In vivo maturation of TH cells in relation to atopy
2011; Elsevier BV; Volume: 128; Issue: 1 Linguagem: Inglês
10.1016/j.jaci.2011.02.033
ISSN1097-6825
AutoresDominique Bullens, Ahmad Kasran, Ellen Dilissen, Kathleen De Swert, Lieve Coorevits, Jacques Van Snick, Jan Ceuppens,
Tópico(s)Immune Cell Function and Interaction
ResumoTo the Editor: The immune system at birth has a default setting toward TH2-type responses. In children with allergic diseases, the TH2 predominance persists.1Prescott S.L. Macaubas C. Smallacombe T. Holt B.J. Sly P.D. Holt P.G. Development of allergen-specific T-cell memory in atopic and normal children.Lancet. 1999; 353: 196-200Abstract Full Text Full Text PDF PubMed Scopus (800) Google Scholar Although the role of regulatory T cells2Bluestone J.A. Mackay C.R. O’Shea J.J. Stockinger B. The functional plasticity of T cell subsets.Nat Rev Immunol. 2009; 9: 811-816Crossref PubMed Scopus (202) Google Scholar in the development of peripheral tolerance and the prevention of allergic sensitization has become more clear, the relation of the more recently identified TH9, TH17, and T follicular helper cell subsets2Bluestone J.A. Mackay C.R. O’Shea J.J. Stockinger B. The functional plasticity of T cell subsets.Nat Rev Immunol. 2009; 9: 811-816Crossref PubMed Scopus (202) Google Scholar to atopy is not unraveled. We studied CD4 TH cell maturation in a Belgian birth cohort (Follow-up of Newborn’s Immune Development in Relation to Allergic Outcome cohort, n = 107). We measured a series of representative cytokines (IL-4, IFN-γ, IL-9, IL-17A, and IL-21) in culture supernatants of anti-CD3/CD80–stimulated cord blood mononuclear cells (CBMCs) and PBMCs obtained at the age of 12 and 24 months (see the Methods and Results sections in this article’s Online Repository at www.jacionline.org). Allergic sensitization and symptoms were assessed at 12 and 24 months and 6 years of age (n = 84, see the Methods section in this article’s Online Repository). Those children who were already allergic before or at the age of 24 months were labeled as “early allergic” (n = 17). Those who had allergy between the age of 2 and 6 years were labeled as “late allergic” (n = 10; see Table E1, Table E2, and E3 in this article’s Online Repository at www.jacionline.org for patients’ characteristics and sensitization profiles). In accordance with our previous findings,3Bullens D.M.A. Rafiq K. Kasran A. Van Gool S.W. Ceuppens J.L. Naive human T cells can be a source of IL-4 during primary immune responses.Clin Exp Immunol. 1999; 118: 384-391Crossref PubMed Scopus (16) Google Scholar IL-4 was detectable in almost all CBMC cultures (see Table E4 in this article’s Online Repository at www.jacionline.org). High neonatal IL-4 production (>50 pg/mL) predicted development of early allergy (relative risk [RR], 3.03; 95% CI, 1.1-8.3) but not late allergy (RR, 0.5; Table I). This is in agreement with the findings of Herberth et al,4Herberth G. Heinrich J. Röder S. Figl A. Weiss M. Diez U. et al.Reduced IFN-gamma- and enhanced IL-4-producing CD4+ cord blood T cells are associated with a higher risk for atopic dermatitis during the first 2 yr of life.Pediatr Allergy Immunol. 2010; 21: 5-13Crossref PubMed Scopus (70) Google Scholar showing an increased risk of atopic dermatitis within the 2 first years of life for children with a high percentage of cord blood IL-4–producing cells.4Herberth G. Heinrich J. Röder S. Figl A. Weiss M. Diez U. et al.Reduced IFN-gamma- and enhanced IL-4-producing CD4+ cord blood T cells are associated with a higher risk for atopic dermatitis during the first 2 yr of life.Pediatr Allergy Immunol. 