Elizabeth J. Ryan

Distinct T-cell subtypes induced with whole cell and acellular pertussis vaccines in children

Recent clinical trials have demonstrated that new generation acellular pertussis vaccines can confer protection against whooping cough. However, the mechanism of protective immunity against Bordetella pertussis infection induced by vaccination remains to be defined. We have examined cellular immune responses in children immunized with a range of acellular and whole cell pertussis vaccines. Immunization of children with a potent whole‐cell vaccine induced B. pertussis‐specific T cells that secreted interferon‐γ (IFN‐γ), but not interleukin‐5 (IL‐5). In contrast, T cells from children immunized with acellular pertussis vaccines secreted IFN‐γ and/or IL‐5 following stimulation with B. pertussis antigens in vitro. These observations suggest that protective immunity conferred by whole‐cell vaccines, like natural immunity, is mediated by type 1 T cells, whereas the mechanism of immune protection generated with acellular vaccines may be more heterogenous, involving T cells that secreted type 1 and type 2 cytokines.

Immunomodulators and delivery systems for vaccination by mucosal routes

Current paediatric immunization programmes include too many injections in the first months of life. Oral or nasal vaccine delivery eliminates the requirement for needles and can induce immunity at the site of infection. However, protein antigens are poorly immunogenic when so delivered and can induce tolerance. Novel ways to enhance immune responses to protein or polysaccharide antigens have opened up new possibilities for the design of effective mucosal vaccines. Here, we discuss the immunological principles underlying mucosal vaccine development and review the application of immunomodulatory molecules and delivery systems to the selective enhancement of protective immune responses at mucosal surfaces.

Altered natural killer cell subset distributions in resolved and persistent hepatitis C virus infection following single source exposure

Background: Natural killer (NK) cells may be impaired in patients with persistent hepatitis C virus (HCV) infection, but studies to date have yielded inconsistent findings due to patient and virus heterogeneity and difficulties obtaining appropriate controls. Aims: To overcome these variables, we have examined numbers, phenotypes, cytotoxic activities and cytokine profiles of circulating NK cells from Irish women who acquired infection through administration of HCV genotype 1b-contaminated anti-D immunoglobulin from a single source and matched controls. Results: Comparing 29 women who developed persistent infection with 21 who spontaneously resolved infection and 26 controls, we found that NK cell numbers were consistently lower in the persistently infected group (p = 0.02 and 0.002). This decrease was due to depletions of NK cells expressing low levels of CD56 (CD56dim NK cells; p = 0.004 and 0.0001), whilst CD56bright NK cells were expanded (p = 0.004 and 0.0001). Compared to HCV resolvers, CD56dim NK cells from persistently infected patients less frequently expressed CD16 and more frequently expressed NKG2A/C/E. These phenotypic changes did not significantly affect natural or interleukin-2-induced cytotoxicity by peripheral blood mononuclear cells against K562 and Daudi targets. Greater frequencies of CD56bright NK cells from chronic HCV patients produced interferon-γ compared with HCV responders (p = 0.05) and controls (p = 0.0001) after phorbol ester stimulation in vitro. Conclusions: Alterations in NK subset distributions in chronic HCV infection may explain why previous reports of impaired NK cell functions were difficult to confirm. Altered NK cell functions may contribute to impaired cellular immune responses and chronicity of disease following HCV infection.

Hepatitis C Virus-Specific Th17 Cells Are Suppressed by Virus-Induced TGF-β

IL-17-secreting T (Th17) cells play a protective role in certain bacterial infections, but they are major mediators of inflammation and are pathogenic in organ-specific autoimmune diseases. However, human Th17 cells appear to be resistant to suppression by CD4+CD25+FoxP3+ regulatory T cells, suggesting that they may be regulated by alternative mechanisms. Herein we show that IL-10 and TGF-β suppressed IL-17 production by anti-CD3-stimulated PBMC from normal individuals. TGF-β also suppressed IL-17 production by purified CD4+ T cells, whereas the inhibitory effect of IL-10 on IL-17 production appears to be mediated predominantly by its effect on APC. An examination of patients infected with hepatitis C virus (HCV) demonstrated that Ag-specific Th17 cells are induced during infection and that these cells are regulated by IL-10 and TGF-β. PBMC from HCV Ab-positive donors secreted IL-17, IFN-γ, IL-10, and TGF-β in response to stimulation with the HCV nonstructural protein 4 (NS4). Furthermore, NS4 induced innate TGF-β and IL-10 expression by monocytes from normal donors and at higher levels from HCV-infected patients. Neutralization of TGF-β, and to a lesser extent IL-10, significantly enhanced NS4-specific IL-17 and IFN-γ production by T cells from HCV-infected donors. Our findings suggest that both HCV-specific Th1 and Th17 cells are suppressed by NS4-induced production of the innate anti-inflammatory cytokines IL-10 and TGF-β. This may represent a novel immune subversion mechanism by the virus to evade host-protective immune responses. Our findings also suggest that TGF-β and IL-10 play important roles in constraining the function of Th17 cells in general.

