Upregulation of T-cell Immunoglobulin and Mucin-Domain Containing-3 (Tim-3) in Monocytes/Macrophages Associates with Gastric Cancer Progression

T-cell immunoglobulin and mucin-domain containing-3 (Tim-3) is an important immune regulatory molecule in cancer immune system. However, expression and function of Tim-3 in monocytes/macrophages in cancer progression mainly remain unclear. In this study, we analyzed Tim-3 levels in peripheral blood mononuclear cells (PBMCs) from 62 gastric cancer patients and 45 healthy controls using flow cytometry and then associated Tim-3 levels with clinical pathological data from patients. We found Tim-3 level was significantly upregulated in monocytes from gastric cancer patients compared with those from healthy controls, and that upregulated Tim-3 levels associated with depth of tumor invasion and tumor lymph node metastasis and advanced clinical stages of gastric cancer patients. Furthermore, tumor-bearing mouse experiments revealed that Tim-3 level on monocytes/macrophages associated with xenograft formation and growth. In addition, culture of monocytes from healthy controls with gastric cancer cell-conditioned medium upregulated Tim-3 expression, but IL-10, TNF-α, IFN-γ, or GM-CSF treatment or T-bet, Eomes, and T-bet/Eomes double gene knockout did not affect Tim-3 levels in blood monocytes/macrophages from human or mouse, respectively. Gal-9/Tim-3 signal was able to significantly stimulate monocyte to secrete IL-6, IL-8, and IL-10, but not IL-1β, IL-12p70, or TNF-α in presence of LPS. In conclusion, our study demonstrated that Tim-3 expressed by monocyte/macrophages might be an important mechanism in gastric cancer progression.     

Tumor-Associated Macrophages as Target for Antitumor Therapy

It is well known that the microenvironment of solid tumors is rich in inflammatory cells that influence tumor growth and development. Macrophages, called tumor-associated macrophages (TAMs), are the most abundant immune cell population present in tumor tissue. Several studies have demonstrated that the density of TAMs is associated with a poor prognosis and positively correlates with tumor growth. Several studies have proved that TAMs may activate and protect tumor stem cells, stimulate their proliferation as well as promote angiogenesis and metastasis. Furthermore, TAMs-derived cytokines and other proteins, such as CCL-17, CCL-22, TGF-β, IL-10, arginase 1, and galectin-3, make a significant contribution to immunosuppression. Since TAMs influence various aspects of cancer progression, there are many attempts to use them as a target for immunotherapy. The numerous studies have shown that the primary tumor growth and the number of metastatic sites can be significantly decreased by decreasing the population of macrophages in tumor tissue, for example, by blocking recruitment of monocytes or eliminating TAMs already present in the tumor tissue. Moreover, there are attempts at reprogramming TAMs into proinflammatory M1 macrophages or neutralizing the protumoral products of TAMs. Another approach uses TAMs for anticancer drug delivery into the tumor environment. In this review, we would like to summarize the clinical and preclinical trials that were focused on macrophages as a target for anticancer therapies.

     

PD-1受体在HBV—ACLF患者外周血CD8^＋T细胞上的表达研究

目的探讨乙型肝炎病毒所致慢加急性肝衰竭（HBV—ACLF）患者外周血中CD8^＋T细胞上PD-1（Programmed death-1）的表达以及对CD8^＋T细胞的影响。方法收集60例HBV—ACLF患者外周血;应用流式细胞仪检测外周血中CD8^＋淋巴细胞上的PD-1、CD95、穿孔素、颗粒酶A、颗粒酶B、CD107a的表达以及CD14细胞上PD—L1的表达;根据不同病情分期和不同预后比较各分子的表达差异;设15名肝硬化患者及15名健康人为对照组。结果（1）HBV-ACLF患者CD8^＋T细胞上PD-1较肝硬化组和健康组表达上调（P〈0．05）;好转组PD-1表达率低于无效组（P〈0．05）;早、中、晚三期PD-1表达率呈上升趋势（P〈0．05）;（2）PD—L1在好转组及病情早期表达相对最低;（3）PD-1与CD95在死亡组表达明显升高;穿孔素、颗粒酶及CD107a在死亡组表达升高,但PD-1在这三类细胞上表达率非常低。结论PD-1在HBV．ACLF患者外周血CD8^＋T细胞上表达上调,且与病情严重程度呈正比。其上调表达可能促进总CD8^＋T细胞的凋亡,但对于具有杀伤活性的CD8^＋T细胞抑制作用相对较弱。     

