Search

The members of the TGFβ superfamily play a key role in regulating developmental and homeostasis programs by controlling differentiation, proliferation, polarization, and survival of different cell types. Although the role of TGFβ1 in inflammation and immunity is well evident, the contribution of other TGFβ family cytokines in the modulation of the antitumor immune response remains less documented. Here we show that activin A triggers SMAD2 and ERK1/2 pathways in dendritic cells (DC) expressing type I and II activin receptors, and upregulates production of the TNFα family cytokines BAFF (TALL-1, TNFSF13B) and APRIL (TALL-2, TNFSF13A), which is blocked by SMAD2 and ERK1/2 inhibitors, respectively. BAFF and APRIL derived from activin A-treated DCs upregulate proliferation and survival of T cells expressing the corresponding receptors, BAFF-R and TACI. In vivo, activin A-stimulated DCs demonstrate a significantly increased ability to induce tumor-specific CTLs and inhibit the growth of melanoma and lung carcinoma, which relies on DC-derived BAFF and APRIL, as knockdown of the BAFF and APRIL gene expression in activin A-treated DCs blocks augmentation of their antitumor potential. Although systemic administration of activin A, BAFF, or APRIL for the therapeutic purposes is not likely due to the pluripotent effects on malignant and nonmalignant cells, our data open a novel opportunity for improving the efficacy of DC vaccines. In fact, a significant augmentation of the antitumor activity of DC pretreated with activin A and the proven role of DC-derived BAFF and APRIL in the induction of antitumor immunity in vivo support this direction. Cancer Res; 76(17); 4959-69. ©2016 AACR., Competing Interests: The authors state that there is no an actual, potential, or perceived conflict of interest with regard to the manuscript submitted for review., (©2016 American Association for Cancer Research.)

Dendritic cells (DC) play unique and diverse roles in the tumor occurrence, development, progression and response to therapy. First of all, DC can actively uptake tumor-associated antigens, process them and present antigenic peptides to T cells inducing and maintaining tumor-specific T cell responses. DC interaction with different immune effector cells may also support innate antitumor immunity, as well as humoral responses also known to inhibit tumor development in certain cases. On the other hand, DC are recruited to the tumor site by specific tumor-derived and stroma-derived factors, which may also impair DC maturation, differentiation and function, thus resulting in the deficient formation of antitumor immune response or development of DC-mediated tolerance and immune suppression. Identification of DC-stimulating and DC-suppressing/polarizing factors in the tumor environment and the mechanism of DC modulation are important for designing effective DC-based vaccines and for recovery of immunodeficient resident DC responsible for maintenance of clinically relevant antitumor immunity in patients with cancer. DC-targeting tumor-derived factors and their effects on resident and administered DC in the tumor milieu are described and discussed in this review.

Protumorigenic activity of immune regulatory cells has been proven to play a major role in precluding immunosurveillance and limiting the efficacy of anticancer therapies. Although several approaches have been offered to deplete myeloid-derived suppressor cells (MDSC) and regulatory T cells, there are no data on how to control suppressive dendritic cell (DC) accumulation or function in the tumor environment. Although immunosuppressive function of DC in cancer was implicated to immature and plasmacytoid DC, details of how conventional DC (cDC) develop immunosuppressive properties remain less understood. Here, we show that the development of lung cancer in mice was associated with fast accumulation of regulatory DC (regDC) prior to the appearance of MDSC. Using the in vitro and in vivo approaches, we demonstrated that (i)both cDC and MDSC could be polarized into protumor regDC in the lung cancer environment; (ii) cDC → regDC polarization was mediated by the small Rho GTPase signaling, which could be controlled by noncytotoxic doses of paclitaxel; and (iii) prevention of regDC appearance increased the antitumor potential of DC vaccine in lung cancer. These findings not only bring new players to the family of myeloid regulatory cells and provide new targets for cancer therapy, but offer novel insights into the immunomodulatory capacity of chemotherapeutic agents used in low, noncytotoxic doses., (© 2013 UICC.)

