[Show abstract][Hide abstract] ABSTRACT: Bullous pemphigoid is a blistering skin disease characterized by autoantibodies against the NC16a domain of bullous pemphigoid 180. This study was performed to characterize and map the fine specifity of T cell responses to NC16a. PBMC from a total of 28 bullous pemphigoid patients and 14 matched controls were tested for proliferative and cytokine responses to recombinant NC16a and a complete panel of 21 overlapping peptides spanning this region of BP180. Proliferative responses to NC16A and the peptide panel in the patients with active disease were similar in frequency and magnitude to those in healthy donors, and included late responses typical of naive cells in about 60% of each group. IL-4 responses were slightly stronger for 6 peptides, and significantly stronger to Nc16a, in patients than controls. Factor analysis identified factors that separate responses to the peptide panel discretely into IL-4(Th2 pattern), IFNγ(Th1 pattern) and IL-10 or TGFβ(Treg pattern). Factors segregating IL-10 versus IFNγ were predicted by active blistering or remission, and TGFβ or IL-10 versus IFNγ by age. Finally, we confirmed a significant upregulation of IgE responses to BP180 in the patients with pemphigoid. This shows the complexity of T cell phenotype and fine autoreactive specificity in responses to NC16A, in patients and normal controls. Important disease associated factors determine the balance of cytokine responses. Of these, specific IL-4 and IgE responses show the strongest associations with pemphigoid, pointing to an important contribution by Th2 cytokines to pathogenesis.
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[Show abstract][Hide abstract] ABSTRACT: Macrophages are antigen presenting cells that can adopt different activation states as directed by microenvironmental stimuli. It is well-recognised how CD4+ T helper (Th) signals drive macrophage activation, but the ability of differentially activated human macrophages to stimulate the major types of CD4+ T helper (Th) response by presenting antigen have not been well defined. Previous studies have focussed on murine cells, undifferentiated human monocytes, or macrophage products, and have been limited to non-physiological mitogenic Th responses. The aim was therefore to compare the Th cell polarising abilities of different human macrophage subsets when presenting specific antigen. We demonstrate for the first time that the way macrophages are activated, while naturally presenting antigen, has profound effects on downstream adaptive immune responses. In autologous co-cultures, LPS-activation was the most potent stimulus for antigen-loaded macrophages to drive Th17 polarisation from both unfractionated CD4+ T-cells and the CD45RO+ memory population, while IFNγ/LPS activated macrophages preferentially induced a Th1 phenotype. By contrast, IL-4-activated macrophages were ineffective in inducing responses by either Th subset. Although antigen-loaded dendritic cells were superior to macrophages in driving Th1 responses, the Th17 polarising capacity of the two antigen-presenting cell types was equivalent, and was strongly dependent on IL-1β secretion. Taken together, these results clearly demonstrate for the first time how differentially activated human macrophages present antigen to bias specific, rather than mitogen-driven, Th responses and lead us to propose that they impact adaptive immunity in vivo, particularly in determining Th17 polarisation within inflamed tissues.
[Show abstract][Hide abstract] ABSTRACT: CTLA-4 is an inhibitory protein that contributes to immune homeostasis and tolerance, a role that has led to its exploitation as a therapeutic in several clinical settings including cancer and autoimmune disease. Development of CTLA-4 therapies focused largely on the full-length receptor isoform but other CTLA-4 isoforms are also expressed, including a secretable form of CTLA-4 (soluble CTLA-4 [sCTLA-4]). The contribution of sCTLA-4 to immune regulation has been less well studied, primarily because it was identified some years after the original description of CTLA-4. Here, we examine how sCTLA-4 might contribute to immune regulation and ask whether it might be a biomarker to inform current CTLA-4 therapies or represent a novel CTLA-4 target for future therapeutics.
