Heterogeneous IL-9 Production by Circulating Skin-Tropic and Extracutaneous Memory T Cells in Atopic Dermatitis Patients

Abstract

Interleukin (IL)-9 is present in atopic dermatitis (AD) lesions and is considered to be mainly produced by skin-homing T cells expressing the cutaneous lymphocyte-associated antigen (CLA). However, its induction by AD-associated triggers remains unexplored. Circulating skin-tropic CLA⁺ and extracutaneous/systemic CLA^- memory T cells cocultured with autologous lesional epidermal cells from AD patients were activated with house dust mite (HDM) and staphylococcal enterotoxin B (SEB). Levels of AD-related mediators in response to both stimuli were measured in supernatants, and the cytokine response was associated with different clinical characteristics. Both HDM and SEB triggered heterogeneous IL-9 production by CLA⁺ and CLA^- T cells in a clinically homogenous group of AD patients, which enabled patient stratification into IL-9 producers and non-producers, with the former group exhibiting heightened HDM-specific and total IgE levels. Upon allergen exposure, IL-9 production depended on the contribution of epidermal cells and class II-mediated presentation; it was the greatest cytokine produced and correlated with HDM-specific IgE levels, whereas SEB mildly induced its release. This study demonstrates that both skin-tropic and extracutaneous memory T cells produce IL-9 and suggests that the degree of allergen sensitization reflects the varied IL-9 responses in vitro, which may allow for patient stratification in a clinically homogenous population.

Keywords: atopic dermatitis; cutaneous lymphocyte-associated antigen; extracutaneous; heterogenous; house dust mite; interleukin-9; skin-tropic; staphylococcal enterotoxin B.

Figure 1

HDM induces IL-9 production by both skin-tropic CLA⁺ and non-tropic CLA⁻ memory T cells at higher level than other proinflammatory cytokines, which correlates with HDM-specific IgE plasma levels. (A) IL-9 (pg/mL) produced in basal conditions (M) or stimulated with HDM in AD- (n = 52) and C-derived (n = 12) CLA⁺/Epi and CLA⁻/Epi cocultures after 5 days. IL-4, IL-5, IL-9, IL-13, IL-17A, IL-31, IFN-γ (n = 52), IL-21 (n = 46), and IL-22 (n = 45) cytokines were simultaneously quantified in (B) CLA⁺/Epi and (C) CLA⁻/Epi AD cocultures. Cytokines were sorted according to the median production and compared in relation to IL-9 production levels. Red line indicates the median. Correlations between IL-9 (pg/mL) and (D) HDM-specific, and (E) total IgE plasma levels (n = 51). AD, atopic dermatitis; C, control subjects; CLA, cutaneous lymphocyte-associated antigen; Epi, epidermal cells; HDM, house dust mite; M, untreated; OD, optical density. ns: p > 0.05; * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Figure 2

HDM-activated memory T cells require cell-to-cell contact with epidermal cells for IL-9 production, which primarily depends on HLA class II molecules. (A) Epidermal cells, CLA^+/− T cells, and CLA^+/−/Epi cocultures were left untreated or stimulated with HDM, and IL-9 (pg/mL) was measured at day 5. (B) HDM-stimulated (n = 4) CLA^+/− T cells and epidermal cells were added in the upper and lower chamber of the transwell plate (right) or cocultured together (left), and IL-9 (pg/mL) was quantified on day 5. (C) HLA class I and (D) class II molecules were neutralized in HDM-activated CLA^+/−/Epi cocultures on day 0, and IL-9 levels (pg/mL) were compared to isotype values on day 5 (n = 8). Cytokine content under HDM stimulation without neutralizing antibodies (M) are shown. CLA^+/−/Epi cocultures were polyclonally activated with (E) PMA/ION and (F) anti-CD3/CD28 beads, and IL-9 (pg/mL) was measured on day 5 (n = 6). Red line indicates the median. Cl, class; CLA, cutaneous lymphocyte-associated antigen; Epi, epidermal cells; HDM, house dust mite; HLA, human leukocyte antigens; ION, ionomycin; M, untreated; PMA, phorbol myristate acetate. ns: p > 0.05; * p < 0.05; ** p < 0.01.

Figure 3

Patients with HDM-induced CLA⁺ T cell IL-9 response have elevated specific and total IgE plasma levels; their IL-9 levels decrease over the course of the disease and, within the CLA⁺ subset, IL-9 correlates with cytokines of Th2- and Th17/22-induced inflammation. (A) AD patients were stratified according to IL-9 production by HDM-stimulated CLA⁺ T (producers n = 30, non-producers n = 22) and CLA⁻ T cells (producers n = 27, non-producers n = 25). (B) Specific IgE (OD) and (C) total IgE (kU/L) plasma levels were compared between IL-9 producers and non-producers in the CLA⁺ (n = 28 and 26, respectively) and CLA⁻ (n = 21 and 23, respectively) subsets in AD patients, and control volunteers (n = 25). Red lines indicate the median. Correlation between IL-9 (pg/mL) produced by HDM-activated CLA^+/− T cell cocultures and (D) years since diagnosis and (E) IL-13, IL-4, IL-5, IL-31, IL-17A, IL-22, IL-21, IFN-γ, CCL17, and CCL22 (pg/mL) within CLA⁺ (n = 11–30) and CLA⁻ (n = 12–27) IL-9 producers. Bold values indicate significant data. AD, atopic dermatitis; C, control subjects; CLA, cutaneous lymphocyte-associated antigen; Epi, epidermal cells; HDM, house dust mite; M, untreated; OD, optical density. ns: p > 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Figure 4

SEB triggers mild IL-9 production in both CLA⁺ and CLA⁻ memory T cells. (A) IL-9 (pg/mL) produced in basal conditions (M) or stimulated with SEB in AD- (n = 50) and C-derived (n = 17) CLA⁺/Epi and CLA⁻/Epi cocultures after 24 h. AD patients were stratified according to SEB-induced IL-9 response. Blue and green boxes show those AD patients producing IL-9 by CLA⁺ (n = 35) and CLA⁻ (n = 17) T cells, respectively. (B) In CLA⁺/Epi and (C) CLA⁻/Epi AD cocultures IL-4, IL-5, IL-9, IL-13, IL-17A, IL-31, IFN-γ (n = 52), IL-21 (n = 46), and IL-22 (n = 45) levels were simultaneously quantified. Cytokines were sorted according to the median production and compared to IL-9 production levels. Red lines indicate the median. Correlations of IL-9 levels (pg/mL) in HDM-activated CLA⁺ and CLA⁻ T cell cocultures and (D) years since diagnosis (n = 33 for CLA⁺, n = 16 for CLA⁻), (E) IL-13, IL-4, IL-5, IL-31, IL-17A, IL-22, IL-21, IFN-γ, CCL17, and CCL22 (pg/mL) within patients with IL-9 production. Bold values indicate significant data. AD, atopic dermatitis; C, control subjects; CLA, cutaneous lymphocyte-associated antigen; Epi, epidermal cells; M, untreated; SEB, staphylococcal enterotoxin B. ns: p > 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Figure 5

Workflow of the study design. AD, atopic dermatitis; CLA, cutaneous lymphocyte-associated antigen; Epi, epidermal cells; HDM, house dust mite; PBMCs, peripheral blood mononuclear cells; SEB, staphylococcal enterotoxin B; Tm, memory T cells. This figure was created using images under a Creative Commons license.