Graphene oxide (GO) and C60 - or C60 -TRIS fullerenes, internalized by murine dendritic cells (DCs), differently affect their abilities to present antigens to T-cells. While C60 -fullerenes stimulate the ovalbumin-specific MHC class I-restricted T-cell response, GO impairs the stimulatory potential of DCs. In contrast to C60 -fullerenes, GO decreases the intracellular levels of LMP7 immunoproteasome subunits required for processing of protein antigens. This is important for the development of DC-based vaccines., (Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Immune effector and regulatory cells in the tumor microenvironment are key factors in tumor development and progression as the pathogenesis of cancer vitally depends on the multifaceted interactions between various microenvironmental stimuli provided by tumor-associated immune cells. Immune regulatory cells participate in all stages of cancer development from the induction of genomic instability to the maintenance of intratumoral angiogenesis, proliferation and spreading of malignant cells, and formation of premetastatic niches in distal tissues. Dendritic cells in the tumor microenvironment serve as a double-edged sword and, in addition to initiating potent anti-tumor immune responses, may mediate genomic damage, support neovascularization, block anti-tumor immunity and stimulate cancerous cell growth and spreading. Regulatory dendritic cells in cancer may directly and indirectly maintain antigen-specific and non-specific T cell unresponsiveness by controlling T cell polarization, MDSC and Treg differentiation and activity, and affecting specific microenvironmental conditions in premalignant niches. Understanding the mechanisms involved in regulatory dendritic cell polarization and operation and revealing pharmacological means for harnessing these pathways will provide additional opportunities for modifying the tumor microenvironment and improving the efficacy of different therapeutic approaches to cancer., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Myeloid cells play a key role in the outcome of anti-tumor immunity and response to anti-cancer therapy, since in the tumor microenvironment they may exert both stimulatory and inhibitory pressures on the proliferative, angiogenic, metastatic, and immunomodulating potential of tumor cells. Therefore, understanding the mechanisms of myeloid regulatory cell differentiation is critical for developing strategies for the therapeutic reversal of myeloid derived suppressor cell (MDSC) accumulation in the tumor-bearing hosts. Here, using an in vitro model system, several potential mechanisms of the direct effect of paclitaxel on MDSC were tested, which might be responsible for the anti-tumor potential of low-dose paclitaxel therapy in mice. It was hypothesized that a decreased level of MDSC in vivo after paclitaxel administration might be due to (i) the blockage of MDSC generation, (ii) an induction of MDSC apoptosis, or (iii) the stimulation of MDSC differentiation. The results revealed that paclitaxel in ultra-low concentrations neither increased MDSC apoptosis nor blocked MDSC generation, but stimulated MDSC differentiation towards dendritic cells. This effect of paclitaxel was TLR4-independent since it was not diminished in cell cultures originated from TLR4-/- mice. These results support a new concept that certain chemotherapeutic agents in ultra-low non-cytotoxic doses may suppress tumor progression by targeting several cell populations in the tumor microenvironment, including MDSC.

The tumor microenvironment is a pivotal factor in tumorigenesis, and especially in progression, as the pathogenesis of cancer critically depends on the complex interactions between various microenvironmental components. A key component of the tumor immunoenvironment is the infiltration of immune cells, which has been proven to play a dual role in tumor growth and progression. This Janus two-faced function of the tumor immunoenvironment is seen in tumor infiltration by T cells, which correlates with improved patient survival, but also with the homing of multiple subsets of immunoregulatory cells that inhibit the antitumor immune response. Regulatory dendritic cells (regDCs) have recently been shown to be induced by tumor-derived factors and represent a new and potentially important player in supporting tumor progression and suppressing the development of antitumor immune responses. Our recent data reveal that different tumor cell lines produce soluble factors that induce polarization of conventional DCs into regDCs, both in vitro and in vivo. These regDCs can suppress the proliferation of pre-activated T cells and are phenotypically and functionally different from their precursors as well as the classical immature conventional DCs. Understanding the biology of regDCs and the mechanisms of their formation in the tumor immunoenvironment will provide a new therapeutic target for re-polarizing protumorigenic immunoregulatory cells into proimmunogenic effector cells able to induce and support effective antitumor immunity.

Pharyngeal aspiration of single-walled carbon nanotubes (SWCNTs) caused inflammation, pulmonary damage, and an altered cytokine network in the lung. Local inflammatory response in vivo was accompanied by modified systemic immunity as documented by decreased proliferation of splenic T cells. Preincubation of naïve T cells in vitro with SWCNT-treated dendritic cells reduced proliferation of T cells. Our data suggest that in vivo exposure to SWCNT modifies systemic immunity by modulating dendritic cell function.

Background: We have recently reported that chemotherapeutic agents in ultra low noncytotoxic concentrations may block the ability of tumor cells to suppress functional activation of dendritic cells (DCs)., Methods: HCT-116 human colon cancer cells were treated with 0.5 nM paclitaxel (PAC) or 2 nM doxorubicin (DOX) with the aim of defining the immunogenic changes induced by ultra low noncytotoxic concentrations of antineoplastic chemotherapeutic agents. Genetic alterations were screened by DNA microarray that revealed increased expression of genes involved in antigen processing and presentation, including the heat-shock protein, calmodulin, and proteasome 26 genes. As the proteins encoded by these genes are involved in the cytosolic route of antigen processing machinery, we next evaluated whether PAC and DOX in noncytotoxic concentrations changed expression of MHC class I antigen processing machinery (APM) components in three different colon cancer cell lines., Results: Our results showed that PAC and DOX increased the intracellular expression of APM proteins, including calmodulin, LMP2, LMP7, TAP1 and tapasin. The biological significance of modulation of antigen processing and presentation proteins in tumor cells by ultra low nontoxic concentrations of chemotherapeutic drugs was revealed when non-treated and treated tumor cells were used as a source of tumor antigens for the generation of tumor-specific cytotoxic T cells (CTLs) in vitro. We demonstrated that (i) DCs that engulf tumor cells pretreated with noncytotoxic concentrations of chemotherapeutic agents induced CTLs with a higher cytotoxic potential than DCs loaded with nontreated tumor cells, and (ii) CTLs induced by tumor lysate-pulsed DCs killed live tumor cells more efficiently if these tumor cells were pretreated with noncytotoxic concentrations of chemotherapeutic drugs., Conclusions: These results demonstrate that chemomodulation of human tumor cells with noncytotoxic concentrations of chemotherapeutic agents increases tumor immunogenicity and results in the generation of more efficient DC vaccines and CTLs, which can be used for cell-based anticancer immunotherapies.

