髓系来源抑制性细胞在肿瘤免疫和肿瘤免疫治疗中作用的研究进展

髓系来源抑制性细胞（MDSCs）是由多种具有抑制功能的细胞构成的群体,是肿瘤免疫逃逸的关键因素.一方面,MDSCs主要通过抑制宿主固有免疫及适应性免疫减弱对肿瘤的免疫监视,促进肿瘤血管形成,参与肿瘤免疫逃逸.另一方面,肿瘤细胞又分泌多种细胞因子诱导MDSCs产生及活化,形成恶性循环.基于MDSCs进行相关靶向治疗,将有助于减弱MDSCs对宿主肿瘤微环境的抑制作用,可能为肿瘤的免疫治疗提供新思路和策略.     

抗CD4单抗增强抗CD3单抗／IL—2活化的肿瘤特异性T细胞的抗瘤活性

目的探讨抗CD4mAb增强抗CD3mAb刺激的肿瘤特异性T细胞增殖和杀瘤活性的作用。方法将肿瘤细胞免疫的小鼠脾细胞,采用4种不同的方案培养1单独加2×104U/LrIL2IL2组2单独加抗CD3mAb抗CD3组3加抗CD3mAb48h后,再加入抗CD3mAb和2×104U/LrIL2抗CD3 IL2组4同时加抗CD3mAb和抗CD4mAb48h后,再加入抗CD3mAb、抗CD4mAb和2×104U/LrIL2抗CD3 IL2 抗CD4组。然后分别检测4组效应细胞的增殖水平、杀瘤活性及表型。结果抗CD3 IL2组细胞的3HTdR掺入量在第6,12和20d分别为:22045、13986和1931;抗CD3 IL2 抗CD4组细胞的3HTdR掺入量在第6、12和20d,分别为46193、31047和7443,后者明显高于前者P0.05。在培养12d时,抗CD3 IL2组的细胞对FBL3细胞株的最大杀伤率为83.6%;抗CD3 IL2 抗CD4组细胞的最大杀伤率为91.7%。细胞表型:FACS分析表明,抗CD3 IL2 抗CD4组培养12d的细胞,99%以上为Thy1.2 细胞,且CD4 、CD25 细胞的百分率均高于抗CD3 IL2组。结论抗CD4mAb对抗CD3mAb刺激、IL2诱导的肿瘤特异性T细胞的增殖和杀瘤活性具有增强作用。     

IL-6对大鼠急性髓系白血病R2细胞的增殖抑制及分化诱导作用

重组人白细胞介素６（ｒｈＩＬ－６）可抑制大鼠急性髓系白血病（ＡＭＬ）Ｒ２细胞的体外生长（ＩＣ５０１００ｎｇ／Ｌ），并在１０ｎｇ／Ｌ～１μｇ／Ｌ的剂量范围内诱导Ｒ２细胞向终末方向分化。诱导后的Ｒ２细胞具有单核巨噬细胞的形态特征；且非特异性酯酶（ＮＳＥ）及酸性醋酸酯酶（ＡＮＡＥ）的活性增强，而过氧化物酶（ＰＯＸ）的活性却一直很低。经典型的ＤＮＡ梯状条带及凋亡小体证实，≥１μｇ／ＬｒｈＩＬ－６亦可诱导Ｒ２细胞凋亡。我们认为，Ｒ２细胞是研究ＩＬ－６信号转导通路及ＡＭＬ细胞分化机制的一个很好模型。     

Experimentally induced psoriatic lesion associates with interleukin (IL)-6 in mast cells and appearance of dermal cells expressing IL-33 and IL-6 receptor