[Show abstract][Hide abstract] ABSTRACT: Although it is widely believed that IL-27 is anti-inflammatory, its role in controlling human immune responses is not fully established. In particular, its interactions with Th17 cytokines are unclear. Our aims were to establish the relationships between IL-27 and pro-inflammatory cytokines, including IL-17A, in human sera and cultures of peripheral blood mononuclear cells. Plasma IL-27 levels in 879 healthy humans from 163 families varied widely, but with relatively low heritability (19%). Despite IL-27 including a subunit encoded by Epstein-Barr virus-induced gene 3 (EBI3), there was no correlation of levels with serological evidence of infection with the virus. Although IL-27 has been reported to inhibit IL-17A production, we demonstrated a strong positive correlation in sera, but lower correlations of IL-27 with other pro-inflammatory cytokines. We verified that IL-27 inhibited IL-17A production by human peripheral blood T cells in vitro, but not that it stimulated IL-10 secretion. Importantly, addition of IL-17A decreased IL-27 production by stimulated T cells but had the opposite effect on resting T cells. Together, these data suggest a model whereby IL-27 and IL-17A exerts complex reciprocal effects to boost inflammatory responses, but restrain resting cells to prevent inappropriate activation.
[Show abstract][Hide abstract] ABSTRACT: Candida albicans remains the fungus most frequently associated with nosocomial bloodstream infection. In disseminated candidiasis, the role of Foxp3(+) regulatory T (Treg) cells remain largely unexplored. Our aims were to characterize Foxp3(+) Treg-cell activation in a murine intravenous challenge model of disseminated C. albicans infection, and determine the contribution to disease. Flow cytometric analyses demonstrated that C. albicans infection drove in vivo expansion of a splenic CD4(+) Foxp3(+) population that correlated positively with fungal burden. Depletion from Foxp3(hCD2) reporter mice in vivo confirmed that Foxp3(+) cells exacerbated fungal burden and inflammatory renal disease. The CD4(+) Foxp3(+) population expanded further after in vitro stimulation with C. albicans antigens, and included at least three cell types. These arose from proliferation of the natural Treg-cell subset, together with conversion of Foxp3(-) cells to the induced Treg-cell form, and to a cell type sharing effector Th17-cell characteristics, expressing ROR-γt and secreting IL-17A. The expanded Foxp3(+) T cells inhibited Th1 and Th2 responses, but enhanced Th17-cell responses to C. albicans antigens in vitro, and in vivo depletion confirmed their ability to enhance the Th17-cell response. These data lead to a model for disseminated candidiasis whereby expansion of Foxp3(+) T cells promotes Th17-cell responses that drive pathology. This article is protected by copyright. All rights reserved.
European Journal of Immunology 04/2014; 44(4). DOI:10.1002/eji.201343604 · 4.03 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Autoreactive CD4(+) helper T cells specific for a range of nucleoprotein-derived autoantigens are an important feature of systemic lupus erythematosus, driving B cell differentiation and autoantibody production and contributing to the inflammatory lesions caused by immune complex deposition. Several peptide epitopes from nucleoprotein antigens have been identified and offer a means selectively to manipulate T cell responses by skewing toward a profile of cytokines that is less pro-inflammatory.Antigen-specific T cell lines and clones can be useful in the study of helper T cell subsets because their life span is prolonged and many individual cells can be generated, allowing particular phenotypes to be studied in detail. Magnetic beads offer a robust and convenient method for the isolation, polarization, and expansion of T cells, which can be adapted for a broad range of applications.
[Show abstract][Hide abstract] ABSTRACT: The offspring from pregnancies of women who have developed anti-D blood group antibodies are at risk of hemolytic disease of the newborn. We have previously mapped four peptides containing immunodominant T-helper cell epitopes from the RhD protein and the purpose of the work was to develop these into a product for suppression of established anti-D responses. A panel of each of the four immunodominant RhD peptides was synthesized with modifications to improve manufacturability and solubility, and screened for retention of recognition by human T-helper cells. A selected version of each sequence was combined in a mixture (RhDPmix), which was tested for suppressive ability in a humanized murine model of established immune responses to RhD protein. After HLA-DR15 transgenic mice had been immunized with RhD protein, a single dose of RhDPmix, given either intranasally (p=0.008, Mann-Whitney rank sum test) or subcutaneously (p=0.043), rapidly and significantly suppressed the ongoing antibody response. This was accompanied by reduced T-helper cell responsiveness, although this change was less marked for subcutaneous RhDPmix delivery, and by the recruitment of cells with a regulatory T-cell phenotype. The results support human trials of RhDPmix peptide immunotherapy in women with established antibody responses to the RhD blood group.