Booster immunization of children with an acellular pertussis vaccine enhances Th2 cytokine production and serum IgE responses against pertussis toxin but not against common allergens

Acellular pertussis vaccines (Pa) protect against severe pertussis in children. However, serum antibody responses decline quickly after immunization. Studies in animal models suggest that cell‐mediated immunity also contributes to protection against Bordetella pertussis, and it has already been demonstrated that Pa induce T cells that secrete type‐1 and type‐2 cytokines in children. In this study we examined the persistence of the T cell response and the effect of booster immunization in 4–6‐year‐old children. Cell‐mediated immunity to B. pertussis antigens was detected in a high proportion of children more than 42 months after their last immunization. Peripheral blood mononuclear cells (PBMC) from the majority of children secreted interferon‐gamma (IFN‐γ) and a smaller proportion IL‐5, in response to specific antigen stimulation in vitro. However, following booster immunization, significantly higher concentrations of IL‐5, but not IFN‐γ, were produced by PBMC in response to B. pertussis antigens. Furthermore, plasma IL‐4 and IL‐5 concentrations were increased, whereas IFN‐γ concentrations were reduced following booster immunization. It has been suggested that childhood immunization with Th2‐inducing vaccines may predispose some children to atopic disease. Although we found that pertussis toxin (PT)‐specific IgE was significantly increased after booster immunization in both atopic and non‐atopic children, the levels of IgE to common allergens and the prevalence of positive skin prick test were unaffected by the booster vaccination. Thus, despite the enhancement of type‐2 responses to B. pertussis antigens, booster vaccination with Pa does not appear to be a risk factor for allergy.

Caspase Activity Is Required for Stimulated B Lymphocytes to Enter the Cell Cycle

Following activation with proliferative stimuli, including ligation of CD40, dense human tonsillar B cells (>98% cells in G0) have increased cleavage and activation of caspase-8 and -6 accompanied by decreased caspase-3 activation and apoptosis. Proliferation was blocked by either a broad specificity caspase inhibitor or inhibitors selective for caspase-6 or caspase-8. In contrast, an inhibitor selective for caspase-3 was without effect. Furthermore, induction of cyclin D and cyclin-dependent kinase 4 mRNA and protein was blocked upon inhibition of caspase-6, but not caspase-3. Thus, caspase-6-like activity is required for quiescent B cells to increase the expression of genes required for entry into G1. In support of this model, the transcriptional suppressor special AT-rich sequence-binding protein 1, a preferred caspase-6 substrate, was cleaved upon B cell stimulation. Caspase activity was not required for all signaling events, as caspase inhibitors did not affect the phosphorylation of p42/44 mitogen-activated protein kinase, the expression of the survival factor cellular inhibitor of apoptosis 2, or the production of IL-6 by stimulated G0 B cells. These findings suggest a mechanism by which caspase-6 may selectively allow entry of quiescent B cells into the cell cycle.

Tumour Tissue Microenvironment Can Inhibit Dendritic Cell Maturation in Colorectal Cancer

Inflammatory mediators in the tumour microenvironment promote tumour growth, vascular development and enable evasion of anti-tumour immune responses, by disabling infiltrating dendritic cells. However, the constituents of the tumour microenvironment that directly influence dendritic cell maturation and function are not well characterised. Our aim was to identify tumour-associated inflammatory mediators which influence the function of dendritic cells. Tumour conditioned media obtained from cultured colorectal tumour explant tissue contained high levels of the chemokines CCL2, CXCL1, CXCL5 in addition to VEGF. Pre-treatment of monocyte derived dendritic cells with this tumour conditioned media inhibited the up-regulation of CD86, CD83, CD54 and HLA-DR in response to LPS, enhancing IL-10 while reducing IL-12p70 secretion. We examined if specific individual components of the tumour conditioned media (CCL2, CXCL1, CXCL5) could modulate dendritic cell maturation or cytokine secretion in response to LPS. VEGF was also assessed as it has a suppressive effect on dendritic cell maturation. Pre-treatment of immature dendritic cells with VEGF inhibited LPS induced upregulation of CD80 and CD54, while CXCL1 inhibited HLA-DR. Interestingly, treatment of dendritic cells with CCL2, CXCL1, CXCL5 or VEGF significantly suppressed their ability to secrete IL-12p70 in response to LPS. In addition, dendritic cells treated with a combination of CXCL1 and VEGF secreted less IL-12p70 in response to LPS compared to pre-treatment with either cytokine alone. In conclusion, tumour conditioned media strongly influences dendritic cell maturation and function.

