Mature moDC Research Articles

Abstract 1363: Blocking inhibitory receptor ILT3 by an antibody with enhanced Fc function promoted adaptive immunity against hematopoietic malignancy independent of ADCC

Abstract Immunoglobulin-like transcript 3 (ILT3) is an inhibitory receptor that is expressed by mature monocytes, dendritic cells (DC), plasma blasts, and the malignant cells arising thereof including acute myeloid leukemia (AML), B cell lymphoma and multiple myeloma (MM). We developed an anti-ILT3 blocking monoclonal antibody (mAb) 17G8 to test the hypothesis that blockade of ILT3 on hematopoietic malignant cells might drive anti-cancer responses through several mechanisms such as cell migration, ADCC, and T cell driven immune responses. Blockade of ILT3 using 17G8 without Fc effector function impaired AML cell line THP1 dissemination into distant sites in vivo and using 17G8 with enhanced Fc function through afucosylation further reduced the tumor burden. This effect of afucosylated 17G8 appears to be mediated by a mechanism other than ADCC, since afucosylated 17G8 showed modest tumor inhibition in ICR mice carrying ILT3 positive AML and MM xenografts. In immune competent mice carrying murine syngeneic AML cell C1498 expressing human ILT3, 17G8 also showed modest anti-tumor effects as a single agent, however, when combined with anti-PD1, afucosylated 17G8 increased CD8+ effector memory T cells, reduced proportion of cells with an exhausted phenotype and significantly improved survival when compared to different controls. Since T cells don’t express ILT3, the effects of 17G8 on T cells are hypothesized to be mediated by blocking ILT3 expressed on myeloid cells. The expression of ILT3 on immature human monocyte-derived DC (moDC) is higher than on mature moDC. Blocking ILT3 with afucosylated 17G8 on immature moDC potentiated maturation of moDC when compared to the controls in vitro. Surprisingly, afucosylated 17G8 stimulated IFNγ production from moDC during maturation. The enhanced DC function induced by afucosylated 17G8 was subsequently found to be translated into enhanced T cell function in an allogeneic mixed lymphocyte reaction (allo-MLR) assay, in which afucosylated 17G8 significantly increased IFNγ production when combined with anti-PD1. The activation of effector T cells was also monitored through a cytotoxicity assay by mixing THP-1 cells with human PBMC in culture. Afucosylated 17G8 induced strong cytotoxicity on THP-1 cells with minimal effects on normal ILT3 positive cells such as monocytes or moDC, suggesting that blockade of ILT3 with afucosylated 17G8 induced more potent T cell mediated allogeneic cytotoxicity than innate cell mediated ADCC killing. In summary, ILT3 is a highly selective M4/M5 AML target. Developing a blocking antibody with enhanced Fc function could add an additional therapeutic strategy of inducing host anti-tumor immunity to current AML treatments which induce direct tumor killing, such as chemotherapy, targeted therapy, and antibody drug conjugate. Citation Format: Shiming Ye, Dong Zhang, Min-Zu Wu, Harini Raghu, Diane Cohen, Mally Romero, Siu-Sze Tan, Donghee Choi, Jonathan Hickson, John Engelhardt, Alex Shoemaker. Blocking inhibitory receptor ILT3 by an antibody with enhanced Fc function promoted adaptive immunity against hematopoietic malignancy independent of ADCC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1363.

Equine Arteritis Virus in Monocytic Cells Suppresses Differentiation and Function of Dendritic Cells

