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Background:The maturation of innate immune responses in health and atopy is still incompletely understood. Methods:We aimed to evaluate age-related trajectories of the TLR3 and TLR7/8 pathways from birth to adulthood and whether these differ between healthy and atopic individuals. Peripheral blood mononuclear cells (PBMCs) were isolated from 39 otherwise healthy, atopic and 39 non-atopic subjects, aged 0-45 years. Selected cytokines involved in antiviral responses were measured by Luminex in culture supernatants of poly(I:C)-and R848-stimulated PBMCs. The non-parametric correlation between age and cytokine expression and differences in developmental trajectories between healthy and atopic subjects were estimated. Patterns of cytokine development were identified with principal component analysis. Results:Normal innate immune maturation entails significant and progressive agerelated changes in the production of IL-1β, TNFα, MIP-1β, MCP-3, IP-10, IL-10, IL-12p70, and IFNγ upon TLR3 and/or TLR7/8 stimulation. Individual cytokines made small contributions to the observed variability; chemokines MCP-3 and IP-10 were key contributors. The development of these pathways deviated in atopic subjects with significant differences observed in the trajectories of IL-1β, MIP-1β, and IL-10 syntheses. Conclusion:TLR3 and TLR7/8 pathways mature during childhood, while atopy is associated with an abnormal maturation pattern. Suboptimal responses in Th1, inflammatory cytokine, and chemokine production may be implicated in poor antiviral immunity in atopics. Moreover, the deficient maturation of IL-10 synthesis may be implicated in the breaking of tolerance, characterizing the onset of atopic disease.

Table of contentsWORKSHOP 4: Challenging clinical scenarios (CS01–CS06)CS01 Bullous lesions in two children: solitary mastocytomaS. Tolga Yavuz, Ozan Koc, Ali Gungor, Faysal GokCS02 Multi-System Allergy (MSA) of cystic fibrosis: our institutional experienceJessica Hawley, Christopher O’Brien, Matthew Thomas, Malcolm Brodlie, Louise MichaelisCS03 Cold urticaria in pediatric age: an invisible cause for severe reactionsInês Mota, Ângela Gaspar, Susana Piedade, Graça Sampaio, José Geraldo Dias, Miguel Paiva, Mário Morais-AlmeidaCS04 Angioedema with C1 inhibitor deficiency in a girl: a challenge diagnosisCristina Madureira, Tânia Lopes, Susana Lopes, Filipa Almeida, Alexandra Sequeira, Fernanda Carvalho, José OliveiraCS05 A child with unusual multiple organ allergy disease: what is the primer?Fabienne Gay-CrosierCS06 A case of uncontrolled asthma in a 6-year-old patientIoana-Valentina Nenciu, Andreia Florina Nita, Alexandru Ulmeanu, Dumitru Oraseanu, Carmen ZapucioiuORAL ABSTRACT SESSION 1: Food allergy (OP01–OP06)OP01 Food protein-induced enterocolitis syndrome: oral food challenge outcomes for tolerance evaluation in a Pediatric HospitalAdrianna Machinena, Olga Domínguez Sánchez, Montserrat Alvaro Lozano, Rosa Jimenez Feijoo, Jaime Lozano Blasco, Mònica Piquer Gibert, Mª Teresa Giner Muñoz, Marcia Dias da Costa, Ana Maria Plaza MartínOP02 Characteristics of infants with food protein-induced enterocolitis syndrome and allergic proctocolitisEbru Arik Yilmaz, Özlem Cavkaytar, Betul Buyuktiryaki, Ozge Soyer, Cansin SackesenOP03 The clinical and immunological outcomes after consumption of baked egg by 1–5 year old egg allergic children: results of a randomised controlled trialMerrynNetting, Adaweyah El-Merhibi, Michael Gold, PatrickQuinn, IrmeliPenttila, Maria MakridesOP04 Oral immunotherapy for treatment of egg allergy using low allergenic, hydrolysed eggStavroula Giavi, Antonella Muraro, Roger Lauener, Annick Mercenier, Eugen Bersuch, Isabella M. Montagner, Maria Passioti, Nicolò Celegato, Selina Summermatter, Sophie Nutten, Tristan Bourdeau, Yvonne M. Vissers, Nikolaos G. PapadopoulosOP05 Chemical modification of a peanut extract results in an increased safety profile while maintaining efficacyHanneke van der Kleij, Hans Warmenhoven, Ronald van Ree, Raymond Pieters, Dirk Jan Opstelten, Hans van Schijndel, Joost SmitOP06 Administration of the yellow fever vaccine in egg allergic childrenRoisin Fitzsimons, Victoria Timms, George Du ToitORAL ABSTRACT SESSION 2: Asthma (OP07–OP12)OP07 Previous exacerbation is the most important risk factor for future exacerbations in school-age children with asthmaS. Tolga Yavuz, Guven Kaya, Mustafa Gulec, Mehmet Saldir, Osman Sener, Faysal GokOP08 Comparative study of degree of severity and laboratory changes between asthmatic children using different acupuncture modalitiesNagwa Hassan, Hala Shaaban, Hazem El-Hariri, Ahmed Kamel Inas E. MahfouzOP09 The concentration of exhaled carbon monoxide in asthmatic children with different controlled stadiumPapp Gabor, Biro Gabor, Kovacs CsabaOP10 ...

