An improved protocol for generation of immuno-potent dendritic cells through direct electroporation of CD14+ monocytes

In this study we demonstrate a novel protocol showing that electroporation of CD14+ monocytes directly isolated from blood with green fluorescent protein (GFP) RNA results in a 3-fold higher yield of antigen presenting dendritic cells (DCs) when compared to conventional methods employing immature DCs for electroporation. We further show a stable electroporation efficacy resulting in 60% of GFP positive cells. Expression of co-stimulatory molecules and maturation markers such as CD80, CD86, CD83 as well of the chemokine receptor 7 (CCR7) was found in 90% of the mature DCs. Importantly, production of IL-12p70 was 10 times higher in cells electroporated at the monocyte stage compared to cells electroporated at the immature DC stage. Stimulation of autologous na?ve lymphocytes by DCs electroporated at monocytes stage elicited proliferation of CD8+ T-cell with 7-fold increase in IFN-gamma release. Blocking of the MHC-Class I molecules significantly inhibited the IFN-gamma release, indicating that antigen presentation was MHC-Class I mediated. In summary, electroporation of CD14+ monocytes with RNA results in a high yield of antigen presenting DCs with high immuno-stimulatory capacity and antigen presentation on MHC-Class I molecules. This improved method may represent an attractive approach for RNA-based DC immunotherapy.     

Galectin-9 induces maturation of human monocyte-derived dendritic cells]

We investigated the role of galectin-9 (Gal-9) in maturation of dendritic cells (DC). Culture of immature DCs with exogenous Gal-9 markedly increased the surface expression of CD40, CD54, CD80, CD83, CD86, and HLA-DR in a concentration-dependent manner, although Gal-9 had no effect on differentiation of human monocytes into immature DCs. Gal-9-treated DCs secreted IL-12 but not IL-10, and they elicited the production of Th1 cytokines (IFN-gamma and IL-2), but not that of the Th2 cytokines (IL-4 and IL-5) by allogeneic CD4(+) T cells. These effects of Gal-9 on immature DCs were not essentially dependent on its lectin properties, given that they were only slightly inhibited by lactose. We further found that a Gal-9 mutant that lacks beta-galactoside binding activity reproduced the above activities, and that an anti-Gal-9 mAb suppressed them. Gal-9 induced phosphorylation of the p38 MAPK and ERK1/2 in DCs, and an inhibitor of p38 signaling, but not inhibitors of signaling by either ERK1/2 or phosphatidylinositol 3-kinase, blocked Gal-9-induced up-regulation of costimulatory molecule expression and IL-12 production. These findings suggest that Gal-9 plays a role not only in innate immunity but also in acquired immunity by inducing DC maturation and promoting Th1 immune responses.     

IL-21 enhances SOCS gene expression and inhibits LPS-induced cytokine production in human monocyte-derived dendritic cells

Dendritic cells (DCs) play an important role in innate and adaptive immune responses. In addition to their phagocytic activity, DCs present foreign antigens to na?ve T cells and regulate the development of adaptive immune responses. Upon contact with DCs, activated T cells produce large quantities of cytokines such as interferon-gamma (IFN-gamma) and interleukin (IL)-21, which have important immunoregulatory functions. Here, we have analyzed the effect of IL-21 and IFN-gamma on lipopolysaccharide (LPS)-induced maturation and cytokine production of human monocyte-derived DCs. IL-21 and IFN-gamma receptor genes were expressed in high levels in immature DCs. Pretreatment of immature DCs with IL-21 inhibited LPS-stimulated DC maturation and expression of CD86 and human leukocyte antigen class II (HLAII). IL-21 pretreatment also dramatically reduced LPS-stimulated production of tumor necrosis factor alpha, IL-12, CC chemokine ligand 5 (CCL5), and CXC chemokine ligand 10 (CXCL10) but not that of CXCL8. In contrast, IFN-gamma had a positive feedback effect on immature DCs, and it enhanced LPS-induced DC maturation and the production of cytokines. IL-21 weakly induced the expression Toll-like receptor 4 (TLR4) and translation initiation region (TIR) domain-containing adaptor protein (TIRAP) genes, whereas the expression of TIR domain-containing adaptor-inducing IFN-beta (TRIF), myeloid differentiation (MyD88) 88 factor, or TRIF-related adaptor molecule (TRAM) genes remained unchanged. However, IL-21 strongly stimulated the expression of suppressor of cytokine signaling (SOCS)-1 and SOCS-3 genes. SOCS are known to suppress DC functions and interfere with TLR4 signaling. Our results demonstrate that IL-21, a cytokine produced by activated T cells, can directly inhibit the activation and cytokine production of myeloid DCs, providing a negative feedback loop between DCs and T lymphocytes.     