2010; 21: 5-13Crossref PubMed Scopus (70) Google Scholar IL-17A was detectable in only 4 of the CBMC cultures, even though IL-4 bioactivity in the cultures was blocked (Fig 1). Notably, the 4 children with measurable IL-17A production by CBMCs remained healthy. IL-9 production was completely absent at birth (<5 pg/mL).Table IRR assessment for early or late allergy by means of evaluation of CBMC and PBMC cytokine productionCutoff value for cytokine production (pg/mL)∗CBMCs (5 × 105) or PBMCs (5 × 105) obtained at 12 and 24 months were stimulated with anti-CD3/CD80 in the absence (IFN-γ or IL-9) or presence (IL-4) of anti–IL-4 receptor mAb. Supernatants were collected after 3 days; cytokine production was measured by means of ELISA. The table represents RRs for early or late allergy (see the Methods section in this article’s Online Repository) and 95% CIs for cytokine production expressed as concentrations in supernatants in picograms per milliliter; the cutoff value was based on the 75th percentile in nonallergic children. Because only 3 children allergic at age 2 years were not allergic at age 1 year, no RR for early allergy was studied at age 1 year.RR95% CIP value, Fisher exact testIL-4 CBMCs50Early: 3.031.1-8.3.03Late: 0.50.06-3.6.4 12-mo PBMCs150Late: 6.30.8-47.03 24-mo PBMCs200Late: 20.5-7.2.2IL-9 CBMCsNA 12-mo PBMCs400Late: 1.90.6-9.2 24-mo PBMCs600Late: 4.481.01-19.72.03IFN-γ CBMCs20,000Early: 0.70.2-2.3.4Late: 0.30.04-2.4.2 12-mo PBMCs70,000Late: 0.40.08-1.6.1 24-mo PBMCs70,000Late: 0.30.04-2.4.2∗ CBMCs (5 × 105) or PBMCs (5 × 105) obtained at 12 and 24 months were stimulated with anti-CD3/CD80 in the absence (IFN-γ or IL-9) or presence (IL-4) of anti–IL-4 receptor mAb. Supernatants were collected after 3 days; cytokine production was measured by means of ELISA. The table represents RRs for early or late allergy (see the Methods section in this article’s Online Repository) and 95% CIs for cytokine production expressed as concentrations in supernatants in picograms per milliliter; the cutoff value was based on the 75th percentile in nonallergic children. Because only 3 children allergic at age 2 years were not allergic at age 1 year, no RR for early allergy was studied at age 1 year. Open table in a new tab IFN-γ (see Table E4) and, surprisingly, IL-21 (Fig 1) were measurable in almost all CBMC cultures, but levels were not related to later allergy development. Low cord blood IFN-γ production has been reported by several authors to be associated with allergy development.4Herberth G. Heinrich J. Röder S. Figl A. Weiss M. Diez U. et al.Reduced IFN-gamma- and enhanced IL-4-producing CD4+ cord blood T cells are associated with a higher risk for atopic dermatitis during the first 2 yr of life.Pediatr Allergy Immunol. 2010; 21: 5-13Crossref PubMed Scopus (70) Google Scholar, 5Hagendorens M.M. Ebo D.G. Bridts C.H. Van de Water L. De Clerck L.S. Stevens W.J. Prenatal exposure to house dust mite allergen (Der p 1), cord blood T cell phenotype and cytokine production and atopic dermatitis during the first year of life.Pediatr Allergy Immunol. 2004; 15: 308-315Crossref PubMed Scopus (40) Google Scholar This discrepancy with our results might be explained by the fact that we measured IFN-γ production by total CBMCs instead of by isolated CD4+ T cells. IFN-γ was present intracellularly in 10% (mean of 3 experiments) of lymphocytes and ±0.6% (mean of 3 experiments) of isolated CD4+ T cells, indicating that other cell fractions beside CD4+ T cells (eg, CD8+ T cells) were predominantly responsible for its accumulation in the supernatants. Moreover, IFN-γ production by cord blood CD8+ T cells was reported not to be defective in atopic children.4Herberth G. Heinrich J. Röder S. Figl A. Weiss M. Diez U. et al.Reduced IFN-gamma- and enhanced IL-4-producing CD4+ cord blood T cells are associated with a higher risk for atopic dermatitis during the first 2 yr of life.Pediatr Allergy Immunol. 2010; 21: 5-13Crossref PubMed Scopus (70) Google Scholar, 5Hagendorens M.M. Ebo D.G. Bridts C.H. Van de Water L. De Clerck L.S. Stevens W.J. Prenatal exposure to house dust mite allergen (Der p 1), cord blood T cell phenotype and cytokine production and atopic dermatitis during the first year of life.Pediatr Allergy Immunol. 