Role of biphasic changes in splenic dendritic cell activity in a mouse model of multiple organ dysfunction syndrome

To analyze the changes in splenic dendritic cell (DC) activity and serum cytokine levels during the progression of multiple organ dysfunction syndrome (MODS). A C57BL/6 mouse model of MODS was established by intraperitoneal injection of zymosan. Immunohistochemistry and flow cytometry were used to detect expression of I-A^b (MHC-II molecules of mice) as well as co-stimulatory and co-inhibitory molecules in spleen and DC surface. The levels of various cytokines in serum and spleen tissue were analyzed 6 h, 12 h, 24 h, 48 h, 5 d and 12 d after injury. Death occurred at 24-48 h and 10-12 d after injury. The expression of I-A^b and CD86 in spleen tissue and on DCs increased 6-12 h after injury, followed by gradual reduction and at 12 d. The inhibitory molecule, PD-L1, was expressed on normal DCs, but expression of PD-1 was undetectable. PD-L1 and PD-1 expression increased and remained high at 5 d and 12 d after injury. In addition, TNF and IL-1 levels increased 6-12 h after injury; HMGB1 and IL-10 levels increased 24 h and 5 d after injury, respectively. In contrast, IL-2 and IL-12 decreased with disease progression. At 12 d after injury, proinflammatory and anti-inflammatory cytokine levels remained high, while IL-2 and IL-12 were significantly reduced. IL-10 and IL-12 changes in spleen were consistent with those in serum. MODS progression was characterized by changes in splenic DC activity as well as altered serum pro-inflammatory and anti-inflammatory cytokine levels, suggesting early immune activation and predominant immune tolerance at the late stage.     

Increased expression of programmed death (PD)‐1 and its ligand PD‐L1 correlates with impaired cell‐mediated immunity in high‐risk human papillomavirus‐related cervical intraepithelial neoplasia

Impaired local cellular immunity contributes to the pathogenesis of persistent high-risk human papillomavirus (HR-HPV) infection and related cervical intraepithelial neoplasia (CIN), but the underlying molecular mechanisms remain unclear. Recently, the programmed death 1/programmed death 1 ligand (PD-1/PD-L1; CD279/CD274) pathway was demonstrated to play a critical role in attenuating T-cell responses and promoting T-cell tolerance during chronic viral infections. In this study, we examined the expression of PD-1 and PD-L1 on cervical T cells and dendritic cells (DCs), respectively, from 40 women who were HR-HPV-negative (−) or HR-HPV-positive (+) with CIN grades 0, I and II–III. We also measured interferon-γ, interleukin-12 (IL-12) and IL-10 in cervical exudates. The most common HPV type was HPV 16, followed by HPV 18, 33, 51 and 58. PD-1 and PD-L1 expression on cervical T cells and DCs, respectively, was associated with HR-HPV positivity and increased in parallel with increasing CIN grade. The opposite pattern was observed for CD80 and CD86 expression on DCs, which decreased in HR-HPV(+) patients in parallel with increasing CIN grade. Similarly, reduced levels of the T helper type 1 cytokines interferon-γ and IL-12 and increased levels of the T helper type 2 cytokine IL-10 in cervical exudates correlated with HR-HPV positivity and CIN grade. Our results suggest that up-regulation of the inhibitory PD-1/PD-L1 pathway may negatively regulate cervical cell-mediated immunity to HPV and contribute to the progression of HR-HPV-related CIN. These results may aid in the development of PD-1/PD-L1 pathway-based strategies for immunotherapy of HR-HPV-related CIN.     

