Peripheral DCs Research Articles

LMAN1, a Negative Regulator of the Response to House Dust Mite

Abstract We have utilized an unbiased receptor capture approach (CaptiRec) to screen for receptors on dendritic cells which directly bind HDM. Using this technique, we have identified LMAN1 (Lectin, Mannose Binding 1) as a candidate receptor for HDM. LMAN1 is a mannose binding lectin which is primarily known to act as a cargo transporter between the ER, ERGIC, and Golgi compartments. The role played by LMAN1 in the recognition of and response to HDM, is currently unknown. We verify the ability of LMAN1 to directly bind HDM proteins biochemically and in a cellular context. Furthermore, we present evidence to demonstrate that LMAN1 downregulates NF-κB responses in response to inflammatory cytokines and to HDM. Loss of LMAN1 on DCs used for allergic sensitization leads to exacerbated airway inflammation in a HDM model of allergic asthma. Lastly, peripheral DCs of asthmatics show a significant reduction in expression of LMAN1 compared to healthy controls. These findings have potential implications for the development of novel therapeutic interventions for allergy and allergic asthma. Supported by American Lung Association Innovation Award IA-695632

Single-Cell Analysis Reveals the Heterogeneity of Monocyte-Derived and Peripheral Type-2 Conventional Dendritic Cells.

Dendritic cells (DCs) are critical for pathogen recognition and Ag processing/presentation. Human monocyte-derived DCs (moDCs) have been extensively used in experimental studies and DC-based immunotherapy approaches. However, the extent of human moDC and peripheral DCs heterogeneity and their interrelationship remain elusive. In this study, we performed single-cell RNA sequencing of human moDCs and blood DCs. We identified seven subtypes within moDCs: five corresponded to type 2 conventional DCs (cDC2s), and the other two were CLEC10A+CD127+ cells with no resemblance to any peripheral DC subpopulations characterized to date. Moreover, we defined five similar subtypes in human cDC2s, revealed the potential differentiation trajectory among them, and unveiled the transcriptomic differences between moDCs and cDC2s. We further studied the transcriptomic changes of each moDC subtype during maturation, demonstrating SLAMF7 and IL15RA as maturation markers and CLEC10A and SIGLEC10 as markers for immature DCs. These findings will enable more accurate functional/developmental analyses of human cDC2s and moDCs.

Haploidentical mixed chimerism cures autoimmunity in established type 1 diabetic mice.

Clinical trials are currently testing whether induction of haploidentical mixed chimerism (Haplo-MC) induces organ transplantation tolerance. Whether Haplo-MC can be used to treat established autoimmune diseases remains unknown. Here, we show that established autoimmunity in euthymic and adult-thymectomized NOD (H-2g7) mice was cured by induction of Haplo-MC under a non-myeloablative anti-thymocyte globulin-based conditioning regimen and infusion of CD4+ T cell-depleted hematopoietic graft from H-2b/g7 F1 donors that expressed autoimmune-resistant H-2b or from H-2s/g7 F1 donors that expressed autoimmune-susceptible H-2s. The cure was associated with enhanced thymic negative selection, increased thymic Treg (tTreg) production, and anergy or exhaustion of residual host-type autoreactive T cells in the periphery. The peripheral tolerance was accompanied by expansion of donor- and host-type CD62L-Helios+ tTregs as well as host-type Helios-Nrp1+ peripheral Tregs (pTregs) and PD-L1hi plasmacytoid DCs (pDCs). Depletion of donor- or host-type Tregs led to reduction of host-type PD-L1hi pDCs and recurrence of autoimmunity, whereas PD-L1 deficiency in host-type DCs led to reduction of host-type pDCs and Helios-Nrp1+ pTregs. Thus, induction of Haplo-MC reestablished both central and peripheral tolerance through mechanisms that depend on allo-MHC+ donor-type DCs, PD-L1hi host-type DCs, and the generation and persistence of donor- and host-type tTregs and pTregs.

