Glenn D. Papworth

Aspirin Inhibits In Vitro Maturation and In Vivo Immunostimulatory Function of Murine Myeloid Dendritic Cells

Aspirin is the most commonly used analgesic and antiinflammatory agent. In this study, at physiological concentrations, it profoundly inhibited CD40, CD80, CD86, and MHC class II expression on murine, GM-CSF + IL-4 stimulated, bone marrow-derived myeloid dendritic cells (DC). CD11c and MHC class I expression were unaffected. The inhibitory action was dose dependent and was evident at concentrations higher than those necessary to inhibit PG synthesis. Experiments with indomethacin revealed that the effects of aspirin on DC maturation were cyclooxygenase independent. Nuclear extracts of purified, aspirin-treated DC revealed a decreased NF-κB DNA-binding activity, whereas Ab supershift analysis indicated that aspirin targeted primarily NF-κB p50. Unexpectedly, aspirin promoted the generation of CD11c+ DC, due to apparent suppression of granulocyte development. The morphological and ultrastructural appearance of aspirin-treated cells was consistent with immaturity. Aspirin-treated DC were highly efficient at Ag capture, via both mannose receptor-mediated endocytosis and macropinocytosis. By contrast, they were poor stimulators of naive allogeneic T cell proliferation and induced lower levels of IL-2 in responding T cells. They also exhibited impaired IL-12 expression and did not produce IL-10 after LPS stimulation. Assessment of the in vivo function of aspirin-treated DC, pulsed with the hapten trinitrobenzenesulfonic acid, revealed an inability to induce normal cell-mediated contact hypersensitivity, despite the ability of the cells to migrate to T cell areas of draining lymphoid tissue. These data provide new insight into the immunopharmacology of aspirin and suggest a novel approach to the manipulation of DC for therapeutic application.

Exosomes As a Short-Range Mechanism to Spread Alloantigen between Dendritic Cells during T Cell Allorecognition

Exosomes are nanovesicles released by different cell types including dendritic cells (DCs). The fact that exosomes express surface MHC-peptide complexes suggests that they could function as Ag-presenting vesicles or as vehicles to spread allogeneic Ags for priming of anti-donor T cells during elicitation of graft rejection or induction/maintenance of transplant tolerance. We demonstrate that circulating exosomes transporting alloantigens are captured by splenic DCs of different lineages. Internalization of host-derived exosomes transporting allopeptides by splenic DCs leads to activation of anti-donor CD4 T cells by the indirect pathway of allorecognition, a phenomenon that requires DC-derived, instead of exosome-derived, MHC class II molecules. By contrast, allogeneic exosomes are unable to stimulate direct-pathway T cells in vivo. We demonstrate in mice that although graft-infiltrating leukocytes release exosomes ex vivo, they do not secrete enough concentrations of exosomes into circulation to stimulate donor-reactive T cells in secondary lymphoid organs. Instead, our findings indicate that migrating DCs (generated in vitro or isolated from allografts), once they home in the spleen, they transfer exosomes expressing the reporter marker GFP to spleen-resident DCs. Our results suggest that exchange of exosomes between DCs in lymphoid organs might constitute a potential mechanism by which passenger leukocytes transfer alloantigens to recipient’s APCs and amplify generation of donor-reactive T cells following transplantation.

Epidermal growth factor receptor-transfected bone marrow stromal cells exhibit enhanced migratory response and therapeutic potential against murine brain tumors