The dose-delivery schedule of conventional chemotherapy, which determines its efficacy and toxicity, is based on the maximum tolerated dose. This strategy has lead to cure and disease control in a significant number of patients but is associated with significant short-term and long-term toxicity. Recent data demonstrate that moderately low-dose chemotherapy may be efficiently combined with immunotherapy, particularly with dendritic cell (DC) vaccines, to improve the overall therapeutic efficacy. However, the direct effects of low and ultra-low concentrations on DCs are still unknown. Here we characterized the effects of low noncytotoxic concentrations of different classes of chemotherapeutic agents on human DCs in vitro. DCs treated with antimicrotubule agents vincristine, vinblastine, and paclitaxel or with antimetabolites 5-aza-2-deoxycytidine and methotrexate, showed increased expression of CD83 and CD40 molecules. Expression of CD80 on DCs was also stimulated by vinblastine, paclitaxel, azacytidine, methotrexate, and mitomycin C used in low nontoxic concentrations. Furthermore, 5-aza-2-deoxycytidine, methotrexate, and mitomycin C increased the ability of human DCs to stimulate proliferation of allogeneic T lymphocytes. Thus, our data demonstrate for the first time that in low noncytotoxic concentrations chemotherapeutic agents do not induce apoptosis of DCs, but directly enhance DC maturation and function. This suggests that modulation of human DCs by noncytotoxic concentrations of antineoplastic drugs, i.e. chemomodulation, might represent a novel approach for up-regulation of functional activity of resident DCs in the tumor microenvironment or improving the efficacy of DCs prepared ex vivo for subsequent vaccinations.

Antineoplastic chemotherapeutic agents may indirectly activate dendritic cells (DCs) by inducing the release of "danger" signals from dying tumor cells. Whereas the direct cytotoxic or inhibitory effect of conventional chemotherapy on DCs has been reported, modulation of DC function by chemotherapeutic agents in low noncytotoxic concentrations has not yet been investigated. We have tested the effects of different classes of antineoplastic chemotherapeutic agents used in low noncytotoxic concentrations on the Ag-presenting function of DCs. We revealed that paclitaxel, doxorubicin, mitomycin C, and methotrexate up-regulated the ability of DCs to present Ags to Ag-specific T cells. Stimulation of DC function was associated with the up-regulation of expression of Ag-processing machinery components and costimulatory molecules on DCs, as well as increased IL-12p70 expression. However, the ability of DCs treated with paclitaxel, methotrexate, doxorubicin, and vinblastine to increase Ag presentation to Ag-specific T cells was abolished in DCs generated from IL-12 knockout mice, indicating that up-regulation of Ag presentation by DCs is IL-12-dependent and mediated by the autocrine or paracrine mechanisms. At the same time, IL-12 knockout and wild-type DCs demonstrated similar capacity to up-regulate OVA presentation after their pretreatment with low concentrations of mitomycin C and vincristine, suggesting that these agents do not utilize IL-12-mediated pathways in DCs for stimulating Ag presentation. These findings reveal a new mechanism of immunopotentiating activity of chemotherapeutic agents-a direct immunostimulatory effect on DCs (chemomodulation)-and thus provide a strong rationale for further assessment of low-dose chemotherapy given with DC vaccines for cancer treatment.

Introduction: Suppression of dendritic cells (DCs) is a crucial mechanism by which tumor cells escape immune recognition and elimination. We have recently reported that MHC class I antigen processing machinery (APM) component expression in human DCs is down-regulated by tumor-derived gangliosides. However, the molecular mechanisms underlying this abnormality were not identified. Thus, the aim of this work was to analyze the role of interferon regulatory factor 8 (IRF-8) in APM protein expression and the antigen presenting capacity of DCs developed in the tumor microenvironment., Results: We demonstrate that the expression of several MHC class I APM components, including delta, MB-1, LMP-10, ERp57, and tapasin, is significantly decreased in murine DCs generated in the presence of prostate cancer cells. APM component down-regulation was associated with decreased ability of DCs to present model antigen to antigen-specific T cells. Notable, impaired antigen-presenting activity of DCs co-cultured with tumor cells was accompanied by decreased levels of IRF-8. Transduction of DCs with the silencing RNA for the IRF-8 gene also led to reduced expression of APM components in DCs and decreased antigen presenting function., Conclusion: Together, our data suggest that tumor-induced inhibition of antigen processing and presenting function of DCs is mediated by IRF-8, a member of the interferon regulatory factor family. These results provide a new molecular target for optimizing the generation of efficient DC vaccines for cancer therapy.

