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Evolution of pathologic T-cell subsets in patients with atopic dermatitis from infancy to adulthood

Published:October 15, 2019DOI:https://doi.org/10.1016/j.jaci.2019.09.031

      Background

      The circulating immune phenotype was defined in adults and young children with early atopic dermatitis (AD), but chronologic changes in the blood of infants and children with AD through adolescence have not been explored.

      Objective

      We sought to compare immune activation and cytokine polarization in the blood of 0- to 5-year-old (n = 39), 6- to 11-year-old (n = 26), 12- to 17-year-old (n = 21) and 18-year-old or older (n = 43) patients with AD versus age-matched control subjects.

      Methods

      Flow cytometry was used to measure IFN-γ, IL-9, IL-13, IL-17, and IL-22 cytokine levels in CD4+/CD8+ T cells, with inducible costimulator molecule and HLA-DR defining midterm and long-term T-cell activation, respectively, within skin-homing/cutaneous lymphocyte antigen (CLA)+ versus systemic/CLA T cells. Unsupervised clustering differentiated patients based on their blood biomarker frequencies.

      Results

      Although CLA+ TH1 frequencies were significantly lower in infants with AD versus all older patients (P < .01), frequencies of CLA+ TH2 T cells were similarly expanded across all AD age groups compared with control subjects (P < .05). After infancy, CLA TH2 frequencies were increased in patients with AD in all age groups, suggesting systemic immune activation with disease chronicity. IL-22 frequencies serially increased from normal levels in infants to highly significant levels in adolescents and adults compared with levels in respective control subjects (P < .01). Unsupervised clustering aligned the AD profiles along an age-related spectrum from infancy to adulthood (eg, inducible costimulator molecule and IL-22).

      Conclusions

      The adult AD phenotype is achieved only in adulthood. Unique cytokine signatures characterizing individual pediatric endotypes might require age-specific therapies. Future longitudinal studies, comparing the profile of patients with cleared versus persistent pediatric AD, might define age-specific changes that predict AD clearance.

      Graphical abstract

      Key words

      Abbreviations used:

      AD (Atopic dermatitis), CLA (Cutaneous lymphocyte antigen), EASI (Eczema Area and Severity Index), FCH (Fold change), ICOS (Inducible costimulator molecule), ILC (Innate lymphoid cell), TC (Cytotoxic T), Tcm (Central memory T), Tem (Effector memory T), TEWL (Transepidermal water loss), Treg (Regulatory T)
      Infancy, childhood, and adolescence are critical periods for immune system maturation.
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      • Hollander G.A.
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      Evolution of the immune system in humans from infancy to old age.
      Early abnormal immune development can cause immune-related disorders. Indeed, 85% of patients with atopic dermatitis (AD) present before 5 years of age.
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      Although young adults have a different AD phenotype from elderly patients,
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      immune changes in patients with AD between early childhood and adulthood are unknown.
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      Blood studies that dissect developmental changes from infancy through adulthood are limited and primarily focused on TH1/TH2 subsets. Some studies with healthy control subjects showed expansion of TH1/TH2/cytotoxic T (TC) 1 subsets with age,
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      whereas others reported no changes in TH2/TC2 subsets over time.
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      Immature IFN-γ response
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      Imbalanced cytokine secretion in newborns.
      and low TH1/TC1 cell frequencies
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      Down-regulation of Th1 responses in human neonates.
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      Age-related changes in intracellular TH1/TH2 cytokine production, immunoproliferative T lymphocyte response and natural killer cell activity in newborns, children and adults.
      • Debock I.
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      Unbalanced neonatal CD4(+) T-cell immunity.
      were seen in early stages of normal development, and abnormal TH1/Th2 ratios seen in cord blood and infants with AD were described.
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      • Laan M.P.
      • Baert M.R.
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      • Savelkoul H.F.
      Selective development of a strong Th2 cytokine profile in high-risk children who develop atopy: risk factors and regulatory role of IFN-gamma, IL-4 and IL-10.
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      • 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.
      • Tang M.L.
      • Kemp A.S.
      • Thorburn J.
      • Hill D.J.
      Reduced interferon-gamma secretion in neonates and subsequent atopy.
      However, few studies compared pediatric and adult AD populations,
      • Kawamoto N.
      • Kaneko H.
      • Takemura M.
      • Seishima M.
      • Sakurai S.
      • Fukao T.
      • et al.
      Age-related changes in intracellular cytokine profiles and Th2 dominance in allergic children.
      ,
      • Kaminishi K.
      • Soma Y.
      • Kawa Y.
      • Mizoguchi M.
      Flow cytometric analysis of IL-4, IL-13 and IFN-gamma expression in peripheral blood mononuclear cells and detection of circulating IL-13 in patients with atopic dermatitis provide evidence for the involvement of type 2 cytokines in the disease.
      • Katsunuma T.
      • Kawahara H.
      • Yuki K.
      • Akasawa A.
      • Saito H.
      Impaired interferon-gamma production in a subset population of severe atopic dermatitis.
      • Jung T.
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      • Gelfand E.W.
      • et al.
      Decreased frequency of interferon-gamma- and interleukin-2-producing cells in patients with atopic diseases measured at the single cell level.
      and none directly compared consecutive age groups of patients with AD with age-matched control subjects, which is critical in understanding normal versus pathologic development of acquired immunity.
      The therapeutic arsenal available for 0- to 12-year-old patients with AD is limited to topical agents, broad systemic immune suppressants, or both.
      • Davidson W.F.
      • Leung D.Y.M.
      • Beck L.A.
      • Berin C.M.
      • Boguniewicz M.
      • Busse W.W.
      • et al.
      Report from the National Institute of Allergy and Infectious Diseases Workshop on “atopic dermatitis and the atopic march: mechanisms and interventions”.
      ,
      • Glines K.R.
      • Stiff K.M.
      • Freeze M.
      • Cline A.
      • Strowd L.C.
      • Feldman S.R.
      An update on the topical and oral therapy options for treating pediatric atopic dermatitis.
      Active development of targeted therapeutics is ongoing for adults and adolescents with AD and will eventually move to children, further necessitating the elucidation of pediatric endotypes at successive preadult age groups to introduce safe, effective, and age-tailored targeted approaches.
      • Siegfried E.C.
      • Igelman S.
      • Jaworsk J.C.
      • Antaya R.J.
      • Cordoro K.M.
      • Eichenfield L.F.
      • et al.
      Use of dupilimab in pediatric atopic dermatitis: access, dosing, and implications for managing severe atopic dermatitis.
      We compared T-cell memory subset activation and polarized CD4/CD8 subset frequencies within the skin-homing/cutaneous lymphocyte antigen (CLA)+ and systemic/CLA compartments in the blood of infants and toddlers (0-5 years old), young children (6 to 11 years old), adolescents (12 to 17 years old), and adults (≥18 years old) with moderate-to-severe AD. Age-matched healthy control subjects were included to differentiate pathologic from physiologic immune maturation. We found that decreased TH1/TH2 ratios were shared across all AD ages, but unique fingerprinting characterizes individual AD age groups, differentiating them from their age-matched peers. Our intracellular and T-cell blood biomarker data grouped the AD cohort, but not the control subjects, into 3 unique age phenotypes aligned along a spectrum.

