Margaret A. Baird

Liposomal delivery of antigen to human dendritic cells

This study investigated whether formulation of antigen in mannosylated liposomes enhanced uptake and activation of dendritic cells (DC) and increased the ability of DC to induce primed T cell proliferation compared to formulation of antigen in unmodified liposomes or in solution. Immature human DC were generated from peripheral blood monocytes cultured with GM-CSF and IL-4. Uptake of antigen by DC and the degree of expression of the cell surface markers MHC class II, CD80, CD86 and the DC maturation marker CD83, was investigated by flow cytometry following incubation with liposomes or solution containing FITC-conjugated antigen. Exposure to liposomes containing FITC-ovalbumin resulted in enhanced expression of cell surface markers when compared to exposure to antigen in solution. Expression was highest following exposure to mannosylated liposomes. Mannosylated liposomes containing tetanus toxoid (TT) stimulated primed T cell proliferation more effectively than TT-neutral liposomes or TT-solution. This work suggests that mannosylated liposomes provide a versatile delivery vehicle for initiating enhanced immune responses to encapsulated peptide or protein vaccines.

Bifidobacterial Species Differentially Affect Expression of Cell Surface Markers and Cytokines of Dendritic Cells Harvested from Cord Blood

ABSTRACT The gut microbiota may be important in the postnatal development of the immune system and hence may influence the prevalence of atopic diseases. Bifidobacteria are the most numerous bacteria in the guts of infants, and the presence or absence of certain species could be important in determining the geographic incidence of atopic diseases. We compared the fecal populations of bifidobacteria from children aged 25 to 35 days in Ghana (which has a low prevalence of atopy), New Zealand, and the United Kingdom (high-prevalence countries). Natal origin influenced the detection of bifidobacterial species in that fecal samples from Ghana almost all contained Bifidobacterium infantis whereas those of the other children did not. Choosing species on the basis of our bacteriological results, we tested bifidobacterial preparations for their effects on cell surface markers and cytokine production by dendritic cells harvested from cord blood. Species-specific effects on the expression of the dendritic-cell activation marker CD83 and the production of interleukin-10 (IL-10) were observed. Whereas CD83 expression was increased and IL-10 production was induced by Bifidobacterium bifidum, Bifidobacterium longum, and Bifidobacterium pseudocatenulatum, B. infantis failed to produce these effects. We concluded that B. infantis does not trigger the activation of dendritic cells to the degree necessary to initiate an immune response but that B. bifidum, B. longum, and B. pseudocatenulatum induce a Th2-driven immune response. A hypothesis is presented to link our observations to the prevalence of atopic diseases in different countries.

Lipid based particulate formulations for the delivery of antigen

Particulate adjuvant systems are largely classified according to their functional characteristics, such as the nature of the typical immune response they induce, or their perceived mode of action. From a formulation science perspective, it is practical to classify antigen delivery systems according to the physical nature of the formulations. This article discusses lipid based particulate systems, grouped according to the nature of their predominant lipid constituent.

Gut commensal Lactobacillus reuteri 100-23 stimulates an immunoregulatory response.

Lactobacillus reuteri 100‐23 is a bacterial commensal of the gastrointestinal tract of mice. Previous studies have shown that colonization of the murine gut by this strain stimulates small‐bowel enterocytes to produce proinflammatory cytokines. This is associated with a mild, transitory inflammatory response 6 days after inoculation of formerly Lactobacillus‐free animals. The inflammation subsides by 21 days after colonization, although lactobacilli continue to be present in the bowel. To determine the immunological mechanisms that underpin tolerance to bowel commensals, we investigated cytokine responses of dendritic cells and T cells after exposure to cells of L. reuteri 100‐23. Interleukin‐10 (IL‐10), IL‐2 and transforming growth factor‐β (TGF‐β) concentrations in supernatants of cultured immune cells, as well as the results of proliferative assays of mesenteric lymph node (MLN) cells and quantification of Foxp3‐positive cells in MLN and spleen, indicated that L. reuteri 100‐23 stimulated the development of an increased number of regulatory T cells.

