Michael R. Shurin

Th1/Th2 balance in cancer, transplantation and pregnancy.

The carefully orchestrated events that regulate homeostasis of the immune system and the development of a protective immune response are coordinated to a large extent by cytokines produced by Thl, Th2, and the nominal Th3 lymphocyte subsets. An imbalance of Thl and Th2 may be responsible for both the occurrence as well as the progression of several diseases and their resultant complications. Patients with advanced cancer often have impaired cell-mediated immunity associated with a switch from Thl to Th2. On the other hand, shifting from one cytokine pattern to another may be highly beneficial in certain physiological conditions. For instance, IL-10, a Th2-type cytokine, may play a role in pregnancy-associated immune tolerance through the establishment of a Th2 cytokine bias at the maternal-fetal interface. Graft-versus-host disease (GVHD) is the major complication after allogeneic bone marrow transplantation (BMT) and is initiated by Thl cytokines and resultant dysregulation of the cytokine network. The balance between type 1 and type 2 cytokines governs the extent to which a cell-mediated immune response and a systemic inflammatory response develop after allogeneic BMT. Successful interventions to regulate Thl/Th2 balance and modify the immune response may thus decrease the risk of development or relapse of malignancy, avoid impairment of donor cell engraftment, and allow successful fetal maturation.

Cyanidin-3-rutinoside, a Natural Polyphenol Antioxidant, Selectively Kills Leukemic Cells by Induction of Oxidative Stress

Anthocyanins are a group of naturally occurring phenolic compounds widely available in fruits and vegetables in human diets. They have broad biological activities including anti-mutagenesis and anticarcinogenesis, which are generally attributed to their antioxidant activities. We studied the effects and the mechanisms of the most common type of anthocyanins, cyanidin-3-rutinoside, in several leukemia and lymphoma cell lines. We found that cyanidin-3-rutinoside extracted and purified from the black raspberry cultivar Jewel induced apoptosis in HL-60 cells in a dose- and time-dependent manner. Paradoxically, this compound induced the accumulation of peroxides, which are involved in the induction of apoptosis in HL-60 cells. In addition, cyanidin-3-rutinoside treatment resulted in reactive oxygen species (ROS)-dependent activation of p38 MAPK and JNK, which contributed to cell death by activating the mitochondrial pathway mediated by Bim. Down-regulation of Bim or overexpression of Bcl-2 or Bcl-xL considerably blocked apoptosis. Notably, cyanidin-3-rutinoside treatment did not lead to increased ROS accumulation in normal human peripheral blood mononuclear cells and had no cytotoxic effects on these cells. These results indicate that cyanidin-3-rutinoside has the potential to be used in leukemia therapy with the advantages of being widely available and selective against tumors.

Loss of New Chemokine CXCL14 in Tumor Tissue Is Associated with Low Infiltration by Dendritic Cells (DC), while Restoration of Human CXCL14 Expression in Tumor Cells Causes Attraction of DC Both In Vitro and In Vivo

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-κB activity. These data suggest that CXCL14 is a potent chemoattractant and activator of DC and might be involved in DC homing in vivo.

Chemotherapeutic Agents in Noncytotoxic Concentrations Increase Antigen Presentation by Dendritic Cells via an IL-12-Dependent Mechanism

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.

Bone marrow-derived dendritic cells pulsed with a tumor-specific peptide elicit effective anti-tumor immunity against intracranial neoplasms.

Although the central nervous system (CNS) is often regarded as an immunologically privileged site, it is well established that specific CNS immunoreactivity can be generated through peripheral vaccination with CNS antigens. Dendritic cells (DC) are potent antigen presenting cells of hematopoietic origin that have emerged as a promising tool for cancer immunotherapy capable of evoking significant anti‐tumor immunity when pulsed with tumor‐associated peptides. To explore a role for DC‐based immunization strategies for the treatment of CNS tumors, we developed a brain tumor model using the C3 sarcoma cell line which expresses the tumor‐specific, major histocompatibility complex (MHC) class I‐restricted peptide epitope E749–57. Syngeneic C57Bl/6 mice receiving intravenous (i.v.) injections of bone marrow‐derived DCs pulsed with E7 peptide were effectively protected against a subsequent intracerebral challenge with C3 tumor cells. More importantly, this systemic immunization strategy was effective in a therapy model as 67% of animals (10 of 15) with established (day 7) intracerebral C3 tumors treated with 3 weekly injections of E7 peptide‐pulsed DCs achieved a long‐term survival (>90 days) while no control animals survived beyond day 41. In vivo depletion of CD8+ cells, but not CD4+ or asialo‐GM1+ cells, abrogated the efficacy of E7 peptide‐pulsed DC therapy of established tumors, indicating a pivotal role of specific CD8+ T‐cell responses in mediating the anti‐tumor effect. Our findings support the hypothesis that effective CNS anti‐tumor immunoreactivity can be generated with DC‐based tumor vaccines. Int. J. Cancer 78:196–201, 1998.© 1998 Wiley‐Liss, Inc.