Fine-tuned expression of programmed death 1 ligands in mature dendritic cells stimulated by CD40 ligand is critical for the induction of an efficient tumor specific immune response

During maturation, murine myeloid dendritic cells （DCs） upregulated the expressions of CDllc, CD25, CD40, CD80, CD86, MHC Ⅱ and programmed death 1 ligands 1 and 2 （PD-L1 and PD-L2）. Differential expression patterns of PD-L1 and PD-L2 were found when DCs were triggered by CD40 ligand and TNF-α. PD-L1 expression was repressed and PD-L2 expression remained unchanged in mature CD40-ligated DCs, whereas TNF-α stimulated DCs kept high expression of PD-L1 and significantly enhanced PD-L2 expression on DCs. Proliferations of T lymphocytes stimulated by immature DCs were enhanced by blockade of the PD-1 and PD-1 ligand interaction. But inhibitive effects were found in T lymphocytes stimulated by CD40-ligated DCs. With the fine-tuned expressions of PD-L1 and PD-L2, CD40-1igated DCs could sustain a longer activation period and elicit a more efficient T lymphocyte activation. Cellular ＆ Molecular Immunology.     

CCR7在多器官功能障碍综合征小鼠脾脏树突状细胞迁移变化中的作用

目的:探讨CCR7在多器官功能障碍综合征(MODS)脾脏中的表达变化及其对树突状细胞(DC)迁移的影响.方法:用酵母多糖腹腔注射复制小鼠MODS模型,分为正常对照组和实验3～6小时组、24～48小时组、5～7天组及10～12天组.运用免疫组化方法检测CD11c和CD205标记阳性DC在各组小鼠脾脏中分布的变化,用流式细胞术检测CD86/CD11c和CCR7/CD11c标记阳性细胞在脾脏中含量的变化.结果:正常小鼠脾脏DC含量较少,主要分布在脾脏边缘区;在3～6小时组CCR7表达率较正常对照组显著增加,DC含量显著增加、活性增高,并向白髓T细胞区大量迁移;24～48小时组T细胞区中DC含量开始减少,而CCR7表达率升高达到峰值;5～7天组DC与CCR7含量接近正常对照组,边缘区和T细胞区均可见DC分布;10～12天组DC含量再次升高,但多呈不成熟状态,且以边缘区分布为主,CCR7表达率下降.结论:在MODS病程中脾脏DC的含量和分布变化与CCR7的表达率密切相关,CCR7可以作为评估脾脏DC迁移能力及功能活性的重要指标.     

TLR-mediated induction of negative regulatory ligands on dendritic cells

Dendritic cells (DCs) shape T-cell response patterns and determine early, intermediate, and late outcomes of immune recognition events. They either facilitate immunostimulation or induce tolerance, possibly determined by initial DC activation signals, such as binding Toll-like receptor (TLR) ligands. Here, we report that DC stimulation through the TLR3 ligand dsRNA [poly(I:C)] limits CD4 T-cell proliferation, curtailing adaptive immune responses. CD4+ T cells instructed by either lipopolysaccharide (LPS) or poly(I:C)-conditioned DCs promptly upregulated the activation marker CD69. Whereas LPS-pretreated DCs subsequently sustained T-cell clonal expansion, proliferation of CD4+ T cells exposed to poly(I:C)-pretreated DCs was markedly suppressed. This proliferative defect required DC-T cell contact, was independent of IFN-alpha, and was overcome by exogenous IL-2, indicating T-cell anergy. Coinciding with the downregulation, CD4+ T cells expressed the inhibitory receptor PD-1. Antibodies blocking the PD-1 ligand PD-L1 restored proliferation. dsRNA-stimulated DCs preferentially induced PD-L1, whereas poly(I:C) and LPS both upregulated the costimulatory molecule CD86 to a comparable extent. Poly(dA-dT), a ligand targeting the cytoplasmic RNA helicase pattern-recognition pathway, failed to selectively induce PD-L1 upregulation, assigning this effect to the TLR3 pathway. Poly(I:C)-conditioned DCs promoted accumulation of phosphorylated SHP-2, the intracellular phosphatase mediating PD-1 inhibitory effects. The ability of dsRNA to bias DC differentiation toward providing inhibitory signals to interacting CD4+ T cells may be instrumental in viral immune evasion. Conversely, TLR3 ligands may have therapeutic value in silencing pathogenic immune responses.     

