Tumoricidal Function Research Articles

Abstract 450: Suppression of innate and adaptive immune responses in the human lung tumor microenvironment via microRNA-183

Abstract Innate and adaptive immunity plays a critical role in defense against cancer but can be undermined by the tumor microenvironment. To understand the underlying mechanism, we set out to analyze the molecular and cellular basis for natural killer (NK) and CD8 T cell suppression by human lung tumor cells. We found that transforming growth factor-beta (TGFb), abundantly produced by tumor cells, effectively reduced tumoricidal function in activated NK cells and CD8 T cells by specifically targeting a key adaptor protein, DAP12, which is required for surface anchoring of multiple NK receptors, including NKp44. For proof of the TGFb-miR-183-DAP12 circuit, flow cytometric analysis demonstrated that TGFb reduced surface levels of NKp44 on activated primary human NK and CD8 T cells, but western blot analysis demonstrated their intact presence in the cytoplasm. This was due to loss of DAP12, which is critical for surface expression of NKp44 and for downstream signaling to mobilize lytic granules to kill tumor cells. Mechanistically, TGFb readily induced miR-183 expression in both primary human NK cells and T cells which could bind specifically to the 3′UTR of human DAP12 to inhibit transcription and translation. Only miR-183 but not miR-185 or control (CT) transfection repressed luciferase activity of DAP12 3′UTR luciferase reporter constructs transferred into HELA cells. Moreover, we overexpressed lentiviral miR-183 in either primary NK and T cells and found that such expression effectively reduced DAP12 levels in both cell types. Flow cytometric analysis of these same cells indicated that loss of DAP12 was accompanied by loss of NKp44 surface expression and by loss of cytotoxic function against tumor cells. As further proof, we showed that lentiviral anti-sense miR-183 transfection into NK cells and T cells prevented TGFb from suppressing DAP12 expression. Thus, we have uncovered a seminal TGFb-miR-183-DAP12 pathway utilized by tumor cells to silence infiltrating NK and T cells, constituting the first report of miR control of an adaptor protein to control both NK and T cell immune function. Citation Format: Melba Marie Tejera, Melba Marie Tejera, Sarah S. Donatelli, Jun-Min Zhou, Danielle L. Gilvary, Xianghong Chen, Erika A.. Suppression of innate and adaptive immune responses in the human lung tumor microenvironment via microRNA-183. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 450. doi:10.1158/1538-7445.AM2015-450

33 Cetuximab paying toll to improve its therapeutic efficacy – Improving cetuximab-mediated head and neck cancer treatment via co-activation of Fc-receptors and toll-like receptors

Cetuximab, a therapeutic antibody used to treat Head and Neck Squamous Cell Carcinoma (HNSCC), blocks binding of epidermal growth factor to its receptor (EGFR) on tumor cells, thereby inhibiting tumor outgrowth. However, antibodies can also activate anti-tumor immune responses through binding of Fcgamma-receptors (FcgRs) on effector cells. In this way, immune effector cells bind to tumor cells, become activated and execute their tumoricidal functions. NK cells, for instance, can eliminate tumors via release of toxic components whereas macrophages phagocytose and kill tumor cells intracellularly. The immune suppressive tumor microenvironment severely hampers the efficacy of therapeutic antibodies like cetuximab to induce adequate anti-tumor responses via patients’ own immune cells. We recently demonstrated that simultaneous activation of FcgRs and pathogen recognizing Toll-like receptors (TLRs) on immune cells induces strong and profound inflammatory responses. Therefore, our aim is to investigate whether co-activation of TLRs during cetuximab treatment overrules the immune suppressive environment inflicted by the tumor. We performed cytotoxicity assays with HNSCC patient-derived tumor cell lines and cetuximab with or without Toll-like receptor co-activation. We tested these treatments with several types of effector immune cells. Furthermore, we investigated changes in cytokine profiles of HNSCC cell lines treated with immune effector cells and cetuximab with or without Toll-like receptor co-activation. We now demonstrate that cetuximab in combination with Toll-like receptor co-activation improves tumoricidal functions of immune effector cells. Furthermore, we demonstrate that this improved capacity to eliminate tumor cells is linked with induction of a pro-inflammatory cytokine profile. Based on our results we anticipate that cetuximab treatment in combination with Toll-like receptor co-activation will overcome the tumor immunosuppressive environment of HNSCC and thus positively affects tumor killing capacity of immune effector cells. This novel strategy may improve current cetuximab treatment of patients with HNSCC.