Dendritic cells (DC) loaded with tumor antigens from apoptotic/necrotic tumor cells are commonly used as vaccines for cancer therapy. However, the use of dead tumor cells may cause both tolerance and immunity, making the effect of vaccination unpredictable. To deliver live tumor "cargoes" into DC, we developed a new approach based on the "labeling" of tumors with a phospholipid "eat-me" signal, phosphatidylserine. Expression of phosphatidylserine on live tumor cells mediated their recognition and endocytosis by DC resulting in the presentation of tumor antigens to antigen-specific T cells. In mice, topical application of phosphatidylserine-containing ointment over melanoma induced tumor-specific CTL, local and systemic antitumor immunity, and inhibited tumor growth. Thus, labeling of tumors with phosphatidylserine is a promising strategy for cancer immunotherapy.

Conventional chemotherapy targets dividing tumor cells and might support antitumor immunity by providing tumor antigens from dying tumor cells to antigen-presenting dendritic cells (DCs). Despite emerging evidence to suggest that phagocytosis of dying tumor cells by DCs requires membrane targeting of specific small Rho guanosine triphosphatases (GTPases), nothing is known with regard to the direct effect of chemotherapeutic agents on low molecular weight Rho GTPases in DCs. Prompted by a recent observation that low-dose chemotherapeutic drug paclitaxel could up-regulate DC maturation and function, here we studied putative regulatory roles for various chemotherapeutic agents in modulating small Rho GTPases in DC. Our results demonstrate that different classes of chemotherapeutic drugs at low nontoxic concentrations regulate activity of Rac, RhoA, and RhoE in murine DC, suggesting that small Rho GTPases might serve as new targets for modulating functional activity of DC vaccines or endogenous DCs in various immunotherapeutic or chemoimmunotherapeutic strategies.

The immune system shows a decline in responsiveness to antigens both with aging, as well as in the presence of tumors. The malfunction of the immune system with age can be attributed to developmental and functional alterations in several cell populations. Previous studies have shown defects in humoral responses and abnormalities in T cell function in aged individuals, but have not distinguished between abnormalities in antigen presentation and intrinsic T cell or B cell defects in aged individuals. Dendritic cells (DC) play a pivotal role in regulating immune responses by presenting antigens to naïve T lymphocytes, modulating Th1/Th2/Th3/Treg balance, producing numerous regulatory cytokines and chemokines, and modifying survival of immune effectors. DC are receiving increased attention due to their involvement in the immunobiology of tolerance and autoimmunity, as well as their potential role as biological adjuvants in tumor vaccines. Recent advances in the molecular and cell biology of different DC populations allow for addressing the issue of DC and aging both in rodents and humans. Since DC play a crucial role in initiating and regulating immune responses, it is reasonable to hypothesize that they are directly involved in altered antitumor immunity in aging. However, the results of studies focusing on DC in the elderly are conflicting. The present review summarizes the available human and experimental animal data on quantitative and qualitative alterations of DC in aging and discusses the potential role of the DC system in the increased incidence of cancer in the elderly.

Purpose: The main goal of this study was to provide the "proof-of-principle" that low-dose paclitaxel is able to change the tumor microenvironment and improve the outcome of intratumoral dendritic cell vaccine in a murine lung cancer model., Experimental Design: We evaluated the antitumor potential and changes in the intratumoral milieu of a combination of low-dose chemotherapy and dendritic cell vaccine in the Lewis lung carcinoma model in vivo., Results: The low-dose paclitaxel, which induced apoptosis in approximately 10% of tumor cells, was not toxic to bone marrow cells and dendritic cells and stimulated dendritic cell maturation and function in vitro. Although tumor cells inhibited dendritic cell differentiation in vitro, this immunosuppressive effect was abrogated by the pretreatment of tumor cells with low-dose paclitaxel. Based on these data, we next tested whether pretreatment of tumor-bearing mice with low-dose paclitaxel in vivo would improve the antitumor potential of dendritic cell vaccine administered intratumorally. Significant inhibition of tumor growth in mice treated with low-dose paclitaxel plus intratumoral dendritic cell vaccine, associated with increased tumor infiltration by CD4(+) and CD8(+) T cells and elevated tumor-specific IFN-gamma production by draining lymph node cells, was revealed. Using a novel intratumoral microdialysis technique and Luminex technology for collecting and characterizing soluble factors released within the tumor bed for several days in live freely moving animals, we showed that low-dose paclitaxel altered the cytokine network at the tumor site., Conclusions: Our data indicate that low-dose chemotherapy before intratumoral delivery of dendritic cells might be associated with beneficial alterations of the intratumoral microenvironment and thus support antitumor immunity.

The generation, maturation, and function of dendritic cells (DC) have been shown to be markedly compromised in the tumor microenvironment in animals and humans. However, the molecular mechanisms and intracellular pathways involved in the regulation of the DC system in cancer are not yet fully understood. Recently, we have reported on the role of the small Rho GTPase family members Cdc42, Rac1, and RhoA in regulating DC adherence, motility, and Ag presentation. To investigate involvement of small Rho GTPases in dysregulation of DC function by tumors, we next evaluated how Cdc42, Rac1, and RhoA regulated endocytic activity of DC in the tumor microenvironment. We revealed a decreased uptake of dextran 40 and polystyrene beads by DC generated in the presence of different tumor cell lines, including RM1 prostate, MC38 colon, 3LL lung, and B7E3 oral squamous cell carcinomas in vitro and by DC prepared from tumor-bearing mice ex vivo. Impaired endocytic activity of DC cocultured with tumor cells was associated with decreased levels of active Cdc42 and Rac1. Transduction of DC with the dominant negative Cdc42 and Rac1 genes also led to reduced phagocytosis and receptor-mediated endocytosis. Furthermore, transduction of DC with the constitutively active Cdc42 and Rac1 genes restored endocytic activity of DC that was inhibited by the tumors. Thus, our results suggest that tumor-induced dysregulation of endocytic activity of DC is mediated by reduced activity of several members of the small Rho GTPase family, which might serve as new targets for improving the efficacy of DC vaccines.