PPD extract induces the maturation of human monocyte-derived dendritic cells

Dendritic cells (DCs) are the most powerful antigen presenting cells, capable of inducing T-dependent immune responses even in naive T cells. DCs are of special interest as cellular adjuvants for immunity induction in clinical settings and several methods for their generation and maturation are recently under investigation. The present study was set out to define the effects of PPD (Purified Protein Derivative), a mycobacterial extract used in the tuberculin skin test, on in vitro differentiation and maturation of human monocyte derived dendritic cells. Immature DCs were prepared from the peripheral blood monocytes of healthy volunteers by culturing in a medium supplemented with granulocyte macrophage colony stimulating factor (GM-CSF) and interleukin-4 (IL-4). The resultant cells were then stimulated with PPD extract and their properties such as cell morphology and the expression of key surface molecules were compared with tumor necrosis factor-alpha (TNF-alpha) stimulated immature DCs. Our results suggest that mycobacterial purified extract is as potent as TNF-alpha, a well-established DC stimulator, in induction of maturation in human monocyte derived DCs. We also ruled out the contribution of lipopolysaccharide (LPS) and beta-glucan contamination in maturation effect of PPD preparations. So, PPD as an examined safe material for in vivo consumption could be used to stimulate DC maturation in DC based immunotherapy protocols.     

Intravenous immunoglobulin modulates the maturation of TLR 4-primed peripheral blood monocytes

Intravenous immunoglobulin (IgIV) has immune modulating effects on the differentiation and function of dendritic cells (DC). Peripheral blood CD14+ monocytes were induced to differentiate into immature DC with IL-4/GM-CSF. DC maturation was analyzed by flow cytometry, and function assessed for antigen uptake and antigen processing. IgIV added during the differentiation process induced immature DC to differentiate into a mature DC with increased expression of CD83 and CCR7. A "priming" step with low concentrations of LPS or other TLR agonists that utilize the myD88 signaling pathway was necessary to observe these changes. These modulated DCs had reduced antigen uptake, but exhibited increased antigen presentation. Treatment of the IgIV with pepsin to generate F(ab')2 fragments abrogated these effects on DC maturation and function. The enhanced differentiation of PBM into DC required two signals: an initial exposure to low concentrations of LPS followed by IVIG. The second signal with IVIG was Fc dependent.     

Composition of MHC class II-enriched lipid microdomains is modified during maturation of primary dendritic cells