Dendritic and stromal cells from the spleen of lupic mice present phenotypic and functional abnormalities

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by the increase in the percentage of autoreactive B and T lymphocytes. Since dendritic cells (DCs) are essential for B cell and T cell function, we hypothesized that changes in DC biology may play a critical role in the pathogenesis of the disease. We analyzed the phenotype and distribution of two main DC subsets, conventional (cDC) and plasmacytoid (pDC), in lupus prone (NZW×NZB)F1 (BWF1) mice and age-matched NZW×BALB/c control mice. Our results show that both subsets of lupic DCs displayed an abnormal phenotype, characterized by an over-expression of the co-stimulatory molecules CD80, CD86, PD-L1 and PD-L2 compared with control mice. Accordingly, spleen CD4⁺ T cells from lupic mice exhibit an activated phenotype characterized by a higher expression of PD-1, CD25, CD69 and increased secretion of IFN-γ and IL-10. Interestingly, lupic mice also present an increase in the percentage of cDC in peripheral blood and an increase in the percentage of pDCs in spleen and mesenteric lymph nodes (MLNs) compared with control and pre-lupic mice. Homing experiments demonstrate that lupic and pre-lupic DCs migrate preferentially to the spleen compared to DCs from control mice. This preferential recruitment and retention of DCs in the spleen is related to an altered expression of different chemokine and chemokine receptors on both, DCs and stromal cells from lupic mice. Our results suggest that this altered phenotype and migratory behavior shown by DCs from lupic mice may account for the abnormal T cell and B cell responses in lupus.     

Blockage of immune checkpoint molecules increases T-cell priming potential of dendritic cell vaccine

Dendritic cell (DC) -based cancer immunotherapy is one of the most important anti-cancer immunotherapies, and has been associated with variable efficiencies in different cancer types. It is well-known that tumor microenvironment plays a key role in the efficacy of various immunotherapies such as DC vaccine. Accordingly, the expression of programmed death ligand 1 (PD-L1) on DCs, which interacts with PD-1 on T cells, leads to inhibition of anti-tumor responses following presentation of tumor antigens by DCs to T cells. Therefore, we hypothesized that down-regulation of PD-L1 in DCs in association with silencing of PD-1 on T cells may lead to the enhancement of T-cell priming by DCs to have efficient anti-tumor T-cell responses. In this study, we silenced the expression of PD-L1 in DCs and programmed cell death protein 1 (PD-1) in T cells by small interfering RNA (siRNA) -loaded chitosan–dextran sulfate nanoparticles (NPs) and evaluated the DC phenotypic and functional characteristics and T-cell functions following tumor antigen recognition on DCs, ex vivo. Our results showed that synthesized NPs had good physicochemical characteristics (size 77·5 nm and zeta potential of 14·3) that were associated with efficient cellular uptake and target gene silencing. Moreover, PD-L1 silencing was associated with stimulatory characteristics of DCs. On the other hand, presentation of tumor antigens by PD-L1-negative DCs to PD-1-silenced T cells led to induction of potent T-cell responses. Our findings imply that PD-L1-silenced DCs can be considered as a potent immunotherapeutic approach in combination with PD-1-siRNA loaded NPs, however; further in vivo investigation is required in animal models.     

PD-L2 modulates asthma severity by directly decreasing dendritic cell IL-12 production