Ataxia telangiectasia mutated kinase inhibition promotes irradiation-induced PD-L1 expression in tumour-associated macrophages through IFN-I/JAK signalling pathway

Tumour-associated macrophages (TAMs) are one of the primary sources of PD-L1 expression in the tumour microenvironment (TME). Ionizing radiation (IR) promotes PD-L1 expression in tumour cells. However, the effect of IR on macrophage PD-L1 expression and the underlying mechanisms remain unclear. ATM kinase, as the key kinase for initiating DNA damage repair (DDR) process, is associated with innate immune STING axis activation. Here, we explored the molecular mechanism implicated in macrophage PD-L1 expression regulated by IR as well as the role of ATM kinase in this process. IR-regulated PD-L1 expression in macrophages and associated signalling pathways were explored by in vitro studies using murine and human macrophage cell lines. A colorectal xenograft murine model was employed to demonstrate the impact of targeting ATM and PD-L1 expression in TAMs following IR on growth of tumour in vivo. IR up-regulated PD-L1 expression in macrophages, which was further augmented by ATM kinase inhibition. ATM inhibition increased IR-induced DNA damage, which activated STING/interferon regulatory factor 3 (IRF3) signalling pathway and up-regulated type I interferon (IFN-I) expression in macrophages. IFN-I bound to the IFN α receptor 1 on macrophages, activated the downstream JAK1 and STAT1/3 signalling and eventually led to PD-L1 up-expression. ATM inhibition augmented IR-induced PD-L1 expression in macrophages and CD8⁺ T cell infiltration, and promoted anti-tumour efficacy in vivo. These results suggested that ATM inhibition promoted IR-induced PD-L1 expression through the activation of innate immunity in TAMs, which provided a novel approach to enhance the anti-tumour efficacy of RT.     

Human NKT cells direct the differentiation of myeloid APCs that regulate T cell responses via expression of programmed cell death ligands

NKT cells are innate lymphocytes that can recognize self or foreign lipids presented by CD1d molecules. NKT cells have been shown to inhibit the development of autoimmunity in murine model systems, however, the pathways by which they foster immune tolerance remain poorly understood. Here we show that autoreactive human NKT cells stimulate monocytes to differentiate into myeloid APCs that have a regulatory phenotype characterized by poor conjugate formation with T cells. The NKT cell instructed myeloid APCs show elevated expression of the inhibitory ligand PD-L2, and blocking PD-L1 and PD-L2 during interactions of the APCs with T cells results in improved cluster formation and significantly increased T cell proliferative responses. The elevated expression of PD-L molecules on NKT-instructed APCs appears to result from exposure to extracellular ATP that is produced during NKT-monocyte interactions, and blocking purinergic signaling during monocyte differentiation results in APCs that form clusters with T cells and stimulate their proliferation. Finally, we show that human monocytes and NKT cells that are injected into immunodeficient mice co-localize together in spleen and liver, and after 3 days in vivo in the presence of NKT cells a fraction of the myeloid cells have upregulated markers associated with differentiation into professional APCs. These results suggest that autoreactive human NKT cells may promote tolerance by inducing the differentiation of regulatory myeloid APCs that limit T cell proliferation through expression of PD-L molecules.     

