Host Effector Cells Research Articles

Galectin-9, a pro-survival factor inducing immunosuppression, leukemic cell transformation and expansion.

Leukemia is a malignancy of the bone marrow and blood originating from self-renewing cancerous immature blast cells or transformed leukocytes. Despite improvements in treatments, leukemia remains still a serious disease with poor prognosis because of disease heterogeneity, drug resistance and relapse. There is emerging evidence that differentially expression of co-signaling molecules play a critical role in tumor immune evasion. Galectin-9 (Gal-9) is one of the key proteins that leukemic cells express, secrete, and use to proliferate, self-renew, and survive. It also suppresses host immune responses controlled by T and NK cells, enabling leukemic cells to evade immune surveillance. The present review provides the molecular mechanisms of Gal-9-induced immune evasion in leukemia. Understanding the complex immune evasion machinery driven by Gal-9 expressing leukemic cells will enable the identification of novel therapeutic strategies for efficient immunotherapy in leukemic patients. Combined treatment approaches targeting T-cell immunoglobulin and mucin domain-3 (Tim-3)/Gal-9 and other immune checkpoint pathways can be considered, which may enhance the efficacy of host effector cells to attack leukemic cells.

Anti-Inflammatory Effects of Encapsulated Human Mesenchymal Stromal/Stem Cells and a Method to Scale-Up Cell Encapsulation.

Mesenchymal stem/stromal cells (MSC) promote recovery in a wide range of animal models of injury and disease. They can act in vivo by differentiating and integrating into tissues, secreting factors that promote cell growth and control inflammation, and interacting directly with host effector cells. We focus here on MSC secreted factors by encapsulating the cells in alginate microspheres, which restrict cells from migrating out while allowing diffusion of factors including cytokines across the capsules. One week after intrathecal lumbar injection of human bone marrow MSC encapsulated in alginate (eMSC), rat IL-10 expression was upregulated in distant rat spinal cord injury sites. Detection of human IL-10 protein in rostrally derived cerebrospinal fluid (CSF) indicated distribution of this human MSC-secreted cytokine throughout rat spinal cord CSF. Intraperitoneal (IP) injection of eMSC in a rat model for endotoxemia reduced serum levels of inflammatory cytokines within 5 h. Detection of human IL-6 in sera after injection of human eMSC indicates rapid systemic distribution of this human MSC-secreted cytokine. Despite proof of concept for eMSC in various disorders using animal models, translation of encapsulation technology has not been feasible primarily because methods for scale-up are not available. To scale-up production of eMSC, we developed a rapid, semi-continuous, capsule collection system coupled to an electrosprayer. This system can produce doses of encapsulated cells sufficient for use in clinical translation.

FcγRIIB controls antibody-mediated target cell depletion by ITIM-independent mechanisms.

Many therapeutic antibodies deplete target cells and elicit immunotherapy by engaging activating Fc gamma receptors (FcγRs) on host effector cells. These antibodies are negatively regulated by the inhibitory FcγRIIB (CD32B). Dogma suggests inhibition is mediated through the FcγRIIB immunoreceptor tyrosine-based inhibition motif (ITIM), negatively regulating immunoreceptor tyrosine-based activation motif (ITAM)-mediated signaling from activating FcγR. To assess this, we generated experimental models expressing human (h)FcγRIIB on targets or effectors, lacking or retaining ITIM signaling capacity. We demonstrate that signaling through the hFcγRIIB ITIM is dispensable for impairing monoclonal antibody (mAb)-mediated depletion of normal and malignant murine target cells through three therapeutically relevant surface receptors (CD20, CD25, and OX40) affecting immunotherapy. We demonstrate that hFcγRIIB competition with activating FcγRs for antibody Fc, rather than ITIM signaling, is sufficient to impair activating FcγR engagement, inhibiting effector function and immunotherapy.

Chimeric antigen receptor T-cell lymphoma immunotherapy: the next questions.