Relationship between Helicobacter pylori Infection and Plasmacytoid and Myeloid Dendritic Cells in Peripheral Blood and Gastric Mucosa of Children.

Purpose To investigate the frequency and activation status of peripheral plasmacytoid DCs (pDCs) and myeloid DCs (mDCs) as well as gastric mucosa DC subset distribution in Helicobacter pylori- (H. pylori-) infected and noninfected children. Materials and Methods Thirty-six children were studied; twenty-one had H. pylori. The frequencies of circulating pDCs (lineage−HLA-DR+CD123+) and mDCs (lineage−HLA-DR+CD11c+) and their activation status (CD83, CD86, and HLA-DR expression) were assessed by flow cytometry. Additionally, the densities of CD11c+, CD123+, CD83+, CD86+, and LAMP3+ cells in the gastric mucosa were determined by immunohistochemistry. Results The frequency of circulating CD83+ mDCs was higher in H. pylori-infected children than in the noninfected controls. The pDCs demonstrated upregulated HLA-DR surface expression, but no change in CD86 expression. Additionally, the densities of gastric lamina propria CD11c+ cells and epithelial pDCs were increased. There was a significant association between frequency of circulating CD83+ mDCs and gastric lamina propria mDC infiltration. Conclusion This study shows that although H. pylori-infected children had an increased population of mature mDCs bearing CD83 in the peripheral blood, they lack mature CD83+ mDCs in the gastric mucosa, which may promote tolerance to local antigens rather than immunity. In addition, this may reduce excessive inflammatory activity as reported for children compared to adults.

IMMU-47. RNA-NANOPARTICLE VACCINES ARE SAFE AND IMMUNOLOGICALLY ACTIVE IN CLIENT-OWNED CANINES WITH TERMINAL GLIOMAS

Abstract BACKGROUND The lack of appropriate preclinical murine glioblastoma models limits comprehensive toxicity/efficacy evaluation of investigational agents. To overcome this challenge, we evaluated the safety and activity of a new immunotherapeutic technology that we have pioneered (composed of tumor mRNA complexed into a custom lipid-nanoparticle formulation) in client-owned canines (pet dogs) diagnosed with malignant gliomas. OBJECTIVE/ METHODS Canine malignant gliomas were biopsied for generation of personalized tumor mRNA loaded into our custom lipid-nanoparticle (NP) vector. The patients received RNA-NPs intravenously beginning two weeks after their biopsy once weekly (x3) and no other anti-tumor therapeutic interventions. RESULTS Within a few hours after administration, tumor specific RNA-NPs elicited margination of peripheral blood mononuclear cells, which increased in the subsequent days/weeks post-treatment; suggesting that RNA-NPs mediate lymphoid honing of immune cell populations before egress. RNA-NPs also elicited increased: 1) serum interferon-α that spiked at 2 hours; 2) CD80 and MHCII on CD11c+ cells (demonstrating activation of peripheral DCs); and 3) interferon-γ + T-cells (i.e. activated T-cells). After receiving weekly RNA-NPs (×3), the canines had a steady course. Aside from low-grade fevers on the vaccination days, personalized tumor RNA-NPs (1x) were well tolerated with stable blood counts, chemistries, and renal/liver function tests. All patients assessed developed immunologic response with pseudoprogression or stable/smaller tumors by MRI. Although we have treated a small cohort, we have observed improvement in median/overall survival in all canine patients with terminal gliomas receiving RNA-NPs (compared with historical controls). CONCLUSION RNA-NPs were feasible, safe and immunologically active in client-owned canines with terminal gliomas. We have not appreciated significant toxicities in canines that would preclude investigation in humans at 1x dosing. Although these results need to be validated in larger canine data sets, these results suggest safety and activity of tumor specific RNA-NPs in canines with terminal gliomas.

Plasmacytoid dendritic cells protect against immune-mediated acute liver injury via IL-35.