Airway epithelial cells modify immune responses by inducing an anti-inflammatory microenvironment

The upper airways are prone to contact with pathogenic as well as non-pathogenic microbes, therefore immune recognition principles have to be tightly controlled. Here we show that human BEAS-2B bronchial epithelial cells inhibited secretion of the pro-inflammatory cytokines TNF-alpha and IL-12 by monocytes, macrophages and dendritic cells. This inhibitory effect could be transferred by supernatant of resting BEAS-2B cells and was also observed when primary murine tracheal epithelial cells were prepared. In contrast to inhibition of pro-inflammatory cytokine secretion epithelial cell-conditioned dendritic cells showed increased expression of IL-10 and arginase-1, thus displaying properties of alternative activation. Accordingly, Toll-like receptor-mediated up-regulation of CD40, CD86 and PD-L2 (CD273) on murine dendritic cells was reduced in the presence of bronchial epithelial cell supernatant. However, expression of negative regulatory PD-L1 (CD274) was increased and dendritic cell induced proliferation of T lymphocytes was diminished. Epithelial cells also showed a direct inhibitory effect on T lymphocyte proliferation and this was due to the constitutive secretion of TGF-beta by bronchial epithelial cells. Moreover, epithelial cell-conditioned T lymphocytes showed increased differentiation towards IL-10-producing Tr1 cells. The results indicate that bronchial epithelial cells induce a non-inflammatory microenvironment that regulates local immune homeostasis.     

Increased IL-10 production during spontaneous apoptosis of monocytes

Monocytes/macrophages undergo apoptosis and are in contact with apoptotic cells both in vitro and in vivo. The data show that monocytes undergoing spontaneous apoptosis in vitro change their cytokine production profile. We demonstrate that the lipopolysaccharide (LPS)-induced production of interleukin-10 (IL-10) is up-regulated, while production of pro-inflammatory cytokines such as tumor necrosis factor-alpha (TNF-alpha) and interleukin-1beta (IL-1beta) is either not affected or reduced. These differences seen both at the protein and mRNA level directly correlate with the appearance of apoptotic cells in the culture. Flow cytometry analysis using double staining, surface with annexin V and intracellular with anti-IL-10, suggested that annexin V-negative monocytes are the predominant source of IL-10. Analysis of sorted populations of monocytes indicated that the increase in IL-10 synthesis appears to result from direct interactions between non-apoptotic and apoptotic cells at the time of stimulation. Also non-apoptotic, freshly isolated monocytes produced more IL-10 upon stimulation with LPS, Staphylococcus aureus or zymosan when apoptotic neutrophils were added to the culture. In contrast, monocyte-derived macrophages did not produce more IL-10 in the presence of apoptotic neutrophils. Finally, we found that the presence of apoptotic monocytes in the culture may influence specific immune responses. The data show that in the presence of annexin V-positive monocytes CD4-positive memory T cells produce less IFN-gamma upon stimulation with purified protein derivative of tuberculin, which could be partially reversed by anti-IL-10 neutralizing antibodies. We conclude that these findings might illustrate the mechanisms operating within an inflammatory site and play an important immunoregulatory role during the resolution of inflammation and specific immune responses.     

Regulation of human CD4(+) alphabeta T-cell-receptor-positive (TCR(+)) and gammadelta TCR(+) T-cell responses to Mycobacterium tuberculosis by interleukin-10 and transforming growth factor beta

Mycobacterium tuberculosis is the etiologic agent of human tuberculosis and is estimated to infect one-third of the world's population. Control of M. tuberculosis requires T cells and macrophages. T-cell function is modulated by the cytokine environment, which in mycobacterial infection is a balance of proinflammatory (interleukin-1 [IL-1], IL-6, IL-8, IL-12, and tumor necrosis factor alpha) and inhibitory (IL-10 and transforming growth factor beta [TGF-beta]) cytokines. IL-10 and TGF-beta are produced by M. tuberculosis-infected macrophages. The effect of IL-10 and TGF-beta on M. tuberculosis-reactive human CD4(+) and gammadelta T cells, the two major human T-cell subsets activated by M. tuberculosis, was investigated. Both IL-10 and TGF-beta inhibited proliferation and gamma interferon production by CD4(+) and gammadelta T cells. IL-10 was a more potent inhibitor than TGF-beta for both T-cell subsets. Combinations of IL-10 and TGF-beta did not result in additive or synergistic inhibition. IL-10 inhibited gammadelta and CD4(+) T cells directly and inhibited monocyte antigen-presenting cell (APC) function for CD4(+) T cells and, to a lesser extent, for gammadelta T cells. TGF-beta inhibited both CD4(+) and gammadelta T cells directly and had little effect on APC function for gammadelta and CD4(+) T cells. IL-10 down-regulated major histocompatibility complex (MHC) class I, MHC class II, CD40, B7-1, and B7-2 expression on M. tuberculosis-infected monocytes to a greater extent than TGF-beta. Neither cytokine affected the uptake of M. tuberculosis by monocytes. Thus, IL-10 and TGF-beta both inhibited CD4(+) and gammadelta T cells but differed in the mechanism used to inhibit T-cell responses to M. tuberculosis.     