Equine viral arteritis is an infectious disease of equids caused by equine arteritis virus (EAV), an RNA virus of the family Arteriviridae. Dendritic cells (DC) are important modulators of the immune response with the ability to present antigen to naïve T cells and can be generated in vitro from monocytes (MoDC). DC are important targets for many viruses and this interaction is crucial for the establishment-or rather not-of an anti-viral immunity. Little is known of the effect EAV has on host immune cells, particularly DC. To study the interaction of eqDC with EAV in vitro, an optimized eqMoDC system was used, which was established in a previous study. MoDC were infected with strains of different genotypes and pathogenicity. Virus replication was determined through titration and qPCR. The effect of the virus on morphology, phenotype and function of cells was assessed using light microscopy, flow cytometry and in vitro assays. This study confirms that EAV replicates in monocytes and MoDC. The replication was most efficient in mature MoDC, but variable between strains. Only the virulent strain caused a significant down-regulation of certain proteins such as CD14 and CD163 on monocytes and of CD83 on mature MoDC. Functional studies conducted after infection showed that EAV inhibited the endocytic and phagocytic capacity of Mo and mature MoDC with minimal effect on immature MoDC. Infected MoDC showed a reduced ability to stimulate T cells. Ultimately, EAV replication resulted in an apoptosis-mediated cell death. Thus, EAV evades the host anti-viral immunity both by inhibition of antigen presentation early after infection and through killing infected DC during replication.

Pharmacological inhibition of the mTOR pathway alters phenotype and cytokine expression in bovine monocyte-derived dendritic cells

Epidemiological studies have long demonstrated the association of nutrient status and immune dysfunction in dairy cows. Postpartum dairy cows experiencing a nutrient deficit show a propensity for increased inflammatory response, decreased pathogen clearance, and increased incidence of infectious disease. Studies in cows and other species show that the nutrient sensing mechanistic target of rapamycin (mTOR) signaling pathway could be one potential causal pathway connecting the deficit in nutrient availability and the heightened inflammatory response. Our objective was to investigate the effects of pharmacological mTOR pathway inhibition on phenotype and cytokine expression of bovine monocyte derived dendritic cells (moDC). We differentiated CD14+ monocytes from dairy cows (n = 14) into moDC in the presence or absence of first- or second-generation mTOR inhibitor rapamycin and PP242 (both 100 nM), respectively. On day seven cells were matured with E. coli lipopolysaccharide (LPS, 100 ng/mL) or left unstimulated to represent naïve moDC. Surface expression of CD14, CD40, CD80, and MHCII was measured via flow cytometry. We measured mRNA expression of IL10, IL12A, IL12B, and TNFα by rt-qPCR, and protein concentrations of IL-10 and TFN-α in cell culture supernatants with a bead-based multiplex assay. Cultures from ten cows successfully developed the moDC phenotype in culture without inhibitors, defined as increased surface expression of CD40, CD80, and MHCII compared with naïve moDC. Only data from these cows were considered for the results on effects of mTOR inhibitors. In naïve and mature moDC mTOR inhibition increased MHCII expression compared to controls. In mature moDC, in addition to MHCII, CD80 expression was increased compared with untreated LPS-stimulated controls. Expression of IL12A mRNA was upregulated in mature, mTOR inhibited moDC compared with untreated controls. In cell culture supernatants mTOR inhibition reduced IL-10 and increased TNF-α concentrations in naïve and mature moDCs compared with untreated controls. Overall rapamycin had a more consistent effect on altering phenotype and cytokine expression of moDC than PP242. In summary we observed an increased expression of co-stimulatory molecules and antigen presentation potential in mature moDC differentiated under mTOR inhibition, and a cytokine pattern that would potentially favor a Th1 type response. This study provides novel data indicating a role for mTOR signaling in bovine moDC phenotype and mediator profile. This proof-of-concept study demonstrates the role of the mTOR pathway in shaping the bovine immune response and may help to provide mechanistic insight and opportunities for modulation of the immune response during the nutrient deficit of early lactation.