Chimeric antigen receptor (CAR) T-cell therapy is an innovative form of adoptive cellular immunotherapy targeting CD19 in its most advanced form. Up to 30% of infused patients achieve long-term survival, meaning that 70% of patients still fail to respond or relapse after therapy. This review will address the unresolved issues relating to responders' characterization, relapse prediction, and prevention, CAR T-cell construct optimization, rational combination with other therapies and treatment toxicity, focusing on the management of relapsed/refractory lymphoma patients. Many new antigenic targets are currently investigated and raise the hope of broader successes. However, literature data report that treatment failure is not only related to CAR T construct and infusion but is also due to hostile tumor microenvironment and poor interaction with the host effector cells. Further research should not only target CAR T structure, toxicity and associated therapies, but also tumor-related and host-related microenvironment interactions that lead to treatment failure in relapsed/refractory lymphoma patients. Poor persistence of CAR T and loss of CD19 antigen are well established mechanisms of relapse in Acute Lymphoblastic Leukemia (ALL). A fourth generation of CAR T construct is currently investigated to overcome this issue. In non-Hodgkin lymphoma, mechanisms of treatment failure remain poorly understood but tumor and host microenvironment are undoubtedly involved and should be further investigated. A deeper understanding of CAR T-cell therapy failure in individuals will help personalize CAR T-cell therapy in the future.

Proteomic analysis revealed T cell hyporesponsiveness induced by Haemonchus contortus excretory and secretory proteins

Haemonchus contortus has evolved highly integrated and sophisticated mechanisms to promote coexistence with hosts. The excretory-secretory (ES) products generated by this parasite contribute to the regulation of the host immune response to facilitate immune evasion and induce chronicity, but the proteins responsible for this process and the exact cellular mechanisms have yet to be defined. In this study, we identified 114 H. contortus ES proteins (HcESPs) interacting with host T cells and 15 T cell binding receptors via co-immunoprecipitation and shotgun liquid chromatography-tandem mass spectrometry analysis. Based on bioinformatics analysis, we demonstrated that HcESPs could inhibit T cell viability, induce cell apoptosis, suppress T cell proliferation and cause cell cycle arrest. Furthermore, the stimulation of HcESPs exerted critical control effects on T cell cytokine production profiles, predominantly promoting the secretion of interleukin (IL)-10, IL-17A and transforming growth factor-β1 and inhibiting IL-2, IL-4 and interferon-γ production. Collectively, these findings may provide insights into the interaction between ES proteins and key host effector cells, enhancing our understanding of the molecular mechanism underlying parasite immune evasion and providing new clues for novel vaccine development.

Dynamic Effects of Two-Time Delays on a Model for Tumor Growth.

In order to understand the dynamic mechanisms of tumor growth and make a contribution to develop anti-cancer treatment strategies, a mathematical model for tumor growth with two-time delays is proposed in this article. First, the relationships among host cells, tumor cells and effector cells, and the biological meaning of two-time delays are explained. Moreover, the system stability is discussed by analyzing the characteristic equation of the model. In addition, the existence and properties of oscillatory dynamic are also researched by using normative theory and central manifold method. Finally, the numerical simulations are performed to further illustrate and support the theoretical results. Both two-time delays in the model can affect the dynamics of tumor growth. Meanwhile, the system can experience a Hopf bifurcation when the delay crosses a series of critical values. Further, a clear formula is deduced to determine the Hopf bifurcation and the direction of stability of the periodic solution. Finally, these results are verified by using numerical simulation. The results demonstrated that the time from identifying tumor cells to making the appropriate response for the immune system and the time needed for competition between host cells and tumor cells for natural resources and living space is significant for tumor growth. These findings in this paper may help us better understand the behaviors of tumors and develop better anti-cancer treatment strategies.

Heart Transplant Tolerance Induction in Mice: Understanding the Complex Interplay Between Tolerizing Cells and Host Immune System by Systematic Depletion of Effector Cells

To induce robust heart transplant tolerance in neonates with fully allogeneic spleen and bone marrow cells (SC/BMC) it is crucial to define the complex interplay amongst the different cells of the neonatal host and adult-derived tolerizing inoculum. We used specific and systematic depletion of donor and host effector cells, studying the effects of each depletion on immune outcome.

Mechanisms and risk factors of thrombosis in cancer.