Expression of galectin-3 in the tumor immune response in colon cancer

The role of tumor-associated macrophages (TAMs) is controversial. Although most studies on different cancer types associate them with a poorer prognosis, interestingly in colon cancer, most articles indicate that TAMs prevent tumor development; patients with high TAMs have better prognosis and survival rate. M1-polarized macrophages produce high level of tumor necrosis factor-alpha, interleukin-1 beta or reactive oxygen species, which can effectively kill susceptible tumor cells. In contrast, M2-polarized macrophages can secrete different factors that promote tumor cell growth and survival or favor angiogenesis and tissue invasion. Considering the beneficial role of TAMs in colon cancer, we speculated that they may not display the M2 polarization commonly observed in tumor microenvironment, but rather develop M1 properties. Therefore, we used an in vitro model to analyze the effects of supernatants from M1-polarized macrophages on DLD-1 colon cancer cells. Our data indicate that the conditioned medium from LPS-activated macrophages (CM-LAM) contains a high level of granulocyte-macrophage colony-stimulating factor, interleukins-1 beta, -6, -8 and tumor necrosis factor-alpha, and that it exerts a marked growth inhibitory activity on DLD-1 cells. Prolonged exposure to CM-LAM results in cell death by apoptosis. Such exposure to CM-LAM leads to the modulation of gal-3 expression: we observed a marked downregulation of gal-3 mRNA and protein expression following CM-LAM treatment. We also describe that the knockdown of gal-3 sensitizes DLD-1 cells to CM-LAM. These data suggest an involvement of gal-3 in the response of colon cancer cells to proinflammatory stimuli, such as the conditioned medium from activated macrophages.     

Arginine metabolism in tumor-associated macrophages in cutaneous malignant melanoma: evidence from human and experimental tumors

Tumor-associated macrophages (TAMs) may elicit contrasting effects on tumor growth, depending on their biological activities. Macrophages use arginine either to synthesize nitric oxide (NO) through the inducible NO synthase (iNOS) or to produce ornithine through arginase activity. Although the effects of NO are primarily cytotoxic, production of ornithine may promote tumor cell proliferation. Thus, iNOS/arginase balance in TAMs may be crucial in tumor progression. The aim of this study was (a) to explore iNOS and arginase expression in TAMs associated with human melanoma at different stages of tumor progression and (b) to explore whether melanoma cells influence iNOS and/or arginase expression in TAMs under basal condition and in the presence of interferon gamma and/or lipopolysaccharide. Immunohistochemical analyses performed on tissue sections from in situ melanoma, invasive melanoma of different pT categories, and metastatic melanoma revealed that (a) the percentage of iNOS-positive TAMs was significantly higher in in situ and thin melanomas in comparison with more advanced, thicker tumors; (b) the percentage of arginase-positive TAMs did not change among the pT categories analyzed; and (c) the percentage of iNOS-positive TAMs was greater than that of arginase-positive TAMs in peritumoral and intratumoral locations of thin melanomas (pT1). Moreover, by the use of an in vitro experimental protocol represented by B16 murine melanoma cells cocultivated with inflammatory macrophages, we found that melanoma cells stimulate iNOS expression and NO production in macrophages. In conclusion, our in vivo and in vitro results suggest that, mainly in early melanoma lesions, iNOS prevails over arginase in TAMs, a phenomenon possibly stimulated by contact with tumor cells. However, macrophages stimulated by murine melanoma cells secreted a level of NO compatible with an antitumor activity only in the presence of interferon gamma.     