Interaction of porcine monocyte-derived dendritic cells with African swine fever viruses of diverse virulence

African swine fever (ASF) is a devastating disease for which there is no vaccine. The ASF virus (ASFV) can infect dendritic cell (DC), but despite the critical role these cells play in induction of adaptive immunity, few studies investigated their response to ASFV infection. We characterized the in vitro interactions of porcine monocyte-derived DCs (moDC) with a virulent (22653/14), a low virulent (NH/P68) and an avirulent (BA71V) ASFV strain. At a high multiplicity of infection (MOI = 1), all three strains infected immature moDC. Maturation of moDC, with IFN-α/TNF-α, increased susceptibility to infection with 22653/14 and other virulent strains, but reduced susceptibility to NH/P68 and BA71V. The reduced moDC susceptibility to BA71V/NH/P68 was IFN-α mediated, whereas increased susceptibility to 22653/14 was induced by TNF-α. Using an MOI of 0.01, we observed that BA71V replicated less efficiently in moDC compared to the other isolates and we detected increased replication of NH/P68 compared to 22653/14. We observed that BA71V and NH/P68, but not 22653/14, downregulated expression of MHC class I on infected cells. All three strains decreased CD16 expression on moDC, whereas ASFV infection resulted in CD80/86 down-regulation and MHC class II DR up-regulation on mature moDC. None of the tested strains induced a strong cytokine response to ASFV and only modest IL-1α was released after BA71V infection. Overall our results revealed differences between strains and suggest that ASFV has evolved mechanisms to replicate covertly in inflammatory DC, which likely impairs the induction of an effective immune response.

Altered Heme-Mediated Modulation of Dendritic Cell Function in Sickle Cell Alloimmunization

Red blood cell alloimmunization can be a life-threatening complication for patients with sickle cell disease (SCD) receiving therapeutic transfusions. However, it remains unknown why only some (20-60%) SCD patients develop alloantibodies whereas others do not. Because of ongoing hemolysis in SCD, we have investigated the effects of toxic hemin on T cell responses of patients with SCD on chronic transfusion therapy. We found impaired monocyte control of proinflammatory T cells in response to hemin in alloimmunized SCD patients, partly due to defective levels of monocyte heme-oxygenase-1 (HO-1), an anti-inflammatory heme degrading enzyme that catabolizes heme into iron, biliverdin and carbon monoxide (CO). Monocytes are precursors of dendritic cells (DCs), which represent the antigen presenting cells most able to initiate and regulate immune responses. However, little is known about the functional properties of DCs in SCD patients. We therefore hypothesized that DCs of alloimmunized SCD patients would also have impaired response to hemin. Monocyte-derived DCs (moDCs) from healthy donor controls (n=11) and a cohort of SCD patients (aged 15-30 on chronic transfusion therapy every 3-4 weeks for at least two years using C,E,K phenotyped-matched, leukodepleted units) grouped either as "non-alloimmunized" (no history of antibody production, n=6), versus "alloimmunized" (with a history of having produced at least one alloantibody, n=7) were matured in the absence or presence of hemin (5uM and 20uM) with TLR agonists: LPS/IFNg (TLR4 agonist+STAT1 activation) or R848 (TLR7/8 agonist). Hemin-exposed mature and immature (no TLR agonist) moDCs were then assayed for HO-1 expression, cytokine production, co-stimulation, and T cell priming. Interestingly, upon hemin exposure, immature moDCs from healthy donors and non-alloimmunized patients upregulated more HO-1 (1056±206 fold; mean fluorescent intensity (MFI): 12283±1818 at 20uM hemin) than alloimmunized patients (fold increase 494±49; MFI: 7422±959, p<0.03). In addition, there were no differences among healthy donors and either patient groups in the following mature moDC associated cytokine/surface marker expression after hemin exposure: IL-12p70, IL-6, and TNFa production and surface expression of activation (CD80 and CD86) and antigen presentation (HLA-DR) markers, suggesting that these mature moDC-associated molecules are unlikely to be associated with alloimmunization status. As with healthy donors, hemin inhibited (by 35%) the upregulation of DC-specific maturation marker CD83 expression on mature moDCs of non-alloimmunized patients (MFI before hemin 5199±689 after 20uM hemin 3471±401, p=0.003). Furthermore, HO-1 breakdown product CO, but not biliverdin, inhibited CD83 upregulation on mature moDCs to a similar degree as hemin, suggesting that hemin-mediated CD83 inhibition in mature MoDCs involves HO-1. Remarkably, however, hemin did not alter CD83 expression on mature moDC in alloimmunized patients (before hemin 4382±949 after hemin 4825±1051, p=0.5). To determine whether hemin modulation of CD83 expression affects mature moDC driven differentiation of naïve CD4+ T cells (ie. priming), we performed mixed lymphocyte reactions: whereas hemin inhibited (roughly 30%) moDC-mediated priming of pro-inflammatory IFNg secreting TH1 cells in healthy donors and non-alloimmunized patients (%CD4+ IFNg+ without hemin 58%±9% with 20uM hemin 41%±8%, p=0.005), TH1 cell responses were not suppressed in the alloimmunized group (without hemin: 52%±10% with 20uM hemin: 54%±10%, p=0.4). In summary, hemin-exposed moDCs from healthy donors and non-alloimmunized SCD patients suppress CD83 upregulation and inhibit ensuing TH1 cell responses, possibly through HO-1 induction and subsequent production of CO. In contrast, CD83 upregulation and proinflammatory T cell priming are not inhibited by hemin in alloimmunized patients, probably representing a mechanism by which heme-associated inflammation is maintained in alloimmunized SCD, thereby increasing their risk of alloimmunization. Deciphering this mechanism not only offers a potential biomarker for alloimmunization, but also opens the way to designing innovative treatments for these patients. DisclosuresNo relevant conflicts of interest to declare.

