Simon M. Barratt-Boyes

Dendritic Cells Acquire Antigens from Live Cells for Cross-Presentation to CTL

Dendritic cells (DC) can readily capture Ag from dead and dying cells for presentation to MHC class I-restricted CTL. We now show by using a primate model that DC also acquire Ag from healthy cells, including other DC. Coculture assays showed that fluorescently labeled plasma membrane was rapidly and efficiently transferred between DC, and transfer of intracellular proteins was observed to a lesser extent. Acquisition of labeled plasma membrane and intracellular protein was cell contact-dependent and was primarily a function of immature DC, whereas both immature and CD40L-matured DC could serve as donors. Moreover, immature DC could acquire labeled plasma membrane and intracellular proteins from a wide range of hemopoietic cells, including macrophages, B cells, and activated T cells. Notably, macrophages, which readily phagocytose apoptotic bodies, were very inefficient at acquiring labeled plasma membrane and intracellular proteins from other live macrophages or DC. With live-cell imaging techniques, we demonstrate that individual DC physically extract plasma membrane from other DC, generating endocytic vesicles of up to 1 μm in diameter. Finally, DC but not macrophages acquired an endogenous melanoma Ag expressed by live DC and cross-presented Ag to MHC class I-restricted CTL, demonstrating the immunological relevance of our finding. These data show for the first time that DC readily acquire Ag from other live cells. We suggest that Ag acquisition from live cells may provide a novel mechanism whereby DC can present Ag in the absence of direct infection, and may serve to expand and regulate the immune response in vivo.

Protection of Mice and Poultry from Lethal H5N1 Avian Influenza Virus through Adenovirus-Based Immunization

ABSTRACT The recent emergence of highly pathogenic avian influenza virus (HPAI) strains in poultry and their subsequent transmission to humans in Southeast Asia have raised concerns about the potential pandemic spread of lethal disease. In this paper we describe the development and testing of an adenovirus-based influenza A virus vaccine directed against the hemagglutinin (HA) protein of the A/Vietnam/1203/2004 (H5N1) (VN/1203/04) strain isolated during the lethal human outbreak in Vietnam from 2003 to 2005. We expressed different portions of HA from a recombinant replication-incompetent adenoviral vector, achieving vaccine production within 36 days of acquiring the virus sequence. BALB/c mice were immunized with a prime-boost vaccine and exposed to a lethal intranasal dose of VN/1203/04 H5N1 virus 70 days later. Vaccination induced both HA-specific antibodies and cellular immunity likely to provide heterotypic immunity. Mice vaccinated with full-length HA were fully protected from challenge with VN/1203/04. We next evaluated the efficacy of adenovirus-based vaccination in domestic chickens, given the critical role of fowl species in the spread of HPAI worldwide. A single subcutaneous immunization completely protected chickens from an intranasal challenge 21 days later with VN/1203/04, which proved lethal to all control-vaccinated chickens within 2 days. These data indicate that the rapid production and subsequent administration of recombinant adenovirus-based vaccines to both birds and high-risk individuals in the face of an outbreak may serve to control the pandemic spread of lethal avian influenza.

Human infection with highly pathogenic H5N1 influenza virus

Highly pathogenic H5N1 influenza A viruses have spread relentlessly across the globe since 2003, and they are associated with widespread death in poultry, substantial economic loss to farmers, and reported infections of more than 300 people with a mortality rate of 60%. The high pathogenicity of H5N1 influenza viruses and their capacity for transmission from birds to human beings has raised worldwide concern about an impending human influenza pandemic similar to the notorious H1N1 Spanish influenza of 1918. Since many aspects of H5N1 influenza research are rapidly evolving, we aim in this Seminar to provide an up-to-date discussion on select topics of interest to influenza clinicians and researchers. We summarise the clinical features and diagnosis of infection and present therapeutic options for H5N1 infection of people. We also discuss ideas relating to virus transmission, host restriction, and pathogenesis. Finally, we discuss vaccine development in view of the probable importance of vaccination in pandemic control.