The major obstacles in using FOXP3+ T regulatory cells as T-cell based immunotherapy against GVHD after allogeneic hematopoietic cell transplantation are their low numbers in the circulation and the lack of specific cell surface markers for efficient purification. DNA methylation has been considered to play a role in the regulation of T-cell effector function and cytokine gene expression, indicating a promising role of hypomethylating agents for immunomodulation. Recently it was shown that in-vitro treatment of conventional T-cells with hypomethylating agent azacitidine (aza) induced FoxP3 expression and converted CD4+CD25- cells into immunosuppressive T-cells the suppressor function of which is independent of FOXP3 expression (Choi et al Blood 116:129;2010), suggesting that aza induced suppressor function depends on the modification of other hypomethylated genes. Human leukocyte antigen-G (HLA-G) is a non-classical HLA class I molecule, shown to exert immunoregulatory functions, the expression of which is epigenetically regulated. In this study we investigated whether hypomethylating agent aza can induce HLA-G+ immunoregulatory T cells. We used CD3+ T cells from peripheral blood of healthy individuals isolated by MACS negative selection. T cells were stimulated with anti-CD3+ plus anti-CD28+ coated magnetic beads and then were treated for 72 hours with aza (0.5-15 mM) in the presence of 50U/ml inteleukin-2 (IL-2). Detailed phenotypical characterization of in-vitro aza treated cells was performed with flow cytomentry. Aza-induced FACS sorted HLA-G+ T cells, were irradiated and then used as third party cells in allogeneic mixed lymphocyte cultures (MLC). For the analysis of the in-vivo effect of aza on HLA-G expression, peripheral blood of patients with myelodysplastic syndrome (MDS) was obtained at baseline and after Vidaza treatment. In-vitro treatment of CD3+ T cells with aza increases the percentage of HLA-G+ cells, with maximum induction at a concentration of aza 7.5 mM in comparison to control (aza-0mM) (n=4, 9,11±0,6% vs 0,76±0,74%, p<0,0001). However optimum aza concentration for the maximum HLA-G induction with the lowest toxicity in CD3 T cells was determined at 5 mM (HLA-G+:6,88±3,9%, p=0,0022). Aza treatment increases the percentage of CD4+CD25highFoxP3+ cells in culture. Maximum HLA-G induction was observed on CD4+CD25+ population (n=2, 9,21±5,5%). Aza treatment of FACS-sorted CD4+CD25negHLA-Gneg cells induced CD4lowCD25+HLA-G positive cells, revealing that aza induced HLA-G positive cells are not the result of a selectively expanded proexisting HLA-G+ population. Strickingly aza induced CD4lowCD25+HLA-G+ are FoxP3 negative. Aza induced HLA-G+ cells showed suppressive function (20% inhibition of proliferation) in allo MLCs, whereas HLA-G- selected cells had no effect.The percentage of peripheral blood HLA-G+ lymphocytes of healthy donors was 1,33±0,36% (n=4) in comparison to MDS patients which was 2,08±0,99% (n=2). In accordance to the in- vitro studies, preliminary data from MDS patients under Vidaza show up to 10 fold increase in CD4+CD25highHLA-G+ cells on day 28 post treatment. In conclusion, aza induces de-novo HLA-G expression on CD4+ T cells in-vitro and in-vivo. By using hypomethylating agent aza in-vitro, we generated a CD4lowHLA-G+ FoxP3neg immunoregulatory population, that is highly possible to be the aza induced FoxP3 independent T regulatory population.This strategy may provide an easy methodology to generate ex-vivo immunoregulatory T cells for adoptive immunotherapy. Disclosures: No relevant conflicts of interest to declare.