Dendritic cells (DCs) are the most potent antigen presenting cells. Major histocompatibility complex (MHC) class II molecule expression changes with maturation; immature DCs concentrate MHC class II molecules intracellularly, whereas maturation increases surface expression of MHC class II and costimulatory molecules to optimize antigen presentation. Signal transduction via MHC class II molecules localized in lipid microdomains has been described in B lymphocytes and in the THP-1 monocyte cell line. We have characterized MHC class II molecules throughout human DC maturation with particular attention to their localization in lipid-rich microdomains. Only immature DCs expressed empty MHC class II molecules, and maturation increased the level of peptide-bound heterodimers. Ligand binding to surface human leukocyte antigen (HLA)-DR induced rapid internalization in immature DCs. The proportion of cell-surface detergent-insoluble glycosphingolipid-enriched microdomain-clustered HLA-DR was higher in immature DCs despite the higher surface expression of HLA-DR in mature DCs. Constituents of HLA-DR containing microdomains included the src kinase Lyn and the cytoskeletal protein tubulin in immature DCs. Maturation modified the composition of the HLA-DR-containing microdomains to include protein kinase C (PKC)-delta, Lyn, and the cytoskeletal protein actin, accompanied by the loss of tubulin. Signaling via HLA-DR redistributed HLA-DR and -DM and PKC-delta as well as enriching the actin content of mature DC microdomains. The increased expression of HLA-DR as a result of DC maturation was therefore accompanied by modification of the spatial organization of HLA-DR. Such regulation could contribute to the distinct responses induced by ligand binding to MHC class II molecules in immature versus mature DCs.     

PPD Extract Induces the Maturation of Human Monocyte-Derived Dendritic Cells

Dendritic cells (DCs) are the most powerful antigen presenting cells, capable of inducing T-dependent immune responses even in naive T cells. DCs are of special interest as cellular adjuvants for immunity induction in clinical settings and several methods for their generation and maturation are recently under investigation. The present study was set out to define the effects of PPD (Purified Protein Derivative), a mycobacterial extract used in the tuberculin skin test, on in vitro differentiation and maturation of human monocyte derived dendritic cells. Immature DCs were prepared from the peripheral blood monocytes of healthy volunteers by culturing in a medium supplemented with granulocyte macrophage colony stimulating factor (GM-CSF) and interleukin-4 (IL-4). The resultant cells were then stimulated with PPD extract and their properties such as cell morphology and the expression of key surface molecules were compared with tumor necrosis factor-α (TNF-α) stimulated immature DCs. Our results suggest that mycobacterial purified extract is as potent as TNF-α, a well-established DC stimulator, in induction of maturation in human monocyte derived DCs. We also ruled out the contribution of lipopolysaccharide (LPS) and beta-glucan contamination in maturation effect of PPD preparations. So, PPD as an examined safe material for in vivo consumption could be used to stimulate DC maturation in DC based immunotherapy protocols.     

Human dendritic cells differentiated in hypoxia down-modulate antigen uptake and change their chemokine expression profile

Dendritic cells (DCs) are the most potent antigen-presenting cells and fine-tune the immune response. We have investigated hypoxia's effects on the differentiation and maturation of DCs from human monocytes in vitro, and have shown that it affects DC functions. Hypoxic immature DCs (H-iDCs) significantly fail to capture antigens through down-modulation of the RhoA/Ezrin-Radixin-Moesin pathway and the expression of CD206. Moreover, H-iDCs released higher levels of CXCL1, VEGF, CCL20, CXCL8, and CXCL10 but decreased levels of CCL2 and CCL18, which predict a different ability to recruit neutrophils rather than monocytes and create a proinflammatory and proangiogenic environment. By contrast, hypoxia has no effect on DC maturation. Hypoxic mature DCs display a mature phenotype and activate both allogeneic and specific T cells like normoxic mDCs. This study provides the first demonstration that hypoxia inhibits antigen uptake by DCs and profoundly changes the DC chemokine expression profile and may have a critical role in DC differentiation, adaptation, and activation in inflamed tissues.     