Mast cells are involved in the development of psoriatic lesion, but it is not known how mast cells are activated or whether mast cell cytokines are expressed during the lesion development. In this study, the Köbner reaction was induced in uninvolved psoriatic skin of 18 patients using the tape‐stripping technique, and a sequence of biopsies was collected at 0 days, 2 h and 3 days or at 0 days, 1 day and 7 days for histochemical analysis. Eight patients developed the Köbner reaction verified at the follow‐up visit 2–2·5 weeks later. No significant differences were observed in total tryptase⁺ mast cells, psoriasis area and severity index and age/sex. Instead, the percentage of tryptase⁺ mast cells showing interleukin (IL)‐6 immunoreactivity was significantly higher in biopsies from Köbner‐positive patients than in those from Köbner‐negative patients. IL‐33 is a known inducer of IL‐6 in mast cells, and the number of IL‐33⁺ cells increased significantly in Köbner‐positive dermal skin at days 3–7. The number of dermal cells with IL‐6 receptor (IL‐6R, CD126) also increased in Köbner‐positive skin at days 3–7. Unexpectedly, the number of IL‐6R⁺ cells was even higher in Köbner‐negative skin at days 3–7. In the chronic plaque of 10 other psoriatic patients, the numbers of IL‐6⁺ mast cells and dermal cells showing IL‐6R were higher than those in the non‐lesional skin. In conclusion, the positive Köbner reaction is associated with IL‐6 in mast cells and appearance of IL‐6R⁺ and IL‐33⁺ dermal cells. This suggests that a previously unrecognized vicious circle may develop in the early psoriatic lesion.     

Interleukin-6 and soluble interleukin-6 receptor serum levels in recurrent spontaneous abortion women immunized with paternal white cells

PROBLEM: Recurrent spontaneous abortion (RSA) could be interpreted as the cause for the incapacity of the mother to recognize paternal antigens to produce the desired protective response. The practise of alloimmunization was introduced in an attempt to induce in the mother the production of an alloimmune response; some authors proposed an association between cytokines and RSA. The production of IL6 and its soluble receptor (sIL6R) before and after lymphocyte immunotherapy was evaluated in sera of 33 patients suffering from two or more RSA, and in sera of 47 women with normal pregnancy. METHOD OF STUDY: The immunization of RSA patients was achieved by injection of four doses of 10(5) mononuclear cells (MNC) from the husband, at weekly intervals, before pregnancy. The IL6 and sIL6R levels were measured using sandwich ELISAs and the results evaluated by Tukey-Kramer multiple comparison-tests. RESULTS: Our data show no significant differences between IL6 and sIL6R serum levels of normal pregnant women and RSA pregnant women with white-cell immunization before pregnancy. In contrast, the sera of pregnant RSA patients without allogeneic therapy show higher values. We also found significant differences between IL6 levels in non-pregnant RSA women with and without immunotherapy. CONCLUSION: These results show that the alloimmunization with paternal white cells leads the serum IL6 and sIL6R-levels to the values observed in the course of normal pregnancy, suggesting a role for IL6 and sIL6R in the modulation of the immune response's quality.     

Interleukin-6 and its receptor during homeostasis,inflammation, and tumor growth

Summary This review focuses on describing the specific role of interleukin-6 within the network of inflammatory mediators in man. Sites of interleukin-6 synthesis, regulation of its expression, and the biological functions of this molecule are here outlined. The potential role of interleukin-6 as a diagnostic monitor is discussed. Particular attention is paid to experimental evidence that interleukin-6 and its receptor may be involved in the pathogenesis of autocrine tumor growth. A recently proposed therapeutical use of cytotoxic interleukin-6 fusion proteins in order to selectively destroy certain interleukin-6 receptor bearing tumor cells is discussed in the light of the finding, that not only hepatocytes, but also normal peripheral blood monocytes express the interleukin-6 receptor.Abbreviations ACTH Adenocorticotropic hormone - BCDF B-cell differentiation factor - BSF-2 B-cell stimulatory factor 2 - bp base pairs - CAT chloramphenicol acetyltransferase - cDNA complementary deoxyribonucleic acid - cAMP cyclic adenosinmonophosphate - CHX cycloheximid - CRP C-reactive protein - DNA deoxyribonucleic acid - EBV Epstein Barr Virus - FACS fluorescence activated cell sorter - GMCSF granulocyte-monocyte colony stimulating factor - HSF hepatocyte stimulating factor - HGF hybridoma growth factor - IFN 2 interferon beta 2 - IL-1 interleukin-1 - IL-3 interleukin-3 - IL-6 interleukin-6 - IL6R interleukin-6 receptor - kDa kilo-dalton - LPS lipopolysaccharide/endotoxin - M-CSF macrophage colony stimulating factor - mRNA messenger ribonucleic acid - PDGF platelet-derived growth factor - RNA ribonucleic acid - SAC Staphylococchus aureus Cowan I - TNF tumor necrosis factor/cachectin Dedicated to Hedwig     