Expression of PD-L1 and PD-L2 on human macrophages is up-regulated by HIV-1 and differentially modulated by IL-10

PD-1 plays an important role in T cell exhaustion during HIV infection. PD-1 has two ligands: PD-L1, expressed on hematopoietic and nonhematopoietic cells, and PD-L2, limited to DCs and macrophages. Little is known about PD-L1 expression and regulation in human macrophages. Previous reports have found few immediate effects of macrophage exposure to HIV, suggesting that macrophages lack PRRs for this virus. Using quantitative confocal microscopy and a multiplexed cytokine bead array, we measured induction of PD-L1, PD-L2, and innate response cytokines in human MDMs in response to chemically inactivated HIV virions. Consistent with previous reports, no cytokines were induced by HIV virion exposure. Whereas PD-L1 and PD-L2 had low baseline expression, TLR ligands (LPS and CL097) up-regulated PD-L1 but not PD-L2. Unlike what we found for cytokine expression, PD-L1 and PD-L2 were up-regulated in response to exposure with inactivated HIV virions or with replication-competent HIV. Expression of PD-L1 was differentially modulated by IL-10, which induced up-regulation of PD-L1 but not of PD-L2, and IL-10 blockade enhanced only PD-L2 expression. We discuss implications for innate recognition of HIV by macrophages and potential, different roles for PD-L1 and PD-L2 in immunity and pathogenesis.     

The tumour stromal features are associated with resistance to 5‐FU‐based chemoradiotherapy and a poor prognosis in patients with oral squamous cell carcinoma

It has been increasingly recognized that the tumour microenvironment is a critical factor involved in cancer progression. However, little is known about the clinical value of the stromal features in oral squamous cell carcinoma (OSCC). The purpose of this study was to determine the clinical significance of cancer‐associated fibroblasts (CAFs) and tumour‐associated macrophages (TAMs) in OSCC. OSCC specimens were obtained from 60 patients who underwent surgery following 5‐fluorouracil‐based chemoradiotherapy. Paraffin‐embedded sections obtained from biopsy specimens were immunohistochemically analysed. The associations among CAFs, TAMs and various clinicopathological features were examined, and the effects of CAFs and TAMs on the prognosis were evaluated. In the group with a high level of CAFs, the incidence of advanced pT‐ and pN‐stage cases was significantly higher than that in the group with the low level. A high TAMs tumour expression was significantly correlated with a poor response to preoperative chemoradiotherapy. A Kaplan–Meier analysis revealed that higher numbers of CAFs and TAMs were significantly correlated with a poor prognosis. These findings suggest that TAMs are a potential biomarker for predicting the clinical response to 5‐FU‐based chemoradiotherapy, and the expression status of the CAFs and TAMs may be useful for making treatment decisions to improve the survival of OSCC patients.     

Characterization of rag1 mutant zebrafish leukocytes

Background

Immunosuppression by gliomas contributes to tumor progression and treatment resistance. It is not known when immunosuppression occurs during tumor development but it likely involves cross-talk among tumor cells, tumor-associated macrophages and microglia (TAMs), and peripheral as well as tumor-infiltrating lymphocytes (TILs).

Results

We have performed a kinetic study of this immunomodulation, assessing the dynamics of immune infiltration and function, within the central nervous system (CNS) and peripherally. PDGF-driven murine glioma cells were injected into the white matter of 13 mice. Four mice were sacrificed 13 days post-injection (dpi), four mice at 26 dpi, and five mice at 40 dpi. Using multiparameter flow cytometry, splenic T cells were assessed for FoxP3 expression to identify regulatory T cells (Tregs) and production of IFN-γ and IL-10 after stimulation with PMA/ionomycin; within the CNS, CD4⁺ TILs were quantified, and TAMs were quantified and assessed for TNF-α and IL-10 production after stimulation with LPS. Peripheral changes associated with tumor development were noted prior to effects within the CNS. The percentage of FoxP3⁺ regulatory T cells (Tregs) increased by day 26, with elevated frequencies throughout the duration of the study. This early increase in Tregs was paralleled by an increase in IL-10 production from Tregs. At the final time points examined (tumor morbidity or 40 dpi), there was an increase in the frequency of TAMs with decreased capacity to secrete TNF-α. An increase in TIL frequency was also observed at these final time points.