DC-SIGN ligation greatly affects dendritic cell differentiation from monocytes compromising their normal function

DC-SIGN is a C-type lectin selectively expressed by certain types of DCs, including monocyte-derived DCs. Many reports have described the impact of DC-SIGN engagement with concomitant TLR signaling in tailoring of the DC maturation process, but so far, none has addressed the importance of DC-SIGN engagement during their differentiation from blood progenitors. We therefore examined the role of DC-SIGN engagement limited to the stage of IL-4-guided differentiation of DCs from human peripheral blood monocytes but not during maturation. We used two different anti-DC-SIGN antibodies with reported DC-SIGN-engaging activities. In cultures with DC-SIGN ligands, the resulting iDCs displayed abrogated expression of differentiation markers CD1a and DC-SIGN. Without further DC-SIGN activation, such DCs matured with low CD80/CD86 and high ILT3 expression, along with the appearance of macrophage marker CD14. Additionally, treated DCs indicated a tolerogenic potential by possessing a low, allostimulatory capacity and inducing na?ve, allogeneic CD4(+) T cells to produce low levels of IFN-γ. Upon activation, IL-10 production was greatly increased by such DCs; however, the use of IL-10-blocking antibodies could not completely reverse alternative DC activation. This suggests an alternative activation response that is a result of a different elementary state of DCs generated with concomitant ligation of DC-SIGN. During differentiation, IL-4-induced pSTAT6 was reduced by DC-SIGN ligands. Furthermore, during LPS-induced maturation, treated DCs displayed lowered activation levels of p38 MAPK, STAT1, as well as STAT6, compared with controls. Collectively, evidence is presented confirming a crucial role for DC-SIGN signaling in DC generation from monocytes.     

C反应蛋白与酶修饰低密度脂蛋白对树突状细胞成熟的影响

目的探讨在动脉粥样斑块硬化进程中C反应蛋白（CRP）和酶修饰低密度脂蛋白（E-LDL）对外周血中树突状细胞（DC）分化成熟的影响。方法梯度离心法分离人外周血单核细胞,用含rhGM-CSF和rhIL-4的Cellgro培养液培养5d,使其分化为DC。分别用CRP、E-LDL及CRP加E-LDL刺激DC48h后,采用流式细胞术检测DC表型（CD1a、CD80、CD86、HLA-DR）的表达,ELISA法检测DC上清液中IL-12、TNFα、IL-2和IL-10的含量,并进行比较。结果CRP抑制DC的CD1a、CD80表达及IL-12、TNFα分泌,E-LDL促进DC的CD1a、CD80、CD86和HLA-DR表达及IL-12、TNFα分泌,CPR加E-LDL结果与E-LDL类似。结论CRP抑制DC的活化,E-LDL可促进DC的分化成熟,E-LDL激活DC的作用强于CRP的抑制作用。     

Differential effects of parainfluenza virus type 3 on human monocytes and dendritic cells.

To understand the lack of protective immunity observed after infection with parainfluenza virus type 3 (PIV3), we tested the effect of the virus on human monocytes and monocyte-derived immature dendritic cells (DCs). Expression of viral antigens on the cell surfaces correlated with replication of the virus, which was marginal in monocytes but extremely efficient in DCs. The virus increased monocyte survival at least in part through the production of granulocyte-macrophage colony-stimulating factor but, in contrast, accelerated DC apoptosis. In addition, PIV3 infection failed to activate monocytes but induced maturation of DCs with increased expression of CD54, HLA-DR, CD86, and CD83 and production of bioactive IL-12. However, PIV3-infected DCs demonstrated low stimulatory properties in DC-T cell cocultures, a finding that could not be attributed to the production of infectious virus or IL-10. These results demonstrate for the first time that PIV3 dramatically modifies the survival and/or the function of antigen-presenting cells and might therefore prevent the development of efficient antiviral immune responses.     