Studies examining the role of programmed death 1 (PD-1) ligand 2 (PD-L2)/PD-1 in asthma have yielded conflicting results. To clarify its role, we examined the PD-L2 expression in biopsies from human asthmatics and the lungs of aeroallergen-treated mice. PD-L2 expression in bronchial biopsies correlated with the severity of asthma. In mice, allergen exposure increased PD-L2 expression on pulmonary myeloid dendritic cells (DCs), and PD-L2 blockade diminished allergen-induced airway hyperresponsiveness (AHR). By contrast, PD-1 blockade had no impact, suggesting that PD-L2 promotes AHR in a PD-1-independent manner. Decreased AHR was associated with enhanced serum interleukin (IL)-12 p40, and in vitro stimulation of DCs with allergen and PD-L2-Fc reduced IL-12 p70 production, suggesting that PD-L2 inhibits allergen-driven IL-12 production. In our model, IL-12 did not diminish T helper type 2 responses but rather directly antagonized IL-13-inducible gene expression, highlighting a novel role for IL-12 in regulation of IL-13 signaling. Thus, allergen-driven enhancement of PD-L2 signaling through a PD-1-independent mechanism limits IL-12 secretion, exacerbating AHR.     

早期凋亡的T淋巴细胞诱导生成耐受性树突状细胞的实验研究

目的： 探讨早期凋亡T淋巴细胞的抑制性免疫调节性能。方法： 采用定时紫外线照射诱导T淋巴细胞早期凋亡，深低温反复冻融获得坏死T淋巴细胞。体外诱导、纯化并培养骨髓源性不成熟树突状细胞（imDCs）,imDCs分别和早期凋亡或坏死T淋巴细胞共培养。用流式细胞仪、双夹心ELISA、［³H］掺入混合淋巴细胞反应等方法分析imDCs吞噬早期凋亡或坏死T淋巴细胞后，在不同处理条件下，MHC-Ⅱ、CD40、CD80、CD86的表达水平、分泌IL-12 p70以及刺激T淋巴细胞增殖能力的差异。结果： imDCs和坏死细胞碎片共培养后明显趋于成熟，其MHCⅡ和CD40、CD80、CD86的表达水平显著上调；分泌较高水平的IL-12 p70；和同种异体处女T淋巴细胞混合培养后显著刺激处女T淋巴细胞增殖。 imDCs和早期凋亡的T淋巴细胞共培养后,其MHC-Ⅱ、CD40、CD80和CD86的表达维持较低水平；仅分泌较低水平IL-12 p70；和同种异体处女T淋巴细胞混合培养后不能刺激淋巴细胞增殖。此外，早期凋亡的T淋巴细胞孵育上清显著抑制了吞噬坏死细胞碎片后的imDCs表达共刺激分子CD40、CD80、CD86。当TGFβ₁中和抗体和早期凋亡T淋巴细胞同加入imDCs，在表达MHC-Ⅱ、CD40、CD80、CD86，分泌IL-12 p70，刺激处女T淋巴细胞增殖等方面和吞噬坏死T淋巴细的DCs相比无显著差异。结论： 早期凋亡T淋巴细胞通过释放免疫抑制性细胞因子TGFβ₁，诱导imDCs呈现出耐受性DCs（TolDCs）的免疫表型及生物学特征，从而发挥抑制性免疫调节作用。     

Programmed death-1 upregulation is correlated with dysfunction of tumor-infiltrating CD8+ T lymphocytes in human non-small cell lung cancer

T-cell tolerance is an important mechanism for tumor escape, but the molecular pathways involved in T-cell tolerance remain poorly understood. It remains unknown whether the inhibitory immunoreceptor programmed death-1 (PD-1) plays a role in conditions of human non-small cell lung cancer (NSCLC). In this study, we detected PD-1 expression on CD8⁺ T cells from healthy control peripheral blood mononuclear cells (PBMCs) and the PBMCs of NSCLC patients as well as NSCLC tissues. Results showed that tumor-infiltrating CD8⁺ T cells had increased PD-1 expression and impaired immune function, including reducing cytokine production capability and impairing capacity to proliferate. Blockade of the PD-1/PD-L1 pathway by the PD-L1-specific antibody partially restored cytokine production and cell proliferation. These data provide direct evidence that the PD-1/PD-L1 pathway is involved in CD8⁺ T-cell dysfunction in NSCLC patients. Moreover, blocking this pathway provides a potential therapy target in lung cancer.     