      Methods

       Patients’ characteristics and blood samples

      Blood was obtained (with signed institutional review board–approved informed consent from parents and patients ≥12 years of age) from 39 infants and toddlers (0-5 years old; mean, 23 months), 26 children (6-11 years old; mean, 7.5 years), 21 adolescents (12-17 years old; mean, 14.9 years), and 43 adults (≥18 years old; mean, 43 years) with moderate-to-severe AD, as well as healthy age-matched control subjects (24-30 in each group; demographic and laboratory data are shown in Table E1 in this article’s Online Repository at www.jacionline.org). Sensitivity analyses on patient subsets who were matched for all demographic parameters yielded similar results to analysis, including all subjects (see Table E2, A and B, in this article’s Online Repository at www.jacionline.org).
      Disease severity was scored by using SCORAD scores for adults and SCORAD scores, Eczema Area and Severity Index (EASI) scores, and Atopic Dermatitis Quickscores in those less than 18 years old. The Atopic Dermatitis Quickscore is a parent-administered tool that assesses involvement and pruritus of 7 body parts and highly correlates with SCORAD.
      • Carel K.
      • Bratton D.L.
      • Miyazawa N.
      • Gyorkos E.
      • Kelsay K.
      • Bender B.
      • et al.
      The Atopic Dermatitis Quickscore (ADQ): validation of a new parent-administered atopic dermatitis scoring tool.
      In subjects less than 18 years old, eosinophil counts and skin barrier assessment through transepidermal water loss (TEWL) of lesional and nonlesional arm skin (AquaFlux Model AF200; Biox, London, United Kingdom)
      • Laudanska H.
      • Reduta T.
      • Szmitkowska D.
      Evaluation of skin barrier function in allergic contact dermatitis and atopic dermatitis using method of the continuous TEWL measurement.
      were measured when feasible. No patients received systemic immunosuppressive treatment within 4 weeks before this study, and thus the data present baseline immune phenotyping of the studied populations. Concomitant allergic manifestations were recorded, and subjects were classified as either having general allergies (allergic rhinitis/asthma/allergic conjunctivitis/environmental), food allergies, both, or neither. Control subjects had no personal history of AD. Only 1 child and 5 adolescent control subjects had histories of noncutaneous atopic manifestations; however, sensitivity analysis excluding these subjects did not alter the results (see Table E2, C).

       Isolation of PBMCs

      PBMCs were isolated from whole blood by using Ficoll-Paque Plus (GE Healthcare, Uppsala, Sweden). Briefly, the blood placed under a Ficoll gradient, and after spinning, PBMCs were collected at the interface between the plasma and the Ficoll gradient (see the Methods section in this article’s Online Repository at www.jacionline.org).

       Stimulation of blood cell populations for cytokine responses

      Ex vivo cell activation is required to detect cytokine production because less than 1% of nonstimulated cells produce cytokines. Whole blood was incubated with phorbol 12-myristate 13-acetete (25 ng/mL) plus ionomycin (2 μg/mL) in the presence of brefeldin A (10 μg/mL) for 4 hours at 37°C to induce cytokine responses. After stimulation, red blood cells were lysed with FACS lysing solution to obtain leukocytes (see the Methods section in this article’s Online Repository).

       Cell-surface and intracellular staining on PBMCs and stimulated and nonstimulated CD4/CD8 T cells

      PBMCs were stained with fluorochrome-labeled antibodies to cell-surface markers (CD3, CD8, CD4, CD45RO, CCR7, inducible costimulator molecule [ICOS], HLA-DR, CLA, CCR4, CD25, and CD127). Stimulated and nonstimulated blood cells were also stained for cell-surface markers (CD3, CD4, and CLA [CD8+ T cells were gated thorough the CD3+CD4 T-cell subpopulation]) and permeabilized with FACS/perm to stain for cytokines, including IL-13, IL-22, IL-9, IFN-γ, and IL-17 (see the Methods section in this article’s Online Repository).