MUC-1 epithelial tumor mucin-based immunity and cancer vaccines

Many obstacles still stand in the way to eliciting an effective immune response against cancer, even though several antigens and antigenic peptides have been identified as potential tumor targets. All of them, including the MUC-1 mucin, share the caveat of being normal cellular proteins. Unlike all the others, however, MUC-1 expressed on tumors can still be considered a truly tumor-specific antigen. Its expression on normal cells is hidden from the immune system, and its aberrant glycosylation on tumors creates new epitopes recognized by the immune system. Moreover, all other tumor targets identified so far are MHC-restricted peptides that can only be recognized by patients who carry a specific HLA type, or on tumors which continue to express particular HLA alleles. MUC-1 is powerfully different. Recognized as a native molecule independent of MHC, it is a universal immunogen and a universal target, and if made effectively immunogenic, it would be expected to elicit immune responses in all patients, and against numerous MUC-1 expressing human tumors. It may, in fact, be the extraordinary solution to an extraordinary problem of cancer immunity and immunotherapy.

Effects of a SARS-associated coronavirus vaccine in monkeys

Summary The causative agent of severe acute respiratory syndrome (SARS) has been identified as a new type of coronavirus. Here, we have investigated the ability of adenoviral delivery of codon-optimised SARS-CoV strain Urbani structural antigens spike protein S1 fragment, membrane protein, and nucleocapsid protein to induce virus-specific broad immunity in rhesus macaques. We immunised rhesus macaques intramuscularly with a combination of the three Ad5-SARS-CoV vectors or a control vector and gave a booster vaccination on day 28. The vaccinated animals all had antibody responses against spike protein S1 fragment and T-cell responses against the nucleocapsid protein. All vaccinated animals showed strong neutralising antibody responses to SARS-CoV infection in vitro. These results show that an adenoviral-based vaccine can induce strong SARS-CoV-specific immune responses in the monkey, and hold promise for development of a protective vaccine against the SARS causal agent.

A Role for Class A Scavenger Receptor in Dendritic Cell Nibbling from Live Cells

Monocyte-derived dendritic cells (DC) possess the unique capacity to capture Ag from live cells through intimate cell contact, a process referred to as nibbling. We sought to define the receptor(s) mediating DC nibbling. Uptake of fluorescently labeled plasma membrane from live cells by DC was inhibited by protease treatment and by a panel of polyanionic ligands, implicating scavenger receptors (SR) in this process. Differential expression of SR on DC and macrophages correlated with the capacity to acquire membrane from live cells. Internalized membrane colocalized with SR ligand and entered the endosomal pathway. DC very efficiently acquired and internalized gp100 tumor Ag expressed at the surface of viable adenocarcinoma cells via recombinant adenoviral infection. Cross-presentation of gp100 by DC to MHC class I-restricted T cells was inhibited by polyanionic SR ligand and an Ab to type A SR (SR-A), whereas Ab to the class B SR CD36, which mediates uptake of apoptotic cells, induced no inhibition. DC capture of fluorescently labeled membrane from live cells was partially blocked by SR-A-specific Ab, suggesting that other SR may also be contributing to nibbling. DC maturation resulted in a switch in expression from type II SR-A (SR-AII) to the SR-AI splice variant. Finally, SR-A was identified on interdigitating DC isolated from monkey lymph nodes. These findings define a novel role for SR-A, and suggest that Ag uptake from live cells by DC may be important in the generation of immunity and in the maintenance of peripheral tolerance in vivo.

Maturation and trafficking of monocyte-derived dendritic cells in monkeys: implications for dendritic cell-based vaccines.

Human dendritic cells (DC) have polarized responses to chemokines as a function of maturation state, but the effect of maturation on DC trafficking in vivo is not known. We have addressed this question in a highly relevant rhesus macaque model. We demonstrate that immature and CD40 ligand-matured monocyte-derived DC have characteristic phenotypic and functional differences in vitro. In particular, immature DC express CC chemokine receptor 5 (CCR5) and migrate in response to macrophage inflammatory protein-1α (MIP-1α), whereas mature DC switch expression to CCR7 and respond exclusively to MIP-3β and 6Ckine. Mature DC transduced to express a marker gene localized to lymph nodes after intradermal injection, constituting 1.5% of lymph node DC. In contrast, cutaneous DC transfected in situ via gene gun were detected in the draining lymph node at a 20-fold lower frequency. Unexpectedly, the state of maturation at the time of injection had no influence on the proportion of DC that localized to draining lymph nodes, as labeled immature and mature DC were detected in equal numbers. Immature DC that trafficked to lymph nodes underwent a significant up-regulation of CD86 expression indicative of spontaneous maturation. Moreover, immature DC exited completely from the dermis within 36 h of injection, whereas mature DC persisted in large numbers associated with a marked inflammatory infiltrate. We conclude that in vitro maturation is not a requirement for effective migration of DC in vivo and suggest that administration of Ag-loaded immature DC that undergo natural maturation following injection may be preferred for DC-based immunotherapy.