2004; 15: 308-315Crossref PubMed Scopus (40) Google Scholar We also confirmed IL-21 production by CD4+ CBMCs using intracellular staining of 3 additional cord blood samples. IL-21 production by cord blood CD4+ T cells was strongly upregulated in the presence of IL-23 and IL-1β (±8.5%) compared with 0.35% in its absence (data not shown). Only a minor fraction of IL-21–producing CD4+ T cells were also IFN-γ producers (see Fig E1 in this article’s Online Repository at www.jacionline.org). The presence of IL-21–producing cells in cord blood was surprising. Whether they represent naive TH0 cells, circulating T follicular helper–like cells, or cells in the early differentiation pathway toward TH17 cells requires further investigation. At the age of 12 and 24 months, we again studied cytokine production in PBMC cultures. IL-4 production increased significantly over time (from birth to age 24 months) in both allergic and nonallergic children (see Table E4). At age 24 months, IL-4 production correlated with the allergic state at the moment of evaluation, and this confirms earlier observations that the TH2 predominance persists in atopic children.1Prescott S.L. Macaubas C. Smallacombe T. Holt B.J. Sly P.D. Holt P.G. Development of allergen-specific T-cell memory in atopic and normal children.Lancet. 1999; 353: 196-200Abstract Full Text Full Text PDF PubMed Scopus (800) Google Scholar However, high IL-4 production at age 12 or 24 months (Table I) did not predict late allergy development. IFN-γ production increased significantly during the first 12 months (see Table E4). Interestingly, in the subpopulation of children with “late allergy” but not in healthy children or “early allergic” children, IFN-γ production then again decreased significantly during the second year of life (P = .004). Decreasing IFN-γ production thus precedes the development of allergy later in life and apparently reflects the predisposition for allergic sensitization. Importantly, IL-9 production was significantly higher at the age of 12 months, as well as at the age of 24 months, in both children with early and those with late allergy when compared with that seen in children without allergy (Fig 1). Moreover, the RR for late allergy in children with high in vitro polyclonal IL-9 production at 24 months is 4.48 (95% CI, 1.01-19.72; P = .03, Fisher exact test; Table I). Thus polyclonal IL-9 responses at young age are predictive for allergy development. The difference in the predictive value of IL-4 and IL-9 production supports the concept that IL-4 and IL-9 are produced by separate T-cell lineages.2Bluestone J.A. Mackay C.R. O’Shea J.J. Stockinger B. The functional plasticity of T cell subsets.Nat Rev Immunol. 2009; 9: 811-816Crossref PubMed Scopus (202) Google Scholar Earlier observations demonstrated that allergen-induced IL-9 production is associated with asthmatic symptoms in 12-year-old allergic children, also pointing toward the important role for IL-9–producing cells in allergy.6Jenmalm M.C. Van Snick J. Cormont F. Salman B. Allergen-induced Th1 and Th2 cytokine secretion in relation to specific allergen sensitization and atopic symptoms in children.Clin Exp Allergy. 2001; 31: 1528-1535Crossref PubMed Scopus (50) Google Scholar Furthermore, allergen-induced IL-9 production in allergic adults has been shown to be an excellent marker for atopy because its production is virtually absent in nonallergic control subjects and correlates significantly with allergen-specific IgE levels in allergic patients.7Devos S. Cormont F. Vrtala S. Hooghe-Peters E. Pirson F. Van Snick J. Allergen-induced interleukin-9 production in vitro: correlation with atopy in human adults and comparison with interleukin-5 and interleukin-13.Clin Exp Allergy. 