Intratumoral cytokines/chemokines/growth factors and tumor infiltrating dendritic cells: friends or enemies?

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.

Dendritic Cells in the Cancer Microenvironment

The complexity of the tumor immunoenvironment is underscored by the emergence and discovery of different subsets of immune effectors and regulatory cells. Tumor-induced polarization of immune cell differentiation and function makes this unique environment even more intricate and variable. Dendritic cells (DCs) represent a special group of cells that display different phenotype and activity at the tumor site and exhibit differential pro-tumorigenic and anti-tumorigenic functions. DCs play a key role in inducing and maintaining the antitumor immunity, but in the tumor environment their antigen-presenting function may be lost or inefficient. DCs might be also polarized into immunosuppressive/tolerogenic regulatory DCs, which limit activity of effector T cells and support tumor growth and progression. Although various factors and signaling pathways have been described to be responsible for abnormal functioning of DCs in cancer, there are still no feasible therapeutic modalities available for preventing or reversing DC malfunction in tumor-bearing hosts. Thus, better understanding of DC immunobiology in cancer is pivotal for designing novel or improved therapeutic approaches that will allow proper functioning of DCs in patients with cancer.

Dendritic cells presenting tumor antigen.

Abstract Since the first identification of dendritic cells by Steinman and Cohn in 1973, progress in understanding their biology has included the development of novel methods of cell culture, recognition of critical aspects of migration and maturation, and appreciation of their major role as antigen-presenting cells (APC), and how this activity is regulated by cytokines and expression of accessory molecules. Dendritic cells are the major APC involved in the initiation of the immune response and the development of tolerance. There is considerable evidence that they can acquire antigen in the peripheral tissues and process, transport, and present it to T cells in secondary lymphoid tissue. A number of studies show that, in vitro or in vivo, antigen-pulsed dendritic cells can directly sensitize T cells and stimulate the development of antigen-specific immune responses, including both protective and therapeutic antitumor responses. In this paper, several important aspects of dendritic cell biology are discussed and a number of studies confirming the role of these professional APC in antitumor immunity are reviewed.

Tumor's other immune targets: dendritic cells.

The induction of apoptosis in T cells is one of several mechanisms by which tumors escape immune recognition. We have investigated whether tumors induce apoptosis in dendritic cells (DC) by co‐culture of murine or human DC with different tumor cell lines for 4–48 h. Analysis of DC morphological features, JAM assay, TUNEL, caspase‐3‐like and transglutaminase activity, Annexin V binding, and DNA fragmentation assays revealed a time‐ and dose‐dependent induction of apoptosis in DC by tumor‐derived factors. This finding is both effector and target specific. The mechanism of tumor‐induced DC apoptosis involved regulation of Bcl‐2 and Bax expression. Double staining of both murine and human tumor tissues confirmed that tumor‐associated DC undergo apoptotic death in vivo. DC isolated from tumor tissue showed significantly higher levels of apoptosis as determined by TUNEL assay when compared with DC isolated from spleen. These findings demonstrate that tumors induce apoptosis in DC and suggest a new mechanism of tumor escape from immune recognition. DC protection from apoptosis will lead to improvement of DC‐based immunotherapies for cancer and other immune diseases. J. Leukoc. Biol. 66: 336–344; 1999.

Antitumor Effect of Paclitaxel Is Mediated by Inhibition of Myeloid-Derived Suppressor Cells and Chronic Inflammation in the Spontaneous Melanoma Model

The antitumor effects of paclitaxel are generally attributed to the suppression of microtubule dynamics resulting in defects in cell division. New data demonstrated that in ultralow noncytotoxic concentrations, paclitaxel modulated in immune cells in vitro the activity of small Rho GTPases, the key regulators of intracellular actin dynamics. However, the immunomodulatory properties of paclitaxel in vivo have not been evaluated. In this study, using the ret transgenic murine melanoma model, which mimics human cutaneous melanoma, we tested effects of ultralow noncytotoxic dose paclitaxel on functions of myeloid-derived suppressor cells (MDSCs), chronic inflammatory mediators, and T cell activities in the tumor microenvironment in vivo. Administration of paclitaxel significantly decreased accumulation and immunosuppressive activities of tumor-infiltrating MDSCs without alterations of the bone marrow hematopoiesis. This was associated with the inhibition of p38 MAPK activity, TNF-α and production, and S100A9 expression in MDSCs. The production of mediators of chronic inflammation in the tumor milieu also was diminished. Importantly, reduced tumor burden and increased animal survival upon paclitaxel application was mediated by the restoration of CD8 T cell effector functions. We suggest that the ability of paclitaxel in a noncytotoxic dose to block the immunosuppressive potential of MDSCs in vivo represents a new therapeutic strategy to downregulate immunosuppression and chronic inflammation in the tumor microenvironment for enhancing the efficacy of concomitant anticancer therapies.