PD-L1和PD-L2在树突状细胞上的表达及其生物学意义

探讨小鼠髓系DC (CD8α )中PD L1和PD L2的表达及其在T淋巴细胞活化中的作用。采用mCD4 0L CHO和TNF α分别刺激凋亡肿瘤细胞负载DC 4 8h ;免疫荧光标记检测DC表型 ;RT PCR和realtime PCR检测PD L1和PD L2mRNA转录水平 ;ELISA测定IL 2的分泌水平 ;3 H TdR掺入试验和51Cr释放试验测定DC体外激发T细胞的增殖和细胞毒杀伤率。结果显示 :PD L1和PD L2随着DC的成熟呈上调表达 ,CD4 0配基化DC的PD L1和PD L2均为中度表达 ,TNF α激发的DC为高度表达 ,二者呈现差异性表达 (P <0 0 5 ) ;CD4 0配基化髓系DC分泌IL 2的量明显高于TNF α组 (P <0 0 5 ) ,体外刺激T增殖和激活CTL能力在CD4 0配基化DC组最高 (P <0 0 5 )。提示CD4 0配基化的小鼠髓系DC呈现PD L1和PD L2的中度表达 ,IL 2大量分泌 ,这些均有助于激发有效的特异性免疫应答     

Rapid and coordinated switch in chemokine receptor expression during dendritic cell maturation

Dendritic cells (DC) migrate into inflamed peripheral tissues where they capture antigens and, following maturation, to lymph nodes where they stimulate T cells. To gain insight into this process we compared chemokine receptor expression in immature and mature DC. Immature DC expressed CCR1, CCR2, CCR5 and CXCR1 and responded to their respective ligands, which are chemokines produced at inflammatory sites. Following stimulation with LPS or TNF-α maturing DC expressed high levels of CCR7 mRNA and acquired responsiveness to the CCR7 ligand EBI1 ligand chemokine (ELC), a chemokine produced in lymphoid organs. Maturation also resulted in up-regulation of CXCR4 and down-regulation of CXCR1 mRNA, while CCR1 and CCR5 mRNA were only marginally affected for up to 40 h. However, CCR1 and CCR5 were lost from the cell surface within 3 h, due to receptor down-regulation mediated by chemokines produced by maturing DC. A complete down-regulation of CCR1 and CCR5 mRNA was observed only after stimulation with CD40 ligand of DC induced to mature by LPS treatment. These different patterns of chemokine receptors are consistent with “inflammatory” and “primary response” phases of DC function.     

低氧对人成熟树突状细胞MMP9/TIMP1及CCR7 表达的影响

目的： 观察低氧对人单核细胞来源成熟树突状细胞（mDCs）趋化因子受体（CCR7）,基质金属蛋白酶-9（MMP-9）及其组织抑制剂（TIMP-1）表达的影响,以期为改进DCs疫苗体内迁移能力低下提供新策略。方法： 分离制备人外周血单核细胞（PBMCs）,采用体外常规培养体系 ［粒-巨噬细胞集落刺激因子（GM-CSF）与白细胞介素-4（IL-4）共同刺激］ 诱导DCs,于诱导后第5 d加入TNF-α促成熟,48 h后将mDCs置低氧环境下（1％ O2、5％ CO2、94％ N2）分别继续培养6 h、12 h,设常氧对照组（21% O2、5% CO2）,收获细胞及培养上清;采用RT-PCR技术检测MMP-9、TIMP-1及CCR7的表达;明胶酶谱法检测培养上清中MMP-9的水平与活性,流式细胞术检测mDCs表面CCR7的表达。结果： RT-PCR及酶谱实验结果显示,低氧6 h、12 h处理组mDCs MMP-9水平显著下降;与对照组比较,各低氧处理组mDCs均未检测到TIMP-1 mRNA表达的变化;转录及细胞表面表达分析结果显示,与常氧对照组比较,低氧6 h处理组趋化因子受体CCR7表达显著降低,而12 h处理组其表达显著回升至近常氧组水平。结论： 低氧下调mDCs MMP-9表达,破坏MMP-9/TIMP-1平衡,可能是DCs疫苗体内迁移能力低下的主要因素之一。趋化因子受体CCR7在mDCs对低氧应答中可能起重要作用。     