2006; 36: 174-182Crossref PubMed Scopus (43) Google Scholar IL-17A production could be induced in the PBMC cultures at the age of 12 months. IL-17A production was not different between healthy and allergic children at any age. At the age of 24 months, no further increase in IL-17A production was noted (Fig 1). The appearance of IL-9 and IL-17 production during the first year of life might represent the differentiation of TH9 and TH17 cells on immune maturation. Because of its early production at birth, IL-21 might contribute to TH17 differentiation (as it does in vitro if associated with TGF-β) and to TH9 differentiation (in association with type I interferons).8Wong M.T. Ye J.J. Alonso M.N. Landrigan A. Cheung R.K. Engleman E. et al.Regulation of human Th9 differentiation by type I interferons and IL-21.Immunol Cell Biol. 2010; 88: 624-631Crossref PubMed Scopus (107) Google Scholar Especially those children allergic before the age of 2 years might have a high risk for the atopic march. Interestingly, our findings on differences in IL-4, IL-9, and IFN-γ production at birth and at young age between children who will become allergic early and those who will become allergic at a later time point suggest that both groups of children are immunologically different; their sensitization pattern is also different (see Tables E2 and E3). The recent findings by Matricardi et al9Matricardi P.M. Bockelbrink A. Grüber C. Keil T. Hamelmann E. Wahn U. et al.Longitudinal trends of total and allergen-specific IgE throughout childhood.Allergy. 2009; 64: 1093-1098Crossref PubMed Scopus (45) Google Scholar indicating that total and specific IgE profiles, as well as clinical characteristics, differ between children sensitized early and those sensitized late also support this hypothesis. Furthermore, recent observations by Simpson et al10Simpson A. Tan V.Y.F. Winn J. Svensén M. Bishop C.M. Heckerman D.E. et al.Beyond atopy. Multiple patterns of sensitization in relation to asthma in a birth cohort study.Am J Respir Crit Care Med. 2010; 181: 1200-1206Crossref PubMed Scopus (337) Google Scholar revealed differences in asthmatic outcomes between infants sensitized early and those sensitized late, pointing to different phenotypes in IgE-mediated allergy. In conclusion, our results show that differences in T-cell maturation between atopic and nonatopic children can be detected in early life. In particular, the data suggest that evaluation of IL-4 and IL-9 production at birth and in early life, respectively, are useful for estimation of allergy risks. Although the source of the cells responsible for this cytokine production still has to be defined, our results strongly point toward a role for TH9 cells in allergy development. Moreover, the detection of IL-21–producing CD4+ T cells in cord blood warrants further study on the subset source and physiological role of this early IL-21. The Follow-up of Newborn’s Immune Development in Relation to Allergic Outcome study is a prospective study of newborns from 139 pregnant women in the province of Flemish-Brabant, Belgium. Pregnant women between the 12th and 38th weeks of pregnancy in 2001 to 2003 answered interview questionnaires in view of potential inclusion. Allergic parents, siblings, or both were defined based on a clinical history of doctor-diagnosed allergic rhinoconjunctivitis, asthma, anaphylaxis, or food allergy. In case of at least 1 allergic first-degree relative of the expected newborn, mothers who consented were all included in the study. When there was no familial risk factor, mothers were randomly included 1 in 2. Randomization was done by a blinded researcher, and the label “in” or ”out“ was placed in consecutively numbered closed envelopes. As a result of this selection, power analysis calculated the chance of the offspring to become atopic to be 30%. Pregnancy complications were an exclusion criterion. Women provided informed consent. In total, 107 newborns born of 106 pregnancies were included. The study was approved by the local ethics committee. Infants were clinically evaluated at 12 and 24 months and 6 years of age based on a detailed history and clinical