Conclusion

These data provide insight into the kinetics of the immunosuppressive state associated with tumor growth in a murine model of human gliomas. Functional impairment of TAMs occurs relatively late in the course of GBM tumor growth, potentially providing a window of opportunity for therapeutic strategies directed towards preventing their functional impairment.     

Macrophage infiltration predicts a poor prognosis for human ewing sarcoma

Ewing sarcoma-primitive neuroectodermal tumor (EWS) is associated with the most unfavorable prognosis of all primary musculoskeletal tumors. The objective of the present study was to investigate whether tumor-associated macrophages (TAMs) affect the development of EWS. TAMs were isolated from mouse xenografts using CD11b magnetic beads and examined for their cytokine expression and osteoclastic differentiation. To evaluate the role of TAMs in xenograft formation, liposome-encapsulated clodronate was used to deplete TAMs in mice. Macrophage infiltration and tumor microvascular density were histologically evaluated in 41 patients with EWS, and association with prognosis was examined using Kaplan-Meier survival analysis. In mouse EWS xenografts, TAMs expressed higher concentrations of cytokines including interleukin-6, keratinocyte-derived chemokine, and monocyte chemotactic protein-1. TAMs were more capable than normal monocytes of differentiating into tartrate-resistant acid phosphatase-positive giant cells. Depleting macrophages using liposome-encapsulated clodronate significantly inhibited development of EWS xenografts. In human EWS samples, higher levels of CD68-positive macrophages were associated with poorer overall survival. In addition, enhanced vascularity, increase in the amount of C-reactive protein, and higher white blood cell counts were also associated with poor prognosis and macrophage infiltration. TAMs seem to enhance the progression of EWS by stimulating both angiogenesis and osteoclastogenesis. Further investigation of the behavior of TAMs may lead to development of biologically targeted therapies for EWS.     

The blockade of PD-1/PD-L1 pathway promotes the apoptosis of CD19+CD25+ Bregs and suppresses the secretion of IL-10 in patients with allergic rhinitis

PD-1/PD-L1 pathway is crucial to immune regulation by controlling the balance between T cell tolerance and activation. However, the association between PD-1/PD-L1 pathway and regulatory B cells has not been fully investigated in allergic rhinitis. In this study, we detected the number of peripheral CD19⁺CD25⁺ Bregs and the expression of IL-10 on this cell subset in healthy control and patients with allergic rhinitis using flow cytometry. Then, we evaluated the level of PD-L1 in CD19⁺CD25⁺ Bregs and investigated the correlation between PD-L1 and CD4⁺ follicular T helper cells. Finally, we studied the effects of anti–PD-L1 on the apoptosis of Bregs and the production of IL-10. Comparing with healthy controls, the percentage of CD19⁺CD25⁺ Bregs and the expression of IL-10 were both significantly decreased in AR group. In addition, the expression of PD-L1 on CD19⁺CD25⁺ Bregs was also lower in allergic rhinitis patients. Interestingly, a negative correlation was found between the expression of PD-L1⁺ Bregs and CD4⁺CXCR5⁺ follicular T helper cells. In vitro assay revealed that anti–PD-L1 promoted Bregs apoptosis and inhibited the expression of IL-10 in CD19⁺CD25⁺ Bregs. Collectively, these results suggest that PD-L1 expressed on CD19⁺CD25⁺ Bregs may be a potential regulator in the treatment of allergic rhinitis. Blockade of PD-1/PD-L1 pathway might be a valuable pathogenic target for allergic rhinitis through inhibiting the secretion of immunosuppressive cytokine and promoting CD19⁺CD25⁺ Bregs apoptosis.     