Nontoxic Shiga toxin derivatives from Escherichia coli possess adjuvant activity for the augmentation of antigen-specific immune responses via dendritic cell activation

Shiga toxin (Stx) derivatives, such as the Stx1 B subunit (StxB1), which mediates toxin binding to the membrane, and mutant Stx1 (mStx1), which is a nontoxic doubly mutated Stx1 harboring amino acid substitutions in the A subunit, possess adjuvant activity via the activation of dendritic cells (DCs). Our results showed that StxB1 and mStx1, but not native Stx1 (nStx1), resulted in enhanced expression of CD86, CD40, and major histocompatibility complex (MHC) class II molecules and, to some extent, also enhanced the expression of CD80 on bone marrow-derived DCs. StxB1-treated DCs exhibited an increase in tumor necrosis factor alpha and interleukin-12 (IL-12) production, a stimulation of DO11.10 T-cell proliferation, and the production of both Th1 and Th2 cytokines, including gamma interferon (IFN-gamma), IL-4, IL-5, IL-6, and IL-10. When mice were given StxB1 subcutaneously, the levels of CD80, CD86, and CD40, as well as MHC class II expression by splenic DCs, were enhanced. The subcutaneous immunization of mice with ovalbumin (OVA) plus mStx1 or StxB1 induced high titers of OVA-specific immunoglobulin M (IgM), IgG1, and IgG2a in serum. OVA-specific CD4+ T cells isolated from mice immunized with OVA plus mStx1 or StxB1 produced IFN-gamma, IL-4, IL-5, IL-6, and IL-10, indicating that mStx1 and StxB1 elicit both Th1- and Th2-type responses. Importantly, mice immunized subcutaneously with tetanus toxoid plus mStx1 or StxB1 were protected from a lethal challenge with tetanus toxin. These results suggest that nontoxic Stx derivatives, including both StxB1 and mStx1, could be effective adjuvants for the induction of mixed Th-type CD4+ T-cell-mediated antigen-specific antibody responses via the activation of DCs.     

The receptor for type I IFNs is highly expressed on peripheral blood B cells and monocytes and mediates a distinct profile of differentiation and activation of these cells.

Type I interferons (IFNs) are potent regulators of both innate and adaptive immunity. All type I IFNs bind to the same heterodimeric cell surface receptor composed of IFN-alpha receptor (IFNAR-1) and IFN-alpha/beta receptor (IFNAR-2) polypeptides. This study revealed that type I IFN receptor levels vary considerably on hematopoietic cells, with monocytes and B cells expressing the highest levels. Overnight treatment of peripheral blood mononuclear cells (PBMCs) with IFN-alpha2b or IFN-beta led to increased expression on monocytes and B cells of surface markers commonly associated with activated antigen-presenting cells (APCs), such as CD38, CD86, MHC class I, and MHC class II. Five-day exposure of adherent monocytes to granulocyte-macrophage colony-stimulating factor (GM-CSF) plus IFN-alpha or IFN-beta caused the development of potent allostimulatory cells with morphology similar to that of myeloid dendritic cells (DCs) obtained from culture with GM-CSF and interleukin-4 (IL-4) but with distinct cell surface marker profiles and activity. In contrast to IL-4-derived DCs, IFN-alpha-derived DCs were CD14+, CD1a-, CD123+, CD32+, and CD38+ and expressed high levels of CD86 and MHC class II. Development of these cells was completely blocked by an antibody to IFNAR-1. Furthermore, activity of the type I IFN-derived DC in a mixed lymphocyte reaction (MLR) was consistently more potent than that of IL-4-derived DCs, especially at high responder/stimulator ratios. This MLR activity was abrogated by the addition of anti-IFNAR-1 antibody at the start of the DC culture. In contrast, there was no effect of anti-IFNAR-1 on IL-4-derived DCs, indicating that this is a distinct pathway of DC differentiation. These results suggest a potential role for anti-IFNAR-1 immunotherapy in autoimmune diseases, such as systemic lupus erythematosus (SLE), in which the action of excessive type I IFN on B cells and myeloid DCs may play a role in disease pathology.     