Tumor produces a number of immunosuppressive factors that block maturation of dendritic cells (DCs). Here, we demonstrated that endogenous factors presented in the serum of patients with prostate cancer (CaP) inhibited the generation of functionally active DCs from CD14+ monocytes in vitro. We have shown a significant inhibitory potential of serum obtained from patients with CaP and benign prostate hyperplasia benign prostatic hyperplasia (BPH) when compared with serum from healthy volunteers. As assessed by flow cytometry, expression of CD83, CD86, and CD40 molecules was strongly inhibited by CaP and BPH serum. In addition, these DCs were weak stimulators of allogeneic T cell proliferation when compared with DCs produced in the presence of healthy volunteer serum. Statistical analysis of the results revealed a positive relationship between the inhibition of expression of DC markers CD83 and CD80 and the levels of serum-free prostate-specific antigen (PSA). These data suggest that the DC system may be impaired in CaP patients.

The tumor microenvironment consists of a variable combination of tumor cells, stromal fibroblasts, endothelial cells and infiltrating leukocytes, such as macrophages, T lymphocytes, and dendritic cells. A variety of cytokines, chemokines and growth factors are produced in the local tumor environment by different cells accounting for a complex cell interaction and regulation of differentiation, activation, function and survival of multiple cell types. The interaction between cytokines, chemokines, growth factors and their receptors forms a comprehensive network at the tumor site, which is primary responsible for overall tumor progression and spreading or induction of antitumor immune responses and tumor rejection. Although the general thought is that dendritic cells are among the first cells migrating to the tumor site and recognizing tumor cells for the induction of specific antitumor immunity, the clinical relevance of dendritic cells at the site of the tumor remains a matter of debate regarding their role in the generation of successful antitumor immune responses in human cancers. While several lines of evidence suggest that intratumoral dendritic cells play an important role in antitumor immune responses, understanding the mechanisms of dendritic cell/tumor cell interaction and modulation of activity and function of different dendritic cell subtypes at the tumor site is incomplete. This review is limited to discussing the role of intratumoral cytokine network in the understanding immunobiology of tumor-associated dendritic cells, which seems to possess different regulatory functions at the tumor site.

We have recently reported that MHC class I Ag-processing machinery (APM) component expression in dendritic cells (DC) might be down-regulated by tumor cells. However, the tumor-derived factors responsible for inhibition of the APM component expression in DC generated in the tumor microenvironment as well as potential protective mechanism have not yet been investigated. In this article, we demonstrate that expression of several MHC class I APM components, including MB1 (beta5), LMP2, LMP7, LMP10, and ERp57, is significantly down-regulated in human DC generated in the presence of primary oral squamous cell carcinoma cell lines or coincubated with purified gangliosides. Suppression of MHC class I APM component expression in DC generated in the presence of tumor cells was significantly attenuated by the inhibition of glucosyl transferase in tumor cells, suggesting that tumor-induced MHC class I APM component down-regulation in DC was mediated in part by oral squamous cell carcinoma-derived gangliosides. Furthermore, rIL-15 restored both tumor cell-induced and ganglioside-induced MHC class I APM component expression in DC, as well as their ability to present Ags to autologous Ag-specific T cells. These results demonstrate that IL-15 restores MHC class I APM component expression in DC down-regulated by tumor-derived gangliosides.

Breast and kidney-expressed chemokine (BRAK) CXCL14 is a new CXC chemokine with unknown function and receptor selectivity. The majority of head and neck squamous cell carcinoma (HNSCC) and some cervical squamous cell carcinoma do not express CXCL14 mRNA, as opposed to constitutive expression by normal oral squamous epithelium. In this study, we demonstrate that the loss of CXCL14 in HNSCC cells and at HNSCC primary tumor sites was correlated with low or no attraction of dendritic cell (DC) in vitro, and decreased infiltration of HNSCC mass by DC at the tumor site in vivo. Next, we found that recombinant human CXCL14 and CXCL14-positive HNSCC cell lines induced DC attraction in vitro, whereas CXCL14-negative HNSCC cells did not chemoattract DC. Transduction of CXCL14-negative HNSCC cell lines with the human CXCL14 gene resulted in stimulation of DC attraction in vitro and increased tumor infiltration by DC in vivo in chimeric animal models. Furthermore, evaluating the biologic effect of CXCL14 on DC, we demonstrated that the addition of recombinant human CXCL14 to DC cultures resulted in up-regulation of the expression of DC maturation markers, as well as enhanced proliferation of allogeneic T cells in MLR. Activation of DC with recombinant human CXCL14 was accompanied by up-regulation of NF-kappaB activity. These data suggest that CXCL14 is a potent chemoattractant and activator of DC and might be involved in DC homing in vivo.

