Flow cytometry imaging identifies rare TH2 cells expressing thymic stromal lymphopoietin receptor in a “proallergic” milieu Amanda J. Reefer, MS, Kathryn.

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Flow cytometry imaging identifies rare TH2 cells expressing thymic stromal lymphopoietin receptor in a “proallergic” milieu Amanda J. Reefer, MS, Kathryn E. Hulse, PhD, Josephine A. Lannigan, MS, Michael D. Solga, MS, Paul W. Wright, BS, Libby A. Kelly, MD, James Patrie, MS, Martin D. Chapman, PhD, Judith A. Woodfolk, MBChB, PhD Journal of Allergy and Clinical Immunology Volume 126, Issue 5, Pages 1049-1058.e10 (November 2010) DOI: 10.1016/j.jaci.2010.07.023 Copyright © 2010 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 1 Validation of anti-TSLP receptor mAbs by image-based flow cytometry. Activated THP-1 monocytes were stained for TSLPr, HLA-DR, and DNA. A, Dot plots represent gated focused single cells with DNA negative cells excluded. Boxes denote TSLPr+ gates determined by visual inspection of single cells inside the gate versus cells outside the gate. Green crosshairs denote single cells shown in B and C. B, Single-cell images of TSLPr+ cells using different anti-TSLPr clones. Images for each stain are overlaid on the brightfield image. C, Composite images corresponding to cells shown in B. FITC, Fluorescein isothiocyanate; PE, phycoerythrin. Journal of Allergy and Clinical Immunology 2010 126, 1049-1058.e10DOI: (10.1016/j.jaci.2010.07.023) Copyright © 2010 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 2 TH2-promoting factors induce activated TH2 cells. A, CD4+ T cells from DC/T-cell cocultures analyzed for expression of CD25 and IL-4 by image-based flow cytometry. Values denote percentages of CD25+IL-4+ T cells for each condition. Dotted line indicates the gate used to delineate IL-4hi from IL-4lo cells. NS, Not stimulated. B, Images of 3 representative T cells showing different staining intensity for IL-4 (green). Right, Composite images. DF, Darkfield. Data are representative of 5 subjects. Journal of Allergy and Clinical Immunology 2010 126, 1049-1058.e10DOI: (10.1016/j.jaci.2010.07.023) Copyright © 2010 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 3 TSLPr+ T cells are rare under TH2-promoting conditions. A, CD4+ T cells were stimulated and analyzed for TSLPr expression by image-based flow cytometry. Values denote the percentage of cells within a gate that captured all TSLPr+ events by visual inspection. Data are representative of 5 subjects. B, Image of a single representative TSLPr+ T cell with corresponding composite image. C, Table of actual frequencies of TSLPr+ T cells confirmed by inspection of cells (data from 5 subjects; ∗P = .015). NS, Not stimulated. Journal of Allergy and Clinical Immunology 2010 126, 1049-1058.e10DOI: (10.1016/j.jaci.2010.07.023) Copyright © 2010 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 4 TSLPr+ T cells are enlarged and express high levels of CD25 and IL-4. A and B, Percentage of TSLPr+ T cells within CD25mid/hi and CD25neg/lo subsets (A) and IL-4hi and IL4neg/lo subsets (B) after stimulation with TSLP (closed symbols) or TSLP + HDM (open symbols). Bars represent the mean (n = 5 subjects). C and D, Area of nuclear staining for IL-4hi, IL-4lo, and IL-4neg subsets induced by TSLP (C) or TSLP+HDM (D) (mean ± SD). Numbers of TSLPr+ cells for each observation are shown. Data for TSLPr-negative subsets were compiled by using 200 cells randomly selected from each subset (n = 4 subjects). Journal of Allergy and Clinical Immunology 2010 126, 1049-1058.e10DOI: (10.1016/j.jaci.2010.07.023) Copyright © 2010 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 5 TSLPr+ T cells are actively dividing. A, Scatter plot showing T cells stimulated with TSLP + allergen analyzed for DNA (7-AAD) and IL-4. Values denote the frequency of TSLPr+ T cells within each gate, and red circles denote TSLPr+ cells shown in corresponding composite images. DNA staining for cells in gate E is shown in bottom row of cell panel. Composite images of representative resting effector cells in gate C are shown for comparison (CD127 [blue] and DNA [red]). B, Nuclear aspect ratios for gates in A. Representative data are shown (AD 1). Journal of Allergy and Clinical Immunology 2010 126, 1049-1058.e10DOI: (10.1016/j.jaci.2010.07.023) Copyright © 2010 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 6 Bacterial superantigen induces rare TSLPr+ TH2 cells. A, Analysis of CD25 and TSLPr on CD4+ T cells stimulated with SEB (day 3). Percentage of T cells within the CD25+ gate (dotted line) is denoted. NS, Not stimulated. B, Frequency of TSLPr+ T cells within IL-4hi, IL-4lo and IL-4neg subsets in SEB-stimulated cultures from a responder patient. Left scatter plot shows gating strategy for IL-4hi(red) and IL-4neg/lo cells (green). C, Images of representative TSLPr+ cells. Journal of Allergy and Clinical Immunology 2010 126, 1049-1058.e10DOI: (10.1016/j.jaci.2010.07.023) Copyright © 2010 