PD-1 upregulation is associated with HBV-specific T cell dysfunction in chronic hepatitis B patients

Programmed death-1 (PD-1) is demonstrated to have an increased expression on antigen-specific T cells during chronic virus infections, and the blockage of PD-1/PD-ligand (PD-L1) pathway could restore the function of exhausted T cells. We measured the PD-1 expression levels on HBV-specific CD8 T cells and investigated the role of PD-1/PD-L1 pathway in T-cell responses of patients with different HBV infection statuses. Compared to the patients with convalescent acute hepatitis B, PD-1 expression on total CD8 T cells from chronic hepatitis B (CHB) patients was significantly upregulated, especially on the HBV pentamer-positive CD8 T cells. And PD-L1, but not PD-L2, was also significantly upregulated on PBMC from CHB patients. In CHB patients, HBV-specific T cells and cellular proliferation could be observed under the recombinant HBV-Ag stimulation in vitro, and blockade of PD-1 pathway significantly enhanced the IFN-gamma production and cellular proliferation of PBMC. Furthermore, PD-1 expression level on HBV-pentamers positive CD8 T cells was positively associated with plasma viral load in CHB patients. Thus, PD-1 upregulation on HBV-specific CD8 T cells is engaged in the dysfunction of T cells and high viraemia in CHB patients, and the antiviral T-cell responses could be improved by the blockade of this inhibitory PD-1/PD-L1 pathway.     

IL-10上调PD-1配体的表达诱导耐受性树突状细胞形成

目的 探讨IL-10诱导耐受性树突状细胞（DCs）形成的机制。方法 用30ng／mL的IL-10处理Balb／c小鼠骨髓DCs，分别用RT-PCR和流式细胞术分析DCs的PD-1配体分子的表达，用MTF法测定DCs激活T细胞的能力，并用IL-10-DCs免疫荷瘤小鼠，观察H22肝癌细胞的生长情况。结果 IL-10-DCs对T细胞的激活能力降低；在注射了IL-10-DCs的小鼠体内，H22肝癌细胞的生长速度快于对照组；免疫抑制性受体PD-1的配体分子在IL-10-DCs上的表达上调。结果提示，在H22肝癌模型上，IL-10上调PD-1的配体分子表达可能是免疫耐受的一个机制。结论 减少IL-10的产生或封闭PD-1／PD-L路径可能是一个很有前景的肝癌生物学治疗策略。     

Delivery of rapamycin by PLGA nanoparticles enhances its suppressive activity on dendritic cells

The purpose of this study was to evaluate the effect of rapamycin delivery by poly (D,L-lactic-co-glycolic acid) (PLGA) nanoparticles on the maturation of dendritic cells (DCs). DCs were generated from mouse bone marrow and exposed to particulate and soluble rapamycin without any additional treatment, or with pre- or posttreatment with lipopolysaccharide (LPS). Annexin V-FITC/PI staining was performed on DC cultures to assess the viability of DCs during study. Surface phenotype of DCs was characterized for the expression of maturation markers, that is, MHC class II, CD86, and CD40 by flow cytometry. Cell culture supernatants were analyzed for the production of TGF-beta, IL-12, and IL-10 cytokines using sandwich ELISA method. DCs from Balb/C mice were cocultured with T cells from C57BL/6 mice and allogenic mixed lymphocyte reaction was assessed by [3H]-Thymidine incorporation. Unlike free rapamycin that has shown little if any effect on the expression of maturation markers in immature DCs, PLGA encapsulated rapamycin decreased the expression of all maturation markers under the study, that is, MHC class II, CD86, and CD40, significantly. LPS pre- or posttreated DCs demonstrated decreased expression of MHC class II, CD86, and CD40 in the presence of soluble or encapsulated rapamycin. The cytokine secretion profiles revealed high levels of TGF-beta and very low levels of IL-10 and IL-12 production. Rapamycin in soluble or encapsulated form significantly inhibited mixed lymphocyte reaction in DCs. The inhibitory effect of rapamycin on the maturation of DCs with respect to DC phenotype, cytokine production, and functional effects on the proliferation of T cells was significantly increased by PLGA delivery.     

