Dendritic cells generated in the presence of interferon-alpha stimulate allogeneic CD4+ T-cell proliferation: modulation by autocrine IL-10, enhanced T-cell apoptosis and T regulatory type 1 cells

Dendritic cells (DCs) generated in the presence of IFN-alpha (IFN-DCs) exhibit high expression of major histocompatibility and co-stimulatory molecules and a potent ability to stimulate CD8(+) T-cell responses. Here, we found that IFN-DCs were more potent stimulators of bulk and purified CD8(+) T-cell proliferation, as compared with IL-4-DCs. In contrast, IFN-DCs were less efficient than IL-4-DCs in stimulating allogeneic CD4(+) T-cell proliferation, due to a weak induction of naive CD4(+)CD45RO(-) T-cell proliferation by these DCs. However, both DC populations induced similar levels of proliferation of memory CD4(+)CD45RO(+) T cells. IFN-DCs and IL-4-DCs exhibited a similar phenotype and production of IL-10 following maturation induced by CD40 ligation. In contrast, IFN-DCs produced higher levels of IL-10 during the first days of differentiation. In addition, neutralization of IL-10 during the differentiation of DCs increased the expression of DC-LAMP and MHC class II by IFN-DCs, and the ability of IFN-DCs to stimulate allogeneic CD4(+) T-cell proliferation at similar levels, than IL-4-DCs. Independently of IL-10 production, IFN-DCs were found to induce higher levels of CD4(+)T-cell apoptosis, this effect being more sticking on naive T cells. Finally, we demonstrated that IFN-DCs induced a differentiation bias of naive CD4(+) T cells towards Th1 and Tr1 cells, compared to IL-4-DCs. Taken together, these results indicate that, despite the induction of Tr1 cells and enhanced apoptosis of naive CD4(+) T cells, IFN-DCs are potent stimulators of CD8(+) and memory CD4(+) T cells, and induce a strong polarization of naive CD4(+) T cells towards Th1 cells, further supporting their use in immune-based therapy.     

Distinct effect of CD40 and TNF-signaling on the chemokine/chemokine receptor expression and function of the human monocyte-derived dendritic cells

A key and limiting step in the process of human monocyte-derived dendritic cells （mDCs） for clinical use is their in vitro maturation and in vivo migration. We previously observed that CD40 signal facilitated human mDC growth and maturation. To further explore this process, mDCs generated with GM-CSF and IL-4 were co-cultured with apoptotic tumor cells for 24 hours, followed by incubating with anti-CD40 monoclonal antibody or TNF-a for 48 hours to generate mature DCs. The chemokine/chemokine receptor expression and functions of mature DCs upon various stimuli were determined. The expression of costimulatory molecules on apoptotic tumor cell-loaded mature DCs co-cultured with either anti-CD40 antibody （anti-CD40-DCs） or TNF-a （TNF-DCs） were up-regulated compared to immature DCs, consistent with the abilities of these cytokine to drive DC maturation in vitro. The mRNA levels of chemokines such as stromal cell-derived factor-1a （SDF-1a）, EBV-induced molecule 1 ligand chemokine （ELC）, and IFN inducible protein-10 （IP-10） in anti-CD40 activated DCs were increased and the dendritic cell-specific chemokine 1 （DC-CK1） was moderately up-regulated as compared with other mature DCs. The corresponding chemokine receptors CXCR4 and CCR7 of anti-CD40-DCs were significantly expressed. The CXCR3 expression on activated T cells stimulated by anti-CD40-DCs was also increased. Moreover, the anti-CD40-DCs had a stronger ability to stimulate T cell proliferation than any other DCs. The NF-xB activity was much higher in anti-CD40-DCs than that of TNF-DCs. These results offer further evidence of the importance of the CD40 signal in developing efficient human DC vaccines for cancer immune therapy. Cellular ＆ Molecular Immunology.     