Structural characterisation of immunoglobulin G glycosylation in multiple myeloma

smoldering MM (SMM). Samples from patients with MGUS and healthy donors were also included in the study. LEN treatment was performed at concentration ranging from 0,1 to 2 M during all the differentiation culture period. Moreover moDC differentiation was performed in the presence of CM of human TERT transfected-hMSCs (hTERT-hMSCs) treated for 5 days with LEN. DC maturation markers (CD83, HLA-DR, CD80, CD86 and CD209) were evaluated by flow cytometry. The levels of soluble factors produced by mo-DCs were measured in the conditioned media (CM) of mo-DC by a Bio-Plex Multiplex System. The expression levels of Ikaros and Aiolos were also evaluated by western blot. LEN treatment induced a reduction of both number and % of mature mo-DC as compared to untreated controls. On the other hand, LEN treatment significantly increased the median intensity expression of CD86 HLA-DR and CD209 but not of CD80 by moDCs derived from BM. Similarly increased CD209 expression has been also seen in mo-DCs derived from PB in both MM and SMM patients. LEN treatment enhanced the production of IL-8 and MCP1 and decreased TNFlevels. Ikaros mediated the effect of LEN on DCs as we observed a significant down-regulation of Ikaros levels in THP1-DCs after LEN treatment with a dose dependent effect. On the other hand, we found that LEN blunted the inhibitory effect of hTERT-hMSCs on mo-DC differentiation in three independent experiments and slightly down-regulated IL-6 and IDO but not TGFB1, IL-8, TNF and HGF gene expression. The effect of LEN on the immunomodulatory properties of hMSC was likely to be not mediated by Ikaros or Aiolos because both transcription factors were not expressed by hMSCs. These evidences underline a possible new effect of IMiDs on the alloreactivity against MM cells.