小鼠骨髓来源树突状细胞的分离与扩增培养

目的 :探讨树突状细胞 (DC)分离纯化及其体外扩增的方法。方法 :无菌制备C57BL/6小鼠骨髓 ;依次用红细胞裂解液去除红细胞 ,通过半粘附法去除T、B细胞 ,又在粒细胞巨噬细胞集落刺激因子 (GM CSF)和白细胞介素 4 (IL 4 )协同诱导下培育 ,DC前体分化发育成DC并扩增。在第 7天用脂多糖 (LPS)和肿瘤坏死因子 a (TNF a)刺激 4 8h ,检测细胞因子白介素 12 (IL 12 )浓度及细胞表面标志CD11c、CD80、CD86和MHCⅡ。结果 :DC细胞数增加 ,其形态在光镜下多为特征性星形 ,也有梭形和多角形 ;至培养第 9天DC细胞表面标志CD11c、CD80、CD86、MHCⅡ阳性率分别为 86 .32± 12 .14 %、76 .4 2± 8.4 5%、77.12± 9.0 5%、6 8.4 5± 6 .84 % ,IL 12浓度较未用LPS和TNF a组明显增加 (P <0 .0 1)。结论 :①结果所得细胞的形态和功能符合DC ;②用LPS和TNF a刺激可以获得成熟DC。     

A combination of check-point blockade and α-galactosylceramide elicits long-lasting suppressive effects on murine hepatoma cell growth in vivo

Immunotherapy for cancer cells induced by interfering with PD-1/PD-L1 engagement via check-point blockades was initiated by tumour-specific PD-1⁺ CD8⁺ cytotoxic T lymphocytes (CTLs) within a tumour mass and eliminate the tumour. Here, we used C57BL/6 (B6) mice implanted with the syngeneic hepatoma cell line Hepa1-6-1, and confirmed that the dendritic cells (DCs) within Hepa1-6-1 tumour mass were tolerogenic with downmodulated co-stimulatory molecules by tumour-derived factors. Although Hepa1-6-1 cells did not prime tumour-specific CTLs within the tumour, specific CTLs primed in the regional lymph nodes seemed to be invaded into the tumour mass. The specific CTLs gained PD-1⁺ expression when associated with PD-L1⁺ Hepa1-6-1 cells within the tumour mass. Their cytotoxic activity in vivo was revitalised after intraperitoneal (i.p.) administration of the anti-PD-1 monoclonal antibody (mAb), indicating that PD-1/PD-L1 engagement within the tumour was abrogated by check-point blockade. Nonetheless, the tolerogenic DCs within the Hepa1-6-1 tumour mass remained tolerogenic even after three shots of PD-1-blockade administration, and the suppressed Hepa1-6-1 growth was revisited. In this study, we show here an excellent therapeutic effect consisting of three injections of anti-PD1 mAb and the sequential administration of the CD1d molecule-restricted ligand α-galactosylceramide (α-GalCer), an immuno-potent lipid/glycolipid, which converts tolerogenic DCs into immunogenic DCs with upregulated expression of co-stimulatory molecules. The α-GalCer-activated DCs secreted a large amount of IL-12, which can activate tumour-specific CTLs in vivo. The check-point blockade was not sufficiently effective, but the dose needed for tumour eradication was reduced by 90% when tumour-bearing mice were also administered i.p. α-GalCer.     