       Statistical analysis

      Statistical analyses were performed with R software (www.R-projets.org). Means and medians were compared by using the Welch t test and the Wilcoxon-Mann-Whitney test, respectively. Unsupervised hierarchical clustering of variables (T-cell subset frequencies, age, and clinical scores) was performed by using the R package “hclust,” with a McQuitty agglomeration algorithm and Spearman coefficient as a similarity metric and presented as a heat map and a dendrogram. Individual scatter plots were constructed that display Spearman coefficients, 95% CIs, and P values for samples from patients with AD and healthy control subjects. We performed k-means unsupervised clustering across principal components of the frequencies of all AD subsets and separately among all healthy control subsets. We found that 3 clusters separated patients with AD, but not control subjects, along a chronologic age spectrum. ANOVA, in conjunction with the Tukey test, was used to find markers that differentiated any 2 clusters.
      We also performed a power calculation based on our previous flow cytometric study on patients with moderate-to-severe adult AD.
      • Czarnowicki T.
      • Gonzalez J.
      • Shemer A.
      • Malajian D.
      • Xu H.
      • Zheng X.
      • et al.
      Severe atopic dermatitis is characterized by selective expansion of circulating TH2/cytotoxic T (TC) 2 and TH22/TC22, but not TH17/TC17, cells within the skin-homing T-cell population.
      We determined that 39 patients with AD would have greater than 90% power (at a significance level of .05) to detect differences versus control subjects in various inflammatory cell subsets (CD4+IL-22+CLA+, CD4+IL-13+CLA, CD8+IL-17+CLA+, and CD8+ central memory T [Tcm] HLA-DR+CLA+ cells).

      Results

      Flow cytometry was used to measure frequencies of IFN-γ–, IL-9–, IL-13–, IL-17A–, and IL-22– polarized T cells, defining TH1/TC1, TH9/TC9, TH2/TC2, TH17/TC17, and TH22/TC22 subsets in CD4+/CD8+ T cells, respectively. Cell-surface staining was used to assess expression of midactivation (ICOS) and late activation (HLA-DR) markers in Tcm (CCR7+CD45RO+) and effector memory T (Tem; CCR7CD45RO+) cells in skin-homing/cutaneous/CLA+ and systemic/CLA compartments.
      Patients and control subjects were divided into 4 consecutive age groups (infants and toddlers 0-5 years old, children 6-11 years old, adolescents 12-17 years old, and adults ≥18 years old). To display both healthy versus pathologic developmental changes and immune abnormalities within each age group versus control subjects, we present 2 types of comparison plots; both contain similar data but focus on either patients with AD versus control subjects for each age group or patients with AD versus control subjects across all ages.
      The comparison plots presented below contain both the mean and median ± SE and their respective P values to better represent the effect of value distribution. Results discuss mean values, whereas median percentages for main comparisons discussed are presented in Table E3 in this article’s Online Repository at www.jacionline.org.

       Skin-homing memory T-cell expansion and ICOS activation feature in early AD

      Tcm and Tem cells are the main components of the adaptive immune system, harboring distinct homing capacities.
      • Sallusto F.
      • Geginat J.
      • Lanzavecchia A.
      Central memory and effector memory T cell subsets: function, generation, and maintenance.
      Although both express the skin-homing marker CLA, only Tcm cells retain CCR7 positivity, which enables them to migrate into lymph nodes and function as an immunologic reserve.
      • Sallusto F.
      • Lenig D.
      • Forster R.
      • Lipp M.
      • Lanzavecchia A.
      Two subsets of memory T lymphocytes with distinct homing potentials and effector functions.
      After gating on CD3+ viable T cells using flow cytometry, CD3+CD4+ and CD3+CD8+ cells were defined and analyzed separately. CCR7 and CD45RO were used to differentiate memory subsets within the CD4 and CD8 populations. CCR7+CD45RO defined naive cells, CCR7+CD45RO+ defined Tcm cells, CCR7CD45RO+ defined Tem cells, and CCR7CD45RO defined effector/Temra/terminally differentiated T cells (see Fig E1, A-C, in this article’s Online Repository at www.jacionline.org). We then further defined the activated ICOS/HLA-DR–activated Tcm/Tem cell subset using CLA to segregate skin-homing (CLA+) versus systemic (CLA) subsets.
      Normal development was characterized by a slight decrease in frequencies of CLA+CD4+ Tem (but not Tcm) cells between infancy and childhood (infants: 26.7% vs children: 19%, P = .01; Fig 1, A and B, and see Fig E1, D and E), but this decrease was significantly more evident in patients with AD (P < .05; Fig 1, A and B, and see Fig E1, D and E). CD4+CLA+ Tem/Tcm cell counts were significantly greater in infants with AD (Tcm cells: 20.4% vs 13.4%, P = .006; Tem cells: 38% vs 26.7%, P = .002; see Fig E1, D and E) and children with AD (Tcm cells: 18.8% vs 11.3%, P = .01; Tem cells: 27.7% vs 19%, P = .03; see Fig E1, D and E) than in control subjects, a difference that diminished with increasing age. T-cell memory subset fluctuations with age are presented in Fig E2 in this article’s Online Repository at www.jacionline.org. Because of increased proportions of effector and naive cells, CD8+ Tcm/Tem cell subset frequencies decreased between infancy and childhood exclusively in control subjects (Tcm cells: 12.8% [infants] vs 5.6% [children], P = .008; Tem cell: 15.4% [infants] vs 5% [children], P < .001; Fig 1, C and D), leading to higher frequencies in children with AD (P < .01), but otherwise, frequencies were overall similar between patients with AD and control subjects over time (see Fig E1, F and G). A significant increase in CD4+/CD8+ effector cells with age characterized patients with AD versus control subjects, who showed a decrease in this subset during the study (see Fig E2, J and L).
      Figure thumbnail gr1
      Fig 1Frequency of CLA+ Tem (CD45RO+CCR7) and Tcm (CD45RO+CCR7+) cells among CD4+/CD8+ T cells (A-D) and ICOS+ activation in CLA+/− CD4+/CD8+ Tcm/Tem cells (E-L) in healthy control subjects and patients with AD across ages (ICOS+CLA+CD45RO+CCR7+/−). Bar plots represent means (black)/medians (red) ± SEMs. *P < .05, **P < .01, ***P < .001, and +P < .1.
      CD4+ Tcm/Tem ICOS levels at midactivation continuously increased in both control subjects and patients with AD; however, they decreased significantly between adolescence and adulthood exclusively in control subjects (Tcm CLA+ cells: 24% vs 9%, P = .003; Tcm CLA cells: 9.2% vs 3.7%, P = .001; Fig 1, E-H). Both skin-homing/CLA+ and systemic/CLA ICOS-activated CD4+ T-cell frequencies were significantly increased in infants and adults with AD versus those in respective control subjects (P < .05; Fig 1, E-H). Skin-homing CD8+ Tcm/Tem cell ICOS activation increased gradually, most notably in patients with AD (Fig 1, I-L), with frequency differences uniquely seen in adults with AD versus control subjects (Tcm cells: 18.4% vs 12.5%, P = .01; Tem cells: 26% vs 13.4%, P < .001; Fig 1, I-L).
      The HLA-DR antigen, indicating chronic activation,
      • Ferenczi K.
      • Burack L.
      • Pope M.
      • Krueger J.G.
      • Austin L.M.
      CD69, HLA-DR and the IL-2R identify persistently activated T cells in psoriasis vulgaris lesional skin: blood and skin comparisons by flow cytometry.
      had similar (or even lower) expression in infants with and without AD (P > .1; see Fig E1, H-K) but started to increase in children with AD (Tcm+CD4+CLA+ AD: 15.8% vs 8.4%, P = .02; CLA: 7.2% vs 2.8%, P = .06; see Fig E1, H and I), reaching consistently high levels across CD4+/CD8+/CLA+/CLA/Tcm/Tem cells in adults with AD compared with healthy subjects (P < .05; CD8+ Tcm/Tem cell data are not shown; see Fig E1, H-K).