PD35 ‐ In vitro effects of atorvastatin on function, proliferation and cytokine production of human peripheral blood mononuclear cells

Statins, which are used as cholesterol-lowering agents, have anti-inflammatory and immune-regulating properties. Little comprehensive analysis has been made to investigate the impact of statins on human T lymphocytes function, their proliferation and production of Th1 and Th2 cytokines. We also investigated if the immunossupresive effects of statins are due to their impact on human T lymphocytes or proffesional antigen-presenting dendritic cells (DC) alone, or both. Human peripheral blood mononuclear cells (MNC) proliferation was induced with phytohemagglutinin. Immature DC were cultivated from monocytes of healthy donors. DC maturation was induced by lipopolysaccharide (LPS; 1 μg/mL). Unstimulated and LPS-stimulated DC were treated with atorvastatin (1-10 μM). Ability to induce T cell proliferation and the secretion of cytokines were performed in mixed leukocyte reaction. Atorvastatin reduced phytohemagglutinin-stimulated MNC proliferation, dose dependently. The results showed that atorvastatin, only in high concentrations, strongly inhibit allogenic lymphocytes proliferation irrespectively which stimulators are used: immature or mature dendritic cells. Low concentrations of atorvastatin even stimulated proliferation when the immature dendritic cells were used as stimulators. Atorvastatin have influence on DC differentiation and maturation by changing expression and mean fluorescence intensity of costimulatory, adhesive and maturation molecules such as HLA-DR, CD80, CD83, CD86, CD40, CD54 and CD25. Atorvastatin affects allostimulatory activity of both immature and mature MoDC but in completely oposite way, using different DC/Ly ratio, and change production of TNF-a by immature DC and IL-18 by mature DC and not affect IL-10 and IL-12 production. Atorvastatin decreased the level of IFN-g, increase the level of IL-10, and not affected the level of IL-4 in DC culture supernatants when the atorvastatin was added during maturation. If the atorvastatin was added in monocite culture, no significant change in cytokine level was observed. Our data provide strong evidence that atorvastatin can act as an immunomodulator by reducing, but in low dosage stimulating T lymphocite proliferation, inhibiting the immune response of Th1, decreasing the expression of co-stimulatory molecules, and changing DC cytokine secretion, which can help in better understanding how to take advantage of these new mechanistic insights in increased use of statins in therapeutic strategy in different immune/inflamatory disorders in future.

Altered phenotype and Stat1 expression in Toll-like receptor 7/8 stimulated monocyte-derived dendritic cells from patients with primary Sjögren's syndrome.

IntroductionDendritic cells (DC) are the most potent antigen-presenting cells of the immune system, involved in both initiating immune responses and maintaining tolerance. Dysfunctional and via toll-like receptor (TLR) ligands activated DC have been implicated in the development of autoimmune diseases, but their role in the etiology of Sjögren’s syndrome, a chronic inflammatory autoimmune disease characterized by progressive mononuclear cell infiltration in the exocrine glands, has not been revealed yet. Therefore, the aim of this study was to investigate phenotype and functional properties of immature and TLR7/8 stimulated monocyte-derived DC (moDC) of patients with primary Sjögren’s syndrome (pSS) and compare them to healthy controls.MethodsThe phenotype, apoptosis susceptibility and endocytic capacity of moDC were analyzed by flow cytometry. Secretion of cytokines was measured by enzyme-linked immunosorbent assay (ELISA) and multiplex Luminex analyses in moDC cell culture supernatants. The expression of TLR7 was analyzed by flow cytometry and real-time quantitative polymerase chain reaction (qPCR). Expression of Ro/Sjögren’s syndrome-associated autoantigen A (Ro52/SSA), interferon regulatory factor 8 (IRF-8), Bim, signal transduction and activators of transcription (Stat) 1, p-Stat1 (Tyrosin 701), p-Stat1 (Serin 727), Stat3, pStat3 (Tyrosin 705) and glyceraldehyde 3-phosphatase dehydrogenase (GAPDH) was measured by Western blotting. Nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) family members were quantified using the ELISA-based TransAM NF-κB family kit.ResultsWe could not detect differences in expression of co-stimulatory molecules and maturation markers such as cluster of differentiation (CD) 86, CD80, CD40 or CD83 on moDC from patients compared to healthy controls. Moreover, we could not observe variations in apoptosis susceptibility, Bim and Ro52/SSA expression and the endocytic capacity of the moDC. However, we found that moDC from pSS patients expressed increased levels of the major histocompatibility complex (MHC) class II molecule human leukocyte antigen (HLA)-DR. We also found significant differences in cytokine production by moDC, where increased interleukin (IL)-12p40 secretion in mature pSS moDC correlated with increased RelB expression. Strikingly, moDC from pSS patients matured for 48 hours with TLR7/8 ligand CL097 expressed significantly less Stat1.ConclusionOur results suggest a role for moDC in the pathogenesis of Sjögren’s syndrome.