Dendritic cells (DC) are involved in the regulation of innate and adaptive immunity. However, the molecular mechanisms maintaining DC function remain to be elucidated. In this study, we report on the role of small Rho GTPases: Cdc42, Rac1, and RhoA in the regulation of DC adherence, Ag presentation, migration, chemotaxis, and endocytosis. Murine DC were transfected with vaccinia virus-based constructs, encoding dominant-negative or constitutively active (ca) mutant forms of Rho GTPases. We demonstrate that Cdc42 plays a major role in the regulation of DC adhesion, because caCdc42-transfected DC had significant up-regulation of adhesion to extracellular matrix, which was blocked by the Rho GTPase inhibitor toxin B (ToxB). In contrast, caRho-transfected DC only modestly elevated DC adhesion, and caRac had no effect. Additionally, caCdc42 and caRho increased the ability of DC to present OVA peptide to specific T cells. This effect was abrogated by ToxB. Activation of Cdc42 in DC significantly inhibited spontaneous and chemokine-induced DC migration. Furthermore, uptake of dextran 40 by DC was significantly enhanced by Rho GTPase activators cytotoxic necrotizing factor 1 and PMA, and reduced by ToxB. caCdc42 also increased endocytotic activity of DC, whereas dominant-negative Cdc42 blocked it. Thus, Rho GTPases Cdc42, RhoA, and Rac1 regulate DC functions that are critical for DC-mediated immune responses in vivo.

Background: There is increasing evidence to suggest that dendritic cells (DC) are functionally impaired in tumor bearing hosts. However there is little or no data on the effects of murine prostate cancer (CaP) on DC generation from bone marrow precursors., Methods: Flow cytometry, mixed leukocyte reactions (MLR), and immunohistochemical analyses were used to characterize DC in CaP., Results: DC generated in the presence of CaP cell lines RM1 and the cell line C2 from the transgenic adenocarcinoma of the mouse prostate (TRAMP) mouse in a Transwell system expressed significantly lower levels of DC differentiation markers. This effect was confirmed when TK-neo-transfected RM1 cells were directly added to DC cultures and eliminated 5 days later using gancyclovir (GCV). Furthermore, co-incubation of DC with CaP cells resulted in a decrease in the stimulatory capacity of DC to induce T cell proliferation in the MLR assay. These results were further confirmed in vivo in two different murine models of CaP: i) DC generated from mice intrafemorally injected with TK-neo-transfected RM1 cells; and ii) in DC generated from TRAMP mice., Conclusions: The generation and function of DC are significantly suppressed in the CaP microenvironment in both in vivo and in vitro murine models., (Copyright 2004 Wileey-Liss, Inc.)

Background: Tumors develop mechanisms to escape recognition by the immune system. It has recently been demonstrated that tumors cause apoptotic death of key immune cells, including the major antigen-presenting cells, dendritic cells (DC). Elimination of DC from the tumor environment significantly diminishes development of specific immunologic responses. We have recently demonstrated that tumor-induced DC apoptosis could be prevented by overexpression of the anti-apoptotic molecule Bcl-x(L). The aim of this study was to identify extrinsic and intrinsic tumor-induced apoptotic pathways in DC by targeting different anti-apoptotic molecules, including FLIP, XIAP/hILP, dominant-negative procaspase-9 and HSP70., Methods: Murine bone marrow derived DC were transduced with adenoviral vectors carrying different anti-apoptotic molecules and co-incubated with tumor cells in a Transwell system. Apoptosis of DC was assessed by Annexin V and PI staining., Results: We have demonstrated that adenoviral infection of DC with genes encoding different anti-apoptotic molecules exhibits different degrees of resistance to melanoma-induced apoptosis. Furthermore, we have shown that anti-apoptotic molecules other than the Bcl-2 family of proteins are able to protect DC and prevent tumor-induced apoptosis in DC., Conclusions: The results show that tumor-induced apoptosis of DC is not limited to the mitochondrial pathway of cell death and open additional possibilities for targeted molecular protection of DC longevity in cancer. Therefore, effective protection of DC from tumor-induced apoptosis may significantly improve the efficacy of DC-based therapies for cancer., (Copyright 2004 John Wiley & Sons, Ltd.)

This report describes age-related alterations of dendritic cells (DC) distribution in nude athymic mice in vivo and reversal of certain age-dependent defects by an in vivo administration of hematopoietic growth factor FLT3 ligand (FLT3L). There are decreased percentages of CD11c(+) DC in the bone marrow and spleen and a reduced expression of MHC class II and CD86 molecules on DC in old nude mice. The decreased levels of CD11c(+) DC were due to the CD8alpha(-) DC subset. The distribution of CD11c(+) CD8alpha(+) DC in the lymphoid tissues was not different in young and old mice. The effect of in vivo administration of FLT3L on the generation and distribution of DC in the lymphoid tissues in young and old nude mice was also evaluated. Although, FLT3L had a higher inductive potential on the expansion of DC from the bone marrow in the elderly mice, the total level of CD11c(+) DC in the young animals was still significantly higher as compared to the old animals. Interestingly, FLT3L induced a pronounced redistribution and accumulation of MHC class II(+) DC in the lymphoid tissues in old mice, markedly increased the accumulation of CD8alpha(-) DC in the bone marrow in both young and old nude mice, and elevated both CD8alpha(-) and CD8alpha(+) DC in the spleen in young mice. However, only the level of CD8alpha(+) DC was up regulated in the spleen in old athymic mice after FLT3L-based therapy. In summary, abnormalities in DC generation and distribution in old athymic mice could be, in part, circumvented by the in vivo administration of FLT3L.