体外LPS刺激及CD40的配基化对sCD40基因修饰DCs TLR4-MD2表达及IL-12分泌的影响

目的： 研究体外LPS刺激及CD40的配基化对可溶性CD40（sCD40）基因修饰树突状细胞TLR4-MD2表达及IL-12分泌的影响,为有效利用树突状细胞诱导特异性移植免疫耐受提供实验依据。方法: 脂质体法将质粒pEGFP-N1/sCD40及空质粒pEGFP-N1转染DC2.4细胞株;应用LPS及抗CD40单抗刺激6 h,流式细胞仪检测DC表面TLR4-MD2的表达,RT-PCR法检测DC 的TLR4 mRNA 表达水平,并用ELISA法检测细胞因子IL-12p70的分泌。结果: LPS刺激下调DC表面TLR4-MD2的表达,同时给予CD40配基化可引起TLR4-MD2的表达显著增高;CD40配基化对DC TLR4mRNA 水平表达无影响，但可部分地增高LPS引起的TLR4mRNA 表达降低;此外,CD40的配基化可显著诱导LPS刺激后IL-12分泌增加。sCD40基因修饰DC可拮抗以上作用。结论: 体外LPS及抗CD40单抗刺激下,sCD40基因修饰树突状细胞可显著下调其表面TLR4-MD2的表达,IL-12p70分泌减少，可能与阻断胞浆内的TLR4-MD2的转运过程有关。     

Quantitative analysis of chemokine expression by dendritic cell subsets in vitro and in vivo

Upon maturation, dendritic cells (DCs) have to adjust their chemokine expression to sequentially attract different leukocyte subsets. We used real-time quantitative polymerase chain reaction analysis to study in detail the expression of 12 chemokines involved in the recruitment of leukocytes into and inside secondary lymphoid organs, by DCs in distinct differentiation stages, both in vitro and in vivo. Monocyte-derived immature DCs expressed high levels of DC chemokine 1 (DC-CK1), EBI1-ligand chemokine (ELC), macrophage-derived chemokine (MDC), macrophage-inflammatory protein (MIP)-1alpha, and thymus and activation-regulated chemokine (TARC). Upon maturation, DCs up-regulated the expression of DC-CK1 (60-fold), ELC (7-fold), and TARC (10-fold). Activation of DCs by CD40 ligand further up-regulated the expression of ELC (25-fold). We found that freshly isolated blood DCs expressed only low levels of interleukin-8, lymphotactin, and MIP-1alpha. It is interesting that the chemokine profile expressed by activated CD11c(-) lymphoid-like as well as CD11c(+) myeloid blood DCs mimics that of monocyte-derived DCS: Additionally, purified Langerhans cells that had migrated out of the epidermis expressed a similar chemokine pattern. These data indicate that different DC subsets in vitro and in vivo can express the same chemokines to attract leukocytes.     

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.     

Lys-des[Arg9]-bradykinin alters migration and production of interleukin-12 in monocyte-derived dendritic cells