We have created a novel cellular vehicle for gene therapy of malignant gliomas by transfection of murine bone marrow stroma cells (MSCs) with a cDNA encoding epidermal growth factor receptor (EGFR). These cells (EGFR-MSCs) demonstrate enhanced migratory responses toward glioma-conditioned media in comparison to primary MSCs in vitro. Enhanced migration of EGFR-MSC was at least partially dependent on EGF-EGFR, PI3-, MAP kinase kinase, and MAP kinases, protein kinase C, and actin polymerization. Unlike primary MSCs, EGFR-MSCs were resistant to FasL-mediated cytotoxicity and were capable of stimulating allogeneic mixed lymphocyte reaction, suggesting EGFR-MSCs possess suitable characteristics as vehicles for brain tumor immuno-gene therapy. Following injection at various sites, including the contralateral hemisphere in the brain of syngeneic mice, EGFR-MSCs were able to migrate toward GL261 gliomas or B16 melanoma in vivo. Finally, intratumoral injection with EGFR-MSC adenovirally engineered to secrete interferon-α to intracranial GL261 resulted in significantly prolonged survival in comparison to controls. These data indicate that EGFR-MSCs may serve as attractive vehicles for infiltrating brain malignancies such as malignant gliomas.

Dendritic cells transduced to express interleukin-4 prevent diabetes in nonobese diabetic mice with advanced insulitis.

Our previous studies demonstrated that adoptive transfer of dendritic cells (DC) prevents diabetes in young nonobese diabetic (NOD) mice by inducing regulatory T(H)2 cells. In this report, as a means of treating NOD mice with more advanced insulitis, we infected DC with adenoviral vectors expressing interleukin (IL)-4 (Ad.IL-4), eGFP (Ad.eGFP), or empty vector (Ad psi 5). DC infected with any of the Ad vectors expressed higher levels of CD40, CD80, and CD86 molecules than uninfected DC and Ad.IL-4 DC produced IL-4 after lipopolysaccharide (LPS) and interferon (IFN)-gamma stimulation. Ad-infected DC efficiently stimulated allogeneic T cells, and cultures of T cells with Ad.IL-4 DC produced lower levels of IFN-gamma and marginally higher levels of IL-4. In vivo studies demonstrated that the Ad.eGFP DC trafficked to the pancreatic lymph nodes within 24 hr of intravenous administration, and could be visualized in the T cell areas of the spleen. The intrapancreatic IFN-gamma:IL-4 or IFN-gamma:IL-10 cytokine ratios were lower in 10-week-old mice treated with Ad.IL-4 DC, and these mice were significantly protected from disease. These results demonstrate, for the first time, that genetically modified DC can prevent diabetes in the context of advanced insulitis.

Novel Three‐Dimensional Organotypic Liver Bioreactor to Directly Visualize Early Events in Metastatic Progression

Metastatic seeding leads to most of the morbidity from carcinomas. However, little is known of this key event as current methods to study the cellular behaviors utilize nonrepresentative in vitro models or follow indirect subsequent developments in vivo. Therefore, we developed a system to visualize over a multiday to multiweek period the interactions between tumor cells and target organ parenchyma. We employ an ex vivo microscale perfusion culture system that provides a tissue-relevant environment to assess metastatic seeding behavior. The bioreactor recreates many features of the fluid flow, scale, and biological functionality of a hepatic parenchyma, a common site of metastatic spread for a wide range of carcinomas. As a test of this model, prostate and breast carcinoma cells were introduced. Tumor cell invasion and expansion could be observed by two-photon microscopy of red fluorescent protein (RFP)- and CellTracker-labeled carcinoma cells against a green fluorescent protein (GFP)-labeled hepatic tissue bed over a 14-day period. Tumors visible to the naked eye could be formed by day 25, without evident necrosis in the >0.3-mm tumor mass. These tumor cells failed to grow in the absence of the supporting three-dimensional (3D) hepatic microtissue, suggesting paracrine or stromal support function for the liver structure in tumor progression. Initial ultrastructural studies suggest that early during the tumor-parenchyma interactions, there are extensive interactions between and accommodations of the cancer and host cells, suggesting that the tumor-related epithelial-mesenchymal transition (EMT) reverts, at least transiently, to promote metastatic seeding. In sum, our 3D ex vivo organotypic liver tissue system presents a critical vehicle to examine tumor-host interactions during cancer metastasis and/or invasion. It also circumvents current limitations in assays to assess early events in metastasis, and provides new approaches to study molecular events during tumor progression.