Interleukin‐10 does not affect phagocytosis of particulate antigen by bonemarrow‐derived dendritic cells but does impair antigen presentation

Dendritic cells (DC) are important initiators of an immune response so understanding the factors controlling antigen acquisition and presentation has important consequences for the use of these cells in vaccines and other forms of immunotherapy. We investigated the factors that influence phagocytosis by immature bone marrow‐derived DC (BMDC) and the effect of interleukin‐10 (IL‐10) on this process. Two sizes of fluorescent particles and recombinant bacillus Calmette–Guèrin expressing the green fluorescent protein (rBCG) were used as particulate antigens. The percentage of cells taking up the antigen was found to be dependent on the size and dose of the particles, and the length of exposure to them. BMDC exposed to IL‐10 at various concentrations for different periods exhibited no distinguishable change in antigen uptake. However, if BMDC treated with IL‐10 and rBCG were then exposed to a second dose of particulate antigen, uptake was increased compared with those BMDC not treated with IL‐10. The expression of major histocompatibility complex class II, CD80, CD86 and CD11c by BMDC after phagocytosing rBCG or inert beads, was inhibited when the BMDC were pretreated with IL‐10. In contrast, the expression of CD25 was increased. BMDC that had taken up BCG or purified protein derivative (PPD) were able to stimulate primed T‐cell proliferation but this was severely inhibited if the BMDC were cultured with IL‐10 before exposure to the antigen. This work suggests that although IL‐10 does not affect the phagocytic capacity of BMDC, it does inhibit maturation of the cells and consequently, T‐cell activation.

Structure and functions of exopolysaccharide produced by gut commensal Lactobacillus reuteri 100-23

Lactobacillus reuteri strain 100-23 together with a Lactobacillus-free mouse model, provides a system with which the molecular traits underpinning bacterial commensalism in vertebrates can be studied. A polysaccharide was extracted from sucrose-containing liquid cultures of strain 100-23. Chemical analysis showed that this exopolysaccharide was a levan (β-2, 6-linked fructan). Mutation of the fructosyl transferase (ftf) gene resulted in loss of exopolysaccharide production. The ftf mutant was able to colonise the murine gastrointestinal tract in the absence of competition, but colonisation was impaired in competition with the wild type. Biofilm formation by the mutant on the forestomach epithelial surface was not impaired and the matrix between cells was indistinguishable from that of the wild type in electron micrographs. Colonisation of the mouse gut by the wild-type strain led to increased proportions of regulatory T cells (Foxp3+) in the spleen, whereas colonisation by the ftf mutant did not. Survival of the mutant in sucrose-containing medium was markedly reduced relative to the wild type. Comparison of the genomic ftf loci of strain 100-23 with other L. reuteri strains suggested that the ftf gene was acquired by lateral gene transfer early in the evolution of the species and subsequently diversified at accelerated rates. Levan production by L. reuteri 100-23 may represent a function acquired by the bacterial species for life in moderate to high-sucrose extra-gastrointestinal environments that has subsequently been diverted to novel uses, including immunomodulation, that aid in colonisation of the murine gut.

Cross-presentation of epitopes on virus-like particles via the MHC I receptor recycling pathway

Effective vaccines and immunotherapies against cancer require professional antigen‐presenting cells to cross‐present exogenous antigen to initiate cytotoxic T‐cell responses to destroy tumors. Virus‐like particles (VLPs), containing tumor antigens, which can immunize against cancers, are cross‐presented by dendritic cell (DC) but the mechanism by which this occurs is not fully understood. Here, we used VLPs, derived from rabbit hemorrhagic disease virus (RHDV) with both murine and human DCs, to elucidate these pathways. We have employed inhibitors to demonstrate that these VLPs are taken up by clathrin‐dependent macropinocytosis and phagocytosis before being degraded in acidic lysosomal compartments. VLP‐derived peptides are loaded onto major histocompatibility complex I that have been recycled from the cell surface. Antigen‐coupled VLPs and murine ovalbumin‐specific and human melanoma‐associated antigen recognized by T cells (MART‐1)‐specific CD8+ T cells were used to demonstrate cross‐presentation via this alternate, receptor recycling pathway, which operated independently of the proteasome and the transporter‐associated with antigen presentation. Finally, we found that cross‐presentation of VLPs in vivo was not confined to CD8α+ DC subsets. These data define the cross‐presentation pathway for RHDV VLPs and may lead to improved cancer immunotherapies.