Parallel loss of myeloid and plasmacytoid dendritic cells from blood and lymphoid tissue in simian AIDS.

The loss of myeloid (mDC) and plasmacytoid dendritic cells (pDC) from the blood of HIV-infected individuals is associated with progressive disease. It has been proposed that DC loss is due to increased recruitment to lymph nodes, although this has not been directly tested. Similarly as in HIV-infected humans, we found that lineage-negative (Lin−) HLA-DR+CD11c+CD123− mDC and Lin−HLA-DR+CD11c−CD123+ pDC were lost from the blood of SIV-infected rhesus macaques with AIDS. In the peripheral lymph nodes of SIV-naive monkeys the majority of mDC were mature cells derived from skin that expressed high levels of HLA-DR, CD83, costimulatory molecules, and the Langerhans cell marker CD1a, whereas pDC expressed low levels of HLA-DR and CD40 and lacked costimulatory molecules, similar to pDC in blood. Surprisingly, both DC subsets were depleted from peripheral and mesenteric lymph nodes and spleens in monkeys with AIDS, although the activation status of the remaining DC subsets was similar to that of DC in health. In peripheral and mesenteric lymph nodes from animals with AIDS there was an accumulation of Lin−HLA-DRmoderateCD11c−CD123− cells that resembled monocytoid cells but failed to acquire a DC phenotype upon culture, suggesting they were not DC precursors. mDC and pDC from the lymphoid tissues of monkeys with AIDS were prone to spontaneous death in culture, indicating that apoptosis may be a mechanism for their loss in disease. These findings demonstrate that DC are lost from rather than recruited to lymphoid tissue in advanced SIV infection, suggesting that systemic DC depletion plays a direct role in the pathophysiology of AIDS.

Making the most of mucin : a novel target for tumor immunotherapy

Throughout this brief review I have emphasized the unique biochemical and immunological properties of MUC-1 mucin that make this tumor-associated antigen a novel, exciting and tangible target for tumor immunotherapy. The tandemly repeating nature of the antigenic epitopes in the mucin polypeptide chain, the under-glycosylation of these epitopes, and the expression of the molecule at the cell surface, are all central to the immune recognition of this antigen and must be acknowledged in the design of MUC-1 vaccines. There are considerable difficulties associated with such a design. Short, immunogenic peptides that associate with MHC class I molecules on the cell surface to induce CTL responses are not useful here; MUC-1 must be expressed at the cell surface and this introduces the numerous problems associated with gene transfer. Furthermore, generating under-glycosylated mucin molecules that resemble tumor-associated antigen is not trivial. Competitive inhibition of glycosylation with PhGalNAc is often incomplete, and merely increasing inhibitor concentration results in cell toxicity. But treating tumors is also not trivial. MUC-1 mucin has many characteristics of the ideal tumor-associated antigen, and our understanding of the unique mechanism of mucin recognition on tumors and the appropriate vaccine designs to target this antigen is now advanced. Transgenic mouse and non-human primate models provide excellent preclinical models to test immunotherapeutic and vaccine strategies, and in vitro studies and early-stage clinical trials in humans provide considerable cause for optimism. The next few years in this field should certainly be productive.

Dendritic Cells: Tools and Targets for Transplant Tolerance

Our knowledge of the role of dendritic cells (DC) in the generation and maintenance of T‐cell tolerance has expanded rapidly and is now a key area of research in basic and applied DC biology. This minireview highlights recent developments in the field that are leading to new avenues for exploiting DC in the promotion of transplant tolerance.