       Decreased TH1/TH2 ratio characterizes AD across ages

      Because T-cell activation leads to cytokine polarization, we next studied different polar T-cell subsets. Representative flow cytometric plots and the gating strategy are presented in Fig E3 in this article’s Online Repository at www.jacionline.org. Congruent with past publications,
      • Marodi L.
      Down-regulation of Th1 responses in human neonates.
      ,
      • Gasparoni A.
      • Ciardelli L.
      • Avanzini A.
      • Castellazzi A.M.
      • Carini R.
      • Rondini G.
      • et al.
      Age-related changes in intracellular TH1/TH2 cytokine production, immunoproliferative T lymphocyte response and natural killer cell activity in newborns, children and adults.
      IFN-γ levels increased with age in both control subjects and patients with AD, with the lowest frequencies seen in infants (Fig 2, A-D). Nevertheless, in patients with AD, IFN-γ did not reach control levels, particularly within the skin-homing compartment (CD4+CLA+: 12% [control infants] vs 7.7% [infants with AD], P =.04; 19.5% [control children] vs 13.6% [children with AD], P = .05; 18% [control adolescents] vs 13% [adolescents with AD], P = .07) until adulthood (P = .2; Fig 2, A-D, and see Fig E4, A-D, in this article’s Online Repository at www.jacionline.org). Even in adulthood, levels trended toward lower frequencies. Interestingly, the only population that showed lower systemic/CLA CD4+/CD8+ IFN-γ levels was the 5- to 12-year-old age group (CD4+: 20% [control subjects] vs 14% [patients with AD], P = .04; CD8+: 39% [control subjects] vs 28% [patients with AD], P = .05; see Fig E4, B and D).
      Figure thumbnail gr2
      Fig 2IFN-γ+ (A-D), IL-13+ (E-H), and IFN-γ+/IL-13+ (I-L) cytokine frequencies in CLA+, CLA, CD4+, and CD8+ T cells in healthy control subjects and patients with AD across ages. The ratio between the percentage of IFN-γ+CLA+CD4+ (or CD8+) T cells and the percentage of their IL-13+ counterparts was calculated for each sample but was not multiplied by 100 and is therefore unitless. Bar plots represent means (black)/medians (red) ± SEMs. *P < .05, **P < .01, ***P < .001, and +P < .1.
      In control subjects IL-13+CD4+CLA+ levels were lowest in infants, reaching a plateau in childhood. Conversely, in patients with AD, levels were similarly increased across all ages (P > .1; Fig 2, E). Systemic/CLA TH2 cell counts were overall low in control subjects (Fig 2, F). Counts were significantly higher in patients with AD than in control subjects among children (2.8% vs 0.6%, P = .005; see Fig E4, F), adolescents (1.5% vs 0.7%, P = .006; see Fig E4, F), and adults (1.3% vs 0.7%, P = .005; see Fig E4, F). Although TC2 cell counts were slightly higher in children with versus those without AD, differences in CD8+ subsets were more prominent in adulthood (Fig 2, G and H, and see Fig E4, G and H). Reflecting the TH1 and TH2 imbalances characterizing AD, the TH1/TH2 ratio was decreased in patients with AD compared with that in control subjects in both CLA+/CLA subsets across the ages. TC1/TC2 cell counts were significantly lower in children and adults with AD versus control subjects (Fig 2, I-L, and see Fig E4, I-L).
      Both healthy control subjects and patients with AD had TH9 cell count increases over time, peaking in adolescence and decreasing in adulthood (Fig 3, A and B, and see Fig E5, A and B, in this article’s Online Repository at www.jacionline.org). No TH9 level differences were observed between patients with AD and control subjects, with the exception of 5- to 12-year-old children with AD, who showed significantly increased CLA levels (0.76% vs 0.3%, P = .04; see Fig E5, B). This AD age group also showed increased CLA+/CLA TC9 cell counts (Fig 3, C and D, and see Fig E5, C and D). CLA+ TH17 cell counts were generally stable and similarly abundant across ages among control subjects and patients with AD (Fig 3, E, and see Fig E5, E) contrary to CLA TH17 cell counts, which showed developmental expansion in both (Fig 3, F, and see Fig E5, F). Contrary to adolescents with AD, who had lower CLA+ TC17 cell counts than control subjects (1% vs 4.7%, P = .04), adults had significantly higher frequencies (2.1% vs 1.4%, P =.05; Fig 3, G and H, and see Fig E5, G and H).
      Figure thumbnail gr3
      Fig 3IL-9+ (A-D), IL-17+ (E-H), and IL-22+ (I-L) frequencies in CLA+, CLA, CD4+, and CD8+ T cells in healthy control subjects and patients with AD across ages. Bar plots represent means (black)/medians (red) ± SEMs. *P < .05, **P < .01, ***P < .001, and +P < .1.
      Systemic/CLA CD4+/CD8+ IL-22+ cell counts similarly increased with age in both control subjects and patients with AD (Fig 3, J and L, and see Fig E5, J and L), whereas skin-homing TH22/TC22 cell counts increased with age primarily in patients with AD (Fig 3, I and K, and see Fig E5, I and K). Starting in childhood, skin-homing TH22/TC22 cell counts were significantly higher in patients with AD versus control subjects and incrementally increased with age (TH22 cell: children, 6.7% vs 4%, P = .07; adolescents, 7.9% vs 3.6%, P = .001; adults, 8.2% vs 4.4%, P < .0001; Fig 3, I and K, and see Fig E5, I and K). Polar T-cell subset development in control subjects and patients with AD is summarized in Table E4 in this article’s Online Repository at www.jacionline.org and shown in the graphical abstract.
      The unsupervised hierarchical clustering heat map in Fig 4 summarizes the above, displaying all polarized T-cell subsets for control subjects and patients with AD across age groups (red, positive/increase; blue, negative/decrease). FCHs of the mean frequencies of patients with AD versus control subjects for each age group are presented. The green cluster includes subsets that were relatively low and stable among control subjects but incrementally increased with age in patients with AD. Most of these subsets were significantly increased in adults with AD versus control subjects (FCH > 1.57, P < .05), whereas younger patients with AD showed lower or no significance. The pink box shows increased IL-9 frequencies in childhood, which decrease in adulthood, particularly in patients with AD. The yellow cluster shows markers that increased in both control subjects and patients with AD, therefore minimizing the differences between groups.
      Figure thumbnail gr4
      Fig 4Unsupervised hierarchical clustering heat map displaying polarized T-cell subsets for control subjects and patients with AD across age groups (red, positive/increase; blue, negative/decrease). FCHs of mean frequencies of patients with AD versus control subjects for each age group are listed at right. The green cluster includes subsets that were relatively low and stable among control subjects but incrementally increased with age in patients with AD. The pink box shows increased IL-9 frequencies in childhood, which decrease in adulthood, particularly in patients with AD. The yellow cluster shows markers with increased levels in both control subjects and patients with AD. *P < .05, **P < .01, ***P < .001, and +P < .1.