C1q regulation of dendritic cell development from monocytes with distinct cytokine production and T cell stimulation

The causative association of complement C1q deficiency with systemic lupus erythematosus (SLE), which inevitably involves the breakdown of tolerance, remains poorly explained. Its non-hepatic, macrophage and dendritic cell (DC) origin may be highly relevant. In tissues, C1q is produced by DCs and macrophages which deposits around these cells and we ask whether this pericellular form of C1q regulates DC development from monocytes. DCs cultured on immobilized C1q (C1q-DCs) show similar MHC, CD40, CD80, CD86, CD83 and CCR7 expression as normal DCs, but these cells exhibit increased phagocytosis of apoptotic cells and elevated IL-10 but reduced IL-12 and IL-23 production. Intracellularly, C1q-DCs exhibit increased ERK, p38 and p70S6 kinase activity. By mixed leukocyte reaction, C1q-DCs show reduced Th1 and Th17 induction from allogeneic CD4(+) T cells. LPS and IFNγ, which cause normal DCs to induce increased CD25 expression on CD4(+) T cells, attenuate C1q-DC induction of CD25. These imply that the DC pericellular C1q may induce tolerogenic properties in developing DCs.     

人参皂苷Ro 促进THP-1 细胞向DC 分化的作用

目的:探讨人参皂苷Ro 联合细胞因子(GM-CSF 与IL-4)促进人单核白血病细胞(THP-1)向树突状细胞(DC)分化的作用。方法:筛选细胞因子诱导白血病源DC 敏感细胞株;采用细胞因子联合人参皂苷Ro 诱导筛选的敏感细胞株THP-1 向DC 分化。培养体系中分别加入小(5 μmol/ L)、中(10 μmol/ L)、大(20 μmol/ L)剂量人参皂苷Ro,流式细胞术检测白血病来源DC 表面标志CD1a、MHCⅠ、共刺激分子CD86 表达;逆转录-聚合酶链式反应(RT-PCR)检测白血病来源DCMHCⅠ、CD86 mRNA 转录水平;酶联免疫吸附法(ELISA)检测培养上清TNF-α、IL-6 蛋白水平。结果:THP-1 是细胞因子诱导DC 分化的敏感白血病细胞株,与单纯细胞因子诱导比较,细胞因子联合人参皂苷Ro 作用后,白血病来源DC 表面标志CD1a、MHCⅠ、共刺激分子CD86 比例显著升高(P<0.05),CD1a、CD86 及MHCⅠ转录水平显著升高(P<0.05),培养上清TNF-α、IL-6 蛋白水平显著升高(P<0.05)。结论:人参皂苷Ro 能明显促进细胞因子诱导THP-1 细胞向DC 分化。     

Dendritic cells activated with products released by schistosome larvae drive Th2-type immune responses, which can be inhibited by manipulation of CD40 costimulation

The early immune events in response to infective larvae of the parasitic helminth Schistosoma mansoni are poorly understood, but here for the first time we report on the potential of products released by schistosome larvae (material released in the first 3 h after transformation [0-3hRP]) to stimulate the maturation of dendritic cells (DC) and alter their T-cell-polarizing function. This was performed in comparison with lipopolysaccharide (LPS) and zymosan A, which classically activate DC to prime for Th1- and Th2-type responses, respectively. In our study, immature bone marrow-derived DC stimulated in vitro with 0-3hRP exhibited up-regulated expression of major histocompatibility complex class II, CD40, and CD86 and increased production of interleukin 12p40 (IL-12p40) and IL-6, albeit at lower levels than in response to LPS or zymosan A. Using an in vitro ovalbumin peptide-restricted priming assay, DC matured with 0-3hRP exhibited a potent capacity to drive Th2 polarization of CD4(+) cells from DO11.10 transgenic mice. This was characterized by increased IL-4 production (but not gamma interferon) of a magnitude similar to that primed by DC matured with zymosan A. Inoculation of DO11.10 mice with 0-3hRP-activated DC pulsed with ovalbumin peptide also led to the development of a Th2-type polarized response in the skin-draining lymph nodes and spleen. However, ligation of CD40 on DC by anti-CD40 antibody treatment reversed the ability of 0-3hRP-activated DC to prime for Th2-type responses and instead caused the induction of a more Th1-type response.