In Situ Mapping of Innate Lymphoid Cells in Human Skin: Evidence for Remarkable Differences between Normal and Inflamed Skin  Marie-Charlotte Brüggen, Wolfgang M. Bauer, Bärbel Reininger, Eduard Clim, Catalin Captarencu, Georg E. Steiner, Patrick M. Brunner, Barbara Meier, Lars E. French, Georg Stingl  Journal of Investigative Dermatology  Volume 136, Issue 12, Pages 2396-2405 (December 2016) DOI: 10.1016/j.jid.2016.07.017 Copyright © 2016 The Authors Terms and Conditions

Figure 1 In situ identification of ILCs. (a) Illustration of the gating strategy and validation algorithm. The ILC gating strategy was based on mean fluorescence measurements. Lin– cells were gated for CD3–AHR+ cells. In the implemented validation step, each cell in the target gate (e.g., Lin–CD3–AHR+) was automatically cropped out and back-gated in the section, allowing a manual validation. Validated cells (n = 3) were displayed in a “confirmation gate.” (b) Alternative gating strategy (of the same sample, consecutive section) for Lin–CD127+AHR+ cells yielding the same number of confirmed ILCs. (c–h) Representative image of IF multicolor stainings. The following marker combinations are displayed (all with DAPI): (c) CD45/AHR/Lin, scale bar = 5 μm, (d) CD3/GATA3/Lin, scale bars = 100 μm and 20 μm, (e) CD45/GATA3/Lin, (f) CD127/GATA3/Lin, (g) CD161/GATA3/Lin, and (h) CRTH2/GATA3/Lin, scale bar = 10 μm. CRTH2, chemoattractant receptor-homologous molecule expressed on T helper type 2; IF, immunofluorescence; ILC, innate lymphoid cell; Lin, lineage. Journal of Investigative Dermatology 2016 136, 2396-2405DOI: (10.1016/j.jid.2016.07.017) Copyright © 2016 The Authors Terms and Conditions

Figure 2 Distinct ILC infiltrates in NHS, AD, and Pso. (a) Results from the automated and validated quantitative in situ analysis of ILCs in the upper dermis of abdominal NHS (n = 10). Data are displayed as mean absolute numbers of cells ± SD per mm basement membrane (BM). (b, c) Distance measurements in NHS (n = 10) of ILC subsets to (b) the epidermis and to (c) BVs. Data are shown as mean minimal distance (μm) ± SD. (d–g) Representative images of IF multicolor stainings. The framed areas are displayed in a higher magnification in the lower panel of the respective images. (d) ILC3 (Lin–CD3–RORC+) in Pso, scale bar = 100 μm, (e) CD117 expression in RORC+ILC3s, scale bar = 10 μm, (f) ILC1s (Lin–CD3–TBET+) in Pso, (g) AHR+ILC3s (Lin–CD3–AHR+) in AD, scale bars = 100 μm and 20 μm. AD, atopic dermatitis; AHR, aryl hydrocarbon receptor; BV, blood vessel; IF, immunofluorescence; ILC, innate lymphoid cell; Lin, lineage; NHS, normal human skin; Pso, psoriasis. Journal of Investigative Dermatology 2016 136, 2396-2405DOI: (10.1016/j.jid.2016.07.017) Copyright © 2016 The Authors Terms and Conditions

Figure 3 No perivascular ILC clustering in Pso and AD lesions. (a) Results from the in situ analysis of ILCs in AD and Pso skin lesions (both n = 13) as well as in abdominal NHS (n = 10). (b) Illustration of the vessel detection algorithm used: detected vessels (white areas, upper row) and “screening” (color gradient, lower row) of their surroundings for ILCs, scale bars = 100 μm. (c) Distances between ILCs and the nearest BV in AD, Pso (both n = 13) and NHS (n = 10). (d, f) Scheme showing a distance scale as well as the terms used in the text to describe/interpret distance measurements. (e) Distances between ILCs and the epidermis in AD, Pso (both n = 13) and NHS (n = 10). *P < 0.05, **P < 0.01, ***P < 0.001. AD, atopic dermatitis; BV, blood vessel; ILC, innate lymphoid cell; NHS, normal human skin; Pso, psoriasis. Journal of Investigative Dermatology 2016 136, 2396-2405DOI: (10.1016/j.jid.2016.07.017) Copyright © 2016 The Authors Terms and Conditions

Figure 4 Close proximity between ILCs and T cells. (a) Distance measurements between the different ILC subsets and T cells in AD, Pso (both n = 13) and NHS (n = 10). (b) ILC2 (Lin–CD3–GATA3+) surrounded by two T cells (CD3+), scale bar = 20 μm. (c) Scheme of the applied software algorithm layer assessing the cellular environment of ILCs. The entire cellular content of the upper dermis was screened and assessed as to its distance to validated ILCs. Scale bars = 50 μm and 10 μm. (d) The cellular environment was assessed in a radius of 3 μm (direct cell-cell contact) and 9 μm (closest cellular environment), scale bar = 5 μm. (e, f) Percentage of T cells among total cells in an environmental radius of (e) 3 μm or (f) 9 μm around validated ILCs for AD, Pso (both n = 13) and NHS (n = 10). *P < 0.05, **P < 0.01, ***P < 0.001. AD, atopic dermatitis; GATA3, trans-acting T-cell-specific transcription factor GATA-3; ILC, innate lymphoid cell; Lin, lineage; NHS, normal human skin; Pso, psoriasis. Journal of Investigative Dermatology 2016 136, 2396-2405DOI: (10.1016/j.jid.2016.07.017) Copyright © 2016 The Authors Terms and Conditions

Figure 5 Characteristic Th-cell infiltrates in AD versus Pso skin lesions. (a) Automated quantitative in situ analysis of the Th-cell infiltrate in AD and Pso skin lesions as well as in NHS. (b) Representative image of a multicolor IF staining of Th2 cells in AD (CD3+CD4+GATA3+), scale bars = 100 μm and 50 μm. (c) Pie charts displaying the relative composition of ILC and Th-cell populations in AD, Pso and NHS, respectively. (d) Representative image of a multicolor IF staining Th17 cells in Pso (CD3+CD4+RORC+), scale bars = 100 μm and 50 μm. (e, f) Percentage of the respective T-cell subsets among total cells in an environmental radius of 3 μm or 9 μm around validated ILCs for AD, Pso (both n = 13) and NHS (n = 10). **P < 0.01, ***P < 0.001. AD, atopic dermatitis; GATA3, trans-acting T-cell-specific transcription factor GATA-3; IF, immunofluorescence; ILC, innate lymphoid cell; NHS, normal human skin; Pso, psoriasis; Th, T helper. Journal of Investigative Dermatology 2016 136, 2396-2405DOI: (10.1016/j.jid.2016.07.017) Copyright © 2016 The Authors Terms and Conditions