Differentiation and activation of equine monocyte‐derived dendritic cells are not correlated with CD206 or CD83 expression

Dendritic cells (DC) are the main immune mediators inducing primary immune responses. DC generated from monocytes (MoDC) are a model system to study the biology of DC in vitro, as they represent inflammatory DC in vivo. Previous studies on the generation of MoDC in horses indicated that there was no distinct difference between immature and mature DC and that the expression profile was distinctly different from humans, where CD206 is expressed on immature MoDC whereas CD83 is expressed on mature MoDC. Here we describe the kinetics of equine MoDC differentiation and activation, analysing both phenotypic and functional characteristics. Blood monocytes were first differentiated with equine granulocyte-macrophage colony-stimulating factor and interleukin-4 generating immature DC (iMoDC). These cells were further activated with a cocktail of cytokines including interferon-γ) but not CD40 ligand to obtain mature DC (mMoDC). To determine the expression of a broad range of markers for which no monoclonal antibodies were available to analyse the protein expression, microarray and quantitative PCR analysis were performed to carry out gene expression analysis. This study demonstrates that equine iMoDC and mMoDC can be distinguished both phenotypically and functionally but the expression pattern of some markers including CD206 and CD83 is dissimilar to the human system.

Decreased antigen-specific T-cell proliferation by moDC among hepatitis B vaccine non-responders on haemodialysis

Patients with end-stage kidney disease, whether or not on renal replacement therapy, have an impaired immune system. This is clinically manifested by a large percentage of patients unresponsive to the standard vaccination procedure for hepatitis B virus (HBV). In this study, the immune response to HBV vaccination is related to the in vitro function of monocyte-derived dendritic cells (moDC). We demonstrate that mature moDC from nonresponders to HBV vaccination have a less mature phenotype, compared to responders and healthy volunteers, although this did not affect their allostimulatory capacity. However, proliferation of autologous T cells in the presence of tetanus toxoid and candida antigen was decreased in non-responders. Also, HLA-matched CD4+ hsp65-specific human T-cell clones showed markedly decreased proliferation in the group of non-responders. Our results indicate that impairment of moDC to stimulate antigen-specific T cells provides an explanation for the clinical immunodeficiency of patients with end-stage kidney disease.

Functional impairment of monocyte-derived dendritic cells in patients with severe chronic kidney disease

Dendritic cells (DCs) are antigen-presenting cells that are pivotal for the initiation of the primary immune response. Patients with chronic kidney disease (CKD) with or without chronic intermittent haemodialysis (CIHD) show an impaired immune response. Dysfunction of DCs may underlie this phenomenon. In this study, several different functions of monocyte-derived DCs (moDC) of patients with CKD class IV-V (glomerular filtration rate <30 ml/min) and patients on CIHD were studied in vitro and compared with age- and sex-matched healthy volunteers. We demonstrate that, independent of the maturation stimulus used, mature moDC from both groups of patients did not acquire the same level of terminal differentiation as moDC from controls, as shown by analysis of cell surface markers and the relative high macropinocytosis activity of moDC. The stimulation of allogeneic T-cells by immature moDC and mature moDC did not differ between patients and controls. However, in the presence of immature moDC or antigen-loaded maturated moDC from patients, less proliferation of autologous T-cells was observed in response to recall antigens. There was no difference between moDC from controls and patients in their ability to activate naive T-cells and to differentiate them into Th1 and Th2 cells. These results show that the terminal differentiation of moDC in patients with severe CKD is impaired. This impairment is not restricted to one maturation stimulus and is independent of treatment with haemodialysis.