Anti-P-selectin lectin-EGF domain monoclonal antibody inhibits the maturation of human immature dendritic cells

Dendritic cells (DCs) are professional antigen-presenting cells with the ability to initiate primary T cell responses. While it is well known that inflammatory stimuli induce the functional maturation of immature DCs, whether adhesion molecule selectins regulate DC maturation is poorly understood. Using anti-P-selectin lectin-EGF domain monoclonal antibody (PsL-EGFmAb) that blocks the adhesion of P-, E-, and L-selectin, we demonstrate herein that selectins play important role in stimulating functional maturation of immature DCs. Immature DCs are generated from human cord blood CD34+ hematopoietic stem/progenitor cells that were cultured in the presence of stem cell factor, Fms-like tyrosine-kinase-3 ligand, granulocyte-macrophage colony stimulating factor, and transform growth factor-beta1. When stimulated with tumor necrosis factor-alpha (TNF-alpha), immature DCs differentiated into mature DCs, producing increased levels of costimulatory molecules and interleukin (IL)-12 and obtaining the ability to potently activate na?ve T cells. Interestingly, in contrast to mature DCs derived from TNF-alpha-induced immature DC cultures without PsL-EGFmAb, immature DCs treated with PsL-EGFmAb for 7 days were completely blocked their maturation, as evidenced by decreased expression of costimulatory molecules CD80, CD86, and CD83, inhibited production of IL-12, and inability to activate na?ve T cells in vitro. Thus, blockade of selectins using PsL-EGFmAb will prove to be a valuable tool for the study of the molecular mechanisms of DC maturation, as well as for the prevention and treatment of DC-mediated autoimmunity.     

Epstein–Barr virus induces the differentiation of semi-mature dendritic cells from cord blood monocytes

Background

Epstein–Barr virus (EBV) is a tumorigenic virus which has effectively infected nearly all human beings with over 95% adult being seropositive. The persistence of latent EBV infection is not fully understood. Recent studies point towards a hypothesis of immune suppression and immune evasion involving regulatory T cells (Tregs) and dendritic cells (DCs). We sought to explore the mechanism of EBV suppression and immune evasion.

Methods

We compared the effects of EBV on cord blood (CB) and adult DCs differentiation and maturation including phenotype by flow cytometry, cytokine by ELISA and RT-PCR. And we evaluated the function of DC by co-culture DC and Treg by detection the expression of Foxp3, the phenotype and the cytokine profile of Tregs by flow cytometry.

Results

CB DCs derived from EBV-infected CB monocytes or from EBV-infected CB immature DCs (iDCs) displayed distinct phenotypes of “semi-mature” DCs with high expression of co-stimulatory molecules, such as CD40, CD80 and CD86 but low cytokine production, related to immune tolerance and homeostasis. While the EBV-infected adult iDCs resemble that of “pathogen-driven regulatory mature DCs” with high expression of co-stimulatory molecules, down-regulation of IL-12 secretion and up-regulation of IL-10 secretion, related to protection of host and immune evasion of pathogens. EBV infected cord blood monocytes-derived DCs drived Tregs development by driving the expression of Foxp3, increasing the expression of CTLA-4, decreasing the expression of GITR and promoted the generation of intracellular IL-2 and IL-10 by Tregs.

Conclusion

Epstein–Barr virus induces the differentiation of semi-mature dendritic cells from cord blood monocytes. The differences between CB and adult DCs suggested that the developmental maturity of the cells may affect their immune responses to EBV infection.     

TLR7/8 agonists impair monocyte-derived dendritic cell differentiation and maturation

Pathogen recognition by TLR activates the innate immune response and is typically followed by the development of an adaptive immune response initiated by antigen presentation. Dendritic cells (DC) are the most efficient APC and express diverse TLRs, including TLR7 and -8, which have been recently identified as targets for ssRNA recognition during viral infection. We have studied the effect of TLR7/8 agonists on DC differentiation and maturation from human monocytes. The synthetic agonist Resiquimod (R-848) or the physiological agonist ssRNA impaired monocyte differentiation to DC phenotypically and functionally. Induced expression of the nonclassical MHC molecules of the CD1 family in DC was inhibited at the protein and mRNA levels, and antigen acquisition was inhibited. Proinflammatory cytokine (including IL-6, IL-8, TNF-alpha, IL-1beta) and IL-10 production were induced during DC differentiation. Cross-talk between TLR4 and TLR7/8 was revealed as immature DC, which had been differentiated in the presence of R-848 were insensitive to LPS-mediated maturation and cytokine production but still induced allostimulation. These data lead us to suggest that ongoing viral activation of TLR7/8 could alter the adaptive immune response by modifying DC differentiation and by down-regulating DC responsiveness to a subsequent bacterial TLR4-mediated signal.     