examination by the same pediatrician. In January 2010, all children reached the age of 6 years. The main allergic outcomes were atopic eczema, persistent or intermittent wheeze, persistent or seasonal rhinoconjunctivitis, anaphylaxis, food allergy (positive symptoms), and allergic sensitization without symptoms. Skin prick tests (SPTs) were performed at 12 months and 6 years. In all children house dust mite, tree pollen, birch pollen, grass pollen, Alternaria species, cat dander, dog dander, egg white, cow’s milk, peanut, and potato (Hal laboratories, Haarlem, The Netherlands) were tested, and depending on clinical history, additional SPTs (latex, tree nut, egg yellow, horse dander, and fish) or supplementary SPTs at age 24 months were performed (Aas’ criteria were used to score the SPT responseE1Aas K. Belin L. Grading of skin prick test by comparison to histamine positive control.Acta Allergol. 1974; 29: 238Google Scholar). Moreover, specific IgE levels were measured at 12 and 24 months and 6 years of age to house dust mite, birch pollen, grass pollen, cat dander, dog dander, peanut, cow’s milk, egg, and potato (lower detection level of 0.1 IU/mL; a kind gift of Phadia, Uppsala, Sweden). A CAP test result of greater than 0.35 IU/mL was considered positive. From birth until the second birthday, parents completed a diary at each visit of their infant to the family doctor or pediatrician. The diagnosis of clinical allergy was based on the presence of symptoms in association with relevant sensitization. Children with any specific IgE level between 0.35 and 0.7 IU/mL but negative SPT responses for all allergens tested and no relevant symptoms were considered healthy (n = 3). Asymptomatic sensitization was defined as sensitization based on a positive SPT responseE1Aas K. Belin L. Grading of skin prick test by comparison to histamine positive control.Acta Allergol. 1974; 29: 238Google Scholar or specific IgE levels of greater than 0.7 IU/mL (for ≥1 allergen) without any relevant symptoms (excluded from analysis, n = 3). Those children who were already allergic before or at the age of 2 years were labeled as “early allergic.” Only 1 of these children was no longer allergic at the age of 6 years. Those who had allergy between the ages of 2 and 6 years were labeled as “late allergic.” One hundred seven babies (1 twin pregnancy) were included at birth. Twenty-three children did not continue their participation in the study (mainly because of refusal of venous puncture, for personal reasons, or because they were lost to follow-up). Results are thus based on data from 84 children (32 male subjects). Cord blood was not obtained if delivery occurred by means of cesarean section or was no longer used for stimulation if transport to our laboratory took longer than 4 hours (n = 66 remaining). Six children completed final allergy evaluation at age 6 years, but their parents refused venous puncture at age 12 and 24 months, whereas venous puncture at age 12 months was technically not possible in 1 additional child. T cells in CMBC and PBMC cultures were stimulated with soluble anti-CD3 mAb (UCHT-1) captured by murine P815/CD80 mastocytoma cellsE2Bullens D.M.A. Rafiq K. Kasran A. Van Gool S.W. Ceuppens J.L. Naive human T cells can be a source of IL-4 during primary immune responses.Clin Exp Immunol. 1999; 118: 384-391Crossref PubMed Scopus (24) Google Scholar at a T/P815 ratio of 1:1 in a 1-mL volume. P815 cells transfected with human CD80 were mitomycin C treated and washed 5 times before use. In culture conditions used to measure IL-4, IL-17A, and IL-21, an anti-human IL-4 receptor α mAb (clone 25463.11 from R&D, Abingdon, United Kingdom) was used at a concentration of 2.5 μg/mL, as reported.E3Bullens D.M.A. Kasran A. Peng X. Lorré K. Ceuppens J.L. Effects of anti-interleukin-4-receptor monoclonal antibody on T cell cytokine levels in vitro: interleukin-4 production by T cells from non-atopic donors.Clin Exp Immunol. 