This study tested the hypothesis that proinflammatory kinin peptides are involved in modulating human dendritic cell (DC) function. Inflammation is accompanied by an increased maturation of DCs and the generation of kinins, particularly Lys-des[Arg(9)]-bradykinin (Lda-BK). We assessed the role of Lda-BK in the activation and migration of human monocyte-derived DCs (hMo-DCs) matured through the use of LPS, TNF-α + IL-1β, or CD40 ligand. Kinin B(1) and B(2) receptor mRNA and protein expression were assessed by confocal microscopy, flow cytometry, and RT-PCR. The effects of Lda-BK on the migration of mature hMo-DCs were assessed in Transwell chambers, whereas the expression of costimulatory molecules and the secretion of IL-12 were assessed by flow cytometry and ELISA, respectively. The expression of the kinin B(1) receptor (B(1)R) was down-regulated during the maturation of hMo-DCs, whereas the expression of B(2)R was unchanged. The B(1)R agonist Lda-BK was not chemotactic for hMo-DCs matured using LPS, TNF-α + IL-1β, or CD40 ligand, but Lda-BK enhanced the secretion of IL-12p70 and inhibited the secretion of IL-12p40 by mature hMo-DCs. However, the exposure of hMo-DCs matured with TNF-α + IL-1β to Lda-BK for 6 hours decreased subsequent migration in response to Lda-BK, the chemokine CCL19, or Lda-BK combined with CCL19. The expression of B(1)R was increased in hMo-DCs from subjects with asthma compared with subjects without asthma, in keeping with a tendency toward increased in vitro migration of asthmatic hMo-DCs in response to Lda-BK. The increased formation of Lda-BK and the enhanced expression of B(1)R as a consequence of inflammation may alter the migration of mature, antigen-laden DCs to regional lymph nodes in response to CCL19, may modulate the secretion of cytokines by these DCs, and may contribute to the accumulation of mature DCs in the lungs of patients with asthma.     

Tumour-loaded α-type 1-polarized dendritic cells from patients with chronic lymphocytic leukaemia produce a superior NK-, NKT- and CD8+ T cell-attracting chemokine profile

Tumour-loaded dendritic cells (DCs) from patients with chronic lymphocytic leukaemia (CLL) matured using an α-type 1-polarized DC cocktail (IL-1β/TNF-α/IFN-α/IFN-γ/poly-I:C;αDC1) were recently shown to induce more functional CD8(+) T cells against autologous tumour cells in vitro than DCs matured with the 'standard' cocktail (IL-1β/TNF-α/IL-6/PGE(2) ;PGE(2) DCs). However, the ability of vaccine DCs to induce a type 1-polarized immune response in vivo probably relies on additional features, including their ability to induce a CXCR3-dependent recruitment of NK cells into vaccine-draining lymph nodes. Moreover, their guiding of rare tumour-specific CD8(+) T cells to sites of DC-CD4(+) T cell interactions by secretion of CCL3 and CCL4 is needed. We therefore analysed the chemokine profile and the lymphocyte-attracting ability in vitro of monocyte-derived PGE(2) DCs and αDC1s from patients with CLL. αDC1s produced much higher levels of CXCR3 ligands (CXCL9/CXCL10/CXCL11) than PGE(2) DCs. Functional studies further demonstrated that αDC1s were superior recruiters of both NK and NKT cells. Moreover, αDC1s produced higher levels of CCL3/CCL4 upon CD40 ligation. These findings suggest that functional αDC1s, derived from patients with CLL, produce a desirable NK-, NKT- and CD8(+) T cell-attracting chemokine profile which may favour a guided and Th1-deviated priming of CD8(+) T cells, supporting the idea that αDC1-based vaccines have a higher immunotherapeutic potential than PGE(2) DCs.     

Campylobacter jejuni-mediated induction of CC and CXC chemokines and chemokine receptors in human dendritic cells