Expression and function of 4-1BB and 4-1BB ligand on murine dendritic cells

4-1BB (CDw137) and its ligand (4-1BBL) have been implicated in cellular immune responses. To further characterize the expression and function of 4-1BBL, we newly generated an anti-mouse 4-1BBL mAb (TKS-1), which can inhibit the interaction of 4-1BBL with 4-1BB. Flow cytometric analyses using TKS-1 and an anti-mouse 4-1BB mAb indicated that 4-1BB was inducible on both CD4(+) and CD8(+) splenic T cells by stimulation with immobilized anti-CD3 mAb, but 4-1BBL was not expressed on resting or activated T cells. 4-1BBL expression was inducible on splenic B cells by stimulation with anti-IgM antibody plus anti-CD40 mAb, on peritoneal macrophages by stimulation with lipopolysaccharide (LPS) and on splenic dendritic cells (DC) by stimulation with anti-CD40 mAb or LPS. Interestingly, splenic DC expressed 4-1BB constitutively, which was down-regulated by anti-CD40 stimulation. Co-culture of splenic DC with 4-1BBL-transfected cells or 4-1BBL-expressing tumor cell lines led to cytokine (IL-6 and IL-12) production and co-stimulatory molecule up-regulation by splenic DC, indicating that 4-1BBL can directly activate DC. Moreover, IL-12 production by anti-CD40-stimulated DC was partially inhibited by TKS-1. These results suggest that 4-1BB expressed on DC may be involved in DC activation through DC--tumor interaction and DC--DC interaction.     

Functional repertoire of dendritic cells generated in granulocyte macrophage-colony stimulating factor and interferon-alpha

Monocyte-derived dendritic cells (DCs) generated in granulocyte macrophage-colony stimulating factor (GM-CSF) and interleukin-4 (IL-4-DCs) are used to enhance antitumor immunity in cancer patients, although recent evidence suggests that their functional repertoire may be incomplete; in particular, IL-4-DCs appear unable to induce type 2 cytokine-producing T helper (Th) cells. To assess whether type 1 interferon (IFN) could replace IL-4 and generate DCs with a more complete repertoire, we characterized in detail DCs generated from human monocytes cultured with GM-CSF and IFN-alpha (IFN-DCs). We found that IFN-alpha induces DC differentiation more efficiently than IL-4, yielding similar numbers of DCs in a shorter time and that this differentiation persists upon removal of cytokines. Although IFN-DCs had a more mature immunophenotype than IL-4-DCs, showing higher expression of CD80, CD86, and CD83, they still preserved comparable endocytic and phagocytic capacities and responsiveness to maturation stimuli. IFN-DCs had strong antigen-presenting capacity, inducing intense proliferation of T cells to alloantigens or influenza virus. Moreover, IFN-DCs produced lower levels of IL-12p70 and higher levels of IFN-alpha, IL-4, and IL-10 than IL-4-DCs. As a consequence of this different pattern of cytokine secretion, IFN-DCs induced T cells to produce type 1 (IFN-gamma) and type 2 (IL-4 and IL-10) cytokines, and as expected, IL-4-DCs induced only Th1 differentiation. As immune responses with extreme Th1 bias are considered inadequate for the induction of optimal, systemic antitumor immunity, the ability of IFN-DCs to promote more balanced cytokine responses may suggest the advisability to consider these cells in the development of future, DC-based immunotherapy trials.     

Effect of interleukin-7 on the in vitro development and maturation of monocyte derived human dendritic cells

We have compared the cell phenotype and functional properties of monocyte/macrophage derived dendritic cells (DCs) obtained by culture of human adherent peripheral blood mononuclear cells (PBMCs) in medium containing granulocyte macrophage colony stimulating factor (GM-CSF) either alone (GM-CSF-DCs), or in combination with interleukin (IL)-4 (IL4-DCs) or IL-7 (IL7-DCs). The cell surface phenotype of GM-CSF-DCs and IL-7-DCs was characterized by a high expression of major histocompatibility complex (MHC) class I and II, CD80, CD86 and CD40. In contrast to 'classical' IL-4-DCs, these two types of DCs expressed CD14 and a CD21-like molecule detected by two out of four CD21-specific monoclonal antibodies (MoAb) tested. The same pattern of reactivity with CD21 specific antibodies was observed in freshly isolated adherent PBMCs but not in B lymphocytes. This reactivity was upregulated by IL-7 in a dose dependent manner. Lipopolysaccharide (LPS) treatment induced the upregulation of CD40, CD80, CD86 and the T-cell stimulatory capacity in IL-4-DCs and, to a lesser extent, in the IL-7-DCs whereas GM-CSF-DCs responded very poorly to such treatment. Our data indicate that, together with GM-CSF, the IL-7 drives macrophage precursors to a differentiation stage that is close to but distinct from the phenotype of IL-4-DCs. Comparison of DC development in the presence of IL-7 or IL-4 may help in dissecting signalling pathways that regulate the expression of functionally relevant DC markers.     