Development of tumors is regulated by tumor-derived neuroendocrine factors, including bombesin-like peptides (BLP). We have evaluated neuroendocrine regulation of dendritic cell (DC) maturation and function by both tumor-derived and purified bombesin (BOM), neuromedin B (NMB), gastrin-releasing peptide (GRP), and a BOM antagonist D-Phe-bombesin (DPB). BOM, NMB and GRP dose-dependently inhibited maturation of DC assessed as down-regulation of CD40, CD80 and CD86 expression on DC. BOM and GRP also inhibited interleukin-12 (IL-12) production by DC and their ability to activate T cells. DPB partly abrogated immunosuppressive effect of tumor cells on DC. These data are a first evidence for the role of BLP in the regulation of DC maturation and function, demonstrating that BLP inhibit DC maturation and longevity in the lung cancer microenvironment. This suggests a new mechanism of tumor escape and provides new targets for the immunopharmacological correction of immune effectors in cancer.

Purpose: Prostate specific antigen (PSA) is a serine protease produced by the prostate gland at high concentrations. Serum PSA may be significantly elevated in prostate cancer and benign prostatic diseases. It has recently become evident that, in addition to being a tissue and/or serum marker, PSA may also have biological effects. Despite the voluminous literature on this biomarker in the diagnosis of prostatic diseases relatively few reports have addressed the issue of the physiological function, biological role and immune effects of PSA in the context of prostate cancer development and progression., Materials and Methods: Human dendritic cell (DC) cultures were generated from CD34+ hematopoietic precursors in the presence of PSA. The DC phenotype was assessed by flow cytometry and DC ability to induce T-cell proliferation was detected by allogeneic mixed lymphocyte reaction assay. DCs were also generated in co-cultures with LNCaP cells in the presence of antiPSA antibodies. The concentrations of PSA in cultures were determined by the AXSYM System (Abbott Laboratories, Wiesbaden, Germany)., Results: We noted that purified and LNCaP derived PSA inhibited the generation and maturation of DC (dendropoiesis) in vitro, which might have a crucial role in the induction and regulation of specific antitumor immune responses. The addition of active PSA to DC cultures significantly inhibited the generation and maturation of DC, as assessed by the levels of expression of CD83, CD80, CD86 and HLA DR. The ability of DC to induce T-cell proliferation, which depends on the expression of co-stimulatory and major histocompatibility complex molecules, was also suppressed in PSA treated DC cultures., Conclusions: The antidendropoietic effect of PSA in vitro suggests a new mechanism of prostate cancer induced immunosuppression and tumor escape, and provides novel evidence of the immunoregulatory properties of PSA.

Multiple observations suggest that suppression of the dendritic cell (DC) system might be one of the mechanisms used by the tumors to escape immune response. However, no in vivo data are available to support these in vitro observations. Here we have shown that murine prostate cancer inhibits DC generation (dendropoiesis) from the bone marrow precursors in the in vivo model in mice injected intrafemorally with RM1 prostate adenocarcinoma cells. Phenotyping of DC, generated from in vivo RM1-treated bone marrow cells, revealed a significant inhibition of dendropoiesis assessed as a percentage of CD11c+MHCII+, CD11c+CD86+, and CD11c+CD80+ cells. The stimulatory capacity of these DCs to induce T cell proliferation was also markedly decreased. Notably, Flt3 ligand-based therapy reversed the inhibitory effects of prostate cancer on dendropoiesis in vivo in wild-type and C.B-17 SCID (T and B cell deficient) mice, but not in SCID beige (T, B, and NK cell deficient) animals, suggesting a key role of NK cells in Flt3 ligand-mediated protection of dendropoiesis from tumor-induced inhibition in vivo. Thus, these data demonstrate that prostate cancer inhibits DC hematopoietic precursors in vivo in the bone marrow and this effect could be abolished by a systemic administration of growth factor Flt3 ligand.