1998; 113: 320-326Crossref PubMed Scopus (39) Google Scholar This mAb blocks the bioactivity of both IL-4 and IL-13, blocks IL-4 consumption,E3Bullens D.M.A. Kasran A. Peng X. Lorré K. Ceuppens J.L. Effects of anti-interleukin-4-receptor monoclonal antibody on T cell cytokine levels in vitro: interleukin-4 production by T cells from non-atopic donors.Clin Exp Immunol. 1998; 113: 320-326Crossref PubMed Scopus (39) Google Scholar and avoids inhibition of TH17 cells by TH2 cytokines. Preliminary experiments on 4 healthy adult donors showed that in vitro polyclonally induced IL-17A production significantly increased, whereas IL-21 production remained unaltered when IL-4 bioactivity was blocked. Three hundred microliters of supernatant in the presence of anti–IL-4 receptor mAb was used to measure these 3 cytokines, whereas 200 μL of separate supernatants in the absence of anti–IL-4 receptor was used to measure IL-9 and IFN-γ. The concentrations of IL-4, IFN-γ, and IL-21 in culture supernatants were measured with a sandwich ELISA technique by using combinations of unlabeled and biotin-coupled mAbs to different epitopes of each cytokine: IL-4 capture antibody clone 8D4-8 (PharMingen, San Diego, Calif) and biotin-labeled clone mp4-25D2 (PharMingen); IFN-γ capture antibody clone 350B10G6 (Medgenix Diagnostics, Fleurus, Belgium) and biotin-labeled clone 67F12A8 (Medgenix Diagnostics); and IL-21 capture antibody clone J148-1134 (PharMingen) and biotin-labeled clone I76-539 (PharMingen). IL-17A was measured with IL-17 Duoset (R&D Systems, Minneapolis, Minn). Capture and detection antibodies to measure IL-9 were previously reported.E4Jenmalm M.C. Van Snick J. Cormont F. Salman B. Allergen-induced Th1 and Th2 cytokine secretion in relation to specific allergen sensitization and atopic symptoms in children.Clin Exp Allergy. 2001; 31: 1528-1535Crossref PubMed Scopus (64) Google Scholar Additional experiments were performed on CBMCs from 3 nonselected newborns born in 2009 (n = 3). Cord blood CD4+ T cells were isolated with the CD4+ isolation kit II Mac (Miltenyi Biotec, Bergisch Gladbach, Germany) by using LD columns. Purity of the cord blood CD4+ T cells was at least 94.5%. CBMCs and isolated CD4+ T cells were cultured with anti-CD3 (2 μg/mL) and P815/CD80-transfected cells or PHA (5 μg/mL) in the absence or presence of IL-23 (10 ng/mL) and IL-1β (10 ng/mL). After 3 days, cells were restimulated at 37°C for 4 hours with 50 ng/mL phorbol 12-myristate 13-acetate and 0.5 μg/mL ionomycin in the presence of brefeldin A (10 μg/mL) in RPMI with 10% bovine calf serum. After membrane staining with anti-CD45–peridinin-chlorophyll-protein complex mAb (BD Biosciences, Mississauga, Ontario, Canada; for CBMCs only), cells were washed with PBS containing 1% FCS and fixed with Reagent A (ADG Fix&Perm, Vienna, Austria) for 15 minutes at room temperature. Cells were washed in PBS/1% FCS and resuspended in Reagent B (ADG Fix&Perm). Cells were then incubated with 10 μL of human plasma and either anti-IgG1 FITC (BD Biosciences), anti-IgG–phycoerythrin (PE; BD Biosciences), anti–IL-17–PE (eBioscience, San Diego, Calif), anti–IFN-γ–FITC (BD Biosciences), or anti–IL-21–PE (BD Biosciences) mAb for 15 minutes at room temperature and washed twice with PBS. Analysis was done with the Cell Quest Program on FACS Sort (BD Biosciences). Results were compared with the nonparametric Kruskall-Wallis test with the Dunn post test, Mann-Whitney U test, or paired Wilcoxon tests, 1-tailed were appropriate. A P value of .05 or less was considered statistically significant. Contingency tables were evaluated by using Fisher exact tests. Calculations, including RR assessments, were performed with Prism Graph 5 software (GraphPad Software, Inc, San Diego, Calif). A small percentage of isolated cord blood CD4+ T cells (0.33%) in one of 3 samples had intracellular IL-17A on stimulation with anti-CD3 and CD80 (data not shown). To further increase