Campylobacter jejuni is a leading worldwide bacterial cause of human diarrheal disease. Although the specific molecular mechanisms of C. jejuni pathogenesis have not been characterized in detail, host inflammatory responses are thought to be major contributing factors to the resulting typical acute colitis. The intestinal mucosal chemokine response is particularly important in the initial stages of bacterium-induced gut inflammation. Chemokines attract blood phagocytes and lymphocytes to the site of infection and regulate immune cell maturation and the development of localized lymphoid tissues. The production of chemokines by dendritic cells (DCs) following Campylobacter infection has not yet been analyzed. In the current study, we infected human monocyte-derived DCs with C. jejuni to examine the production of key proinflammatory chemokines and chemokine receptors. The chemokines, including CC families (macrophage inflammatory protein 1α [MIP-1α], MIP-1β, RANTES) and CXC families (growth-related oncogene α [GRO-α], IP-10, and monokine induced by gamma interferon [MIG]), were upregulated in Campylobacter-infected DCs. Chemokine receptors CCR6 and CCR7, with roles in DC trafficking, were also induced in Campylobacter-infected DCs. Further, Campylobacter infection stimulated the phosphorylation of P38, P44/42, and stress-activated protein kinase/Jun N-terminal kinase (SAPK/JNK) mitogen-activated protein kinases (MAPKs) in DCs. NF-κB activation was specifically involved in chemokine induction in DCs infected with C. jejuni. Additionally, STAT3 was significantly increased in Campylobacter-infected DCs compared to that in uninfected DCs. These results suggest that DCs play a significant role in the initiation and modulation of the inflammatory response by enlisting monocytes, neutrophils, and T lymphocytes during human intestinal infection with Campylobacter.     

趋化因子MIP-1α和MIP-3α募集树突状细胞前体细胞的研究

体外获得树突状细胞(dendritic cells,DC)或其前体对基于DC的免疫治疗至关重要。之前有报道注射MIP-1α可使一群F4/80-B220-CD11c+DC前体通过表达CCR1和CCR5招募至外周血。本研究中给小鼠注射MIP-1α之后鉴定了一种新的亚群细胞,即CCR6+CCR1-CCR5-B220-CD11c+细胞。当加入GM-CSF、IL-4和TNF-α培养时,这群细胞可分化为具有典型形态学特征、表型及抗原提呈能力的成熟DC,称为CCR+DC前体。虽然MIP-1α并不驱动CCR+DC前体招募,但其可通过诱导外周血MIP-3α表达的上调来驱动B220-CD11c+DC前体中CCR+DC前体亚群的招募。此外,外源性地给予MIP-3α可显著增强MIP-1α诱导的DC前体招募,联合应用MIP-1α和MIP-3α招募的DC前体数量显著高于单独使用MIP-1α或MIP-3α。本研究进一步证明了MIP-1α诱导DC前体招募的机制,即除了通过CCR1和CCR5表达驱动DC招募之外,MIP-1α可通过诱导MIP-3α,间接驱动CCR+DC前体招募,扩大了B220-CD11c+DC前体细胞数量。因此,联合使用MIP-1α和MIP-3α可能成为大量收集适合于免疫治疗用DC的有效方法。     

Distribution and kinetics of SR-PSOX/CXCL16 and CXCR6 expression on human dendritic cell subsets and CD4+ T cells

Dendritic cells (DCs) coordinate T cell responses by producing T cell-attracting chemokines and by inducing the expression of chemokine receptors on T cells. Scavenger receptor for phosphatidylserine and oxidized lipoprotein (SR-PSOX)/CXC chemokine ligand 16 (CXCL16) is a unique chemokine that also functions as an endocytic receptor and an adhesion molecule in its membrane-bound form. SR-PSOX/CXCL16 is the only known ligand of CXC chemokine receptor 6 (CXCR6) that is expressed on activated T cells and thus, may play an important role in enhancing effector functions of T cells. Here, we investigated the expression of SR-PSOX/CXCL16 on human DC subsets and that of CXCR6 on T cell subpopulations to elucidate the dynamics of CXCL16/CXCR6 interaction in DC/T cell responses. Membrane-bound SR-PSOX/CXCL16 was expressed on macrophages, monocyte-derived DCs, and blood myeloid DCs, and the expression increased after DC maturation. Myeloid antigen-presenting cells constitutively secreted SR-PSOX/CXCL16 for an extended period, suggesting the involvement of CXCL16 in peripheral and lymphoid tissues. Plasmacytoid DCs hardly expressed SR-PSOX/CXCL16 on their surfaces but secreted significant amounts of SR-PSOX/CXCL16. A subset of CD4+ effector memory T (T(EM)) cells constitutively expressed CXCR6, whereas central memory T cells (T(CM)) and naive T cells did not. Upon stimulation with mature DCs, however, the expression of CXCR6 on T(CM) cells was markedly up-regulated, whereas the expression on naive T cells was induced only weakly. These results suggest that the interaction between SR-PSOX/CXCL16 and CXCR6 plays an important role in enhancing T(CM) cell responses by mature DCs in lymphoid tissues and in augmenting T(EM) cell responses by macrophages in peripheral inflamed tissues.     