Olfactory ecto-mesenchymal stem cell-derived exosomes ameliorate murine Sjögren’s syndrome by modulating the function of myeloid-derived suppressor cells

Sjögren’s syndrome (SS) is a systemic autoimmune disease characterized by progressive inflammation and tissue damage in salivary glands and lacrimal glands. Our previous studies showed that myeloid-derived suppressor cells (MDSCs) exhibited impaired immunosuppressive function during disease progression in patients with SS and mice with experimental Sjögren’s syndrome (ESS), but it remains unclear whether restoring the function of MDSCs can effectively ameliorate the development of ESS. In this study, we found that murine olfactory ecto-mesenchymal stem cell-derived exosomes (OE-MSC-Exos) significantly enhanced the suppressive function of MDSCs by upregulating arginase expression and increasing ROS and NO levels. Moreover, treatment with OE-MSC-Exos via intravenous injection markedly attenuated disease progression and restored MDSC function in ESS mice. Mechanistically, OE-MSC-Exo-secreted IL-6 activated the Jak2/Stat3 pathway in MDSCs. In addition, the abundant S100A4 in OE-MSC-Exos acted as a key factor in mediating the endogenous production of IL-6 by MDSCs via TLR4 signaling, indicating an autocrine pathway of MDSC functional modulation by IL-6. Taken together, our results demonstrated that OE-MSC-Exos possess therapeutic potential to attenuate ESS progression by enhancing the immunosuppressive function of MDSCs, possibly constituting a new strategy for the treatment of Sjögren’s syndrome and other autoimmune diseases.     

Exosomes from heat‐stressed tumour cells inhibit tumour growth by converting regulatory T cells to Th17 cells via IL‐6

Exosomes derived from heat‐stressed tumour cells (HS‐TEXs), which contain abundant heat shock protein (HSP) 70, strongly induce antitumour immune responses. HSP70‐induced interleukin (IL)‐6 promotes IL‐17 expression and causes rejection of established prostate tumours. However, it remains unclear whether HS‐TEXs exhibit antitumour effects by converting regulatory T cells (T_regs) into T helper type 17 (Th17) cells. In this study, we found that compared with TEXs, HS‐TEXs were more potent in stimulating secretion of IL‐6 from dendritic cells. In vitro, IL‐6 blocked tumour cell‐derived transforming growth factor beta 1‐induced T_reg differentiation and promoted Th17 cell differentiation. HS‐TEXs exerted strong antitumour effects, converting T_regs into Th17 cells with high efficiency, a process that was entirely dependent upon IL‐6. Neutralization of IL‐17 completely abolished the antitumour effect of TEXs, but only partially inhibited that of HS‐TEXs. In addition, we found higher levels of IL‐6 and IL‐17 in serum from tumour patients treated with hyperthermia, and an increase in Th17 cells and a decrease in T_regs was detected in peripheral blood mononuclear cells isolated from these patients after hyperthermia. Therefore, our results demonstrate that HS‐TEXs possess a powerful capacity to convert immunosuppressive T_regs into Th17 cells via IL‐6, which contributes to their potent antitumour effect.     