[Show abstract][Hide abstract] ABSTRACT: Macrophages respond to their microenvironment and develop polarized functions critical for orchestrating appropriate inflammatory responses. Classical (M1) activation eliminates pathogens while alternative (M2) activation promotes regulation and repair. M1 macrophage activation is strongly associated with suppressor of cytokine signalling 3 (SOCS3) expression in vitro, but the functional consequences of this are unclear and the role of SOCS3 in M1-macrophage polarization in vivo remains controversial. To address these questions, we defined the characteristics and function of SOCS3-expressing macrophages in vivo and identified potential mechanisms of SOCS3 action. Macrophages infiltrating inflamed glomeruli in a model of acute nephritis show significant up-regulation of SOCS3 that co-localizes with the M1-activation marker, inducible nitric oxide synthase. Numbers of SOCS3hi-expressing, but not SOCS1hi-expressing, macrophages correlate strongly with the severity of renal injury, supporting their inflammatory role in vivo. Adoptive transfer of SOCS3-short interfering RNA-silenced macrophages into a peritonitis model demonstrated the importance of SOCS3 in driving production of pro-inflammatory IL-6 and nitric oxide, while curtailing expression of anti-inflammatory IL-10 and SOCS1. SOCS3-induced pro-inflammatory effects were due, at least in part, to its role in controlling activation and nuclear accumulation of nuclear factor-κB and activity of phosphatidylinositol 3-kinase. We show for the first time that SOCS3 also directs the functions of human monocyte-derived macrophages, including efficient M1-induced cytokine production (IL-1β, IL-6, IL-23, IL-12), attenuated signal transducer and activator of transcription 3 activity and ability of antigen-loaded macrophages to drive T-cell responses. Hence, M1-associated SOCS3 was a positive regulator of pro-inflammatory responses in our rodent models and up-regulated SOCS3 is essential for effective M1-macrophage activation and function in human macrophages.
[Show abstract][Hide abstract] ABSTRACT: Low maternal dietary vitamin E (but not vitamin C) intake during pregnancy has been associated with increased in vitro cord blood mononuclear cell (CBMC) proliferative responses, childhood wheezing and asthma. We investigated whether these associations reflect direct effects of vitamin E by investigating the effects of supplementing CBMC cultures with physiological concentrations of vitamin E. CBMC from seventy neonates were cultured supplemented with either nothing, α-tocopherol or ascorbic acid. Proliferative, IFN-γ, IL-4, IL-10 and TGF-β responses were measured. In general, vitamin E supplementation was associated with a trend for reduced proliferative responses after stimulation with antigens and house dust mite, and with increased proliferation after stimulation with timothy grass allergen. There was a trend for CBMC cultures to exhibit decreased secretion of IFN-γ, IL-10 and IL-4. Supplementation with vitamin C had no effect on CBMC proliferation, but increased IFN-γ and IL-4 production, and decreased IL-10 production. In conclusion, in vitro vitamin E and C supplementation of CBMC modifies neonatal immune function, but not in a manner predicted by observational epidemiological studies. The observed associations between vitamin E and childhood respiratory disease are complex, and the nature and form of nutritional intervention need to be carefully considered before inclusion in trials.
[Show abstract][Hide abstract] ABSTRACT: CTLA-4 is a crucial immune regulator that mediates both negative costimulation signals to T cells, and regulatory T (Treg)-cell extrinsic control of effector responses. Here we present evidence supporting a novel mechanism for this extrinsic suppression, executed by the alternatively spliced soluble CTLA-4 isoform (sCTLA-4). Analyses of human T cells in vitro show that sCTLA-4 secretion can be increased during responses, and has potent inhibitory properties, since isoform-specific blockade of its activity significantly increased Ag-driven proliferation and cytokine (IFN-γ, IL-17) secretion. Treg cells were demonstrated to be a prominent source of sCTLA-4, which contributed to suppression in vitro when their numbers were limiting. The soluble isoform was also produced by, and inhibited, murine T cells responding to Ag in vitro, and blockade of its activity in vivo protected against metastatic spread of melanoma in mice. We conclude that sCTLA-4 is an important immune regulator, responsible for at least some of the inhibitory effects previously ascribed to the membrane-bound isoform. These results suggest that the immune system exploits the different CTLA-4 isoforms for either intrinsic or extrinsic regulation of T-cell activity.