       T-cell activation, clinical measures, and IFN-γ levels are associated with age and AD chronicity

      We also evaluated how clinical characteristics, including AD severity (SCORAD and EASI scores), patient age, disease duration, eosinophil counts, pruritus, and TEWL relate to different polar T-cell subsets. Unsupervised hierarchical clustering of all T-cell subset frequencies, clinical scores, AD duration, and age was performed by using Spearman correlations as a similarity metric, as displayed in the correlation heat map and dendrogram in Fig 5 (red, positive correlation; blue, negative correlation; stars and plus signs display significance). Congruent with recent AD data,
      • Czarnowicki T.
      • Gonzalez J.
      • Shemer A.
      • Malajian D.
      • Xu H.
      • Zheng X.
      • et al.
      Severe atopic dermatitis is characterized by selective expansion of circulating TH2/cytotoxic T (TC) 2 and TH22/TC22, but not TH17/TC17, cells within the skin-homing T-cell population.
      ,
      • Czarnowicki T.
      • He H.Y.
      • Wen H.C.
      • Hashim P.W.
      • Nia J.K.
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      • et al.
      Alopecia areata is characterized by expansion of circulating Th2/Tc2/Th22, within the skin-homing and systemic T-cell populations.
      which showed positive correlations between IL-13– and IL-22–producing and IL-17– and IL-22–producing T cells,
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      Severe atopic dermatitis is characterized by selective expansion of circulating TH2/cytotoxic T (TC) 2 and TH22/TC22, but not TH17/TC17, cells within the skin-homing T-cell population.
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      Alopecia areata is characterized by expansion of circulating Th2/Tc2/Th22, within the skin-homing and systemic T-cell populations.
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      these cytokines largely grouped together. IFN-γ–producing T-cells clustered together with AD duration and patient age. A tight cluster gathering multiple clinical measures was located adjacent to IL-9/IL-13–producing T cells. Significant correlations depicted in this heat map are listed in Table E5 in this article’s Online Repository at www.jacionline.org, with selected individual scatter plots presented in Fig 6, Fig 7 and Figs E6 to E8 in this article’s Online Repository at www.jacionline.org.
      Figure thumbnail gr5
      Fig 5Unsupervised hierarchical clustering of polarized cytokine subset frequencies (percentages) with AD clinical measures using Spearman correlation as a similarity metric. CD4+/CD8+ IL-13+, IL-17+, and IL-22+ subsets clustered together (turquoise box). IFN-γ+–producing T-cell subsets grouped together with age and disease duration (yellow box). IL-9+ and some IL-13+ and IL-22+ T cells (purple box) clustered adjacent to AD clinical measures (green box). The heat map shows positive (red) or negative (blue) correlations of all parameters, with color intensity reflecting the strength of the correlation (−1 to +1). Dendrograms are shown as a tree, representing the distance between variables. *P < .05, **P < .01, ***P < .001, and +P < .1. LS, Lesional; NL, nonlesional.
      Figure thumbnail gr6
      Fig 6Spearman correlation scatter plots (linear regression [red, AD; blue, control line] with their 95% CIs [gray]) for SCORAD score (A-E) and age (F-L) versus clinical measures and Tem/Tcm cell subset frequencies (percentages). Dot colors in Fig 6, A-D and F, designate different AD patient ages from infancy to adulthood, as shown in . ADQ, Atopic Dermatitis Quickscore; LS, lesional; NL, nonlesional.
      Figure thumbnail gr7
      Fig 7Spearman correlation scatter plots (linear regression [red, AD; blue, control line] with their 95% CIs [gray]) for CD4+/CD8+ and CLA+/CLA IFN-γ+/IL-13+–producing T-cell ratio (A-D) and IL-13+–producing (E-H) and IL-22+–producing (I-L) T-cell frequencies (percentages) versus age in patients with AD (red dots/line) and control subjects (blue dots/line).
      SCORAD and EASI scores were positively correlated (r = 0.74, P < .001; Fig 6, A). SCORAD scores also correlated with skin-homing CD4+ cell counts (r = 0.24, P = .0016; Fig 6, B), pruritus (r = 0.54, P < .0001; Fig 6, C), and eosinophil counts (r = 0.25, P = .001; Fig 6, D). To evaluate normal versus pathologic development with age, we comprehensively assessed age correlations in control subjects and patients with AD, presenting both on the same scatter plots for clarity, when applicable. Age correlated positively with severity based on SCORAD (r = 0.25, P = .0016; Fig 6, E) and EASI (r = 0.34, P < .001; Fig 6, F) scores and with all other clinical measures, including pruritus (Fig 6, G), eosinophil counts (Fig 6, H), and lesional TEWL (Fig 6, I and J).