Chemokine receptor expression on dendritic cells is normal in HIV-infected patients with a stable response to art, but chemokine levels remain elevated

The development of strategies to optimize T‐cell responses in previously immunodeficient HIV patients with a stable virological response to ART requires an understanding of the factors that affect responsiveness. Chemokines direct the migration of dendritic cells (DC) to non‐lymphoid tissues infected by secondary pathogens and to lymph nodes where they prime T‐cells. Quantitation of mRNA is a sensitive technique enabling assessment of chemokine receptors by CD14⁺ monocytes, myeloid (m)DCs, plasmacytoid (p)DCs, and M‐DC8⁺ cells. MDC8⁺ cells invariably expressed less CCR2, CCR5, and CXCR4 than the other cells, but expression of CCR2, CCR5, CCR6, CCR7, CXCR3, and CXCR4 was similar in patients and healthy controls. However plasma levels of CXCL10, CCL5, and CCL2 remained higher in patients than controls. Overall, it appears that chemokine directed migration of DC may not limit immune responses in these patients. J. Med. Virol. 83:1128–1133, 2011. © 2011 Wiley‐Liss, Inc.     

Interaction between the CCR5 chemokine receptors and microbial HSP70

Evidence is presented that the microbial 70-kD heat shock protein (HSP70) binds to CCR5 chemokine receptors in CCR5-transfected cell lines and in primary human cells. Significant CCR5-mediated calcium mobilization was stimulated by HSP70 and inhibited with TAK 779, which is a specific CCR5 antagonist. HSP70-mediated activation of the p38 MAPK phosphorylation signaling pathway was also demonstrated in CCR5-transfected HEK 293 cells. Direct binding of three extracellular peptides of CCR5 to HSP70 was demonstrated by surface plasmon resonance. Functional evidence of an interaction between HSP70, CCR5 and CD40 was shown by enhanced production of CCL5 by HEK 293 cells transfected with both CD40 and CCR5. Primary monocyte-derived immature DC stimulated with HSP70 produced IL-12 p40, which showed dose-dependent inhibition of >90% on treatment with both TAK 779 and anti-CD40 mAb. Stimulation of IL-12 p40 or TNF-alpha by HSP70 was related to the differential cell surface expression of CCR5 in primary human immature and mature DC, and those with the homozygous triangle DeltaDelta32 CCR5 mutation. These findings may be of significance in the interaction between HSP70 and immune responses of CCR5+ T cells in HIV-1 infection, as well as in inflammatory bowel disease.     

Human CD8⁺ T cells drive Th1 responses through the differentiation of TNF/iNOS-producing dendritic cells

TNF/iNOS-producing dendritic cells (Tip-DCs) have been shown to arise during inflammation and are important mediators of immune defense. However, it is still relatively unclear which cell types contribute to their differentiation. Here we show that CD8(+) T cells, through the interaction with DCs, can induce the rapid development of human monocytes into Tip-DCs that express high levels of TNF-α and iNOS. Tip-DCs exhibited T-cell priming ability, expressed high levels of MHC class II, upregulated co-stimulatory molecules CD40, CD80, CD86, toll-like receptors TLR2, TLR3, TLR4, chemokine receptors CCR1 and CX3CR1 and expressed the classical mature DC marker, CD83. Differentiation of monocytes into Tip-DCs was partially dependent on IFN-γ as blocking the IFN-γ receptor on monocytes resulted in a significant decrease in CD40 and CD83 expression and in TNF-α production. Importantly, these Tip-DCs were capable of further driving Th1 responses by priming naive CD4(+) T cells for proliferation and IFN-γ production and this was partially dependent on Tip-DC production of TNF-α and NO. Our study hence identifies a role for CD8(+) T cells in orchestrating Th1-mediating signals through the differentiation of monocytes into Th1-inducing Tip-DCs.     