the potential for IL-17 production, we added IL-23, the main inducer of IL-17A in human subjects,E5Lyakh L. Trinchieri G. Provezza L. Carra G. Gerosa F. Regulation of interleukin-12/interleukin-23 production and the T-helper 17 response in humans.Immunol Rev. 2008; 226: 112-131Crossref PubMed Scopus (173) Google Scholar and IL-1β, which is essential for human TH17 cell differentiation,E6Acosta-Rodriguez E.V. Napolitani G. Lanzavecchia A. Sallusto F. Interleukins 1β and 6 but not transforming growth factor-β are essential for the differentiation of interleukin 17-producing human T helper cells.Nat Immunol. 2007; 8: 942-949Crossref PubMed Scopus (1534) Google Scholar to the in vitro cultures, but this had no effect (Fig E1). Tabled 1Subjects’ characteristicsNonallergic at 6 y of ageSymptomatic allergic before 2 y of ageSymptomatic allergic at 6 y but not yet at 2 y of ageTotal no.541710≥1 First-degree relative with atopy47/54 (87%)17/17 (100%)9/10 (90%)Infant’s sex, male17/54 (31%)11/17 (64%)4/10 (40%)Neonatal parameters (mean ± SEM) Birth weight (g)3404 ± 673473 ± 973275 ± 166 Head circumference (cm)34.7 ± 0.235.1 ± 0.334.3 ± 0.6 Open table in a new tab Tabled 1Sensitization∗Sensitization was determined as defined in the Methods section in this article’s Online Repository. profiles in children with early allergy12 mo24 mo6 y005Egg, catEgg, nuts, peanuts, catNuts, HDM, cat, dog, horse, birch pollen, Alternaria species022EggHDMHDM, grass pollen, rabbit, Alternaria species025PotatoNegativeHDM040PotatoPotatoHDM, birch pollen041NegativeHDMHDM, cat, dog044Egg, HDMHDM, cat, birch pollenHDM, cat, birch pollen054EggNot doneHDM, cat, dog, grass pollen056Egg, cow’s milk, HDMCow’s milkHDM065NegativeHDMHDM, dog, birch pollen, grass pollen066Egg, HDMHDMHDM, birch pollen075Egg, cow’s milkNegativeNegative077Egg, cow’s milk, HDMEgg, cow’s milk, HDMEgg, HDM, dog, grass pollen086Egg, potatoEggHDM, horse, rabbit, birch pollen, grass pollen088NegativePeanutEgg, HDM, grass pollen089Potato, cow’s milkNegativeHDM, cat, dog, birch pollen, grass pollen097Egg, cow’s milkCow’s milkCow’s milk, HDM, cat, dog, grass pollen104NegativeHDMHDMHDM, House dust mite.∗ Sensitization was determined as defined in the Methods section in this article’s Online Repository. Open table in a new tab HDM, House dust mite. Tabled 1Sensitization∗Sensitization at age 6 years was determined as defined in the Methods section in this article’s Online Repository. profiles in children with late allergy12 mo24 mo6 y002NegativeNegativeHDM, grass pollen027NegativeNegativeGrass pollen045NegativeNegativeHDM, grass pollen046NegativeNegativeHDM050NegativeNegativeHDM, dog, grass pollen, birch pollen055NegativeNegativeHDM, cat, dog, grass pollen060NegativeNegativePeanut068NegativeNegativeGrass pollen078NegativeNegativeBirch, cow’s milk105NegativeNegativeAlternaria speciesHDM, House dust mite.∗ Sensitization at age 6 years was determined as defined in the Methods section in this article’s Online Repository. Open table in a new tab HDM, House dust mite. Tabled 1Polyclonal IL-4 and IFN-γ production by CBMCs and PBMCs at a young ageNonallergic∗Sensitization and clinical symptoms were evaluated as explained in the Methods section in this article’s Online Repository.Early allergy∗Sensitization and clinical symptoms were evaluated as explained in the Methods section in this article’s Online Repository.Late allergy only∗Sensitization and clinical symptoms were evaluated as explained in the Methods section in this article’s Online Repository.P value‡Kruskall-Wallis test.A. CBMC IL-4 production†CBMCs (5 × 105) or PBMCs (5 × 105) obtained at 12 and 24 months were stimulated with anti-CD3/CD80 in the absence (IFN-γ) or presence (IL-4) of anti–IL-4 receptor mAb. Supernatants were collected after 3 days; cytokine production was measured by means of ELISA and expressed as concentrations in supernatants in picograms per milliliter. Values shown represent p25-median-p75 