β‐Glucan enhances antitumor immune responses by regulating differentiation and function of monocytic myeloid‐derived suppressor cells

Myeloid‐derived suppressor cells (MDSCs) accumulate in tumor‐bearing hosts and play a major role in tumor‐induced immunosuppression, which hampers effective immuno‐therapeutic approaches. β‐Glucans have been reported to function as potent immuno‐modulators to stimulate innate and adaptive immune responses, which contributes to their antitumor property. Here, we investigated the effect of particulate β‐glucans on MDSCs and found that β‐glucan treatment could promote the differentiation of M‐MDSCs (monocytic MDSCs) into a more mature CD11c⁺ F4/80⁺ Ly6C^low population via dectin‐1 pathway in vitro, which is NF‐κB dependent, and the suppressive function of M‐MDSCs was significantly decreased. Treatment of orally administered yeast‐derived particulate β‐glucan drastically downregulated MDSCs but increased the infiltrated DCs and macrophages in tumor‐bearing mice, thus eliciting CTL and Th1 responses, inhibiting the suppressive activity of regulatory T cells, thereby leading to the delayed tumor progression. We show here for the first time that β‐glucans induce the differentiation of MDSCs and inhibit the regulatory function of MDSCs, therefore revealing a novel mechanism for β‐glucans in immunotherapy and suggesting their potential clinical benefit.     

Antitumor effector B cells directly kill tumor cells via the Fas/FasL pathway and are regulated by IL‐10

We have previously reported that adoptive transfer of tumor‐draining lymph node (TDLN) B cells confers tumor regression in a spontaneous pulmonary metastasis mouse model of breast cancer. In this study, we identified IL‐10‐producing cells within these B cells, and found that IL‐10 removal, either by using IL‐10^?/? TDLN B cells or by systemic neutralization of IL‐10, significantly augmented the therapeutic efficacy of adoptively transferred TDLN B cells. Depletion of IL‐10 in B‐cell adoptive transfers significantly increased CTLs and B‐cell activity of PBMCs and splenic cells in the recipient. Activated TDLN B cells express Fas ligand, which was further enhanced by coculture of these TDLN B cells with 4T1 tumor cells. Effector B cells killed tumor cells directly in vitro in an antigen specific and Fas ligand‐dependent manner. Trafficking of TDLN B cells in vivo suggested that they were recruited to the tumor and lung as well as secondary lymphoid organs. These findings further define the biological function of antitumor effector B cells, which may offer alternative cellular therapies to cancer.     

The siRNA cocktail targeting interleukin 10 receptor and transforming growth factor‐β receptor on dendritic cells potentiates tumour antigen‐specific CD8+ T cell immunity

Dendritic cells (DCs) are promising therapeutic agents in the field of cancer immunotherapy due to their intrinsic immune‐priming capacity. The potency of DCs, however, is readily attenuated immediately after their administration in patients as tumours and various immune cells, including DCs, produce various immunosuppressive factors such as interleukin (IL)‐10 and transforming growth factor (TGF)‐β that hamper the function of DCs. In this study, we used small interfering RNA (siRNA) to silence the expression of endogenous molecules in DCs, which can sense immunosuppressive factors. Among the siRNAs targeting various immunosuppressive molecules, we observed that DCs transfected with siRNA targeting IL‐10 receptor alpha (siIL‐10RA) initiated the strongest antigen‐specific CD8⁺ T cell immune responses. The potency of siIL‐10RA was enhanced further by combining it with siRNA targeting TGF‐β receptor (siTGF‐βR), which was the next best option during the screening of this study, or the previously selected immunoadjuvant siRNA targeting phosphatase and tensin homologue deleted on chromosome 10 (PTEN) or Bcl‐2‐like protein 11 (BIM). In the midst of sorting out the siRNA cocktails, the cocktail of siIL‐10RA and siTGF‐βR generated the strongest antigen‐specific CD8⁺ T cell immunity. Concordantly, the knock‐down of both IL‐10RA and TGF‐βR in DCs induced the strongest anti‐tumour effects in the TC‐1 P0 tumour model, a cervical cancer model expressing the human papillomavirus (HPV)‐16 E7 antigen, and even in the immune‐resistant TC‐1 (P3) tumour model that secretes more IL‐10 and TGF‐β than the parental tumour cells (TC‐1 P0). These results provide the groundwork for future clinical development of the siRNA cocktail‐mediated strategy by co‐targeting immunosuppressive molecules to enhance the potency of DC‐based vaccines.     