Lysophosphatidic acid modulates the activation of human monocyte-derived dendritic cells

Lysophosphatidic acid (LPA) is a biologically active lysophospholipid that can regulate immune activation. LPA can activate T cells and dendritic cells (DCs), but the effects of LPA on the ability of DCs to influence T cell polarization are not well understood. Human monocyte-derived DCs were differentiated in vitro in the presence of interleukin-4 (IL-4) and granulocyte-macrophage colonystimulating factor (GM-CSF), and matured with LPA and lipopolysaccharide (LPS) alone or in combination. DC activation was monitored by analyzing cell-surface expression of co-stimulatory receptors and cytokine production. The ability of DCs to influence T cell activation was determined using two models of DC:T cell co-culture. Maturation with LPS induced dose-dependent DC activation characterized by enhanced expression of co-stimulatory molecules (e.g., CD86) and production of cytokines including IL-6 and IL-10. Co-incubation with LPA attenuated the LPS-induced production of IL-6, without significantly affecting IL-10 secretion or the ability of DC to promote T cell proliferation. DCs matured in the presence of both LPA and LPS enhanced the production of interferon-gamma (IFN-gamma) when co-cultured with allogeneic T cells, compared with DC matured by LPS alone. Similar results were found using a model of allogeneic na?ve T cell differentiation, where LPA- plus LPS-matured DC enhanced IFN-gamma as well as IL-4 secretion after restimulation. Lysophosphatidic acid fine-tunes the effects of LPS on human myeloid DC maturation, but does not exert a dominant effect on the ability of DC to influence Th cell polarization.     

Monocytes differentiated with GM-CSF and IL-15 initiate Th17 and Th1 responses that are contact-dependent and mediated by IL-15

Distinct types of DCs are generated from monocytes using GM-CSF with IL-4 (IL4-DC) or IL-15 (IL15-DC). IL15-DCs are potent inducers of antigen-specific CD8(+) T cells, display a phenotype similar to CD14(+) cells commonly described in chronically inflamed tissues, and produce high levels of IL-1β and IL-15 in response to TLR4 stimulation. As these cytokines promote Th17 responses, which are also associated with inflammatory diseases, I hypothesized that TLR-primed IL15-DCs favor Th17 activation over IL4-DCs. Compared with IL4-DCs, IL15-DCs stimulated with TLR agonists secreted significantly higher concentrations of the Th17-promoting factors, IL-1β, IL-6, IL-23, and CCL20, and lower levels of the Th1 cytokine, IL-12. In addition, IL15-DCs and not IL4-DCs up-regulated IL-15 on the cell surface in response to TLR agonists. IL15-DCs primed with TLR3 or TLR4 agonists triggered Th17 (IL-17, IL-22, and/or IFN-γ) and Th1 (IFN-γ) responses, whereas IL4-DCs primed with the same TLR agonists activated Th1 (IFN-γ) responses. Secretion of IL-17 and IFN-γ required contact with TLR-primed IL15-DC, and IFN-γ production was mediated by membrane-bound IL-15. These findings identify key differences in monocyte-derived DCs, which impact adaptive immunity, and provide primary evidence that IL-15 promotes Th17 and Th1 responses by skewing monocytes into IL15-DC.     