Colorectal cancer is one of the most common fatal malignancies in the United States, with an incidence second only to lung cancer. The liver is the most common site of colorectal metastases and frequently the only affected organ once the primary tumor has been surgically removed. The only potentially curative treatment for metastatic colorectal cancer in the liver is surgery, although most patients are not eligible for resection. We have therefore, evaluated the therapeutic efficacy of dendritic cells (DCs) engineered to express IL-12 in a liver metastasis model. Direct administration of DCs into the portal vein significantly inhibited the growth of established MC38 colon carcinoma in the liver in C57BL/6 mice. This effect was accompanied by an intratumoral accumulation of CD4+, CD8+, and NLDC-145+ immune effector cells, and also resulted in a systemic immune response as determined by enhanced production of IFN-gamma by T lymphocytes isolated from both spleen and draining lymph nodes. Evaluation of homing of Cy3-labeled DCs following the portal vein injection confirmed their distribution in the liver and lymphoid tissue. Thus, a local delivery of DCs transduced with the IL-12 gene can not only inhibit colorectal tumor growth in vivo but also mount systemic antitumor immune responses. This approach is likely to improve the outcome of immunotherapy for metastatic colorectal cancer since high numbers of tumor-associated DCs positively correlate with a more favorable prognosis. Simultaneous local gene therapy with IL-12 will further improve clinical efficacy without placing the patient at risk for systemic toxicity.

It has been recently demonstrated that dendritic cells (DC) coincubated with interleukin (IL)-15 express high levels of the Bcl-2 family of proteins and display an increased resistance to tumor-induced apoptotic death. Here, the phenotype, functions, and survival of human DC transduced with adenoviral vector encoding the human IL-15 gene were studied. The transduction of DC with the IL-15 gene resulted in a significant elevation of expression of CD83, CD86, and CD40 molecules, which was blocked by anti-IL-15 monoclonal antibodies. This effect was also accompanied by an increased production of IL-12 and stimulated ability of DC to induce T cell proliferation. Furthermore, transduction of DC with the IL-15 gene significantly increased their resistance to prostate cancer-induced apoptosis: Overexpression of IL-15 on DC blocked tumor-induced inhibition of Bcl-2 expression and prolonged DC survival after coincubation with tumor cells. Finally, overexpression of IL-15 in DC was associated with a higher level of expression of IL-15 receptor alpha chain mRNA. In summary, these results suggest that transduction of DC with the IL-15 gene markedly stimulates DC function and protects them from tumor-induced apoptosis.

As CD40 plays a key role in both antitumor immunity and DC maturation, we have studied the regulation of its expression during DC hematopoiesis (dendropoiesis) in vitro and in vivo in the tumor microenvironment. Using MC38 colon adenocarcinoma tumor models, we have demonstrated that DCs generated in vitro from bone marrow precursors obtained from tumor-bearers have significantly lower expression of CD40 molecules compared to DCs generated from tumor-free mice. Furthermore, CD40 expression on DCs isolated from the spleens of tumor-bearing mice was also significantly reduced, suggesting that tumor-derived factors inhibit CD40 expression on DCs during dendropoiesis both in vitro and in vivo. Interestingly, CD40 ligation on DCs generated from tumor-bearers did not result in inducible expression of IL-12 protein or IL-12 p40 mRNA. However, Staphylococcus aureus-induced IL-12 production by DCs was not altered in tumor-bearers, confirming that inhibition of IL-12 production by DCs generated in vitro from tumor-bearing mice was due to reduced expression of CD40 on DCs. We have also shown that MC38 tumor cells produce IL-10 and that exogenous IL-10 causes downregulation of CD40 expression on DCs. In addition, endogenous IL-10 produced by colon carcinoma cells inhibited CD40-dependent IL-12 production by DCs since tumor-induced inhibition of IL-12 production was abrogated by neutralizing anti-IL-10 antibody. Finally, systemic administration of FLT3L and/or CD40L reversed CD40 and IL-12 (p40) deficiency of DCs in tumor-bearing mice in vivo. These findings thus demonstrate that tumor-derived factors, including IL-10, inhibit CD40 expression on DCs and DC precursors and suppress their maturation and function., (Copyright 2002 Wiley-Liss, Inc.)

Dendritic cells (DCs) are important for the induction of primary T-cell responses and may serve as "biologic adjuvants" in therapeutic protocols. However, given the "plasticity" of this antigen-presenting cell, it remains unclear which DC type (source, subtype, and stage of differentiation) should be applied clinically. To provide additional insight in this selection process, we have, for the first time, analyzed the in vitro differentiation of CD34(+) precursor-derived and monocyte-derived DCs for ultrastructure, phenotype, and function. The ultrastructural intracytoplasmic differentiation of DCs correlated with increasing T-cell stimulatory activity of these cells. "Early-stage"-DCs proliferate, exhibit high levels of soluble antigen uptake, and moderate T-cell stimulatory capacity, and are characterized by centrally located nuclei and numerous enlarged mitochondria. "Intermediate-stage"-DCs are enlarged cells with enhanced T-cell stimulatory activity and pronounced cytoplasmic protein synthesis machinery. "Late-stage" (LS)-DCs exhibit a mature secretory cell phenotype and low proliferative index. They express high levels of the HLA-DR, CD40L, B7-1, and B7-2 molecules and CD83, a specific marker of mature DCs, and appear maximally stimulatory to T cells. Ultrastructurally, LS-DCs feature an accentric nucleus, an enlarged cytoplasm, containing numerous secretory storage vesicles, along with a fully developed Golgi complex. LS-DCs exhibited numerous multivesicular and multilaminar structures containing major histocompatibility complex class II molecules, consistent with the MIIC (peptide-loading) compartment. In extended studies, cultured CD14(+) monocyte-derived DCs displayed a similar, but accelerated, temporal differentiation staging pattern.