The close relationship between cancer and thrombosis is known since more than a century. Venous thromboembolism (VTE) may be the first manifestation of an occult malignancy in an otherwise healthy individual. Cancer patients commonly present with abnormalities of laboratory coagulation tests, indicating an ongoing subclinical hypercoagulable condition. The results of laboratory tests demonstrate that a process of fibrin formation and removal parallels the development of malignancy, which is of particular interest since fibrin and other clotting products are important for both thrombogenesis and tumor progression. Besides general clinical risk factors (i.e. age, previous VTE, immobility, etc.), other factors typical of cancer can increase the thrombotic risk in these patients, including the type of cancer, advanced disease stage, and cancer therapies. In addition, biological factors, including tumor cell-specific prothrombotic properties and the host cell inflammatory response to the tumor, play a central role in the pathogenesis of cancer-associated thrombosis. Cancer cells produce and release procoagulant and fibrinolytic proteins, as well as inflammatory cytokines. In addition, they are capable of directly adhering to host cells (i.e. endothelial cells, monocytes, platelets, and neutrophils), thereby stimulating additional prothrombotic properties of the host effector cells. Tumor-shed procoagulant microparticles also contribute to the patient hypercoagulable state. Finally, the changes of stromal cells of the tumor 'niche' induced by tissue factor (TF) highlight new interactions between hemostasis and cancer. Of interest, most of these mechanisms, besides activating the hemostatic system, also promote tumor growth and metastasis, and are regulated by oncogenic events. Indeed, molecular studies demonstrate that oncogenes responsible for the cellular neoplastic transformation drive the programs of hemostatic protein expression and microparticle liberation by cancer tissues. Human and animal experimental models demonstrate that activation of cancer-associated prothrombotic mechanisms parallels the development of overt thrombotic syndromes in vivo.

Paracoccidioides spp. catalases and their role in antioxidant defense against host defense responses

Dimorphic human pathogenic fungi interact with host effector cells resisting their microbicidal mechanisms. Yeast cells are able of surviving within the tough environment of the phagolysosome by expressing an antioxidant defense system that provides protection against host-derived reactive oxygen species (ROS). This includes the production of catalases (CATs). Here we identified and analyzed the role of CAT isoforms in Paracoccidioides, the etiological agent of paracoccidioidomycosis. Firstly, we found that one of these isoforms was absent in the closely related dimorphic pathogen Coccidioides and dermatophytes, but all of them were conserved in Paracoccidioides, Histoplasma and Blastomyces species. We probed the contribution of CATs in Paracoccidioides by determining the gene expression levels of each isoform through quantitative RT-qPCR, in both the yeast and mycelia phases, and during the morphological switch (transition and germination), as well as in response to oxidative agents and during interaction with neutrophils. PbCATP was preferentially expressed in the pathogenic yeast phase, and was associated to the response against exogenous H2O2. Therefore, we created and analyzed the virulence defects of a knockdown strain for this isoform, and found that CATP protects yeast cells from H2O2 generated in vitro and is relevant during lung infection. On the other hand, CATA and CATB seem to contribute to ROS homeostasis in Paracoccidioides cells, during endogenous oxidative stress. CAT isoforms in Paracoccidioides might be coordinately regulated during development and dimorphism, and differentially expressed in response to different stresses to control ROS homeostasis during the infectious process, contributing to the virulence of Paracoccidioides.

Identification and Analysis of the Role of Superoxide Dismutases Isoforms in the Pathogenesis of Paracoccidioides spp.

The ability of Paracoccidioides to defend itself against reactive oxygen species (ROS) produced by host effector cells is a prerequisite to survive. To counteract these radicals, Paracoccidioides expresses, among different antioxidant enzymes, superoxide dismutases (SODs). In this study, we identified six SODs isoforms encoded by the Paracoccidioides genome. We determined gene expression levels of representative isolates of the phylogenetic lineages of Paracoccidioides spp. (S1, PS2, PS3 and Pb01-like) using quantitative RT-PCR. Assays were carried out to analyze SOD gene expression of yeast cells, mycelia cells, the mycelia-to-yeast transition and the yeast-to-mycelia germination, as well as under treatment with oxidative agents and during interaction with phagocytic cells. We observed an increased expression of PbSOD1 and PbSOD3 during the transition process, exposure to oxidative agents and interaction with phagocytic cells, suggesting that these proteins could assist in combating the superoxide radicals generated during the host-pathogen interaction. Using PbSOD1 and PbSOD3 knockdown strains we showed these genes are involved in the response of the fungus against host effector cells, particularly the oxidative stress response, and in a mouse model of infection. Protein sequence analysis together with functional analysis of knockdown strains seem to suggest that PbSOD3 expression is linked with a pronounced extracellular activity while PbSOD1 seems more related to intracellular requirements of the fungus. Altogether, our data suggests that P. brasiliensis actively responds to the radicals generated endogenously during metabolism and counteracts the oxidative burst of immune cells by inducing the expression of SOD isoforms.