Phenotype and Function of Monocyte-Derived Dendritic Cells from Chinese Rhesus Macaques

Dendritic cells （DCs） play a pivotal role in linking the innate immunity and acquired immunity in responses to pathogen. Non-human primates such as Chinese Rhesus Macaque （CRM） are the favorable models for preclinical study of potential therapeutic drugs, vaccines and mechanisms of human diseases. However, the phenotypical characterization of monocyte-derived dendritic cells （MDDCs） from CRM has not been elucidated. Monocytes from CRM were cultured with GM-CSF and IL-4 in RPMI-1640. Six days later, these cells were differentiated with typical dendritical morphology. CDllc and DC-SIGN were highly expressed. The immature MDDCs expressed the low levels of CD25, CD80, CD83, moderate CD40, CD86, and high MHC. After stimulation, the mature MDDCs increased expression of mature molecules CD25 and CD83, co-stimulatory molecules such as CD80, CD86 and CD40, and kept a high level of MHC. The capacity of endocytosis decreased with maturation. The mature MDDCs have strong ability of inducing allogeneic T cell proliferation and producing IL-12. In conclusion, we have characterized the phenotype and ultimate function of MDDCs from CRM for the first time.     

Amplification of tumor immunity by gene transfer of the co-stimulatory 4-1BB ligand: synergy with the CD28 co-stimulatory pathway

We have explored the role of an activation-induced T cell molecule, 4-1BB (CDw137), in the amplification of tumor immunity by retrovirus-mediated transduction of the 4-1BB ligand (4-1BBL) into tumor cells. Mice inoculated with P815 tumor cells expressing 4-1BBL developed a strong cytotoxic T lymphocyte (CTL) response and long-term immunity against wild-type tumor. The optimal effect of 4-1BBL in CTL stimulation required B7-CD28 interaction since blockade of this interaction by antibodies down-regulated the expression of 4-1BB on T cells and decreased CTL activity. Furthermore, co-expression of 4-1BBL and B7-1 in the poorly immunogenic AG104A sarcoma enhanced the induction of effector CTL and the rejection of the wild-type tumor while neither 4-1BBL nor B7-1 single transfectants were effective, suggesting a synergistic effect between the 4-1BB and the CD28 co-stimulatory pathways. Our results underscore the importance of the 4-1BB T cell stimulation pathway in the amplification of an antitumor immune response.     

Late dendritic cells are still able to evoke a potent alloreactive CTL response

On exposure to maturation stimuli, immature dendritic cells (DCs) undergo changes that turn them into potent amplifiers of innate immunity and into antigen-presenting cells (APCs) able to prime na?ve T cells. However, their progression through the maturation process is very rapid and finally ends in apoptosis. The aim of our study was to investigate the importance of the maturation stage of DCs, defined by morphology, expression of surface markers and IL-12 production, for their immunostimulatory capacity. DCs were matured with LPS, monocyte-conditioned medium (MCM) or TNF-alpha, sampled several times during a 3-day long maturation period and used as stimulators of allogeneic T cells over a wide range of DC/T cell ratios. T-cell response was assessed by cell proliferation, CTL generation and IFN-gamma production. Our results indicate that the in vitro T cell response is determined mainly by the level of expression of co-stimulatory molecules on DCs and the DC/T cell ratio in the culture. Thus, DCs matured for over 20h, with high expression of co-stimulatory molecules, can still induce a potent CTL response at DC/T cell ratios of 1:10 and 1:20, although their IL-12 production, as well as their ability to induce IFN-gamma production by T cells, are both decreased. In contrast, the CTL response at DC/T cell ratios of 1:2 and 1:5 can be profoundly decreased. Notably, the proportion of proliferating CD4+ T cells in these cultures is reduced. This could well be the reason for the absence of CTL response, since we showed that, even in the case of high expression of co-stimulatory molecules on DCs, generation of CTLs still depends on CD4+ T cells. Our study emphasizes the importance of strong expression of co-stimulatory molecules on DCs and of their ability to activate CD8+ and CD4+ T cells concomitantly in order to initiate a potent cell-mediated immune response. We therefore suggest that a combination of early DCs, which are strong producers of cytokines, and late DCs, which have high expression of co-stimulatory molecules, could prove beneficial in the attempt to initiate in vitro and in vivo cell-mediated immune responses for therapeutic purposes.     