Acute liver failure (ALF) is a life-threatening condition, and liver transplantation is the only therapeutic option. Although immune dysregulation is central to its pathogenesis, the precise mechanism remains unclear. Here, we show that the number of peripheral and hepatic plasmacytoid DCs (pDCs) decrease during acute liver injury in both humans and mice. Selective depletion of pDCs in Siglechdtr/+ mice exacerbated concanavalin A-induced acute liver injury. In contrast, adoptively transferred BM-derived pDCs preferentially accumulated in the inflamed liver and protected against liver injury. This protective effect was independent of TLR7 and TLR9 signaling, since a similar effect occurred following transfer of MyD88-deficient pDCs. Alternatively, we found an unexpected immunosuppressive role of pDCs in an IL-35-dependent manner. Both Il12a and Ebi3, heterodimeric components of IL-35, were highly expressed in transferred pDCs and CD4+CD25+ Tregs. However, the protective effect of pDC transfer was completely lost in mice depleted of Tregs by anti-CD25 antibody. Moreover, pDCs derived from IL-35-deficient mice had less of a protective effect both in vivo and in vitro even in the presence of Tregs. These results highlight a unique aspect of pDCs in association with Tregs, serving as a guide for immunotherapeutic options in ALF.

Intranodal dendritic cell relocalization during inflammation impacts T cell immunity

Abstract Conventional dendritic cells (cDCs) are the critical antigen-presenting cells that initiate adaptive immunity in response to external inflammatory stimuli. We have previously shown that LN-resident cDC2s, a DC subset critical for generating CD4 T cell responses, reside in regions highly proximal to the lymphatic sinuses, enabling superior acquisition of draining soluble and particulate antigens. However, given that this peripheral positioning is distal from the deep LN paracortex and the T cell zone, we hypothesized that for optimal generation of adaptive immunity, LN-resident cDC2s must migrate intranodally from peripheral sites into the deep T zone in order to engage in cognate DC-T cell interactions during priming. Here, we show that s.c immunization with TLR-agonists, as well as infection with West Nile Virus, induces rapid relocalization of cDC2s into the deep T zone of the LN. This cDC2 relocalization requires Type 1 interferon in some, but not all, adjuvant immunizations, and is predominantly driven by increased CCR7 expression on cDCs after maturation. When lacking CCR7, LN-resident cDC2s failed to reposition to the T cell zone across multiple adjuvant comparisons, and this in turn led to a marked reduction in the activation and proliferation of CD4+ T cells. A decrease in the differentiation of Tfh cells was also observed, suggesting a possible impact on humoral immunity. Collectively, this work identifies a novel CCR7-mediated spatial reorganization of LN-resident cDC2s during inflammation, independent of peripheral migratory DCs, and its role in the generation of adaptive immunity.

Peripherally Induced Tolerance Depends on Peripheral Regulatory T Cells That Require Hopx To Inhibit Intrinsic IL-2 Expression.

Dendritic cells (DCs) can induce peripheral immune tolerance that prevents autoimmune responses. Ag presentation by peripheral DCs under steady-state conditions leads to a conversion of some peripheral CD4(+) T cells into regulatory T cells (Tregs) that require homeodomain-only protein (Hopx) to mediate T cell unresponsiveness. However, the roles of these peripheral Tregs (pTregs) in averting autoimmune responses, as well as immunological mechanisms of Hopx, remain unknown. We report that Hopx(+) pTregs converted by DCs from Hopx(-) T cells are indispensible to sustain tolerance that prevents autoimmune responses directed at self-Ags during experimental acute encephalomyelitis. Our studies further reveal that Hopx inhibits intrinsic IL-2 expression in pTregs after antigenic rechallenge. In the absence of Hopx, increased levels of IL-2 lead to death and decreased numbers of pTregs. Therefore, formation of Hopx(+) pTregs represents a crucial pathway of sustained tolerance induced by peripheral DCs, and the maintenance of such pTregs and tolerance requires functions of Hopx to block intrinsic IL-2 production in pTregs.