Macrophage heterogeneity in liver injury and fibrosis

Hepatic macrophages are central in the pathogenesis of chronic liver injury and have been proposed as potential targets in combatting fibrosis. Recent experimental studies in animal models revealed that hepatic macrophages are a remarkably heterogeneous population of immune cells that fulfill diverse functions in homeostasis, disease progression, and regression from injury. These range from clearance of pathogens or cellular debris and maintenance of immunological tolerance in steady state conditions; central roles in initiating and perpetuating inflammation in response to injury; promoting liver fibrosis via activating hepatic stellate cells in chronic liver damage; and, finally, resolution of inflammation and fibrosis by degradation of extracellular matrix and release of anti-inflammatory cytokines. Cellular heterogeneity in the liver is partly explained by the origin of macrophages. Hepatic macrophages can either arise from circulating monocytes, which are recruited to the injured liver via chemokine signals, or from self-renewing embryo-derived local macrophages, termed Kupffer cells. Kupffer cells appear essential for sensing tissue injury and initiating inflammatory responses, while infiltrating Ly-6C(+) monocyte-derived macrophages are linked to chronic inflammation and fibrogenesis. In addition, proliferation of local or recruited macrophages may possibly further contribute to their accumulation in injured liver. During fibrosis regression, monocyte-derived cells differentiate into Ly-6C (Ly6C, Gr1) low expressing 'restorative' macrophages and promote resolution from injury. Understanding the mechanisms that regulate hepatic macrophage heterogeneity, either by monocyte subset recruitment, by promoting restorative macrophage polarization or by impacting distinctive macrophage effector functions, may help to develop novel macrophage subset-targeted therapies for liver injury and fibrosis.

Abstract 1252: Random unrelated donor peripheral blood mononuclear cells (PBMC) stimulate cytolytic host T cell activity against leukemia.

Abstract Introduction: In the treatment of acute leukemia, much of the therapeutic effect of allogeneic stem cell transplantation is centered on engraftment of donor cells into the host, with a resultant graft versus leukemia effect. Work previously done by our group and others has shown the infusion of haploidentical stem cells in patients with acute leukemia results in a cytokine storm and exhibits an anti-tumor effect in the absence of engraftment. Whether the underlying clinical response was due to this cytokine storm or secondary to host or donor effector cell interactions was not determined. We present in vitro data collected from leukemia patients in which patient CD3+ cells are stimulated with randomly selected unrelated normal donor peripheral blood mononuclear cells (PBMC). The stimulated patient CD3+ cells are then collected and tested for their ability to lyse leukemia cells from the patient. Methods: New leukemia patients were identified through standard pathologic diagnostic criteria. Patient samples were collected and separated into CD3+ and CD3- fractions. Patient CD3+ cells were then placed in a mixed lymphocyte culture and stimulated with mitomycin C treated unrelated normal donor PBMC. Cell proliferation was measured on Day 5. On Day 7, the stimulated patient CD3+ cells were collected and then cultured with Chromium 51 labeled CD3- cells, the bulk of which were leukemic blast cells. Cytolytic activity was measured through Cr51 release assays. Cell lysis was recorded as lytic units/million cells (LU/106), which is inversely related to the effector to target ratio at which 30% cell lysis occurs. Patient demographic data including sex and age in addition to leukemia type, cytogenetics, and molecular markers were recorded. Results: 10 total patients were enrolled in the study; 8 AML, 1 CML, and 1 CMML. 9 patients underwent ex vivo CD3+ stimulation with 2 separate donor PBMC samples while 1 sample underwent only 1 donor stimulation for 19 total assays. The average white blood cell count was 57,640 cells/uL (range 7,100-336,500 cells/ul) with 4-80% peripheral blasts. 7 out of 19 (37%) assays showed leukemia cell cytolytic activity greater than 1 lytic unit. All 7 successful assays were derived from patients with AML (Table 1). Conclusions: Patient CD3+ cells stimulated with random unrelated donor PBMC are able to generate a cytolytic anti-leukemia response in a donor dependant fashion. This cytolytic activity appears to be unrelated to host lymphocyte proliferation upon donor antigenic stimulation. Interestingly, of the seven assays with cytolytic activity greater than one lytic unit, four were performed in the two patients with known FLT3 ITD positive leukemia, which has a notoriously poor prognosis. Further studies to elucidate a mechanism of action for cellular therapy are warranted. Citation Format: John L. Reagan, Loren D. Fast, Martha Nevola, Peter J. Quesenberry. Random unrelated donor peripheral blood mononuclear cells (PBMC) stimulate cytolytic host T cell activity against leukemia. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1252. doi:10.1158/1538-7445.AM2013-1252