重组大鼠4-1BBL对树突状细胞免疫学功能的影响

目的:探讨大鼠4-1BBL对骨髓来源的树突状细胞(DC)表面分子及免疫功能的影响.方法:构建真核表达质粒pIRES2-EGFP- 4-1BBL并利用脂质体介导转染HepG2细胞, G418筛选出阳性克隆, RT-PCR和荧光显微镜分别检测4-1BBL基因转录和EGFP的表达; 流式细胞术(FCM)检测不同HepG2细胞与DC共培养2 d后表面CD80及MHC-Ⅱ的表达, ELISA测定共培养上清中IL-6和IL-12的含量.结果:成功构建pIRES2-EGFP- 4-1BBL重组表达载体并在HepG2细胞中获得稳定表达, 重组大鼠4-1BBL能促进骨髓来源DC表面MHC-Ⅱ、 CD80分子表达和分泌IL-6、 IL-12(P<0.05).结论:重组大鼠4-1BBL具有促进骨髓来源DC成熟的功能.     

4-1BB在人外周血γδT细胞活化中的协同刺激作用

采用结核杆菌(Mtb)低分子多肽刺激人外周血单个核细胞,流式细胞术(FCM)检测不同活化时相γδT细胞膜表面4-1BB分子的表达;用阻断型4-1BB配体(4-1BBL)单抗阻断4-1BB/4-1BBL信号,FCM检测γδT细胞的增殖比率和细胞内产生IFN-γ的情况,同时与阻断CD28/B7-1信号相比较。结果显示,静止的γδT细胞膜表面不表达4-1BB分子,Mtb抗原刺激后6 h,4-1BB即有明显表达(29.71%),48 h达到高峰(49.79%);与未阻断组相比,阻断4-1BB/4-1BBL信号,γδT细胞的增殖效应和细胞内IFN-γ的产生均明显下降(P<0.01),与CD28/B7-1信号阻断组相比,差异无显著性(P>0.05)。提示4-1BB/4-1BBL信号同CD28/B7-1信号一样,可为γδT细胞活化提供协同刺激作用。     

小鼠髓系树突状细胞4-1BB及4-1BBL表达调节

探讨小鼠髓系树突状细胞(bone marrow-derived dendritic cells,BMDC)共刺激分子4-1BB及其配体4-1BBL表达的变化。将促DC成熟活化因子CD40L-CHO、TNF-α、LPS和IFN-γ,免疫负性调节因子IL-10以及各成熟活化因子与IL-10联合加入BMDC中,观察BMDC上4-1BB及4-1BBL表达的变化。结果显示,加入成熟活化因子的各组BMDC上4-1BB及4-1BBL表达与对照组相比有显著上调(P<0.05)。而IL-10组与对照组相比两者的表达显著下调(P<0.05),且各成熟活化因子与IL-10联合应用与单用IL-10组相比,BMDC上4-1BB和4-1BBL表达上调,有显著性差异(P<0.05)。提示成熟活化因子不仅能上调BMDC上4-1BB和4-1BBL的表达并且能有效拮抗mIL-10对BMDC上4-1BB和4-1BBL表达的下调作用。     

IL-4 is more effective than IL-13 for in vitro differentiation of dendritic cells from peripheral blood mononuclear cells

Dendritic cells (DCs) are the most potent professional antigen-presenting cells which can activate T cells to induce the primary immune response. For clinical studies, DCs are often differentiated in vitro from peripheral blood mononuclear cells (PBMCs) through treatment with granulocyte macrophage colony-stimulating factor (GM-CSF) and IL-4. However, IL-13, a cytokine closely related to IL-4, has also been reported to induce differentiation equally or more efficiently when used with GM-CSF. For the present study, we compared the DC characteristics exhibited by iDCs and LPS-matured DCs differentiated from PBMCs using GM-CSF and IL-4 or IL-13. Physical characteristics examined include cellular morphology and surface phenotype. Functional traits investigated include FITC-dextran uptake, IL-10 and IL-12 production, allostimulation and cytokine production by stimulated T cells and antigen-specific T cell stimulation. Compared with IL-13-derived DCs, IL-4 treatment yielded more differentiated DCs, with extensive dendrites and higher expression of DC-SIGN, DEC-205, CD86 and HLA-DR. In addition, IL-4 DCs were more efficient at inducing allogeneic T cell proliferation and immature IL-4 DCs had higher endocytic activity at low FITC-dextran concentrations (1 microg ml(-1)). Although IL-13 was capable of generating DCs from PBMCs, it was not as effective as IL-4 in generating DC phenotype and functionality. Thus, the use of GM-CSF and IL-4 is the more efficient treatment for inducing DC differentiation from PBMCs.     