Oral vaccination of mice with lipid-encapsulated Mycobacterium bovis BCG: Anatomical sites of bacterial replication and immune activity

Lipid microencapsulation of Mycobacterium bovis bacille Calmette–Guérin (BCG) produces an oral delivery vaccine that can establish systemic cell‐mediated immune reactivity and protection against aerosol mycobacterial challenge in mice. Here, we describe the lymphatic and mucosal sites of bacterial replication, and location of Mycobacterium‐specific IFN‐γ‐secreting cell populations, following oral vaccination of BALB/c mice. Eight weeks following a single oral dose of lipid‐encapsulated BCG, viable BCG organisms were recovered from the mesenteric lymph nodes (MLN) of 11/12 mice investigated (93%). Live bacteria were also occasionally recovered from the cervical lymph nodes (17%) and Peyers patches (8%), but not from homogenates of the lungs or spleen. Strong Mycobacterium‐specific IFN‐γ production was recorded among isolated splenocytes, but not among populations of mononuclear cells derived from the MLN or lungs. Oral vaccination of mice with lipid‐encapsulated BCG thus appears to promote a state of systemic immunological reactivity more akin to that observed following parenteral rather than conventional oral vaccination, despite the fact that replicating bacilli are restricted to lymphatic tissues of the alimentary tract. Possible patterns of lymphocyte sensitization and trafficking are discussed.

p53-mediated apoptosis prevents the accumulation of progenitor B cells and B-cell tumors

We propose that the apoptotic function of p53 has an important role in B-cell homeostasis, which is important for the prevention of B-cell lymphomas. We created a mouse model (mΔpro) that lacked residues 58–88 of the proline-rich domain of p53. mΔpro is defective for apoptosis, but is able to arrest cell-cycle progression in hematopoietic tissues. mΔpro develops late-onset B-cell lymphoma, but not the thymic T-cell tumors found in p53-null mice. Interestingly, mΔpro lymphomas comprised incorrectly differentiated B cells. B-cell irregularities were also detected in mΔpro before tumor onset, in which aged mice showed an increased population of inappropriately differentiated B cells in the bone marrow and spleen. We predict that by keeping B-cell populations in check, p53-dependent apoptosis prevents irregular B cells from eventuating in lymphomas.

The immune response to autologous bacteroides in ankylosing spondylitis is characterized by reduced interleukin 10 production.

Objective. Ileocolitis is a recognized feature of ankylosing spondylitis (AS) and is likely to play a role in the pathogenesis of AS, in conjunction with the normal intestinal microbiota. In order to investigate the host immune response in AS, we measured cytokines in tissue culture following exposure of peripheral blood mononuclear cells (PBMC) to autologous colonic bacteria. Methods. Twenty-one patients with AS and 21 matched controls were recruited. Subjects in the AS group were assessed clinically. Bacteroides species belonging to the B. fragilis group were selectively cultured from stool samples and paired with blood samples from each participant. Ten cultures of autologous Bacteroides were randomly selected from cultures grown from the fecal specimens of each of the 21 patients with AS and 21 controls. These were then tested for reactivity with PBMC and the cytokines produced by proliferating lymphocytes [interleukin 10 (IL-10), IL-17, interferon-γ, tumor necrosis factor-α] were measured in cell culture supernatants. Differences between groups were analyzed using censored normal regression analysis. Results. The patients with AS had severe active AS with Bath AS Disease Activity Index 5.5 (± 1.6) and C-reactive protein (mg/l) 13.8 (± 12.2) (mean ± standard deviation). IL-10 concentrations in ex vivo assay supernatants were lower in the AS group compared with controls (p = 0.047). There were no statistically significant differences between the groups for other cytokines. Conclusion. In AS, reduced IL-10 production in response to stimulation with autologous Bacteroides cultures may represent a mechanism by which intestinal inflammation develops and persists, a situation analogous to inflammatory bowel disease.