The influence of adhesive and invasive properties of Staphylococcus aureus defective in fibronectin-binding proteins on secretion of interleukin-6 by human endothelial cells

Fibronectin-binding proteins (FnBP) are surface adhesins of Staphylococcus aureus documented to be virulence attributes in, for example, endovascular infections. By using mutants of S. aureus defective in the FnBPA and B genes we have investigated whether these adhesins affect cytokine expression in human umbilical vein endothelial cells (HUVEC). S. aureus expressing FnBPA and B adhered to and were internalized into HUVEC to a greater extent compared to mutants defective in expression of FnBP. Production and release of IL-6 was higher from endothelial cells infected with the parent FnBP-expressing strain compared to the FnBP-defective mutants. These results indicate that adhesion to and invasion of S. aureus into endothelial cells are important regulators of cytokine expression.     

Ca2+ influx-mediated histamine synthesis and IL-6 release in mast cells activated by monomeric IgE

We previously demonstrated that histamine synthesis is drastically induced upon sensitization with an anti-DNP IgE clone, SPE-7, in IL-3-dependent mouse bone marrow derived mast cells (BMMC). We found that Ca2+ mobilization induced by SPE-7 exhibited a similar profile to the capacitative Ca2+ entry evoked by thapsigargin. Potentials for activation of mast cells were found to vary between different IgE clones, and a monovalent hapten, DNP-lysine, suppressed the activation induced by SPE-7. Ca2+ mobilization induced by SPE-7 was suppressed potently by the specific store-operated Ca2+ channel inhibitor, SK&F 96365, but not at all by Ca2+ channel inhibitors with more broad spectrum, La3+ and Gd3+, whereas the Ca2+ mobilization induced by Ag stimulation was suppressed by these inhibitors. Ca2+ mobilization was also induced by SPE-7 in in vitro differentiated mast cells, although the increases in histamine synthesis and IL-6 release were smaller than those in BMMC. These results suggest that Ca2+ influx operated by a distinct mechanism from that in Ag stimulation is essential for increased histamine synthesis and IL-6 release in mast cells.     

Isoniazid‐induced control of Mycobacterium tuberculosis by primary human cells requires interleukin‐1 receptor and tumor necrosis factor

Proinflammatory cytokines are critical mediators that control Mycobacterium tuberculosis (Mtb) growth during active tuberculosis (ATB). To further inhibit bacterial proliferation in diseased individuals, drug inhibitors of cell wall synthesis such as isoniazid (INH) are employed. However, whether INH presents an indirect effect on bacterial growth by regulating host cytokines during ATB is not well known. To examine this hypothesis, we used an in vitro human granuloma system generated with primary leukocytes from healthy donors adapted to model ATB. Intense Mtb proliferation in cell cultures was associated with monocyte/macrophage activation and secretion of IL‐1β and TNF. Treatment with INH significantly reduced Mtb survival, but altered neither T‐cell‐mediated Mtb killing, nor production of IL‐1β and TNF. However, blockade of both IL‐1R1 and TNF signaling rescued INH‐induced killing, suggesting synergistic roles of these cytokines in mediating control of Mtb proliferation. Additionally, mycobacterial killing by INH was highly dependent upon drug activation by the pathogen catalase‐peroxidase KatG and involved a host PI3K‐dependent pathway. Finally, experiments using coinfected (KatG‐mutated and H37Rv strains) cells suggested that active INH does not directly enhance host‐mediated killing of Mtb. Our results thus indicate that Mtb‐stimulated host IL‐1 and TNF have potential roles in TB chemotherapy.     

Costunolide inhibits production of tumor necrosis factor-α and interleukin-6 by inducing heme oxygenase-1 in RAW264.7 macrophages