Inhibition of breast cancer resistance protein (ABCG2) in human myeloid dendritic cells induces potent tolerogenic functions during LPS stimulation.

Breast cancer resistance protein (ABCG2), a member of the ATP-binding cassette transporters has been identified as a major determinant of multidrug resistance (MDR) in cancer cells, but ABC transporter inhibition has limited therapeutic value in vivo. In this research, we demonstrated that inhibition of efflux transporters ABCG2 induced the generation of tolerogenic DCs from human peripheral blood myeloid DCs (mDCs). ABCG2 expression was present in mDCs and was further increased by LPS stimulation. Treatment of CD1c+ mDCs with an ABCG2 inhibitor, Ko143, during LPS stimulation caused increased production of IL-10 and decreased production of pro-inflammatory cytokines and decreased expression of CD83 and CD86. Moreover, inhibition of ABCG2 in monocyte-derived DCs (MDDCs) abrogated the up-regulation of co-stimulatory molecules and production of pro-inflammatory cytokines in these cells in response to LPS. Furthermore, CD1c+ mDCs stimulated with LPS plus Ko143 inhibited the proliferation of allogeneic and superantigen-specific syngenic CD4+ T cells and promoted expansion of CD25+FOXP3+ regulatory T (Treg) cells in an IL-10-dependent fashion. These tolerogenic effects of ABCG2 inhibition could be abolished by ERK inhibition. Thus, we demonstrated that inhibition of ABCG2 in LPS-stimulated mDCs can potently induce tolerogenic potentials in these cells, providing crucial new information that could lead to development of better strategies to combat MDR cancer.

Hypomorphic mutation in the RAG2 gene affects dendritic cell distribution and migration

OS is a severe combined immunodeficiency characterized by erythrodermia and protracted diarrhea as a result of infiltration of oligoclonal-activated T cells, caused by hypomorphic mutations in RAGs. The RAG2(R229Q) mouse model fully recapitulates the clinical OS phenotype. We evaluated whether T and B cell defects, together with the abnormal lymphoid structure, could affect DC homeostasis and function. High density of LCs was observed in skin biopsies of Omenn patients and in the derma of RAG2(R229Q) mice, correlating with the presence of erythrodermia. In vivo models of cutaneous skin painting and CHS demonstrated a decreased migration of RAG2(R229Q) DCs-in particular, LCs-into draining LNs. Interestingly, at steady state, RAG2(R229Q) mice showed a reduction in DC number in all hematopoietic organs except LNs. Analysis of the MHCII marker revealed a diminished expression also upon the LPS-driven inflammatory condition. Despite the decreased number of peripheral DCs, BM pre-cDCs were present in normal number compared with RAG2(+/+) controls, whereas pDCs and monocytes were reduced significantly. Overall, these results point to a secondary defect in the DC compartment, which contributes to clinical manifestations and autoimmunity in OS.

Aryl hydrocarbon receptor contributes to the MEK/ERK-dependent maintenance of the immature state of human dendritic cells

AbstractDendritic cells (DCs) promote tolerance or immunity depending on their maturation state, which is enhanced or accelerated upon MEK-ERK signaling pathway inhibition. We have determined the contribution of MEK-ERK activation to the profile of gene expression of human immature monocyte-derived dendritic cells (MDDCs) and peripheral blood myeloid DCs. ERK inhibition altered the expression of genes that mediate Chemokine (C-C motif) ligand 19 (CCL19)–directed migration (CCR7) and low-density lipoprotein (LDL) binding (CD36, SCARB1, OLR1, CXCL16) by immature DCs. In addition, ERK upregulated CCL2 expression while impairing the expression of DC maturation markers (RUNX3, ITGB7, IDO1). MEK-ERK–regulated genes exhibited an overrepresentation of cognate sequences for the aryl hydrocarbon receptor (AhR) transcription factor, whose transcriptional and DNA-binding activities increased in MDDCs upon exposure to the MEK1/2 inhibitor U0126. Therefore, the MEK-ERK signaling pathway regulates antigen capture, lymph node homing, and acquisition of maturation-associated genes, and its contribution to the maintenance of the immature state of MDDCs and myeloid DCs is partly dependent on the activity of AhR. Since pharmacologic modulation of the MEK-ERK signaling pathway has been proposed as a potential therapeutic strategy for cancer, our findings indicate that ERK inhibitors might influence antitumor responses through regulation of critical DC effector functions.