Artesunate possesses anti-leukemia properties that can be enhanced by arsenic trioxide

Artesunate (ART), an effective and safe anti-malaria drug, also exhibits anticancer activity. We studied the effects of ART on proliferation and apoptosis of human K562 and U937 leukemia cell lines. MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay demonstrated that ART inhibits cell growth in a dose- and time-dependent manner. Based on the findings obtained from light, fluorescence and transmission electron microscopy and propidium iodide staining, the effect of ART was shown to be mediated through apoptosis. In parallel, ART treatment increased Fas expression, while it decreased the c-Fos level in K562 cells. Furthermore, co-treatment with arsenic trioxide (ATO) significantly facilitated ART-induced K562 cell apoptosis. These findings demonstrated that ART had an antitumor activity against K562 and U937 leukemia cells, largely due to inhibition of proliferation and induction of apoptosis via the intrinsic pathway; and this tumoricidal function could be enhanced by ATO.

1: Chemokine networks regulate the balance of pro-and anti-tumor granulocytes that determine metastatic progression in breast cancer models

Cancer progression is a complex process that involves tumor growth at the primary site coupled with dissemination of cancer cells to vital organs and outgrowth of metastasis. Majority of the cancer-related deaths are due to metastatic outgrowth commonly occurring in the bone, lung, liver and brain. Although progress has been made in deciphering the process of tumor initiation and primary tumor growth, the molecular underpinnings and mechanisms of metastasis remain poorly understood. Given the role of the tumor microenvironment in cancer progression, there has been a growing interest in the function of myeloid cells, an abundant component of the peripheral blood and tumor microenvironment. Using multiple models of breast cancer metastasis, we uncovered a network of paracrine signals between carcinoma and bone marrow derived granulocytic myeloid cells that drive metastasis in breast cancer. We have recently identified two distinct subpopulations of CD11b + Ly6G + granulocytic myeloid cells by RNA sequencing, proteomic profiling and functional analysis with contrasting roles in metastasis. The first subpopulation is abundant early in the peripheral blood before any detectable metastasis. Adoptive transfer of the early population prior to tumor cell injection attenuates metastatic seeding. A second subpopulation is abundant late in the peripheral blood coinciding with systemic metastasis, which promotes metastatic colonization upon adoptive transfer. Additionally, we have identified specific signals and cues that entrain and recruit these two types of granulocytic cells in the tumor microenvironment. We find that CXCL1/2 chemokines from metastatic cells attract pro-tumor CD11b + Ly6G + granulocytic myeloid cells, which in turn produce survival factors called S100A8/9 that promotes metastatic progression. CCL2 on the other hand is involved in entraining the early granulocytic population to perform tumoricidal functions in early stages of metastasis. We envision that a delicate balance exists between the two granulocytic populations that function as a determinant of metastatic progression.

Extracts of Crinum latifolium inhibit the cell viability of mouse lymph oma cell line EL4 and induce activation of anti-tumour activity of macrophages in vitro

Ethnopharmacological relevanceCrinum latifolium L. (CL) leaf extracts have been traditionally used in Vietnam and are now used all over the world for the treatment of prostate cancer. However, the precise cellular mechanisms of the action of CL extracts remain unclear. Aim of the studyTo examine the effects of CL samples on the anti-tumour activity of peritoneal murine macrophages. Materials and methodsThe properties of three extracts (aqueous, flavonoid, alkaloid), one fraction (alkaloid), and one pure compound (6-hydroxycrinamidine) obtained from CL, were studied (i) for redox capacities (DPPH and bleaching beta-carotene assays), (ii) on murine peritoneal macrophages (MTT assay) and on lymphoma EL4-luc2 cells (luciferine assay) for cytotoxicity, (iii) on macrophage polarization (production of ROS and gene expression by PCR), and (iv) on the tumoricidal functions of murine peritoneal macrophages (lymphoma cytotoxicity by co-culture with syngeneic macrophages). ResultsThe total flavonoid extract with a high antioxidant activity (IC50=107.36mg/L, DPPH assay) showed an inhibitory action on cancer cells. Alkaloid extracts inhibited the proliferation of lymphoma cells either by directly acting on tumour cells or by activating of the tumoricidal functions of syngeneic macrophages. The aqueous extract induced mRNA expression of tumour necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and interleukin 6 (IL-6) indicating differentiation of macrophages into pro-inflammatory M1 polarized macrophages. The total flavonoid, alkaloid extracts and an alkaloid fraction induced the expression of the formyl peptide receptor (FPR) on the surface of the polarized macrophages that could lead to the activation of macrophages towards the M1 phenotype. Aqueous and flavonoid extracts enhanced NADPH quinine oxido-reductase 1 (NQO1) mRNA expression in polarized macrophages which could play an important role in cancer chemoprevention. All the samples studied were non-toxic to normal living cells and the pure alkaloid tested, 6-hydroxycrinamidine, was not active in any of the models investigated. ConclusionsOur results indicate that CL extracts and alkaloid fraction (but not pure 6-hydroxycrinamidine) inhibit the proliferation of lymphoma cells in multiple pathways. Our results are in accordance with traditional usage and encourage further studies and in vivo assays.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