Objectives: Heme oxygenase (HO)-1 expression via nuclear factor-erythroid 2-related factor 2 (Nrf2) activation has an ability to inhibit tumor necrosis factor (TNF)-α and interleukin (IL)-6 production. Costunolide has been reported to inhibit IL-1 production, but whether other cytokines could be inhibited remains to be confirmed. We investigated the effects of costunolide and its components (α-methylene-γ-butyrolactone; CH2-BL, α-methyl-γ-butyrolactone; CH3-BL, and γ-butyrolactone; BL) on HO-1 expression as well as TNF-α and IL-6 production in RAW264.7 macrophages. Methods: HO-1 expression and Nrf2 nuclear accumulation were analyzed by Western blot analysis. The production of TNF-α and IL-6 in RAW264.7 macrophages stimulated with lipopolysaccharide (LPS) was assayed by ELISA. Results: Costunolide and CH2-BL induced HO-1 expression and Nrf2 nuclear accumulation, whereas CH3-BL and BL did not. Pre-incubation with costunolide inhibited LPS-induced production of TNF-α and IL-6. The inhibitory effects of costunolide on TNF-α and IL-6 production were abrogated by tin protoporphyrin, an HO inhibitor. Conclusions: Costunolide is an effective HO-1 inducer capable of inhibiting macrophage-derived pro-inflammatory cytokines. CH2-BL moiety of costunolide is essential for Nrf2 activation leading to HO-1 expression. Received 28 January 2007; returned for revision 4 April 2007; returned for final revision 25 June 2007; accepted by A. Falus 14 July 2007     

KIR downregulation by IL‐12/15/18 unleashes human NK cells from KIR/HLA‐I inhibition and enhances killing of tumor cells

To exploit autologous NK cells for cancer immunotherapy, it is highly relevant to circumvent killer cell immunoglobulin‐like receptor (KIR)‐mediated self‐inhibition of human NK cells by HLA‐I–expressing tumor cells. Here, we show that stimulation of NK cells with IL‐12/15/18 for two days led to downregulation of surface expression of the inhibitory KIR2DL2/L3, KIR2DL1 and KIR3DL1 receptors on peripheral blood NK cells. Downregulation of KIR expression was attributed to decreased KIR mRNA levels which could be re‐induced already 3 days after re‐culture in IL‐2. Reduced KIR2DL2/L3 expression on IL‐12/15/18–activated NK cells resulted in less inhibition upon antibody‐mediated KIR engagement and increased CD16‐dependent cytotoxicity in redirected lysis assays. Most importantly, downregulated KIR2DL2/L3 expression enabled enhanced cytotoxicity of IL‐12/15/18–stimulated NK cells against tumor cells expressing cognate HLA‐I molecules. NK cells pre‐activated with IL‐12/15/18 were previously shown to exert potent anti‐tumor activity and memory‐like long‐lived functionality, mediating remission in a subset of acute myeloid leukemia (AML) patients in a clinical trial. Our study reveals a novel mechanism of IL‐12/15/18 in improving the cytotoxicity of NK cells by reducing their sensitivity to inhibition by self–HLA‐I due to decreased KIR expression, highlighting the potency of IL‐12/15/18–activated NK cells for anti‐tumor immunotherapy protocols.     

Tumor expression of CD200 inhibits IL‐10 production by tumor‐associated myeloid cells and prevents tumor immune evasion of CTL therapy

CD200 is a cell‐surface glycoprotein that functions through interaction with the CD200 receptor on myeloid lineage cells to regulate myeloid cell functions. Expression of CD200 has been implicated in multiple types of human cancer; however, the impact of tumor expression of CD200 on tumor immunity remains poorly understood. To evaluate this issue, we generated CD200‐positive mouse plasmacytoma J558 and mastocytoma P815 cells. We found that established CD200‐positive tumors were often completely rejected by adoptively transferred CTL without tumor recurrence; in contrast, CD200‐negative tumors were initially rejected by adoptively transferred CTL but the majority of tumors recurred. Tumor expression of CD200 significantly inhibited suppressive activity and IL‐10 production by tumor‐associated myeloid cells (TAMC), and as a result, more CTL accumulated in the tumor and exhibited a greater capacity to produce IFN‐γ in CD200‐positive tumors than in CD200‐negative tumors. Neutralization of IL‐10 significantly inhibited the suppressor activity of TAMC, and IL‐10‐deficiency allowed TAMC to kill cancer cells and their antigenic variants, which prevented tumor recurrence during CTL therapy. Thus, tumor expression of CD200 prevents tumor recurrence via inhibiting IL‐10 production by TAMC.