P2X7 receptor regulation of non-classical secretion from immune effector cells

P2X7 receptors (P2X7R) are extracellular ATP-gated ion channels expressed in the immune effector cells that carry out critical protective responses during the early phases of microbial infection or acute tissue trauma. P2X7R-positive cells include monocytes, macrophages, dendritic cells and T cells. Given its presence in all host and pathogen cell types, ATP can be readily released into extracellular compartments at local sites of tissue damage and microbial invasion. Thus, extracellular ATP and its target receptors on host effector cells can be considered as additional elements of the innate immune system. In this regard, stimulation of P2X7R rapidly triggers a key step of the inflammatory response: induction of NLRP3/caspase-1 inflammasome signalling complexes that drive the proteolytic maturation and secretion of the proinflammatory cytokines interleukin-1β (IL-1β) and interleukin-18 (IL-18). IL-1β (and IL-18) lacks a signal sequence for compartmentation within the Golgi and classical secretory vesicles and the proIL-1β precursor accumulates within the cytosol following translation on free ribosomes. Thus, ATP-induced accumulation of the mature IL-1β cytokine within extracellular compartments requires non-classical mechanisms of export from the cytosolic compartment. Five proposed mechanisms include: (i) exocytosis of secretory lysosomes that accumulate cytosolic IL-1β via undefined protein transporters; (ii) release of membrane-delimited microvesicles derived from plasma membrane blebs formed by evaginationsof the surface membrane that entrap cytosolic IL-β; (iii) release of membrane-delimited exosomes secondary to the exocytosis of multivesicular bodies formed by invaginations of recycling endosomes that entrap cytosolic IL-β; (iv) exocytosis of autophagosomes or autophagolysosomes that accumulate cytosolic IL-1β via entrapment during formation of the initial autophagic isolation membrane or omegasome and (v) direct release of cytosolic IL-1β secondary to regulated cell death by pyroptosis or necroptosis. These mechanisms are not mutually exclusive and may represent engagement of parallel or intersecting membrane trafficking responses to P2X7R activation.

Abstract 4404: Microparticle generation and activation after treatment with paclitaxel and bavituximab combination therapy in metastatic breast cancer

Abstract Background: Breast cancer is the leading cause of life-threatening malignancy affecting women and the second leading cause of female cancer death in the United States. Weekly paclitaxel, alone or in combination with other chemotherapies, has proven to be an effective and well-tolerated therapy for patients with metastatic disease. Due to the tumor's dependence on vascularization for progression, survival and dissemination, targeting a tumor's vascular network is an important additional cancer management strategy. Bavituximab, a monoclonal antibody developed by Peregrine Pharmaceuticals against phosphatidylserine (PS), is a vascular disrupting agent designed to selectively attack tumor blood vessels already in existence. Under normal conditions, PS is localized to the internal surface of the plasma membrane. However, PS is preferentially localized on the outer surface of vascular endothelial cells in tumors, possibly due to stress conditions in the tumor microenvironment. Bavituximab is able to localize and bind specifically to PS in tumor vessels in a α2-glycoprotein 1 dependent manner, mediating the binding of host effector cells to the tumor vascular endothelium. This leads to vessel damage, vascularity reduction, necrosis, and growth retardation within the tumor. Microparticles are vesicles that are released from cells after activation, stress, death, or malignant transformation. Elevated levels of microparticles derived from platelets, endothelial cells, and/or leukocytes have been associated with several disease states, including cancer, however, the effect of anti-angiogenic or vascular disrupting agents on microparticle production is unclear. We investigated microparticle formation in our patients at baseline, in response to Paclitaxel chemotherapy, and after addition of Bavituximab. Patients and Methods: We initiated a phase I clinical trial in patients with Her-2 negative metastatic breast cancer treated with paclitaxel (80 mg/m2)on a weekly schedule for 3 out of 4 weeks, and weekly bavituximab (3 mg/kg) for 4 out of 4 weeks. Each cycle was defined as 4 weeks of therapy. Microparticles were isolated from peripheral blood and examined by flow cytometric phenotyping. Leukocyte, endothelial cell, and activated platelet microparticles were quantified and longitudinal trends analyzed. Five patients have been enrolled to date with additional patients accruing. At presentation, the longitudinal trends in microparticle formation from platelets, endothelial cells, and leukocytes will be presented in response to weekly Paclitaxel versus combined Paclitaxel and Bavituximab therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4404. doi:1538-7445.AM2012-4404