IgG4 subclass antibodies impair antitumor immunity in melanoma

Host-induced antibodies and their contributions to cancer inflammation are largely unexplored. IgG4 subclass antibodies are present in IL-10-driven Th2 immune responses in some inflammatory conditions. Since Th2-biased inflammation is a hallmark of tumor microenvironments, we investigated the presence and functional implications of IgG4 in malignant melanoma. Consistent with Th2 inflammation, CD22+ B cells and IgG4(+)-infiltrating cells accumulated in tumors, and IL-10, IL-4, and tumor-reactive IgG4 were expressed in situ. When compared with B cells from patient lymph nodes and blood, tumor-associated B cells were polarized to produce IgG4. Secreted B cells increased VEGF and IgG4, and tumor cells enhanced IL-10 secretion in cocultures. Unlike IgG1, an engineered tumor antigen-specific IgG4 was ineffective in triggering effector cell-mediated tumor killing in vitro. Antigen-specific and nonspecific IgG4 inhibited IgG1-mediated tumoricidal functions. IgG4 blockade was mediated through reduction of FcγRI activation. Additionally, IgG4 significantly impaired the potency of tumoricidal IgG1 in a human melanoma xenograft mouse model. Furthermore, serum IgG4 was inversely correlated with patient survival. These findings suggest that IgG4 promoted by tumor-induced Th2-biased inflammation may restrict effector cell functions against tumors, providing a previously unexplored aspect of tumor-induced immune escape and a basis for biomarker development and patient-specific therapeutic approaches.

Differential Interaction of Bovine Alpha-Lactalbumin with Membranes: Interplay of Negatively Charged Lipids and Cholesterol

Many soluble proteins are known to interact with membranes and the mechanism of such interactions in cellular processes is beginning to be understood. Bovine alpha-lactalbumin (BLA) is a soluble protein and yet it interacts with membranes. We recently reported the specific binding of apo-BLA with negatively charged membranes using a variety of fluorescence approaches [1]. A novel finding is that BLA exhibits an enhanced binding to negatively charged membranes in the presence of cholesterol and it possesses cholesterol recognition/interaction amino acid consensus (CRAC), a motif recognized for preferential association with cholesterol in many proteins. We monitored the specificity of sterol interaction by replacing cholesterol with 7-dehydrocholesterol (an immediate biosynthetic precursor of cholesterol), and observed that BLA-sterol interaction is specific to membrane cholesterol. Significant Fluorescence Resonance Energy Transfer (FRET) was observed between the tryptophan residues of BLA and dehydroergosterol (a naturally occurring fluorescent analog of cholesterol) in presence of negatively charged membranes, indicating close proximity between them. Dipole potential measurements upon BLA binding to membranes and docking studies of cholesterol with BLA provide useful insight into the lipid selectivity of BLA binding to membranes. Depth analysis by the parallax approach provides evidence for interfacial localization of tryptophans of BLA when bound to membranes. Taken together, our results assume significance in the light of tumoricidal and antimicrobial functions of α-lactalbumin [2,3].