values for cytokines. A versus B, B versus C, D versus E, and E versus F were tested by using the paired Wilcoxon test.15-30-5511-55-12210-11-30.2B. 12-mo PBMC IL-4 production†CBMCs (5 × 105) or PBMCs (5 × 105) obtained at 12 and 24 months were stimulated with anti-CD3/CD80 in the absence (IFN-γ) or presence (IL-4) of anti–IL-4 receptor mAb. Supernatants were collected after 3 days; cytokine production was measured by means of ELISA and expressed as concentrations in supernatants in picograms per milliliter. Values shown represent p25-median-p75 values for cytokines. A versus B, B versus C, D versus E, and E versus F were tested by using the paired Wilcoxon test.104-151-219 (A vs B: P ≤ .0001)122-159-293 (A vs B: P = .03)162-188-231 (A vs B: P = .008).7C. 24-mo PBMC IL-4 production†CBMCs (5 × 105) or PBMCs (5 × 105) obtained at 12 and 24 months were stimulated with anti-CD3/CD80 in the absence (IFN-γ) or presence (IL-4) of anti–IL-4 receptor mAb. Supernatants were collected after 3 days; cytokine production was measured by means of ELISA and expressed as concentrations in supernatants in picograms per milliliter. Values shown represent p25-median-p75 values for cytokines. A versus B, B versus C, D versus E, and E versus F were tested by using the paired Wilcoxon test.107-181-273 (B vs C: P = .01)180-219-340 (B vs C: P = .009)108-238-345 (B vs C: P = .4).3D. CBMC IFN-γ production†CBMCs (5 × 105) or PBMCs (5 × 105) obtained at 12 and 24 months were stimulated with anti-CD3/CD80 in the absence (IFN-γ) or presence (IL-4) of anti–IL-4 receptor mAb. Supernatants were collected after 3 days; cytokine production was measured by means of ELISA and expressed as concentrations in supernatants in picograms per milliliter. Values shown represent p25-median-p75 values for cytokines. A versus B, B versus C, D versus E, and E versus F were tested by using the paired Wilcoxon test.2,569-6,419-37,7871,872-8,118-24,8581,105-5,289-15,813.9E. 12-mo PBMC IFN-γ production†CBMCs (5 × 105) or PBMCs (5 × 105) obtained at 12 and 24 months were stimulated with anti-CD3/CD80 in the absence (IFN-γ) or presence (IL-4) of anti–IL-4 receptor mAb. Supernatants were collected after 3 days; cytokine production was measured by means of ELISA and expressed as concentrations in supernatants in picograms per milliliter. Values shown represent p25-median-p75 values for cytokines. A versus B, B versus C, D versus E, and E versus F were tested by using the paired Wilcoxon test.15,238-61,317-110,345 (D vs E: P = .0003)21,014-50,863-152,540 (D vs E: P = .008)24,466-50,345-70,118 (D vs E: P = .04).9F. 24-mo PBMC IFN-γ production†CBMCs (5 × 105) or PBMCs (5 × 105) obtained at 12 and 24 months were stimulated with anti-CD3/CD80 in the absence (IFN-γ) or presence (IL-4) of anti–IL-4 receptor mAb. Supernatants were collected after 3 days; cytokine production was measured by means of ELISA and expressed as concentrations in supernatants in picograms per milliliter. Values shown represent p25-median-p75 values for cytokines. A versus B, B versus C, D versus E, and E versus F were tested by using the paired Wilcoxon test.13,829-39,216-88,454 (E vs F: P = .6)36,720-75,726-156,400 (E vs F: P = .8)4,777-47,664-58,551 (E vs F: P = .004).2∗ Sensitization and clinical symptoms were evaluated as explained in the Methods section in this article’s Online Repository.† CBMCs (5 × 105) or PBMCs (5 × 105) obtained at 12 and 24 months were stimulated with anti-CD3/CD80 in the absence (IFN-γ) or presence (IL-4) of anti–IL-4 receptor mAb. Supernatants were collected after 3 days; cytokine production was measured by means of ELISA and expressed as concentrations in supernatants in picograms per milliliter. Values shown represent p25-median-p75 values for cytokines. A versus B, B versus C, D versus E, and E versus F were tested by using the paired Wilcoxon test.‡ Kruskall-Wallis test. Open table in a new tab
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