      Although skin-homing Tcm/Tem cell counts correlated negatively with age (Fig 6, K and L; red, patients with AD; blue, control subjects), the proportion of ICOS- and HLA-DR–activated skin-homing T cells increased exclusively in patients with AD (see Fig E6, A-D). AD duration, which highly correlated with patient age (r = 0.98, P < .0001; see Fig E6, E), also demonstrated significant positive correlations with EASI score and pruritus (see Fig E6, F-H). Among the cytokine subsets (see Fig E6, I-L), the most significant correlation was noted between AD duration and IFN-γ–producing cell counts (r = 0.58, P < .0001; see Fig E6, L).
      Overall, IFN-γ levels increased with age in both patients with AD and control subjects (see Fig E7, A-D); however, although the TH1/TH2 (but not TC1/TC2) ratio, particularly its skin-homing component, increased with age in both groups, it remained significantly decreased in patients with AD across all ages versus control subjects (Fig 7, A-D). Conversely, differences between patients with AD and control subjects were observed in skin-homing TH2/TC2 cell counts throughout development, with control subjects never reaching the levels seen in patients with AD (Fig 7, E-H). CLA+ TH22/TC22 cell counts showed significantly higher developmental increases in patients with AD (Fig 7, I and K), whereas systemic subsets generally overlapped (Fig 7, J and L). Similar negative trends of IL-9+ cells (see Fig E7, E-H) and expansion of systemic TH17 cells were seen in control subjects and patients with AD (see Fig E7, I-L).
      Positive correlations between SCORAD scores and IL-13–, IL-9–, and IL-22–producing cells were recorded (P < .02; see Fig E8, A-C, in this article’s Online Repository at www.jacionline.org). EASI scores correlated with TH9 and TH22 cell counts and pruritus (P < .03; see Fig E8, D-F), whereas pruritus was associated with TH22 cell and eosinophil counts (P < .032; see Fig E8, G and H). TEWL correlated with CD4+CLA+ cell counts only in nonlesional skin (Fig E8, I and J). TH22 cell counts positively correlated with TEWL in lesional tissues (see Fig E8, K and L), and AD severity correlated with TEWL in nonlesional skin (see Fig E8, M-P).
      Because forkhead box P3 staining requires cell permeabilization, surface markers were used for regulatory T (Treg) cell identification. Ninety percent of CD25+CD127CCR4+ cells coexpress forkhead box P3, and therefore the CD25+CD127CCR4+ phenotype defined Treg cells.
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      In analyzing Treg cells, both total Treg cell (CD25+CD127) and skin-homing (CCR4+CLA+ fraction) Treg cell frequencies were captured. The only AD age group that showed significant increases in both total (see Fig E9, A, in this article’s Online Repository at www.jacionline.org) and skin-homing (see Fig E9, B) Treg cells versus control subjects was the 5 to 12-year-old group (P < .01). Developmentally, patients with AD showed similar total Treg cell trends as control subjects, whereas discrepancies were more evident in skin-homing subsets (see Fig E9, C and D).

       Cytokine polarization and T-cell activation differentiate patients with AD into separate age clusters along a spectrum

      We integrated T-cell and cytokine biomarkers to differentiate the entire AD cohort based on their blood phenotype. The principal components of all biomarker data for all subjects were analyzed by using unsupervised k-means clustering separately for patients with AD and control subjects. As shown in Fig 8, in the AD cohort the frequencies of different markers defined 3 meaningful clusters aligning along a spectrum. Although infants clustered on the far left and adults clustered on the right, children and adolescents generally clustered together between the infants and adults. The markers that best distinguished between each set of clusters appear in the boxes between the 2 cohorts and are summarized in Table E6 in this article’s Online Repository at www.jacionline.org. The TH1/TH2 ratio, CD8+ activation, and IFN-γ–producing T-cell counts differentiated adults from younger groups. Treg cells, T-cell activation, and different cytokine subsets were able to differentiate distinctive AD age groups. Applying the same model to the control population did not distinguish between age groups (Fig 8).
      Figure thumbnail gr8
      Fig 8Unsupervised clustering of patients with AD and healthy control subjects across all principal components of the blood flow cytometric marker frequencies (percentages) by using k-means analysis. In patients with AD, frequencies of different markers defined 3 meaningful age clusters aligning along a spectrum. Although infants (pink ellipse) clustered on the far left and adults (green ellipse) clustered on the right, children and adolescents (blue ellipse) clustered together between the other age cohorts. Markers that best distinguished between clusters appear in the boxes between 2 cohorts (colors of markers parallel colors of the relative age group). Arrows designate increased frequencies of a given marker among the specific age group. In healthy control subjects clusters did not clearly align patients along an age spectrum.