European Journal of Immunology 05/2013; 43(5). DOI:10.1002/eji.201242529 · 4.03 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Autoimmune haemolytic anaemia (AIHA) is caused by autoantibodies against RBC surface antigens that render RBC susceptible to Fc-mediated phagocytosis and complement-mediated lysis. Experimental AIHA can be induced by injection of rat RBC to naïve mice but a lymphocyte-mediated regulatory mechanism eventually suppresses the production of autoantibodies specific for mouse RBC. Critically, this tolerogenic response can be transferred to naïve mice by splenocytes from the rat RBC immunized mouse. Here we investigate whether Indoleamine 2,3 dioxygenase (IDO) or the initiators of IDO cascade, including the CTLA-4 receptor and its soluble isoform contribute to this tolerogenic mechanism. Splenocytes from experimental AIHA mice were adoptively transferred to naive mice under the cover of anti-CTLA-4, anti-soluble CTLA-4 antibodies or IDO inhibitor 1-Methyl Tryptophan (1-MT). Recipient mice were immunized with rat RBC and levels of antibody against self-RBC and rat-RBC were monitored. Our results indicate that transfer of tolerance to naïve recipients is dependent on IDO mediated immunosuppression as mice receiving previously tolerized splenocytes under the cover of 1-MT were refractory to tolerance and developed haemolytic disease upon further challenge with rat RBC. Initiators of IDO activity, CTLA-4 or soluble CTLA-4 did not mediate this tolerogenic process but on their blockade, boosted antigen-specific effector immune responses.
[Show abstract][Hide abstract] ABSTRACT: Cytokines are small secreted ‘messenger proteins’ that allow communication between cells. They contribute to a chemical signalling language that controls many processes in multicellular organisms, including haematopoiesis, chemoattraction, tissue repair, embryogenesis and virtually all aspects of immunity and inflammation. Cytokine functions are mediated by their binding to specific receptors to initiate intracellular signalling cascades that increase or decrease expression of transcription factors and genes regulating, for example, secretion of effector molecules (including other cytokines), cell proliferation and expression of membrane receptors. They play an important role in many pathologies, and indeed measurement of cytokine profiles in patients can provide a useful indicator of disease. Owing to their multiple functions in disease, therapeutic interference with cytokines, cytokine receptors or their signal transduction pathways offers new treatment options for a range of disorders. Key Concepts: Cytokines can be extremely potent, acting at picomolar or femtomolar concentrations.Cytokines are not produced constitutively but are expressed on stimulation of cells, usually acting in an autocrine or paracrine fashion.Individual cytokines can have many functions and can act synergistically or antagonistically.Chemokines are a family of cytokines that are not only principally involved in chemoattraction but also participate in cell activation and angiogenesis.Different cytokine receptors can share similar subunit structures, which divide them into families.Most cytokine receptors are heterodimers, but some can be homodimers or heterotrimers.Cytokines exert their effects by activating specific intracellular signalling pathways.By sharing receptor subunits and signal transducing molecules, many cytokines are known to play redundant and pleiotropic roles.Excessive cytokine production or activity can lead to several pathologies, and cytokine modifiers are proving to be a therapeutically effective approach in modifying such diseases.Cytokine synthesis and activity is regulated by many mechanisms including signalling inhibitors, epigenetic modifications and cleavage of precursors.Keywords:cytokines;chemokines;cytokine receptors;cytokine signalling;cytokine/anticytokine therapy
[Show abstract][Hide abstract] ABSTRACT: Recent evidence points to the T helper subset Th17 as key in the pathogenesis of psoriasis, but cells of this type in lesions remain to be fully characterized. Here we isolated, enumerated, functionally tested, and clonotyped, the CD4(+) Th cell population ex vivo from lesional biopsies and paired peripheral blood samples from psoriasis patients. Th17 cells were dramatically over-represented in lesions from all patients, representing 49 to 93% of CD4(+) Th cells compared with 3 to 18% in blood. Most lesional Th17 cells produced IL-17A ex vivo without further stimulation and expressed the CD45RO(+) phenotype characteristic of activated or memory cells. There was no increase in "natural" (CD25(hi) Foxp3(+) ) regulatory T cells in lesions versus peripheral blood, but there was enrichment of "induced" IL-10(+) regulatory T cell numbers in biopsies from some patients. The lesional Th17 cells exhibited a bias in T cell receptor Vβ chain usage, suggestive of specific expansion by antigen. The therapeutic challenge is to overcome the dominance of overwhelming numbers of such antigen-specific Th17 cells in psoriatic lesions.