Mite allergen decreases DC-SIGN expression and modulates human dendritic cell differentiation and function in allergic asthma

House dust mites (HDMs) induce allergic asthma in sensitized individuals, although how HDMs activate immature mucosal dendritic cells (DCs) to render the T helper cell type 2 (Th2)-mediated immune response is unclear. In this study, our results showed a significant calcium-dependent lectin binding of Dermatophagoides pteronyssinus (Der p) extracts to DC-specific intercellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN), the C-type lectin receptors (CLRs) of DCs. Moreover, monocyte-derived DCs (MDDCs) of Der p-sensitized asthmatics (AS) exhibited decreased expression of DC-SIGN, increased endocytosis, and impaired differentiation of DC precursors. The Der p-induced downregulation of DC-SIGN expression in the differentiation of immature MDDCs may be because of the internalization of Der p-DC-SIGN complex. MDDCs of AS produced more interleukin (IL)-6 and less IL-12p70 cytokines when stimulated with lipopolysaccharide (LPS) or Der p than those of nonallergic controls (NC). In the co-culture experiments, MDDCs pretreated with Der p induced GATA-3 expression and IL-4 cytokine productions in naive CD4(+) T cells. These effects of Der p on the differentiation and function of MDDCs could be partially blocked by anti-DC-SIGN antibodies. In conclusion, our results suggest a critical step of allergen sensitization that involves CLRs in the innate immune response of DCs, which may provide a therapeutic or preventive potential for allergic asthma.     

Dendritic cell subsets generated from CD34+ hematopoietic progenitors can be transfected with mRNA and induce antigen-specific cytotoxic T cell responses

Human dendritic cells (DCs) generated in culture from either monocytes or CD34+ hematopoietic progenitor cells (CD34-HPCs) have been used in cancer immunotherapy protocols with encouraging results. Yet an optimal strategy for the delivery of antigen(s) to DCs still remains to be established. Recent studies demonstrated the feasibility of mRNA transfection to load monocyte-derived DCs. It is not known, however, whether DCs derived by culturing CD34-HPC with GM-CSF and TNF-alpha for 9 days (CD34-DCs) can be efficiently transduced with mRNA. Here we show that clinical-grade CD34-DCs generated after 8 days of culture can be transfected with mRNA without significant alteration of cell viability. About 90% of cells transfected with GFP-RNA express GFP 24 h post-transfection. Remarkably, transfected CD34-DCs retain high levels of GFP expression for at least 14 days. CD34-DCs transfected with Flu-MP RNA were highly efficient in inducing the proliferation of Flu-MP-specific CD8+ T cells as measured by tetramer staining. Furthermore, the stimulated CD8+ T cells produced IFN-gamma upon antigenic stimulation and were able to kill targets pulsed with Flu-MP peptide. Both DC subsets in CD34-DCs, CD1a+-DC (Langerhans cells) and CD14+-DC (interstitial DC), were equally transfected with GFP-RNA, and yielded Flu-specific cytotoxic T cells upon transfection with Flu-MP RNA. Thus, RNA can be used to deliver antigens to two distinct myeloid DC subsets in CD34-DC cultures.     

IL-10-conditioned dendritic cells prevent autoimmune diabetes in NOD and humanized HLA-DQ8/RIP-B7.1 mice

This study was to determine whether BMDCs cultured in the presence of IL-10 (G/10-DCs) could promote T cell tolerance and prevent autoimmune diabetes in two different animal models of T1D. Our results showed that G/10-DCs suppressed both insulitis and spontaneous diabetes in NOD and HLA-DQ8/RIP-B7.1 mice. The suppression was likely to be mediated by T cells, as we found that regulatory CD4(+)CD25(+)Foxp3(+) cells were significantly increased in G/10-DC treated animals. In vivo, the G/10-DCs inhibited diabetogenic T cell proliferation; in vitro, they had reduced expression of costimulatory molecules and produced little IL-12/23 p40 or IL-6 but a large amount of IL-10 when compared with DCs matured in the presence of IL-4 (G/4-DC). We conclude that IL-10-treated DCs are tolerogenic and induce islet-directed immune tolerance, which was likely to be mediated by T regulatory cells. This non-antigen-specific DC-based approach offers potential for a new therapeutic intervention in T1D.     