人外周血树突状细胞培养和地塞米松对其分化的影响作用

目的分离培养和鉴定人外周血树突状细胞（DC）,以及探讨地塞米松对其分化的影响作用。方法密度梯度离心法分离人外周血单个核细胞,贴壁后加入GM-CSF、IL-4和LPS培养,部分组另加入地塞米松,观察细胞形态学、流式标志和DC与T淋巴细胞共培养后的增殖变化。结果外周血单核细胞诱导培养后具有DC形态学特征,CD83表达上调,CD14表达下调,DC与T淋巴细胞共培养后呈增殖反应。培养液中加入地塞米松后CD83表达下调,CD14表达上调,DC与T淋巴细胞共培养后增殖反应减弱。结论外周血单核细胞经联合细胞因子可诱导为DC;地塞米松可使DC在功能上处于不成熟状态。     

CD40 ligation and phagocytosis differently affect the differentiation of monocytes into dendritic cells

That monocytes can differentiate into macrophages or dendritic cells (DCs) makes them an essential link between innate and adaptive immunity. However, little is known about how interactions with pathogens or T cells influence monocyte engagement toward DCs. We approached this point in cultures where granulocyte macrophage-colony stimulating factor (GM-CSF) and interleukin (IL)-4 induced monocytes to differentiate into immature DCs. Activating monocytes with soluble CD40 ligand (CD40L) led to accelerated differentiation toward mature CD83(+) DCs with up-regulated human leukocyte antigen-DR, costimulatory molecules and CD116 (GM-CSF receptor), and down-regulation of molecules involved in antigen capture. Monocytes primed by phagocytosis of antibody-opsonized, killed Escherichia coli differentiated into DCs with an immature phenotype, whereas Zymosan priming yielded active DCs with an intermediate phenotype. Accordingly, DCs obtained from cultures with CD40L or after Zymosan priming had a decreased capacity to endocytose dextran, but only DCs cultured with CD40L had increased capacity to stimulate allogeneic T cells. DCs obtained after E. coli or Zymosan priming of monocytes produced high levels of proinflammatory tumor necrosis factor alpha and IL-6 as well as of regulatory IL-10, but they produced IL-12p70 only after secondary CD40 ligation. Thus, CD40 ligation on monocytes accelerates the maturation of DCs in the presence of GM-CSF/IL-4, whereas phagocytosis of different microorganisms does not alter and even facilitates their potential to differentiate into immature or active DCs, the maturation of which can be completed upon CD40 ligation. In vivo, such differences may correspond to DCs with different trafficking and T helper cell-stimulating capacities that could differently affect induction of adaptive immune responses to infections.     

Bacillus anthracis lethal toxin attenuates lipoteichoic acid-induced maturation and activation of dendritic cells through a unique mechanism