Low Resolution Solution Structure of HAMLET and the Importance of Its Alpha-Domains in Tumoricidal Activity

HAMLET (Human Alpha-lactalbumin Made LEthal to Tumor cells) is the first member in a new family of protein-lipid complexes with broad tumoricidal activity. Elucidating the molecular structure and the domains crucial for HAMLET formation is fundamental for understanding its tumoricidal function. Here we present the low-resolution solution structure of the complex of oleic acid bound HAMLET, derived from small angle X-ray scattering data. HAMLET shows a two-domain conformation with a large globular domain and an extended part of about 2.22 nm in length and 1.29 nm width. The structure has been superimposed into the related crystallographic structure of human α-lactalbumin, revealing that the major part of α-lactalbumin accommodates well in the shape of HAMLET. However, the C-terminal residues from L105 to L123 of the crystal structure of the human α-lactalbumin do not fit well into the HAMLET structure, resulting in an extended conformation in HAMLET, proposed to be required to form the tumoricidal active HAMLET complex with oleic acid. Consistent with this low resolution structure, we identified biologically active peptide epitopes in the globular as well as the extended domains of HAMLET. Peptides covering the alpha1 and alpha2 domains of the protein triggered rapid ion fluxes in the presence of sodium oleate and were internalized by tumor cells, causing rapid and sustained changes in cell morphology. The alpha peptide-oleate bound forms also triggered tumor cell death with comparable efficiency as HAMLET. In addition, shorter peptides corresponding to those domains are biologically active. These findings provide novel insights into the structural prerequisites for the dramatic effects of HAMLET on tumor cells.

Tumor-Associated Macrophages Promote Invasion while Retaining Fc-Dependent Anti-Tumor Function

Tumor-associated macrophages (TAMs) have been shown to promote tumor progression, and increased TAM infiltration often correlates with poor prognosis. However, questions remain regarding the phenotype of macrophages within the tumor and their role in mAb-dependent cytotoxicity. This study demonstrates that whereas TAMs have protumor properties, they maintain Fc-dependent anti-tumor function. CD11b(+)CD14(+) TAMs isolated from primary human breast tumors expressed activating FcγRs. To model breast cancer TAMs in vitro, conditioned medium from breast cancer cells was used to drive human peripheral monocyte differentiation into macrophages. Tumor-conditioned macrophages were compared with in vitro derived M1 and M2a macrophages and were found to promote tumor cell invasion and express M2a markers, confirming their protumor potential. However, unlike M2a macrophages, tumor-conditioned macrophages expressed FcγRs and phagocytosed tumor cells in the presence of a tumor Ag-targeting mAb, unmasking an underappreciated tumoricidal capacity of TAMs. In vivo macrophage depletion reduced the efficacy of anti-CD142 against MDA-MB-231 xenograft growth and metastasis in SCID/beige mice, implicating a critical role for macrophages in Fc-dependent cell killing. M-CSF was identified in tumor-conditioned media and shown to be capable of differentiating macrophages with both pro- and anti-tumor properties. These results highlight the plasticity of TAMs, which are capable of promoting tumor progression and invasion while still retaining tumoricidal function in the presence of tumor-targeting mAbs.

Induction of tumoricidal function in CD4+ T cells is associated with concomitant memory and terminally differentiated phenotype

Harnessing the adaptive immune response to treat malignancy is now a clinical reality. Several strategies are used to treat melanoma; however, very few result in a complete response. CD4(+) T cells are important and potent mediators of anti-tumor immunity and adoptive transfer of specific CD4(+) T cells can promote tumor regression in mice and patients. OX40, a costimulatory molecule expressed primarily on activated CD4(+) T cells, promotes and enhances anti-tumor immunity with limited success on large tumors in mice. We show that OX40 engagement, in the context of chemotherapy-induced lymphopenia, induces a novel CD4(+) T cell population characterized by the expression of the master regulator eomesodermin that leads to both terminal differentiation and central memory phenotype, with concomitant secretion of Th1 and Th2 cytokines. This subpopulation of CD4(+) T cells eradicates very advanced melanomas in mice, and an analogous population of human tumor-specific CD4(+) T cells can kill melanoma in an in vitro system. The potency of the therapy extends to support a bystander killing effect of antigen loss variants. Our results show that these uniquely programmed effector CD4(+) T cells have a distinctive phenotype with increased tumoricidal capability and support the use of immune modulation in reprogramming the phenotype of CD4(+) T cells.