Transgenic Galectin-1 Induces Maturation of Dendritic Cells That Elicit Contrasting Responses in Naive and Activated T Cells

Dendritic cells (DC) are professional APC that control the balance between T cell immunity and tolerance. Genetic engineering of DC to regulate the outcome of the immune response is an area of intense research. Galectin (gal)-1 is an endogenous lectin that binds to glycoproteins and exerts potent regulatory effects on T cells. Consequently, gal-1 participates in central deletion of thymocytes and exerts therapeutic effects on experimental models of T cell-mediated autoimmune disorders and graft-vs-host disease. Together, these observations strongly indicate that engineering DC to express transgenic (tg) gal-1 may be beneficial to treat T cell-mediated disorders. In this study, we have investigated the impact of the expression of high levels of tg gal-1 on maturation/activation of DC and on their T cell stimulatory function. Murine DC were transduced with a recombinant adenovirus encoding hu gal-1 (gal-1-DC). Tg gal-1 was exported by a nonclassical pathway through exosomes and was retained on the DC surface inducing segregation of its ligand CD43. Expression of tg gal-1 triggered activation of DC determined by induction of a more mature phenotype, increased levels of mRNA for proinflammatory cytokines, and enhanced ability to stimulate naive T cells. Conversely, gal-1-DC induced rapid apoptosis of activated T cells. In vivo, gal-1-DC increased significantly the sensitization phase of contact hypersensitivity assays while inducing a drastic inhibition of the elicitation phase by triggering apoptosis of activated T cells in the dermis. Gal-1-DC represent a novel tool to control differentially the afferent and efferent arms of the T cell response.

Delivery of Dendritic Cells Engineered to Secrete IFN-α into Central Nervous System Tumors Enhances the Efficacy of Peripheral Tumor Cell Vaccines: Dependence on Apoptotic Pathways

We tested whether modulation of the CNS-tumor microenvironment by delivery of IFN-α-transduced dendritic cells (DCs: DC-IFN-α) would enhance the therapeutic efficacy of peripheral vaccinations with cytokine-gene transduced tumor cells. Mice bearing intracranial GL261 glioma or MCA205 sarcoma received peripheral immunizations with corresponding irradiated tumor cells engineered to express IL-4 or GM-CSFs, respectively, as well as intratumoral delivery of DC-IFN-α. This regimen prolonged survival of the animals and induced tumor-specific CTLs that expressed TRAIL, which in concert with perforin and Fas ligand (FasL) was involved in the tumor-specific CTL activity of these cells. The in vivo antitumor activity associated with this approach was abrogated by administration of neutralizing mAbs against TRAIL or FasL and was not observed in perforin−/−, IFN-γ−/−, or FasL−/− mice. Transduction of the tumor cells with antiapoptotic protein cellular FLIP rendered the gene-modified cells resistant to TRAIL- or FasL-mediated apoptosis and to CTL killing activity in vitro. Furthermore, the combination therapeutic regimen was ineffective in an intracranial cellular FLIP-transduced MCA205 brain tumor model. These results suggest that the combination of intratumoral delivery of DC-IFN-α and peripheral immunization with cytokine-gene transduced tumor cells may be an effective therapy for brain tumors that are sensitive to apoptotic signaling pathways.

Imaging Secretory Vesicles by Fluorescent Protein Insertion in Propeptide Rather Than Mature Secreted Peptide

We combined confocal and live‐cell imaging with a novel molecular strategy aimed at revealing mechanisms underlying glucose‐regulated insulin vesicle secretion. The ‘Ins‐C‐GFP’ reporter monitors secretory peptide targeting, trafficking, and exocytosis without directly tagging the mature secreted peptide. We trapped a green fluorescent protein (GFP) reporter in equimolar quantity within the secretory vesicle by fusing it within the C peptide of proinsulin which only after nascent vesicle sealing and acidification is cleaved from the mature secreted A and B chains of insulin. Ins‐C‐GFP expression in mouse islets without fail exhibited punctate distribution of green fluorescence by confocal microscopy. Ins‐C‐GFP colocalized GFP with insulin at vesicle dense cores by immuno‐electron microscopy. Glucose stimulation decreased vesicle fluorescence coordinately with enhanced secretion from islets of C‐GFP detected by anti‐GFP Western blots, and of insulin detected by anti‐insulin radioimmunoassay. An insulin secretagogue with a red fluorescent label, glibenclamide BODIPY®TR, was applied to islets expressing Ins‐C‐GFP. The stimulus response was imaged as a rise in red secretagogue leading to marked loss in green granules. Since neuropeptides as well as peptide hormones are processed from propeptides after sealing of secretory granules, vesicle trapping likely is widely applicable for studies on targeting, trafficking, and regulated release of secretory peptides.