      Discussion

      This is the first comprehensive study that compares systemic immune profiles of different AD age groups (0-5, 6-11, 12-17, and ≥18 years old) with appropriate comparisons with control subjects. Because circulating CLA+ T cells have been suggested as peripheral biomarkers in patients with AD,
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      Lower proportions of CD8+ versus CD4+ cells in early childhood
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      Age-related changes in the cellular composition of the thymus in children.
      and the fact that IL-22 marks AD chronicity
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      • Malajian D.
      • Shemer A.
      • Noda S.
      • et al.
      Early pediatric atopic dermatitis shows only a cutaneous lymphocyte antigen (CLA)(+) TH2/TH1 cell imbalance, whereas adults acquire CLA(+) TH22/TC22 cell subsets.
      might explain why skin-homing TH22/TC22 cell frequencies are highest in adolescents and adults. Increased IL-22 levels, which gave been shown to negatively regulate IFN-γ,
      • Pennino D.
      • Bhavsar P.K.
      • Effner R.
      • Avitabile S.
      • Venn P.
      • Quaranta M.
      • et al.
      IL-22 suppresses IFN-gamma-mediated lung inflammation in asthmatic patients.
      might also contribute to the low TH1 frequencies seen in patients with AD. Systemic/CLA TH22/TC22 subset development was similar in patients with AD and control subjects, potentially suggesting a greater pathogenic relevance of IL-22 in skin of patients with AD
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      • Lu J.
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      • Jeong M.
      • Barmettler S.
      • et al.
      Expression of IL-22 in the skin causes Th2-biased immunity, epidermal barrier dysfunction, and pruritus via stimulating epithelial Th2 cytokines and the GRP pathway.
      and the impaired skin barrier.
      • Fujita H.
      The role of IL-22 and Th22 cells in human skin diseases.
      Studies have shown positive correlations and cellular coproduction of IL-13 and IL-22
      • Czarnowicki T.
      • Gonzalez J.
      • Shemer A.
      • Malajian D.
      • Xu H.
      • Zheng X.
      • et al.
      Severe atopic dermatitis is characterized by selective expansion of circulating TH2/cytotoxic T (TC) 2 and TH22/TC22, but not TH17/TC17, cells within the skin-homing T-cell population.
      ,
      • Teraki Y.
      • Sakurai A.
      • Izaki S.
      IL-13/IL-22-coproducing T cells, a novel subset, are increased in atopic dermatitis.
      and of IL-17 and IL-22.
      • Czarnowicki T.
      • He H.
      • Leonard A.
      • Kim H.J.
      • Kameyama N.
      • Pavel A.B.
      • et al.
      Blood endotyping distinguishes the profile of vitiligo from that of other inflammatory and autoimmune skin diseases.
      ,
      • Liang S.C.
      • Tan X.Y.
      • Luxenberg D.P.
      • Karim R.
      • Dunussi-Joannopoulos K.
      • Collins M.
      • et al.
      Interleukin (IL)-22 and IL-17 are coexpressed by Th17 cells and cooperatively enhance expression of antimicrobial peptides.
      Indeed, these markers cluster together in the correlation heat map. IFN-γ clusters with patient age and disease duration, differentiating infants from children and adolescents and adolescents from adults with AD. The association of IFN-γ with disease chronicity agrees with previous chronic adult AD data, suggesting that IFN-γ possibly plays a role in inflammatory disorder chronicity rather than in disease intiation.
      • Czarnowicki T.
      • He H.Y.
      • Wen H.C.
      • Hashim P.W.
      • Nia J.K.
      • Malik K.
      • et al.
      Alopecia areata is characterized by expansion of circulating Th2/Tc2/Th22, within the skin-homing and systemic T-cell populations.
      ,
      • Czarnowicki T.
      • Esaki H.
      • Gonzalez J.
      • Malajian D.
      • Shemer A.
      • Noda S.
      • et al.
      Early pediatric atopic dermatitis shows only a cutaneous lymphocyte antigen (CLA)(+) TH2/TH1 cell imbalance, whereas adults acquire CLA(+) TH22/TC22 cell subsets.
      ,
      • Gittler J.K.
      • Shemer A.
      • Suarez-Farinas M.
      • Fuentes-Duculan J.
      • Gulewicz K.J.
      • Wang C.Q.
      • et al.
      Progressive activation of T(H)2/T(H)22 cytokines and selective epidermal proteins characterizes acute and chronic atopic dermatitis.
      Expansion of CD8+ memory cells, increases in CLA+ IL-22–producing cells, and intensification of ICOS+/HLA-DR+ activation with maturity might account for the positive correlations between age, disease duration, and severity measures.
      Increasing severity with age was not seen in a recent study that followed infants with AD only between 0 and 11 months.
      • Hulshof L.
      • Overbeek S.A.
      • Wyllie A.L.
      • Chu M.
      • Bogaert D.
      • de Jager W.
      • et al.
      Exploring immune development in infants with moderate to severe atopic dermatitis.
      TEWL increases with age in patients with AD and correlates with IL-22 (which also increases with age), likely again reflecting the contribution of IL-22 to the barrier impairment in patients with AD.
      In addition to maintaining immune tolerance,
      • Long S.A.
      • Buckner J.H.
      CD4+FOXP3+ T regulatory cells in human autoimmunity: more than a numbers game.
      Treg cells influence activation of effector cells.
      • McHugh R.S.
      • Shevach E.M.
      The role of suppressor T cells in regulation of immune responses.
      ,
      • Hall B.M.
      T cells: soldiers and spies—the surveillance and control of effector T cells by regulatory T cells.