Human monocyte-derived dendritic cells express TLR9 and react directly to the CpG-A oligonucleotide D19

Oligodeoxynucleotides (ODNs) containing unmethylated CpG exhibit their immunostimulatory activities by binding to TLR. Here, we show that human monocyte-derived dendritic cells (moDC) contain TLR9 protein, surprisingly, in amounts comparable with plasmacytoid DC (pDC). Immature moDC but not mature moDC nor monocytes captured CpG-ODNs. moDC stimulation with the CpG-A ODN D19 up-regulated CD83, CD86, and HLA-DR. Without CD40 ligand costimulation, full maturation was not achieved. D19-stimulated moDC primed allogeneic CD4(+)-T cells for proliferation and differentiation into IFN-gamma-secreting Th1 cells. Neither IL-12 nor IL-6 or TNF-alpha was involved. Microarray analysis pointed to a participation of Type I IFNs. In fact, D19-stimulated moDC secreted considerable amounts of IFN-alpha. This indicates that moDC themselves sense viral and bacterial DNA and do not need help from pDC.

Toxoplasma gondii: Comparison of human CD34+ and monocyte-derived dendritic cells after parasite infection

Human dendritic cells (DC) obtained in vitro from CD34+ progenitors (CD34-DC) or blood monocytes (mo-DC) are different DC which may be used in a model of T. gondii infection. We compared the survival, infection rate and cell surface receptor expression of both DC types after living T. gondii tachyzoite infection. CD34-DC appeared less resistant to the parasite than mo-DC. At 48h post-infection, chemokine receptors responsible for DC homing and migration were absent in mo-DC, while down regulation of CCR6 and up regulation of CCR7 was observed in CD34-DC. This result, suggesting migration ability of CD34-DC, was confirmed by in vitro migration experiments against different chemokines. Tachyzoite supernatant, used as chemokine, attracted immature CD34-DC as observed by MIP3α, while MIP3β, as expected, attracted mature CD34-DC. Under similar conditions, no significant difference was noticed between mature or immature mo-DC. These data indicated that CD34-DC represent an alternative model that allows migration assay of infected DC by T. gondii.

Eminent role of ICOS costimulation for T cells interacting with plasmacytoid dendritic cells

CD4+ CD45RO+ T cells could mature freshly isolated human plasmacytoid dendritic cells (PDC) in a superantigen-driven culture in a similar way to recombinant interleukin-3 (IL-3). Mature PDC expressed significantly higher levels of inducible costimulator ligand (ICOS-L), but similar levels of CD80 and CD86, when compared to mature monocyte-derived DC (moDC). We therefore directly compared the capacities of mature PDC and moDC to activate T cells. A similar T helper type 1 (Th1)/Th2 pattern of cytokines was generated in both systems, but significantly higher levels of IL-3, IL-4 and IL-10 were induced by PDC. In T cells interacting with PDC, the ICOS/ICOS-L costimulatory pathway played a pre-eminent role in the generation of IL-3 and IL-10, CD28 was central to the induction of IL-2, and both pathways were equally important for the generation of other cytokines. In cocultures with moDC, the CD28 pathway was dominant over ICOS under all circumstances, except for the ICOS-mediated release of IL-10. In general, our data demonstrate an eminent role of ICOS in the interaction of T cells with PDC, and thus modify the current paradigm of CD28 dominance for the costimulation of T cells interacting with professional antigen-presenting cells. In particular, our data highlight the role of ICOS in the generation of IL-3, a factor central to the biology of human PDC.