Plasmacytoid and myeloid dendritic cells with a partial activation phenotype accumulate in lymphoid tissue during asymptomatic chronic HIV-1 infection

Dendritic cells (DCs) from HIV-1-infected individuals display numeric and functional defects, and recent evidence suggests that HIV-1 can directly and indirectly activate DCs in vitro. The in vivo activation state and compartmentalization of DC subsets during HIV-1 infection remain poorly understood, however. We evaluated phenotypic and functional characteristics of myeloid dendritic cells (mDCs) and plasmacytoid dendritic cells (pDCs) directly ex vivo in peripheral blood and lymphoid tissue from HIV-1-infected and HIV-seronegative individuals. Analysis of a wide range of chemokine receptors and activation/maturation markers on circulating DCs from viremic HIV-1-infected donors revealed a phenotype indicative of partial activation. Yet, blood DCs from viremic subjects still achieved full maturation when stimulated in vitro. In addition, blood pDCs from viremic individuals had a reduced capacity to migrate to CXCL12 in vitro. Total numbers of both DC subsets were increased in lymph nodes of asymptomatic untreated HIV-1-infected subjects, consistent with DC accumulation in the lymphoid compartment. Lymph node DCs also expressed high levels of CD40 in the absence of increases of other typical activation/maturation markers. Activation and depletion of DCs in blood with accumulation in lymphoid tissue may contribute to HIV-associated chronic immune activation and T-cell dysfunction.     

Anti-tumoral capabilities of effector cells after IFN-α or CpG-motif treatment of cocultured dendritic cells

Introduction Ex vivo expansion of monocyte-derived dendritic cells (mDCs) and subsequent coculture with autologous cytokine-induced killer (CIK) cells is an established system to create specific and non-specific anti-tumoral immunity. mDCs constitute the most frequently applied DC subset in clinical studies. One recently published approach to optimize the immunological functions of the DC/CIK cell system is the replacement of interleukin (IL)-4 by interferon (IFN)-α in the maturation process of the DCs. Materials and Methods The expressions of relevant surface antigens of IL-4-DCs and IFNα-DCs by flow cytometry and the anti-tumoral activation of effector cells cocultured with both types of DCs using cytotoxicity assays were compared. In addition, short-term coculture experiments with both types of DCs and IFNγ-LAK effector cells were performed and compared with standard CIK cell coculture experiments. Results Regarding the expressions of functionally relevant surface markers, no differences could be detected for CD80, CD83, and HLA-DR between IFNα-DCs and IL-4-DCs, whereas the mean fluorescence intensities of CD40, CD86, CD54, and HLA-ABC were decreased and the expression of CD14 was increased for IFNγ-DCs. Moreover, no enhancement of cytotoxicity of cocultured CIK cells against tumor cell lines (A498 and SW480) was detected by the use of IFNα-DCs. Additionally, coculture experiments with IFNγ-LAK cells were performed and unexpectedly higher lysis rates in comparison with the established IL-4-DC/CIK coculture model was observed. Early incubation of the mDCs with several CpG-ODNs failed to increase the anti-tumoral cytotoxicity of the cocultured IFNγ-LAK cells. Conclusions These results demonstrate that in the mDC/CIK cell system, IFNα-DCs are not superior in inducing anti-tumoral cytotoxicity and even moderately inferior regarding the expression of functionally relevant surface markers compared with IL-4-DCs. Michael Erhardt and Ingo G. H. Schmidt-Wolf contributed equally.     

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.     