Dendritic Cell-Associated Lectin-1: A Novel Dendritic Cell-Associated, C-Type Lectin-Like Molecule Enhances T Cell Secretion of IL-4

We have characterized dendritic cell (DC)-associated lectin-1 (DCAL-1), a novel, type II, transmembrane, C-type lectin-like protein. DCAL-1 has restricted expression in hemopoietic cells, in particular, DCs and B cells, but T cells and monocytes do not express it. The DCAL-1 locus is within a cluster of C-type lectin-like loci on human chromosome 12p12–13 just 3′ to the CD69 locus. The consensus sequence of the DCAL-1 gene was confirmed by RACE-PCR; however, based on sequence alignment with genomic DNA and with various human expressed sequence tags, we predict that DCAL-1 has two splice variants. C-type lectins share a common sequence motif of 14 invariable and 18 highly conserved aa residues known as the carbohydrate recognition domain. DCAL-1, however, is missing three of the cysteine residues required to form the standard carbohydrate recognition domain. DCAL-1 mRNA and protein expression are increased upon the differentiation of monocytes to CD1a+ DCs. B cells also express high levels of DCAL-1 on their cell surface. Using a DCAL-1 fusion protein we identified a population of CD4+ CD45RA+ T cells that express DCAL-1 ligand. Coincubation with soluble DCAL-1 enhanced the proliferation of CD4+ T cells in response to CD3 ligation and significantly increased IL-4 secretion. In contrast, coincubation with soluble DC-specific ICAM-3-grabbing nonintegrin (CD209) fusion protein as a control had no effect on CD4+ T cell proliferation or IL-4 and IFN-γ secretion. Therefore, the function of DCAL-1 on DCs and B cells may act as a T cell costimulatory molecule, which skews CD4+ T cells toward a Th2 response by enhancing their secretion of IL-4.

CD141+ myeloid dendritic cells are enriched in healthy human liver

BACKGROUND & AIMS Extensive populations of liver immune cells detect and respond to homeostatic perturbation caused by damage, infection or malignancy. Dendritic cells (DCs) are central to these activities, governing the balance between tolerance and immunity. Most of our knowledge about human liver DCs is derived from studies on peritumoral tissue. Little is known about the phenotype and function of DCs, in particular the recently described CD141(+) subset, in healthy human liver and how this profile is altered in liver disease. METHODS During liver transplantation, healthy donor and diseased explant livers were perfused and hepatic mononuclear cells isolated. Dendritic cell subset frequency and phenotype were characterised in liver perfusates by flow cytometry and the function of CD141(+) DCs was evaluated by mixed lymphocyte reactions (MLRs) and measuring cytokine secretion. RESULTS Almost one third of liver CD11c(+) myeloid DCs (mDCs) expressed CD141 compared to <5% of circulating mDCs. Hepatic CD141(+) DCs demonstrated pro-inflammatory function in allogeneic MLRs, inducing T cell production of interferon gamma (IFN-γ) and interleukin (IL)-17. While CD123(+) plasmacytoid DCs (pDCs) and CD1c(+) mDCs were expanded in diseased liver perfusates, CD141(+) DCs were significantly depleted. Despite their depletion, CD141(+) DCs from explant livers produced markedly increased poly(I:C)-induced IFN lambda (IFN-λ) compared with donor DCs. CONCLUSIONS Accumulation of CD141(+) DCs in healthy liver, which are significantly depleted in liver disease, suggests differential involvement of mDC subsets in liver immunity.

Estrogen Receptors and Their Implications in Colorectal Carcinogenesis

Upon binding their cognate receptors, ERα (ESR1) and ERβ (ESR2), estrogens activate intracellular signaling cascades that have important consequences for cellular behavior. Historically linked to carcinogenesis in reproductive organs, estrogens have also been implicated in the pathogenesis of different cancer types of non-reproductive tissues including the colon. ERβ is the predominant estrogen receptor expressed in both normal and malignant colonic epithelium. However, during colon cancer progression, ERβ expression is lost, suggesting that estrogen signaling may play a role in disease progression. Estrogens may in fact exert an anti-tumor effect through selective activation of pro-apoptotic signaling mediated by ERβ, inhibition of inflammatory signals and modulation of the tumor microenvironment. In this review, we analyze the estrogen pathway as a possible therapeutic avenue in colorectal cancer, we report the most recent experimental evidence to explain the cellular and molecular mechanisms of estrogen-mediated protection against colorectal tumorigenesis, and we discuss future challenges and potential avenues for targeted therapy.