Macrophages: Central regulators of hepatic fibrogenesis and fibrosis resolution

Hepatic fibrosis is the common end point to chronic injury of varied aetiology. There is now excellent evidence in both human studies and animal models that liver fibrosis is a bidirectional process with a significant reversible component. The hepatic stellate cell (HSC), following activation to a myofibroblast phenotype, is the principal cell producing extracellular matrix (ECM) during fibrogenesis and is the main source of TIMP-1, which inhibits the endogenous matrix-degrading activity of matrix metalloproteinases (MMPs), thus promoting scar deposition. Furthermore, apoptosis of activated HSCs is a critical feature of scar resolution. However, emerging evidence indicates that it is the hepatic macrophage that is the master regulator of this dynamic fibrogenesis–fibrosis resolution paradigm.

The Identification of Gene Duplication and the Role of Secreted Aspartyl Proteinase 1 in Candida parapsilosis Virulence

In this study, we analyzed the role of Candida parapsilosis-secreted aspartyl proteinase isoenzyme 1 (SAPP1) in virulence. The in silico analysis of SAPP1 sequence revealed a 2871 base pair-duplicated region (SAPP1a and SAPP1b) in the genome of C. parapsilosis. We generated homozygous ΔΔsapp1a, ΔΔsapp1b, and ΔΔsapp1a-ΔΔsapp1b mutants. Notably, Sapp1 production in an inducer medium was reduced by approximately 50% in the ΔΔsapp1a and ΔΔsapp1b mutants, but the other validated SAPP gene (SAPP2) was not affected. In contrast, Sapp2 production was increased in the ΔΔsapp1a-ΔΔsapp1b mutant relative to wild-type (WT) yeast. The ΔΔsapp1a-ΔΔsapp1b strain was hypersusceptible to human serum and was attenuated in its capacity to damage host-effector cells. The phagocytosis and killing of ΔΔsapp1a-ΔΔsapp1b yeasts by human peripheral blood mononuclear cells (PBMCs) and PBMC-derived macrophages (PBMC-DM) was significantly enhanced relative to WT. Phagolysosomal fusion in PBMC-DMs occurred more than twice as frequently with ingested ΔΔsapp1a-ΔΔsapp1b yeast cells compared with WT.

Macrophage therapy for murine liver fibrosis recruits host effector cells improving fibrosis, regeneration, and function

Clinical studies of bone marrow (BM) cell therapy for liver cirrhosis are under way but the mechanisms of benefit remain undefined. Cells of the monocyte-macrophage lineage have key roles in the development and resolution of liver fibrosis. Therefore, we tested the therapeutic effects of these cells on murine liver fibrosis. Advanced liver fibrosis was induced in female mice by chronic administration of carbon tetrachloride. Unmanipulated, syngeneic macrophages, their specific BM precursors, or unfractionated BM cells were delivered during liver injury. Mediators of inflammation, fibrosis, and regeneration were measured. Donor cells were tracked by sex-mismatch and green fluorescent protein expression. BM-derived macrophage (BMM) delivery resulted in early chemokine up-regulation with hepatic recruitment of endogenous macrophages and neutrophils. These cells delivered matrix metalloproteinases-13 and -9, respectively, into the hepatic scar. The effector cell infiltrate was accompanied by increased levels of the antiinflammatory cytokine interleukin 10. A reduction in hepatic myofibroblasts was followed by reduced fibrosis detected 4 weeks after macrophage infusion. Serum albumin levels were elevated at this time. Up- regulation of the liver progenitor cell mitogen tumor necrosis factor-like weak inducer of apoptosis (TWEAK) preceded expansion of the progenitor cell compartment. Increased expression of colony stimulating factor-1, insulin-like growth factor-1, and vascular endothelial growth factor also followed BMM delivery. In contrast to the effects of differentiated macrophages, liver fibrosis was not significantly altered by the application of macrophage precursors and was exacerbated by whole BM. Macrophage cell therapy improves clinically relevant parameters in experimental chronic liver injury. Paracrine signaling to endogenous cells amplifies the effect. The benefits from this single, defined cell type suggest clinical potential.