The innate immune adaptor MyD88 is dispensable for spontaneous autoimmune demyelination in a mouse model of multiple sclerosis

Multiple sclerosis (MS) is an autoimmune disease that is mediated by myelin-reactive T cells resulting in CNS demyelination, however the mechanisms that control their activation are unclear. Mice that are transgenic for a myelin proteolipid protein (PLP)-specific TCR spontaneously develop experimental autoimmune encephalomyelitis (EAE), the animal model of MS. They mimic the spontaneous onset of MS and thus offer the unique opportunity to investigate the mechanisms that may contribute to the development of spontaneous CNS autoimmunity. MyD88 is an adaptor protein that mediates signal transduction by TLRs, IL-1R and IL-18R, resulting in the activation of innate immune cells, including DCs. We investigated the requirement of MyD88 in the pathogenesis of spontaneous EAE in PLP TCR transgenic SJL mice. We show that genetic loss of MyD88 does not intrinsically preclude development of spontaneous EAE and autoimmune demyelination in these mice. EAE was associated with functionally mature peripheral DCs that promoted superior PLP-specific Th1 and Th17 responses compared to those from disease-free mice. Together, our data suggest that MyD88-independent innate immune signaling critically contributes to priming of myelin-reactive T cells and development of spontaneous EAE in MyD88-deficient PLP TCR transgenic mice.

Extracorporeal Photopheresis (ECP) in Kidney Allograft Recipients: 1-Year Followup

Background: ECP is considered a promising immunomodulatory therapy used for treatment of acute allograft rejection in solid organ transplant patients (pts) and GVHD. We report on early results of cadaver KTx in 20 paired recipients, all of them on standard immunosuppression (MPA+CNI+prednisone), and 10 of them additionally treated with 12-16 ECP procedures in the first 3 months (M) following KTx. Methods: There was no significant differences between controls and ECP group in term of age (47 11 vs 43,4 13y), gender of pts (3 vs 4 F), cause of end stage kidney disease, cold ischemia time, diabetes, hypertension, CsA/TAC concentrations, MPA dose, BMI (24,8 2 vs 26,2 5), mean HLA mismatch (2,6 vs 2,75) and sensitization. ECP procedures were conducted using UVAR XTS (Therakos, Exton, PA), an automated system for leukocyte separation and photoactivation (with injection of Methoxsalen). All pts were followed by means of eGFR and peripheral WBCs, T-cell,B,NK, Treg, DCs counts and phenotype. Results: In ECP group a positive tendency to higher GFR at M 6 (67,5 ±10 vs 53,6± 3; p=0.03) and M 12 (64,4 ±8 vs 59,5± 3; p=0.09 in Wilcoxon test) was observed. Transplant related complications are summarized in table1. All rejections (all cellular) were successfully treated with steroid pulse in both groups. One recipient from ECP group who experienced rejection have had unstable CNI levels in the first 6 months and might not response to ECP due to permanent low number of WBCs.Table: [Transplant related complications]Conclusions: An addition of ECP to standard immunosuppression in the first post-KTx year was associated with favorable kidney allograft function and less opportunistic infections. Acute rejections in ECP treated recipients appeared after 6 M and were reversible.