Apoptotic cells selectively suppress the Th1 cytokine interferon γ in stimulated human peripheral blood mononuclear cells and shift the Th1/Th2 balance towards Th2

Apoptotic cells are readily recognized and engulfed by phagocytes and usually do not induce inflammation or tissue damage. Furthermore, they can actively suppress a pro-inflammatory response in phagocytes: In the presence of apoptotic cells, activated monocytes/macrophages produce more of the anti-inflammatory and immunoregulatory cytokines IL-10 and TGF-β, but less of the pro-inflammatory cytokines TNFα, IL-1β and IL-12. This immunoregulatory effect is most likely mediated by several receptors on monocytes/macrophages including the thrombospondin receptor (CD36). In addition to the modulation of cytokine secretion, apoptotic cell material inhibited the expression of MHC class II molecules on the surface of monocytes/macrophages. Decreased MHC II expression appeared to be mediated predominantly by increased IL-10 secretion in a para-/autocrine manner. Here, we show that the functional modulation of antigen-presenting monocytes/macrophages by apoptotic cells also influences T cell activation and function. When human peripheral blood mononuclear cells were stimulated with recall antigens in the presence of apoptotic cells, interferonγ (IFNγ) secretion was markedly suppressed, whereas secretion of the Th2 cytokine IL-4 was not significantly altered. Hence, apoptotic cells shift the T cell cytokine secretion pattern towards a Th2-like response. This Th2 shift can largely be prevented by neutralizing IL-10, indicating an important role of this cytokine for modulating T cell cytokine secretion patterns.     

Apoptotic cells selectively suppress the Th1 cytokine interferon gamma in stimulated human peripheral blood mononuclear cells and shift the Th1/Th2 balance towards Th2

Apoptotic cells are readily recognized and engulfed by phagocytes and usually do not induce inflammation or tissue damage. Furthermore, they can actively suppress a pro-inflammatory response in phagocytes: In the presence of apoptotic cells, activated monocytes/macrophages produce more of the anti-inflammatory and immunoregulatory cytokines IL-10 and TGF-beta, but less of the pro-inflammatory cytokines TNFalpha, IL-1beta and IL-12. This immunoregulatory effect is most likely mediated by several receptors on monocytes/macrophages including the thrombospondin receptor (CD36). In addition to the modulation of cytokine secretion, apoptotic cell material inhibited the expression of MHC class II molecules on the surface of monocytes/macrophages. Decreased MHC II expression appeared to be mediated predominantly by increased IL-10 secretion in a para-/autocrine manner. Here, we show that the functional modulation of antigen-presenting monocytes/macrophages by apoptotic cells also influences T cell activation and function. When human peripheral blood mononuclear cells were stimulated with recall antigens in the presence of apoptotic cells, interferon gamma (IFN gamma) secretion was markedly suppressed, whereas secretion of the Th2 cytokine IL-4 was not significantly altered. Hence, apoptotic cells shift the T cell cytokine secretion pattern towards a Th2-like response. This Th2 shift can largely be prevented by neutralizing IL-10, indicating an important role of this cytokine for modulating T cell cytokine secretion patterns.     

Oncogenic circDHTKD1 promotes tumor growth and metastasis of oral squamous cell carcinoma in vitro and in vivo via upregulating miR-326-mediated GAB1