Lethal toxin (LT), produced by the gram-positive bacterium Bacillus anthracis, was identified as a major etiologic agent causing anthrax due to its strong immunotoxicity. Gram-positive bacteria express lipoteichoic acid (LTA), which is considered as a counterpart to lipopolysaccharide (LPS) of gram-negative bacteria, but differs from LPS in the structure and function. Since dendritic cells (DCs) are essential for the appropriate initiation of immune response, we investigated the effect of LT on LTA-induced DC maturation using immature DCs prepared by differentiation of C57BL/6 mouse bone marrow cells. When immature DCs were matured with LTA in the presence of LT, the expression of representative markers for DC maturation such as CD80, CD83, and CD86 together with MHC class I and II molecules was inhibited. LT ameliorated the attenuation of endocytic capacity during DC maturation by LTA while such effect was not observed in LPS-matured DCs. Furthermore, exposure to LT resulted in a decrease in the expression of pro-inflammatory cytokines including IL-6, TNF-α, and IL-12p40 in LTA-stimulated DCs as in LPS-stimulated DCs. Interestingly, LT showed a minimal change in LTA-induced IL-1β expression while LT highly enhanced the LPS-induced IL-1β expression. Those inhibitory effects might be associated with LT interference of LTA-signaling pathways mediated through mitogen-activated protein kinases (MAPKs) since LT suppressed phosphorylation of MAPK, which was induced by LTA. Meanwhile, no change was observed in the expression of putative anthrax toxin receptors, TEM8 and CMG2, or Toll-like receptor 2. These results suggest that LT suppresses the maturation and activation of DCs stimulated with LTA, similar to the suppression in the LPS-stimulated DCs, but via a distinct mechanism.     

The STATus of PD-L1 (B7-H1) on tolerogenic APCs

Expression by DCs of co-inhibitory molecules such as programmed death ligand-1 (PD-L1/B7-H1/CD274), a member of the B7 superfamily, is crucial for the downregulation of T-cell responses and the maintenance of immune homeostasis. Exposure of immature DCs to danger-associated molecular patterns (DAMPS) or pathogen-associated molecular patterns (PAMPs) generally results in their maturation and acquisition of immunostimulatory function. However, exposure of DCs to TLR ligands early during their differentiation can inhibit further differentiation and confer tolerogenic properties on these APCs. A report in this issue of The European Journal of Immunology reveals that early inhibition of human DC differentiation from blood monocytes by TLR agonists is associated with a tolerogenic phenotype and Treg generation. The tolerogenic function of these APCs is dependent on MAPK-induced IL-6 and IL-10 production, which drives STAT-3-mediated PD-L1 expression. These observations link IL-10 and IL-6 to PD-L1 expression, providing a new dimension to the anti-inflammatory properties of these cytokines. These findings also have implications for understanding the inherent function of DCs in non-lymphoid tissues such as the liver and lung, where they are exposed to PAMPs that are found constitutively in the local microenvironment.     

Thymosin-alpha1 modulates dendritic cell differentiation and functional maturation from human peripheral blood CD14+ monocytes

Although thymosins have been demonstrated to have immunomodulatory effects, it is still not clear whether they could affect dendritic cells (DCs), the most professional antigen-presenting cells. The objective of this study was to determine the effect and potential mechanisms of thymosin-alpha1 (Talpha1) on DC differentiation and functional maturation. Human peripheral blood CD14(+) monocytes were purified by using a magnetic separation column and cultured with GM-CSF and IL-4 to differentiate into immature DCs (iDCs). In the presence of Talpha1, iDC surface markers CD40, CD80, MHC class I and class II molecules were significantly upregulated as measured by flow cytemotry analysis. However, Tbeta4 or Tbeta10 did not show these effects on iDCs. There was an approximately 30% reduction in antigen (FITC-conjugated dextran)-uptake by Talpha1-treated iDCs as compared with non-Talpha1-treated iDCs. In addition, Talpha1-treated matured DCs (mDCs) showed an increased stimulation of allogeneic CD3(+) T-cell proliferation as measured by a mixed-lymphocyte reaction assay. Talpha1-treated mDCs also increased the production of several Th1- and Th2-type cytokines as measured by a Bio-Plex cytokine assay. Furthermore, rapid activation of p38 MAPK and NFkappaB was seen in Talpha1-treated iDCs as measured by a Bio-Plex phosphoprotein assay. Thus, Talpha1 significantly enhances DC differentiation, activation, and functions from human peripheral blood CD14(+) monocytes possibly through a mechanism of the activation of p38 MAPK and NFkappaB pathways. This study provides a basis to further evaluate Talpha1 as a possible adjuvant for a DC-directed vaccine or therapy.