shedding rates. Similarly, pDC number and functional responses to HSV-2 were analyzed in 40 persons. CD4 responses and pDC concentrations and responses ranged as much as 100-fold between persons while displaying moderate within-person consistency over time. No correlations were observed between these immune response parameters and genital HSV-2 severity. Cytomegalovirus (CMV) coinfection was not correlated with differences in HSV-2-specific CD4 T-cell responses. The CD4 T-cell response to HSV-2 was much more polyfunctional than was the response to CMV. These data suggest that other immune cell subsets with alternate phenotypes or anatomical locations may be responsible for genital herpes control in chronically infected individuals.

Pre‐transplant myeloid dendritic cell deficiency associated with cytomegalovirus infection and death after kidney transplantation

Dendritic cells (DCs) are potent antigen-presenting cells critical for immunity. We previously demonstrated a significant association between pre-transplant blood myeloid dendritic cell (mDC) and plasmacytoid dendritic cell (pDC) deficiency and post-transplant BK viremia in renal transplant recipients. In the current post-hoc analysis, we studied the association of these same pre-transplant DC levels with other post-transplant outcomes. Pre-transplant peripheral blood mDC and pDC levels were quantified using flow cytometry in 78 patients undergoing kidney transplantation. Post-transplant outcomes were analyzed, including infection, rejection, and patient death, with a median follow-up of 5.3years. Associations between DC levels and outcomes were assessed using logistic regression analysis and Cox proportional hazards models. An independent association of mDC levels with post-transplant cytomegalovirus infection (adjusted odds ratio 7.0, P=0.01) and patient death (adjusted hazard ratio 13.0, P=0.015) was found. No associations were demonstrated between levels of either DC subtype and bacterial infections or rejection. Pre-transplant mDC deficiency is significantly associated with CMV infection and death after kidney transplantation.

Post-transcriptional down-regulation of Toll-like receptor signaling pathway in umbilical cord blood plasmacytoid dendritic cells

Plasmacytoid dendritic cells (PDCs) from human umbilical cord blood (UCB) produce lower amounts of IFN-α upon TLR stimulation compared with adult counterparts. This difference may play a role in the low graft-versus-host disease rate after UCB transplantation and in the impaired immune response of the neonate to pathogens. Comparing UCB PDC to their adults counterparts, we found that they exhibited a mature surface phenotype and a normal antigen uptake. They upregulated costimulatory molecules upon activation, although with delayed kinetics. Protein, but not ARN, levels of TLR-9, MyD88, IRAK1 and IRF-7, involved in the TLR-9 signaling pathway were reduced. The expression levels of miR-146a and miR-155, known to be involved in the post-transcriptional down-regulation of immune responses, were higher. These data point out a post-transcriptional down-regulation of the TLR-9/IRF-7 signaling pathway in UCB PDC.

HIV-1 infection and induction of interferon alpha in plasmacytoid dendritic cells

Loss of blood plasmacytoid dendritic cell (pDC) in HIV-1 infection is thought to impact on adaptive immune responses whilst the virus also induces aberrant interferon alpha (IFN-α) production that may fuel chronic immune activation and drive disease progression. Recent attention has focussed on the pathway of HIV-induced IFN-α production by pDC and the new data are reviewed here together with the pathway leading to infection. Attachment to CD4 and chemokine co-receptors is essential for HIV-1 infection. Although CD4, but not co-receptor binding, is a major route for passage to endosomes and triggering of IFN-α secretion this may also occur by CD4-independent mechanisms involving other receptors. In contrast to other Toll-like receptor (TLR)-7 ligands and RNA viruses that stimulate pDC to secrete IFN-α for 2-3 h, HIV-1-stimulated pDC can give sustained IFN-α production for up to 48 h which may contribute to chronic immune activation. This may reflect retention of HIV in early endosomes which also seems to be associated with incomplete maturation induced by HIV. HIV-1-pDC interactions contribute to pathogenesis through depletion and aberrant IFN-α production. New data on the pathway of pDC HIV-stimulated IFN-α secretion may facilitate therapy to reduce chronic immune activation and slow disease progression.