Expression of aberrantly glycosylated tumor mucin-1 on human DC after transduction with a fiber-modified adenoviral vector

DC-presenting tumor Ag are currently being developed to be used as a vaccine in human cancer immunotherapy. To increase chances for successful therapy it is important to deliver full-length tumor Ag instead of loading single peptides. In this study we used a fiber-modified adenoviral vector (rAd5F35) containing full-length tumor Ag cDNA to transduce human monocyte (Mo)-derived DC in vitro. Cells were efficiently transduced and survived for at least 3 days after adenoviral transduction. Phenotype and function after maturation of Mo-DC were not impaired by infection with adenovirus particles. Expression of the tumor-associated Ag mucin-1 (MUC1) was detected using MAb defining different MUC1 glycoforms. Non-transduced mature Mo-DC express endogenous MUC1 with normal glycosylation. After transduction with the rAd5F35-MUC1 adenoviral vector, Mo-DC also expressed MUC1 with tumor-associated glycosylation (Tn and T glycoforms), although no changes in mRNA levels of relevant glycosyltransferases could be demonstrated. The presence of aberrantly glycosylated MUC1 may influence Ag presentation of the tumor glycoforms of MUC1 to immune cells, affecting tumor cell killing. These findings could be highly relevant to developing strategies for cancer immunotherapy based on DC vaccines using MUC1 as tumor Ag.

Recruitment of dendritic cells to the cerebrospinal fluid in bacterial neuroinfections

Dendritic cells (DC) accumulate in the CNS during inflammation and may contribute to local immune responses. Two DC subsets present in human cerebrospinal fluid (CSF) are probably recruited from myeloid (CD11c +CD123 dim) and plasmacytoid (CD11c −CD123 high) blood DC. In bacterial meningitis and especially in Lyme meningoencephalitis, numbers of myeloid and plasmacytoid DC in CSF were increased, compared to non-inflammatory neurological diseases, and correlated with chemotactic activity of CSF for immature monocyte-derived DC (moDC). Multiple DC chemoattractants, including macrophage inflammatory protein (MIP)-1β, monocyte chemotactic protein (MCP)-1, MCP-3, RANTES and stromal cell-derived factor (SDF)-1α were elevated in CSF in these two neuroinfections. Chemotaxis of immature moDC induced by these CSFs could be partially inhibited by mAbs against CXCR4, the receptor for SDF-1α, and CD88, the receptor for C5a. SDF-1α present in CSF also chemoattracted mature moDC, which in vivo could correspond to a diminished migration of antigen-bearing DC from the CSF to secondary lymphoid organs. Regulation of DC trafficking to and from the CSF may represent a mechanism of controlling the CNS inflammation.

Potent expansion of human natural killer T cells using α-galactosylceramide (KRN7000)-loaded monocyte-derived dendritic cells, cultured in the presence of IL-7 and IL-15

Natural killer T (NKT) cells have an extremely restricted T-cell receptor repertoire, in man consisting of a Vα24 chain preferentially paired with a Vβ11 chain, and play crucial roles in various immune responses. Characterization of circulating Vα24 +Vβ11 +-T cells is hampered by their low frequencies. The α-galactosylceramide KRN7000 was reported to be presented by CD1d to NKT cells. Since dendritic cells (DC) are potent antigen presenting cells, and have been shown to express CD1d, we analyzed whether these cells could efficiently mediate expansion of Vα24 +Vβ11 +-T cells. During a 7-day co-culture of peripheral blood mononuclear cells and KRN7000-loaded mature monocyte derived DC (moDC) in the presence of interleukin-7 (IL-7) and IL-15, we observed up to 76-fold expansion of Vα24 +Vβ11 +-T cells. The expanded Vα24 +Vβ11 +-T cells expressed the cytotoxic molecule granzyme B, showed negligible expression of Fas ligand and could be induced to express high levels of interferon-γ, while retaining the capacity to produce IL-4. B cells, expressing CD1d, could also present KRN7000, but Vα24 +Vβ11 +-T cell expansion was only observed in the presence of IL-7 and/or IL-15. Considering the low frequency of circulating Vα24 +Vβ11 +-T cells, the present method for expansion of Vα24 +Vβ11 +-T cells using KRN7000-loaded mature moDC will be of value for the further characterization of this unique T cell subset.