Distribution of subpopulations of dendritic cells in peripheral blood of patients treated with exogenous thyrotropin

BackgroundDendritic cells (DCs) play a major role as regulators of inflammatory events associated with thyroid pathology. The immunoregulatory function of DCs depends strongly on their subtype, as well as maturation and activation status. Numerous hormonal factors modulate the immune properties of DCs, however, little is known about effects exerted by the hypothalamus-pituitary-thyroid-axis. Recently, we have shown a direct regulatory influence of thyroid hormones (TH) on human DCs function. The aim of the present study was to analyze the effect of systemically administered thyrotropin (TSH) on human blood DCs ex vivo.MethodsBlood samples for the cytometric analysis of peripheral blood plasmacytoid and myeloid DCs subtypes were collected from patients subjected to total thyroidectomy because of differentiated thyroid carcinoma at 2 time points: (i) directly before the commencement of TSH administration and (ii) 5 days after first TSH injection. The whole blood quantitative and phenotypic analysis of plasmacytoid and myeloid DCs subtypes was performed by flow cytometry.ResultsAdministration of TSH did not influence the percentage of plasmacytoid DCs in peripheral blood of study participants. Also the percentage of the two main myeloid DCs subpopulations – CD1c/BDCA1+ DCs and CD141/BDCA3+ DCs did not change significantly. TSH administration had no effect on the surface expression of CD86 – one of the major costimulatory molecules – neither in the whole peripheral blood mononuclear cell (PBMC) fraction nor in particular DCs subtypes.ConclusionsIn the present study, we demonstrated no influence of systemic TSH administration on human peripheral blood DCs subtypes. These results are in accordance with our previous work suggesting the direct effect of TH on human DCs ex vivo.

Thymic stromal lymphopoietin from trophoblasts induces dendritic cell–mediated regulatory TH2 bias in the decidua during early gestation in humans

AbstractThymic stromal lymphopoietins (TSLPs) play critical roles in dendritic cell–mediated immune responses. In this study, we found that human trophoblasts and decidual epithelial cells in maternal-fetal interface of early placentas express TSLP mRNA and protein, but only trophoblast cells secret soluble TSLP. Human decidual CD1c+ DCs (dDCs) highly express the functional TSLP receptor complex TSLP receptor and interleukin-7 receptor-α. Recombinant human TSLP activates CD1C+ decidual DCs and peripheral monocyte-derived DCs with increased costimulatory molecules, major histocompatibility complex class II, and OX-40L. Human TSLP or supernatants from human trophoblasts specifically stimulate dDCs to highly produce interleukin-10 and TH2-attracting chemokine CCL-17. The TSLP-activated dDCs prime decidual CD4+ T cells for TH2 cell differentiation, involved in maternal-fetal immunotolerance. Interestingly, the protein expression of TSLP in normal pregnancy with significant TH2 bias is much higher than that of miscarriage showing TH1 bias at the maternal-fetal interface. Therefore, human trophoblasts may contribute to maternal-fetal tolerance by instructing dDCs to induce regulatory TH2 bias in human early pregnancy via TSLP.

Ligand surface density is important for efficient capture of immunoglobulin and phosphatidylcholine coated particles by human peripheral dendritic cells

A unique property of dealuminated zeolite particles is the exceptional ability to bind both hydrophilic and hydrophobic biomolecules without any covalent linkages. By adsorbing phospholipids onto the particle surface, capture of particles by human peripheral myeloid dendritic cells (mDCs) was observed. Capture of zeolite particles was only seen when a low density of phosphatidylcholine was present on the particles, indicating a specific recognition of the structural features realised by phosphatidylcholine after adsorption on the particle. Adsorbing IgG on the particles revealed capture by mDCs that was dependent upon the density of the IgG molecules. To obtain a smaller particle exposing a high density of IgG molecules, immune complexes (ICs) were formed and both mDCs and pDCs (peripheral plasmacytoid DCs) captured immune complexes, although the mDCs showed a more efficient capture of ICs. As expected, mDCs captured and internalized ICs, whereas pDCs captured ICs but showed no internalization of ICs.