A novel accessory molecule Trim59 involved in cytotoxicity of BCG-activated macrophages.

BCG-activated macrophages (BAM) could kill the tumor cells through cell-cell contact. In this process membrane proteins play an important role. However, up to date, few membrane proteins were revealed. In this study, we selected a surface molecule named Trim59, which was specifically expressed on BAM membrane (compared with the negative control). We cloned and prokaryoticly expressed the extracellular domain of Trim59, purified the recombinant protein and generated polyclonal antibodies. Immunohistochemistry showed that Trim59 abundantly expressed in spleen, stomach and ovary; intermediately expressed in brain, lung, kidney, muscle and intestine; but not in thymus, liver, heart, uterus. Using the antibodies to block Trim59 on BAM significantly reduced BAM cytotoxicity against MCA207 cells. This demonstrated that Trim59 serves as an indispensable molecule in maintaining BAM activity. Overexpression of Trim59 in Raw264.7 cell line failed to lyse target MCA207 cells, which potentiated Trim59 per se could not enhance macrophage cytotoxicity; on another hand, overexpression of Trim59 enhance the pinocytosis and Phagocytosis activity of Raw-264.7, which imply Trim59 might mediate the cell-molecule interaction. Our results indicate Trim59 might be an essential accessory molecule in mediating BAM tumoricidal functions; and Trim59 is a phagocytosis-correlated molecule.

An In Vivo Immunotherapy Screen of Costimulatory Molecules Identifies Fc-OX40L as a Potent Reagent for the Treatment of Established Murine Gliomas

We tested the combination of a tumor lysate vaccine with a panel of costimulatory molecules to identify an immunotherapeutic approach capable of curing established murine gliomas. Glioma-bearing mice were primed with a tumor lysate vaccine, followed by systemic administration of the following costimulatory ligands: OX40L, CD80, 4-1BBL, and GITRL, which were fused to the Fc portion of human immunoglobulin. Lymphocytes and mRNA were purified from the brain tumor site for immune monitoring studies. Numerous variations of the vaccine and Fc-OX40L regimen were tested alone or in combination with temozolomide. Lysate vaccinations combined with Fc-OX40L led to the best overall survival, yielding cure rates of 50% to 100% depending on the timing, regimen, and combination with temozolomide. Cured mice that were rechallenged with glioma cells rejected the challenge, showing immunologic memory. Lymphocytes isolated from the draining lymph nodes of vaccine/Fc-OX40L-treated mice had superior tumoricidal function relative to all other groups. Vaccine/Fc-OX40L-treated mice exhibited a significant increase in proliferation of brain-infiltrating CD4 and CD8 T cells, as indicated by Ki67 staining. Fc-OX40L had single-agent activity in transplanted and spontaneous glioma models, and the pattern of inflammatory gene expression in the tumor predicted the degree of therapeutic response. These data show that Fc-OX40L has unique and potent activity against experimental gliomas and warrants further testing.

In Vitro Generation of Monocyte-Derived Macrophages under Serum-Free Conditions Improves Their Tumor Promoting Functions

The tumor promoting role of M2 macrophages has been described in in vivo models and the presence of macrophages in certain tumor types has been linked to a poor clinical outcome. In light of burgeoning activities to clinically develop new therapies targeting tumor-associated macrophages (TAMs), reliable in vitro models faithfully mimicking the tumor promoting functions of TAMs are required. Generation and activation of human monocyte-derived macrophages (MDM) in vitro, described as M1 or M2 macrophages attributed with tumoricidal or tumor-promoting functions, respectively, has been widely reported using mainly serum containing culture methods. In this study, we compared the properties of macrophages originating from monocytes cultured either in media containing serum together with M-CSF for M2 and GM-CSF for M1 macrophages or in serum-free media supplemented with M-CSF or GM-CSF and cytokines such as IL-4, IL-10 to induce activated M2 or LPS together with IFN-γ to generate activated M1 phenotype. We observed differences in cell morphology as well as increased surface receptor expression levels in serum-containing culture whereas similar or higher cytokine production levels were detected under serum-free culture conditions. More importantly, MDM differentiated under serum-free conditions displayed enhanced tumoricidal activity for M1 and tumor promoting property for M2 macrophages in contrast to MDM differentiated in the presence of serum. Moreover, evaluation of MDM phagocytic activity in serum free condition resulted in greater phagocytic properties of M2 compared to M1. Our data therefore confirm the tumor promoting properties of M2 macrophages in vitro and encourage the targeting of TAMs for cancer therapy.