A potential mechanism of rituximab-induced inhibition of tumor growth through its sensitization to tumor necrosis factor-related apoptosis-inducing ligand-expressing host cytotoxic cells

Rituximab (anti-CD20 mAb) mediates antibody-dependent cell-mediated cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), and apoptosis in B-NHL cells. The contribution of other host-mediated cytotoxic effects has not been examined. The expression of death-inducing ligands (e.g. TRAIL) by host effector cells may contribute to the mechanism of tumor cell destruction in vivo by rituximab-mediated sensitization of resistant B-cell non-Hodgkin lymphoma (B-NHL) cells. We have examined the sensitizing activity of rituximab on B-NHL cell lines resistant to TRAIL (as model) and natural killer (NK)-induced apoptosis. Treatment of TRAIL-resistant B-NHL cell lines with rituximab sensitized the cells to TRAIL apoptosis and synergy was achieved via activation of the type II mitochondrial pathway for apoptosis. Further, rituximab (Fab′)2-treated tumor cells were killed by purified peripheral blood-derived NK cells via TRAIL. Treatment of B-NHL cells with rituximab inhibited both YY1 DNA-binding activity and expression. Rituximab-mediated sensitization to TRAIL apoptosis was due, in large part, to rituximab-mediated inhibition of the transcription factor Yin Yang 1 (YY1). The direct role of YY1 in TRAIL sensitization by rituximab was shown in cells transfected with YY1 siRNA, and such cells mimicked rituximab and became sensitive to TRAIL-induced apoptosis. These data suggest that, in vivo, host effector cells expressing TRAIL may contribute to rituximab-mediated depletion of B-NHL cells.

OP07 Macrophage cell therapy causes the hepatic recruitment of host effector cells and improves structure and function in a murine model of chronic liver disease

IntroductionBone marrow (BM) cell populations have a number of roles in the development and resolution of chronic liver disease. Clinical trials of BM cell therapy have already begun. These have...

Abstract 5308: Targeting the Cox2 pathway prostaglandin EP receptors to regulate NK cell functions

Abstract Breast malignancies often have high levels of Cox-2. The Cox-2 product prostaglandin E2 (PGE2) contributes to the high metastatic capacity of breast tumors. Our published data indicates that inhibiting either PGE2 production or PGE2-mediated signaling through the PGE2 receptor (EP4) reduces metastasis by a mechanism that requires Natural Killer (NK) cells. In order to better understand the mechanism by which inhibiting PGE2 production or signaling reduces metastatic disease, we examined the role of PGE2 in regulating protective host effector cells. It is known that NK cell function is compromised by PGE2, but very little is known about the mechanism by which PGE2 affects NK effector activity. NK effector function is regulated by complex interactions of recognition receptors expressed on NK cells and cognate ligands expressed on target cells. Both stimulatory and inhibitory signals are delivered through these receptor-ligand interactions. We have previously shown that both Cox inhibitors and antagonists of the EP4 receptor modulate the expression of NK cell stimulatory (H60) and inhibitory (MHC class I) ligands on the tumor target cell in favor of NK recognition and killing. We now report the direct effects of PGE2 on the NK cell. Murine splenic NK cells express all four PGE2 receptors (EP1-4). We examined the role of EP receptors in three NK cell functions; cytokine release, cytotoxicity, and migration. Activating all four NK-EP receptors with PGE2 leads to inhibited IFNγ secretion, induced TNFα production, less migration of NK cells and reduced ability to lyse tumor target cells. Using agonists and antagonists of individual EP receptors, we showed that only certain EP receptors contribute to loss of function. Next, we showed that all NK-EP receptor expression is downregulated during progressive tumor growth, which produced an environment that induced a less mature NK phenotype. EP2 and EP4 are linked to protein kinase A and elevations in intracellular cAMP. NK cells from tumor bearing mice respond poorly to stimulation with 10µM PGE2 and intracellular cAMP levels were increased by only 1.45-1.78 fold. In contrast, cAMP was induced by 6.08-10.58-fold in NK cells from normal mice. Thus, the tumor milieu results in changes in both NK-EP expression levels and EP receptor functions. NK mediated cytotoxicity, cytokine release, and migration were also compromised in tumor bearing mice. These results taken together support a mechanism whereby inhibiting PGE2 production or preventing signaling through some, but not all EP receptors may prevent suppression of NK functions that are critical to control breast cancer metastasis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5308.