Loss of Cabin Pressure in a Military Transport: A Mass Casualty with Decompression Illnesses

Presented here is the sudden cabin depressurization of a military C-130 aircraft carrying 66 personnel. They suffered a depressurization from 2134 to 7317 m, resulting in a potential 66-person mass casualty. The aircrew were able to descend to below 3049 m in less than 5 min. They landed in the Kingdom of Bahrain--the nearest hyperbaric recompression facility. Three cases of peripheral neurologic DCS and one case of spinal DCS were identified. Limited manning, unique host nation concerns, and limited available assets led to difficulties in triage, patient transport, and asset allocation. These led to difficult decisions regarding when and for whom to initiate ground level oxygen or hyperbaric recompression therapy.

A compass that points to lupus: genetic studies on type I interferon pathway

It was more than 20 years ago that patients with systemic lupus erythematosus (SLE) were first reported to display elevated serum levels of type I interferon (IFN). Since then, extensive studies revealed a crucial role for type I IFN in SLE pathogenesis. The current model proposes that small increase of type I IFN production by plasmacytoid dendritic cells (pDCs) is sufficient to induce unabated activation of immature peripheral DCs. IFN-matured DCs select and activate autoreactive T cells and B cells, rather than deleting them, resulting in peripheral tolerance breakdown, a characteristic feature of SLE. Furthermore, immune complexes provide an amplification loop to pDCs for further IFN production. In the past 5 years, high-throughput technologies such as expression profiling and single-nucleotide polymorphism (SNP) typing established the role of altered type I IFN system in SLE, and a detailed picture of its molecular mechanisms is beginning to emerge. In this review, we discuss two major lines of genetics studies on type I IFN pathway related to human SLE: (1) expression profiling of IFN-responsive genes and (2) disease-associated SNPs of IFN-related genes, especially IRF5 (IFN-regulatory factor 5). Lastly, we discuss how such genetic alterations in type I IFN pathway fit in the current model of SLE pathogenesis.

Quantitative and Functional Differences between Peripheral Blood Myeloid Dendritic Cells from Patients with Pleural and Parenchymal Lung Tuberculosis

Dendritic cells (DCs) play a pivotal role in generating protective host immunity to Mycobacterium tuberculosis. Few studies have addressed DC function in the context of active tuberculosis (TB), largely due to technical constraints in obtaining adequate numbers of DC from sick patients. We quantitated peripheral blood myeloid DCs (mDCs) and plasmacytoid DCs (pDCs) in the whole blood of patients with active TB and show that blood from patients with pleural TB was characterized by high circulating levels of mDCs. We also developed and optimized a novel whole-blood assay to study mDC production of the Th1-promoting cytokine interleukin 12 (IL-12) and upregulation of the maturation marker CCR7 after incubation with mycobacteria. We found that pleural TB was associated with increased IL-12 production and CCR7 expression compared to lung parenchymal disease. Our findings suggest important differences in innate immunity between patients with different forms of active TB, and this may contribute to the differences in natural history observed between the two groups.

In vitro antitumor immune response induced by fusion of dendritic cells and Ewing's sarcoma cells

Human Ewing sarcoma A673 cells and human peripheral blood-derived DCs were fused to induce an antitumor activity against human EW. EW A673 cells and human peripheral blood-derived DCs were fused with polyethylene glycol (PEG). Mature DCs with highly expressed surface markers (CD80, CD86, CD83 and HLA-DR) were generated in vitro and flow cytometry. It showed that the highest fusion efficiency was 23.01%. T cell proliferation assay indicated that the novel dendritomas in fused DCs/A673 cells were the most potent in activation of autologous T cell proliferation. The IFN-gamma assay showed that The IFN-gamma secretion by CTLs activated by the novel dendritomas increased more than by other stimulators. CTL assay demonstrated that the novel dendritomas induced A673 cell-specific cytotoxic responses to lyse the A673 cells in the context of MHC class I. The data indicates that fusion of tumor cells with DCs is an attractive strategy to induce tumor rejection.