Neem leaf glycoprotein suppresses regulatory T cell mediated suppression of monocyte/macrophage functions

We have shown that neem leaf glycoprotein (NLGP) inhibits the regulatory T cell (Tregs) induced suppression of tumoricidal functions of CD14+CD68+ monocyte/macrophages (MO/Mφ) from human peripheral blood. Cytotoxic efficacy of MO/Mφ toward macrophage sensitive cells, U937, is decreased in presence of Tregs (induced), however, it was increased further by supplementation of NLGP in culture. Associated Treg mediated inhibition of perforin/granzyme B expression and nitric oxide release from MO/Mφ was normalized by NLGP. Altered status of signature cytokines, like, IL-12, IL-10, IL-6, TNFα from MO/Mφ under influence of Tregs is also rectified by NLGP. Tregs significantly enhanced the expression of altered marker, mannose receptor (CD206) on CD68+ cells that was downregulated upon NLGP exposure. In addition to tumoricidal functions, antigen presenting ability of MO/Mφ is hampered by Treg induced downregulation of CD80, CD86 and HLA-ABC. NLGP upregulated these molecules in MO/Mφ even in the presence of Tregs. Treg mediated inhibition of MO/Mφ chemotaxis in contact dependent manner was also normalized partially by NLGP, where participation of CCR5 was documented. Overall results suggest that Treg influenced pro-tumor MO/Mφ functions are rectified in a significant extent by NLGP to create an anti-tumor immune environment.

Dysregulated CC receptor/ligand in monocytes/macrophages from tongue squamous cell carcinoma patients is partially rectified by interferon α-2b

In an aim to rectify dysregulated CC chemokine receptor (CCR5)/ligand (RANTES, MIP-1α, MIP-1β) status of monocytes/macrophages in tongue squamous cell carcinoma (TSCC; n = 12) patients, we have tested interferon α2b (IFNα2b), a novel immunomodulator with wide use in the management of several forms of cancer. IFNα2b can upregulate reduced CCR5 expression and increases the suppressed secretory status of its ligands, as evidenced from in vitro studies on monocytes/macrophages from the peripheral blood of TSCC patients as well as healthy individuals. Isolated monocytes of TSCC patients (n = 5) undergoing chemotherapeutic treatment along with IFNα2b immunotherapy demonstrated significant upregulation in CCR5 expression and secretion of corresponding ligands. These rectifications in receptor/ligand levels are reflected in improved CCR5-dependent migration of monocytes/macrophages after IFNα2b treatment. The rectified chemokine profile and cellular migration translate into better tumoricidal and antigen-presenting functions of these cells. Accordingly, enhanced T-cell-mediated tumor cell killing is demonstrated upon IFNα2b treatment. Translating dual benefits on monocyte/macrophage functions, IFNα2b may emerge as a potential form of immunotherapy for TSCC patients that may be combined with standard chemotherapy for better clinical outcome.

CD134 Plus CD137 Dual Costimulation Induces Eomesodermin in CD4 T Cells To Program Cytotoxic Th1 Differentiation

Cytotoxic CD4 Th1 cells are emerging as a therapeutically useful T cell lineage that can effectively target tumors, but until now the pathways that govern their differentiation have been poorly understood. We demonstrate that CD134 (OX40) costimulation programs naive self- and virus-reactive CD4 T cells to undergo in vivo differentiation into cytotoxic Th1 effectors. CD137 (4-1BB) costimulation maximized clonal expansion, and IL-2 was necessary for cytotoxic Th1 differentiation. Importantly, the T-box transcription factor Eomesodermin was critical for inducing the cytotoxic marker granzyme B. CD134 plus CD137 dual costimulation also imprinted a cytotoxic phenotype on bystanding CD4 T cells. Thus, to our knowledge, the current study identifies for the first time a specific costimulatory pathway and an intracellular mechanism relying on Eomesodermin that induces both Ag-specific and bystander cytotoxic CD4 Th1 cells. This mechanism might be therapeutically useful because CD134 plus CD137 dual costimulation induced CD4 T cell-dependent tumoricidal function in a mouse melanoma model.