Characterization and functional study of five novel monoclonal antibodies against human OX40L highlight reverse signalling: enhancement of IgG production of B cells and promotion of maturation of DCs

OX40 ligand (OX40L), a molecule originally identified as human gp34, is an important co-stimulatory molecule during immune response. In this study, we report on five functional mouse anti-human OX40L monoclonal antibodies named as 9H10, 4C12, 8D10, 4H4 and 1G1, characterized by means of flow cytometry, Western blot and competition assay. These monoclonal antibodies bound to distinct OX40L epitopes on activated B cells and dendritic cells (DCs) and two of them could suppress the proliferation of T lymphocytes co-stimulated by mature DCs. Furthermore, we demonstrated that our monoclonal antibodies, such as 9H10 and 4C12, could trigger OX40L reverse signal that enhanced IgG production of B cells and promoted maturation of DCs as evidenced by the upexpression of CD80, CD86, CD83 and CXCR4 and monoclonal antibody 9H10 could also promote anti-CD40 monoclonal-antibody-stimulated DCs in order to induce T cells to secrete more interleukin-2 and interferon-gamma, which suggested that OX40L signals could strengthen the effect of CD40 signals on promoting Th1 differentiation.     

Effect of intrauterine HIV-1 exposure on the frequency and function of uninfected newborns' dendritic cells

Immaturity of the neonatal immune system is considered an underlying factor for enhanced severity of infections in newborns. Functional defects of neonatal antigen-presenting cells lead to defective T-cell responses. T cells from uninfected neonates exposed in utero to HIV-1 (EU) exhibit phenotypic and functional alterations; however, the function of their circulating dendritic cells (DCs) has not been characterized. We hypothesized that an HIV-1-infected maternal environment may influence the infants' DC number, phenotype and function. EU exhibited a higher percentage of myeloid DCs (mDCs) than unexposed neonates, although this frequency remained lower than that observed in adults. Plasmacytoid DC (pDC) frequencies were similar in all groups, although both groups of infants tended to have lower frequencies than adults. After LPS stimulation, mDCs from EU up-regulated CD80, CD86 and B7-H1, whereas mDCs from unexposed infants upregulated B7-H1, but not CD80/CD86, and adult mDCs up-regulated mainly CD80 and CD86. IFN-alpha production was similar in all groups, indicating a normal pDC function. Therefore, in utero exposure to HIV-1 induces quantitative and qualitative changes in neonatal DCs, particularly in mDCs, which might be associated with alterations observed in T cells from these EU.     

CD34-derived dendritic cells transfected ex vivo with HIV-Gag mRNA induce polyfunctional T-cell responses in nonhuman primates

The pivotal role of DCs in initiating immune responses led to their use as vaccine vectors. However, the relationship between DC subsets involved in antigen presentation and the type of elicited immune responses underlined the need for the characterization of the DCs generated in vitro. The phenotypes of tissue-derived APCs from a cynomolgus macaque model for human vaccine development were compared with ex vivo-derived DCs. Monocyte/macrophages predominated in bone marrow (BM) and blood. Myeloid DCs (mDCs) were present in all tested tissues and were more highly represented than plasmacytoid DCs (pDCs). As in human skin, Langerhans cells (LCs) resided exclusively in the macaque epidermis, expressing CD11c, high levels of CD1a and langerin (CD207). Most DC subsets were endowed with tissue-specific combinations of PRRs. DCs generated from CD34(+) BM cells (CD34-DCs) were heterogeneous in phenotype. CD34-DCs shared properties (differentiation and PRR) of dermal and epidermal DCs. After injection into macaques, CD34-DCs expressing HIV-Gag induced Gag-specific CD4(+) and CD8(+) T cells producing IFN-γ, TNF-α, MIP-1β, or IL-2. In high responding animals, the numbers of polyfunctional CD8(+) T cells increased with the number of booster injections. This DC-based vaccine strategy elicited immune responses relevant to the DC subsets generated in vitro.