      Only 5- to 12-year-old children with AD have significantly higher total and skin-homing Treg cells than control subjects. This age group is characterized by other unique features, including increased skin-homing Tcm/Tem cell counts, decreased systemic IFN-γ levels, and increased systemic IL-9 frequencies. Additionally, skin-homing IFN-γ levels are almost doubled from infancy to childhood in patients with AD, although they are still lower than those in age-matched control subjects. Multiple immunologic changes occurring during these years might be involved in AD clearance or development of noncutaneous manifestations commonly occurring during these years.
      • Burr M.L.
      • Dunstan F.D.
      • Hand S.
      • Ingram J.R.
      • Jones K.P.
      The natural history of eczema from birth to adult life: a cohort study.
      ,
      • Carlsten C.
      • Dimich-Ward H.
      • Ferguson A.
      • Watson W.
      • Rousseau R.
      • Dybuncio A.
      • et al.
      Atopic dermatitis in a high-risk cohort: natural history, associated allergic outcomes, and risk factors.
      Profiling AD across ages is imperative for targeted therapeutic development. Unlike psoriasis, in which targeted treatments lead to remarkable responses in most patients, AD responses to targeted therapeutics are much lower.
      • Guttman-Yassky E.
      • Krueger J.G.
      Atopic dermatitis and psoriasis: two different immune diseases or one spectrum?.
      This disparity can be attributed to the multicytokine activation seen in patients with AD, despite the shared TH2 activation, versus the TH17-centered responses in patients with psoriasis.
      • Guttman-Yassky E.
      • Krueger J.G.
      Atopic dermatitis and psoriasis: two different immune diseases or one spectrum?.
      Additionally, AD has a highly varied endotype repertoire, with different immune polarizations.
      • Zhou L.
      • Leonard A.
      • Pavel A.B.
      • Malik K.
      • Raja A.
      • Glickman J.
      • et al.
      Age-specific changes in the molecular phenotype of patients with moderate-to-severe atopic dermatitis.
      ,
      • Czarnowicki T.
      • He H.
      • Krueger J.G.
      • Guttman-Yassky E.
      Atopic dermatitis endotypes and implications for targeted therapeutics.
      ,
      • Leung D.Y.
      • Guttman-Yassky E.
      Deciphering the complexities of atopic dermatitis: shifting paradigms in treatment approaches.
      Despite common features, particularly increased TH2 expression, AD is endotypically different across ages, and treatments should be tailored to the unique age endotype. Although one could hypothesize that the immune changes merely reflect developmental phenomena that are age related, the lack of clear clustering in control subjects implicates AD as the driver of the distinct, progressive, age-related endotypic characteristics rather than age alone. The clustering model, based on flow cytometric biomarkers, splits the entire cohort into 3 separate age clusters only in patients with AD. Expectedly, IFN-γ, IL-22, and HLA-DR levels increased chronologically, distinguishing older subjects from infants with AD. Skin-homing Tcm/Tem cell counts and systemic ICOS activation, both of which were higher in infants with AD, separate infants from other groups. This model demonstrates that a limited group of blood biomarkers can distinguish among various AD endotypes based on patient age, with a spectrum of age-dependent versus only “infantile” or “adult” phenotypes. Because our data show that the “adult” or “stable” AD phenotype is only achieved in adulthood, it might be possible to intervene before adulthood and prevent establishment of the adult AD phenotype.
      This study has a few limitations. Despite inclusion of different age groups, this study was not longitudinal and thus did not follow the same cohort with time. Additionally, studies in blood do not allow one to measure T-cell subsets in skin, such as tissue-resident memory T cells. Furthermore, the study characterized polyclonal T-cell responses and not antigen-specific responses induced by culprit triggers. Finally, the pathogenicity of immune axes presented here cannot be further dissected without future targeted therapeutic studies to check mechanisms.
      AD was initially considered an early-onset pediatric disease, with 75% "outgrowing" their disease by age 10 years.
      • Bieber T.
      Atopic dermatitis.
      ,
      • Burr M.L.
      • Dunstan F.D.
      • Hand S.
      • Ingram J.R.
      • Jones K.P.
      The natural history of eczema from birth to adult life: a cohort study.
      ,
      • Carlsten C.
      • Dimich-Ward H.
      • Ferguson A.
      • Watson W.
      • Rousseau R.
      • Dybuncio A.
      • et al.
      Atopic dermatitis in a high-risk cohort: natural history, associated allergic outcomes, and risk factors.
      ,
      • Guttman-Yassky E.
      • Nograles K.E.
      • Krueger J.G.
      Contrasting pathogenesis of atopic dermatitis and psoriasis—part I: clinical and pathologic concepts.
      More recent studies have established AD as a disorder that often persists into adulthood.
      • Margolis J.S.
      • Abuabara K.
      • Bilker W.
      • Hoffstad O.
      • Margolis D.J.
      Persistence of mild to moderate atopic dermatitis.
      ,
      • Abuabara K.
      • Margolis D.J.
      Do children really outgrow their eczema, or is there more than one eczema?.
      Comparing the profile of cleared versus persistent pediatric AD, ideally through longitudinal studies, will better define age-specific characteristics that predict AD clearance.
      Clinical implications
      Diverse immune signatures in different pediatric and adult AD age groups argue for age-specific, rather than uniform, therapeutic interventions.

      Supplementary data

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