T Regulatory Cell Activity as a Disease and a Prognostic Marker in Children with Eczema

T regulatory cell activity as a disease and a prognostic marker in children with eczema Hana Alachkar Manchester University UK Outlines • • • • Hypothesis The objectives of the Study Background Experimental design and methods T-regulatory lymphocytes • Tregs are important in preventing immune hypersensitivity (allergic and autoimmune) reactions by dampening down proinflammatory responses • Hypothesis: aberrant activity of Treg cells (natural and/or induced) is a hallmark and a valuable clinical marker of atopic dermatitis The objectives of the Study 1. To show that Treg cell activity is altered in children with moderate to severe atopic dermatitis 2. To demonstrate that abnormalities of Treg activity are useful in vitro markers of disease severity and natural history in childhood eczema Background • Evidence of deficient Treg activity in immune hypersensitivity diseases • Evidence of alterations in Treg activity in atopic eczema Evidence of deficient Treg activity in immune hypersensitivity diseases • Animal knock-out and transfer experiments • Human Foxp3 knock-out (IPEX) • Treg cell activity in type I and IV hypersensitivity disorders • Effect of immunotherapy on Treg activity Evidence of alterations in Treg activity in atopic eczema Immunohistochemistry of skin lesions of 10 adults with AD • Natural Tregs (CD4+CD25+FoxP3+) were not found in the AD lesions • Induced Tregs, IL-10, TGF-β and, and their receptors were significantly expressed Verhagen et al 2006 Evidence of alterations in Treg activity in atopic eczema Impaired nTreg cell infiltration despite the expression of iTreg in the dermis Verhagen et al 2006 Evidence of alterations in Treg activity in atopic eczema ♣ nTreg cells in PB significantly increased in patients with AD ♣ nTregs have normal immunosuppressive activity ♣after stimulation with Staph antigen, nTreg cells lose their immunosuppressive activity Ou et al. 2004 Alterations in Treg activity in atopic eczema • Treg activity in AD is probably inherently normal • But inability of Tregs to leave the circulation and enter skin results in the exaggerated cutaneous inflammatory response characteristic of AD -Local factors prevent trafficking of these cells into the skin, -Superantigens inhibit nTreg activity -Or subtle functional defects in these cells might prevent normal homing into the skin of AD patients -There are no detailed studies of Treg activity in children with AD Experimental design and methods Detailed investigations of Treg activity in a cohort of 30 children with severe eczema, compared with a nonatopic age-matched control: 1) surface and intra-cellular Treg markers and functional suppression of proliferation by FACS, 2) quantitation of Foxp3 mRNA in blood by RT-PCR, and 3) in vitro production of IL-10 and TGF-β by purified Treg cells measured by ELISA Study Design Recruitment of eczema patients and controls Assessment of clinical disease severity 10ml of heparinised and 10ml EDTA blood FACS-based assay of Treg suppression of proliferation polymorphism studies mRNA RT-PCR: Foxp3, IL-10,TGFβ In vitro unstim and stim (steroid, VitD3) IL-10, TGF-β production Flow cytometric assessment of nTreg and iTreg cells -intracellular Foxp3, IL-10, TGF-β Treg phenotyping by FACS FACS to examine cell surface and intracellular expression of Treg markers - nTreg cells (CD4+CD25brightFoxp3+) will be identified by intracellular Foxp3 staining - iTreg cells CD4+CD25+ will be identified by intracellular and membranous TGF-β and intracellular IL-10 staining - Percentage of these cells which home to the skin (CLA+) Quantitative assessment of mRNA of Foxp3 and cytokines • RT-PCR to quantify -Foxp3 mRNA in PB using fluorescence resonance energy transfer (FRET) hybridisation probes -mRNA levels of TGF-β, IL-10, IL-4, IL-13 and IFN-γ Assessment of IL-10 and TGF-β production • ELISA to measure spontaneous and steroid-induced in vitro IL-10 and TGF-β production of Treg cells -PBMC will be cultured with anti-CD3 and IL-2 in a medium alone or after stimulation with dexamethasone ± calcitriol (Vit D3) -Supernatants harvested at 48 hours for cytokine analysis by ELISA Treg suppressive capacity using a co-culture assay -CD4+CD25– T cells cultured with anti-CD3, staphylococcal enterotoxin B or house dust mite allergen, and with and without CD4+CD25+ nTreg cells (which have been removed from PBMC using immunomagnetic beads) for 72 hours at 37°C -Proliferation will be assessed using a FACS based assay -PBMCs are labelled at the beginning of culture with CFSE and changes in staining of propidium iodide negative cells used as a measure of cell division -Proliferation index will be generated using ModFit LT software PB Treg activity correlates with severity and natural history of childhood eczema The following parameters (a) The percentages of nTreg and iTreg cells (b) Foxp3 mRNA levels by RT-PCR (c) Levels of produced IL-10 and TGF-β (d) Suppressive activity of Treg cells will be compared with (i) the severity of AD measured using the standardised SCORAD eczema severity scale, and (ii) changes in disease activity over the subsequent 12 months Functional polymorphisms in Foxp3, IL-10 and TGF-β -Single-nucleotide polymorphisms in these immunoregulatory genes will be screened for using WAVE technology -differences between 100 eczema patients and 100 non-atopic controls will be compared -Abnormal profiles will be confirmed by direct sequencing and compared with functional assays detailed above Statistics • Correlation between immunological phenotype and genotype and clinical phenotype will be analysed statistically using SPSS Clinical Implications • Treg cells are increasingly recognised as playing a key role in immune hypersensitivity reactions • This study is therefore timely and critical step in developing more focused preventative and curative treatments for this condition