[Show abstract][Hide abstract] ABSTRACT: CD22, an inhibitory co-receptor of the BCR, has been identified as a potential candidate gene for the development of autoimmune haemolytic anaemia in mice. In this study, we have examined Cd22(tm1Msn) CD22-deficient mice and identified an increase in RBC turnover and stress erythropoiesis, which might be consistent with haemolysis. We then, however, eliminated CD22 deficiency as the cause of accelerated RBC turnover and established that enhanced RBC turnover occurs independently of B cells and anti-RBC autoanti-bodies. Accelerated RBC turnover in this particular strain of CD22-deficient mice is red cell intrinsic and appears to be the consequence of a defective allele of glucose phosphate isomerase, Gpi1(c) . This form of Gpi1 was originally derived from wild mice and results in a substantial reduction in enzyme activity. We have identified the polymorphism that causes impaired catalytic activity in the Gpi1(c) allele, and biochemically confirmed an approximate 75% reduction of GPI1 activity in Cd22(-/-) RBCs. The Cd22(-/-) .Gpi1(c) congenic mouse provides a novel animal model of GPI1-deficiency, which is one of the most common causes of chronic non-spherocytic haemolytic anaemia in humans.
European Journal of Immunology 12/2012; 42(12). DOI:10.1002/eji.201242633 · 4.03 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Many immune-mediated diseases are associated with low levels of vitamin D and sunlight. UV light or supplementation with vitamin D can increase regulatory T-cell activity and prevent animal models of autoimmune disease. Increasing population vitamin D levels may therefore alleviate the burden of human immune-mediated disease.
To determine the responses of circulating 25-hydroxyvitamin D [25(OH)D] levels, regulatory T-cell numbers, and immune function to UV light exposure in patients being treated for skin disease.
Twenty-four subjects with skin disease from the North of Scotland were recruited between December and March. At baseline, and after 2 and 4 weeks of narrowband UV light exposure, we measured peripheral blood 25(OH)D level, numbers of regulatory T cells (CD4(+)CD25(hi)FoxP3(+)), and T-cell proliferative and cytokine responses to anti-CD3/CD28 stimulation.
Median (interquartile range) narrowband UV-B received during the study was 39.1 (30.9) as standard erythema dose, comparable to a quarter of the median summer sunlight exposure received locally. This increased the 25(OH)D level from a mean ± SD of 34 ± 17 nmol/L to 58 ± 16 nmol/L after 2 weeks and 78 ± 19 nmol/L after 4 weeks. The mean proportion of circulating regulatory T cells increased from 0.5% to 1.6% CD3(+) cells, which significantly correlated with the increased 25(OH)D level. UV treatment was also followed by reduced proliferative and IL-10 responses to anti-CD3/CD28 independent of the 25(OH)D level.
Narrowband UV light reduces systemic immune responsiveness via the induction of regulatory T cells. Light and 25(OH)D levels may affect particular immune functions independently. The levels of serum 25(OH)D over which these effects are apparent should guide future interventions.
The Journal of allergy and clinical immunology 04/2012; 129(6):1554-61. DOI:10.1016/j.jaci.2012.03.001 · 11.48 Impact Factor