Mature dendritic cells differentiated in the presence of interferon-beta and interleukin-3 prime functional antigen-specific CD8 T cells

Dendritic cell (DC)-based immunization represents a promising approach for the immunotherapy of cancer. The optimal conditions required to prepare DCs remain to be defined. Monocytes incubated in the presence of interferon (IFN)-beta and interleukin (IL)-3 give rise to a distinct type of DCs (IFN-beta/IL-3 DCs) that are particularly efficient at eliciting IFN-gamma and IL-5 production by allogeneic helper T cells. We assessed the capacity of this new type of DCs to prime antigen-specific naive CD8(+) T cells and compared them to the conventional DCs differentiated in the presence of granulocyte-macrophage colony stimulating factor (GM-CSF) and IL-4 (GM-CSF/IL-4 DCs). We demonstrate that IFN-beta/IL-3 DCs matured by TLR3 or CD40 ligation efficiently prime Melan-A(26-35)-specific CD8(+) T cells in vitro, at a similar level as GM-CSF/IL-4 DCs. Activated antigen-specific CD8(+) T cells produced IFN-gamma and displayed potent cytotoxic activity against peptide-pulsed target cells. Expansion of CD8(+) T cell numbers was generally higher following priming with CD40-L than with polyinosinic-polycytidylic acid (poly I:C) matured DCs. Cytolytic activity was induced by both maturing agents. These data indicate that IFN-beta/IL-3 DCs represent a promising cell population for the immunotherapy of cancer.     

Human monocyte-derived dendritic cells differentiated in the presence of IL-2 produce proinflammatory cytokines and prime Th1 immune response

Interleukin (IL)-2 plays an important role in the control of the immune responses, and it is released in a variety of tissues in response to inflammatory stimuli. As monocytes and mature dendritic cells (DCs) express CD25, the high-affinity subunit of IL-2 receptor, we examined the effect of exogenous IL-2 on the in vitro generation and maturation of DCs from monocytes. Human monocyte-derived DCs (MDDCs) were generated by culturing monocytes with granulocyte macrophage-colony stimulating factor (GM-CSF) and IL-4 in the presence or absence of IL-2. The cytokine was added at the beginning and after 5 days of culture. Our findings indicate that IL-2 does induce monocytes to differentiate into DCs with the same morphology and phenotype of that obtained in the presence of GM-CSF and IL-4 alone, but with some distinctive functional properties. DCs differentiated in the presence of IL-2 secreted significantly more IL-1beta, TNF-alpha, and IL-12 p70 in response to lipopolysaccharide stimulation and induced allogeneic, na?ve T cells to release a significantly higher amount of interferon-gamma if compared with DCs obtained by culturing monocytes with GM-CSF and IL-4. These results indicate unrecognized effects of IL-2 on human MDDCs and suggest that an IL-2-rich environment during differentiation and maturation of DCs can modify their T helper cell-inducing properties.     

Expression and function of 4-1BB during CD4 versus CD8 T cell responses in vivo

4-1BBL(-/-) mice have a defect in recall CD8+ T cell responses to viruses, whereas CD4+ T cell responses to virus are unimpaired in these mice. In contrast, both CD4+ and CD8+ T cells respond to 4-1BB ligand (4-1BBL) in vitro. To clarify the role of 4-1BB/4-1BBL in CD4+ versus CD8+ T cell responses in vivo, we compared CD4 (OT-II) and CD8 (OT-I) TCR transgenic T cells responding to the same antigen in an in vivo adoptive transfer model in 4-1BBL(+/+) versus 4-1BBL(-/-) mice. During primary and secondary responses, expression of 4-1BB on in vivo-activated TCR transgenic T cells was earlier and more transient than previously observed in vitro, correlating with expression of the early activation antigen CD69 and preceding the transition to the CD44hi state. Although 4-1BB is expressed early in the primary response, there was no effect of 4-1BBL deficiency on initial CD8 T cell expansion and only a minor effect on initial CD4 T cell expansion. The major effect of 4-1BB/4-1BBL interaction is on the T cell recall response. This is due to effects of 4-1BBL on maintenance of T cell numbers at the end of the primary response with additional effects of 4-1BBL on secondary expansion of T cells.