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 7 Direct effects of TSLP on purified T cells induced in a “proallergic” milieu. A, Purified T cells that had been preactivated with TSLP + allergen were not stimulated or restimulated with TSLP or IL-2 for 0 to 30 minutes and analyzed for phosphorylated STAT5. Values represent the percentage of cells positive for phospho-STAT5 after restimulation (values for nonstimulated cells are shown in parentheses for each corresponding time point). Data are representative of 3 experiments. B, Culture supernatants from purified T cells that had been preactivated sequentially with TSLP + allergen followed by anti-CD3 + anti-CD28 were assayed for cytokines after restimulation with TSLP or IL-2 for up to 6 hours. NS, Not stimulated; FMO, fluorescence-minus-one control. Journal of Allergy and Clinical Immunology 2010 126, 1049-1058.e10DOI: (10.1016/j.jaci.2010.07.023) Copyright © 2010 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 8 Analysis of TSLPr on skin T cells. A, Cells isolated from normal skin obtained from a nonatopic donor were cultured for 7 days with TSLP + allergen and analyzed by standard flow cytometry. B, Cells isolated directly ex vivo from lesional or nonlesional skin from an AD patient (AD1) were analyzed for TSLPr. Both panels show density plots for cells in the CD3+ T lymphocyte gate. Journal of Allergy and Clinical Immunology 2010 126, 1049-1058.e10DOI: (10.1016/j.jaci.2010.07.023) Copyright © 2010 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Induction of TH2 cytokines by TSLP and HDM Induction of TH2 cytokines by TSLP and HDM. A, CD4+ T cells were cocultured with monocyte-derived DCs that had been primed for 48 hours with allergen alone, TSLP alone, or both stimuli. Intracellular cytokines were measured on day 10. Values represent the means ± SDs for 10 subjects with dust mite allergy (5 AD and 5 non-AD). B, Cytokines in supernatants harvested from DC/T-cell cocultures that were analyzed by image-based flow cytometry. NS, Not stimulated. Journal of Allergy and Clinical Immunology 2010 126, 1049-1058.e10DOI: (10.1016/j.jaci.2010.07.023) Copyright © 2010 American Academy of Allergy, Asthma & Immunology Terms and Conditions

False-positive TSLPr+ T cells False-positive TSLPr+ T cells. Images of 4 representative cells within the TSLPr+ gate that were confirmed as false-positives on visual inspection. Journal of Allergy and Clinical Immunology 2010 126, 1049-1058.e10DOI: (10.1016/j.jaci.2010.07.023) Copyright © 2010 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Kinetics of TSLP receptor expression Kinetics of TSLP receptor expression. CD4+ T cells were cocultured with autologous DCs in the presence of TSLP + allergen and analyzed for expression of TSLPr and CD25 at different time points by standard flow cytometry. Journal of Allergy and Clinical Immunology 2010 126, 1049-1058.e10DOI: (10.1016/j.jaci.2010.07.023) Copyright © 2010 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Gating strategies for defining T-cell subsets that express different levels of CD25 and IL-4. Differences in staining intensity for CD25 and IL-4 were confirmed by visual inspection. The same gating strategy was applied to data from 5 subjects. NS, Not stimulated. Journal of Allergy and Clinical Immunology 2010 126, 1049-1058.e10DOI: (10.1016/j.jaci.2010.07.023) Copyright © 2010 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Cytokines induced by SEB Cytokines induced by SEB. A, Secreted cytokines detected at 3 days after stimulation with SEB in patients with AD. B, Frequency of IL-4+ T cells expressing other cytokines after stimulation with SEB. NS, Not stimulated. Journal of Allergy and Clinical Immunology 2010 126, 1049-1058.e10DOI: (10.1016/j.jaci.2010.07.023) Copyright © 2010 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Images of CD127+ and CD127neg T cells that express TSLPr Images of CD127+ and CD127neg T cells that express TSLPr. Data are shown for representative cells from a single subject after stimulation with TSLP + HDM and are representative of 4 subjects. DF, Darkfield. Journal of Allergy and Clinical Immunology 2010 126, 1049-1058.e10DOI: (10.1016/j.jaci.2010.07.023) Copyright © 2010 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Gating strategy for identifying CD127+ T cells Gating strategy for identifying CD127+ T cells. Upper panel shows gated CD127+ T cells (blue) for different stimuli. Lower panel shows the gate for CD25+CD127+ T cells (blue). Values denote the percentage of cells within the CD127+(upper panel) or CD25+CD127+(lower panel) gates. Values in parentheses denote the percentage of CD25+ T cells that are CD127+. Dotted line denotes the CD25+ gate. The same gating strategy was applied to data from 4 different subjects. NS, Not stimulated. Journal of Allergy and Clinical Immunology 2010 126, 1049-1058.e10DOI: (10.1016/j.jaci.2010.07.023) Copyright © 2010 American Academy of Allergy, Asthma & Immunology Terms and Conditions