An Fcγ Receptor-Dependent Mechanism Drives Antibody-Mediated Target-Receptor Signaling in Cancer Cells

Antibodies to cell-surface antigens trigger activatory Fcγ receptor (FcγR)-mediated retrograde signals in leukocytes to control immune effector functions. Here, we uncover an FcγR mechanism that drives antibody-dependent forward signaling in target cells. Agonistic antibodies to death receptor 5 (DR5) induce cancer-cell apoptosis and are in clinical trials; however, their mechanism of action invivo is not fully defined. Interaction of the DR5-agonistic antibody drozitumab with leukocyte FcγRs promoted DR5-mediated tumor-cell apoptosis. Whereas the anti-CD20 antibody rituximab required activatory FcγRs for tumoricidal function, drozitumab was effective in the context of either activatory or inhibitory FcγRs. A CD40-agonistic antibody required similar FcγR interactions to stimulate nuclear factor-κB activity in B cells. Thus, FcγRs can drive antibody-mediated receptor signaling in target cells.

Superior Efficacy of Tumor Cell Vaccines Grown in Physiologic Oxygen

Atmospheric oxygen (∼20% O(2)) has been the universal condition employed to culture tumor cells used as vaccine antigen. We tested the hypothesis that reducing oxygen tension would increase the efficacy of tumor cell lysate vaccines. GL261 glioma cells and EMT6 breast carcinoma cells were grown in 5% or 20% O(2). Syngeneic tumor-bearing mice were vaccinated with these tumor cell lysates mixed with CpG oligodeoxynucleotides as an adjuvant. Tumor infiltrating T cells and apoptotic GL261 cells were quantified by immunohistochemistry. Tumor-reactive immunoglobulin was detected by Western blot. Ovalbumin and gp100-derived peptides were mixed with GL261 lysates as marker antigens to detect changes in presentation of exogenous antigen on MHC class I in vitro, and in vivo following adoptive transfer of gp100-specific CD8(+) T cells. Mice bearing orthotopic glioma and breast carcinoma survived significantly longer when vaccinated with 5% O(2) lysates. Antigen-specific CTL activation was significantly enhanced following stimulation with lysates derived from GL261 cells grown in 5% O(2) versus 20% O(2) through a mechanism that involved enhanced cross-presentation of exogenous antigen on MHC I. Vaccination with 5% O(2) GL261 cell lysates caused a significant increase in CTL proliferation, tumoricidal function, and trafficking into brain tumor sites, whereas 20% O(2) lysate vaccines predominantly evoked an antibody response. Tissue culture oxygen functions as an "immunologic switch" by dictating the cellular and humoral immune responses elicited by tumor cell lysates. These results have profound implications for cancer vaccines that utilize tumor cells as the source of antigen.

SIRT1 Suppresses Activator Protein-1 Transcriptional Activity and Cyclooxygenase-2 Expression in Macrophages

SIRT1 (Sirtuin type 1), a mammalian orthologue of yeast SIR2 (silent information regulator 2), has been shown to mediate a variety of calorie restriction (CR)-induced physiological events, such as cell fate regulation via deacetylation of the substrate proteins. However, whether SIRT1 deacetylates activator protein-1 (AP-1) to influence its transcriptional activity and target gene expression is still unknown. Here we demonstrate that SIRT1 directly interacts with the basic leucine zipper domains of c-Fos and c-Jun, the major components of AP-1, by which SIRT1 suppressed the transcriptional activity of AP-1. This process requires the deacetylase activity of SIRT1. Notably, SIRT1 reduced the expression of COX-2, a typical AP-1 target gene, and decreased prostaglandin E(2) (PGE(2)) production of peritoneal macrophages (pMPhis). pMPhis with SIRT1 overexpression displayed improved phagocytosis and tumoricidal functions, which are associated with depressed PGE(2). Furthermore, SIRT1 protein level was up-regulated in CR mouse pMPhis, whereas elevated SIRT1 decreased COX-2 expression and improved PGE(2)-related macrophage functions that were reversed following inhibition of SIRT1 deacetylase activity. Thus, our results indicate that SIRT1 may be a mediator of CR-induced macrophage regulation, and its deacetylase activity contributes to the inhibition of AP-1 transcriptional activity and COX-2 expression leading to amelioration of macrophage function.