Circular RNAs (circRNAs) have been extensively elucidated with regard to their significant implications in oral squamous cell carcinoma (OSCC). This study performed the functional investigation of circRNA dehydrogenase E1 and transketolase domain containing 1 (circDHTKD1) in OSCC. RNA expression levels of different molecules were measured via quantitative real-time polymerase chain reaction (qRT-PCR). Cellular behaviors were detected by 3-(4, 5-dimethylthiazol-2-y1)-2,5-diphenyl tetrazolium bromide (MTT) for cell viability, colony formation assay for clonal capacity, flow cytometry for cell apoptosis, wound healing assay for migration, and transwell assay for migration/invasion. Western blot was used for analyzing protein expression. RNA pull-down and dual-luciferase reporter assays were applied to assess the binding between targets. A xenograft tumor model was established in nude mice for in vivo experiments. Our expression analysis revealed that circDHTKD1 was upregulated in OSCC tissues and cells. circDHTKD1 knockdown was shown to impede OSCC cell growth and metastasis but motivate apoptosis. Additionally, circDHTKD1 served as a microRNA-326 (miR-326) sponge and the function of circDHTKD1 was achieved by sponging miR-326 in OSCC cells. Also, miR-326 inhibited OSCC development via targeting GRB2-associated-binding protein 1 (GAB1). circDHTKD1 could sponge miR-326 to alter GAB1 expression. Furthermore, circDHTKD1 contributed to OSCC progression in vivo via the miR-326/GAB1 axis. These data disclosed a specific circDHTKD1/miR-326/GAB1 signal axis in governing the malignant progression of OSCC, showing the considerable possibility of circDHTKD1 as a predictive and therapeutic target for clinical diagnosis and treatment of OSCC.     

负性刺激分子PD-L对T细胞介导小鼠L929肿瘤细胞免疫效应的影响

目的：探讨负性刺激分子在肿瘤细胞逃避免疫效应中的作用.方法：以L929细胞为靶细胞,体内致敏C57BL/6小鼠,注入PD-L1、PD-L2抗体阻断,并设立未致敏对照组.分离小鼠脾细胞,体外采用3H-TdR掺入法、荧光标记双染色FCM以及PI染色FCM法分别检测淋巴细胞增殖反应、活化诱导的细胞凋亡以及脾细胞及其培养上清对肿瘤细胞的杀伤效应.结果：L929细胞表达PD-L1,而不表达PD-L2.体内与体外联合应用PD-L1抗体,能显著增强L929肿瘤细胞诱导的致敏组小鼠脾细胞的增殖活性和特异性杀伤效应,并可抑制活化诱导的T细胞凋亡;PD-L1抗体亦可增加L929细胞诱导的未致敏组小鼠脾细胞的增殖活性和促进其脾细胞培养上清对L929细胞的杀伤效应.结论：PD-L1抗体可通过阻断PD1/PDL途径促进初始T细胞和致敏T细胞介导的抗瘤效应.     

Tumour‐associated macrophage‐mediated survival of myeloma cells through STAT3 activation

Overcoming drug resistance is one of the greatest challenges in the treatment of multiple myeloma (MM). The interaction of myeloma cells with the bone marrow (BM) microenvironment is a major factor contributing to drug resistance. Tumour‐associated macrophages (TAMs) with different polarization states constitute an important component of this microenvironment. Previous studies have revealed a role of TAMs in MM cell survival and drug resistance; however, the impact of macrophage polarization (anti‐tumoural ‘M1’ versus pro‐tumoural ‘M2’‐like phenotype) in this process has not yet been described. Here, the presence of TAMs was confirmed in BM sections from MM patients, both at diagnosis and relapse, with two M2 markers, CD163 and CD206. By following different TAM subpopulations during disease progression in the syngeneic murine 5T33MM model, we demonstrated a decrease in the number of inflammatory monocytes and an increase in the number of M2‐oriented TAMs in BM. Co‐culture experiments demonstrated that macrophages provide a survival benefit to myeloma cells that is maintained after treatment with several classes of anti‐myeloma agent (melphalan and bortezomib); the greatest effect was observed with M2‐polarized macrophages. The pro‐survival effect was associated with activation of the STAT3 pathway in 5T33MM cells, less cleavage of caspase‐3, and thus less apoptosis. AZD1480, an ATP‐competitive JAK2 inhibitor, abrogated the observed TAM‐mediated MM cell survival, and partially inhibited resistance to bortezomib. Despite having only a small quantitative impact on myeloid cells in vivo, AZD1480 treatment alone and in combination with bortezomib significantly reduced tumour load. In conclusion, M2 TAMs are present in the MM microenvironment, and contribute to MM cell survival and protection from drug‐induced apoptosis. As a result of TAM‐induced activation of the STAT3 pathway, 5T33MM cells are sensitized to AZD1480 treatment. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.