Delivery of Interferon-α Transfected Dendritic Cells into Central Nervous System Tumors Enhances the Antitumor Efficacy of Peripheral Peptide-Based Vaccines

We evaluated the effects, on immunity and survival, of injection of interferon (IFN)-α-transfected dendritic cells (DC-IFN-α) into intracranial tumors in mice immunized previously with syngeneic dendritic cells (DCs) pulsed either with ovalbumin-derived CTL or T helper epitopes. These immunizations protected animals from s.c. challenge with ovalbumin-expressing M05 melanoma (class I+ and class II-negative). Notably, antiovalbumin CTL responses were observed in animals vaccinated with an ovalbumin-derived T helper epitope but only after the mice were challenged with M05 cells. This cross-priming of CTL was dependent on both CD4+ and CD8+ T cells. Because we observed that s.c., but not intracranial, tumors were infiltrated with CD11c+ DCs, and because IFN-α promotes the activation and survival of both DCs and T cells, we evaluated the combinational antitumor effects of injecting adenoviral (Ad)-IFN-α-engineered DCs into intracranial M05 tumors in preimmunized mice. Delivery of DC-IFN-α prolonged survival. This was most notable for animals prevaccinated with both the CTL and T helper ovalbumin epitopes, with 60% (6 of 10) of mice (versus 0 of 10 of control animals) surviving for >80 days after tumor challenge. DC-IFN-α appeared to persist longer than mock-transfected DCs within the intracranial tumor microenvironment, and DC-IFN-α-treated mice exhibited enhanced levels of ovalbumin-specific CTL in draining cervical lymph nodes. On the basis of these results, we believe that local expression of IFN-α by DCs within the intracranial tumor site may enhance the clinical efficacy of peripheral vaccine approaches for brain tumors.

Relative contribution of direct and indirect allorecognition in developing tolerance after liver transplantation.

The interaction of donor passenger leukocytes and host leukocytes in recipient secondary lymphoid tissues during the early posttransplantation period is crucial in directing host immune reactions toward allograft rejection or acceptance. Responsible T cell clones could be activated through the direct and indirect pathways of allorecognition. We examined the role of the indirect pathway in liver transplantation (LT) tolerance by depleting host antigen‐presenting cells (APC) with phagocytic activity [e.g., cluster domain (CD)68+/CD163+ macrophages, CD11c+ dendritic cells (DC)] using liposome‐encapsulating clodronate (LP‐CL). After Lewis rat cell or liver graft transplantation, Brown Norway (BN) rat recipients pretreated with LP‐CL showed a significantly reduced type 1 helper T cell cytokine up‐regulation than control‐LP‐treated recipients. In the LT model, LP‐CL treatment and host APC depletion abrogated hepatic tolerance; Lewis liver grafts in LP‐CL‐treated‐BN recipients developed mild allograft rejection, failed to maintain donor major histocompatibility complex (MHC) class II+ leukocytes, and developed chronic rejection in challenged donor heart allografts, while control‐LP‐treated BN recipients maintained tolerance status and donor MHC class II+ hepatic leukocytes. Furthermore, in the BN to Lewis LT model, LP‐CL recipient treatment abrogated spontaneous hepatic allograft acceptance, and graft survival rate was reduced to 43% from 100% in the control‐LP group. In conclusion, the study suggests that host cells with phagocytic activity could play significant roles in developing LT tolerance. Liver Transpl 14:346–357, 2008.