Absence of γ-Chain in Keratinocytes Alters Chemokine Secretion, Resulting in Reduced Immune Cell Recruitment  Karolin Nowak, Daniela Linzner, Adrian J. Thrasher, Paul F. Lambert, Wei-Li Di, Siobhan O. Burns  Journal of Investigative Dermatology  Volume 137, Issue 10, Pages 2120-2130 (October 2017) DOI: 10.1016/j.jid.2017.05.024 Copyright © 2017 The Authors Terms and Conditions

Figure 1 Keratinocytes express functional γc and its co-receptors. (a) RNA of ED-7R cell lines, NIKS, and primary keratinocytes (KCs) was isolated, and reverse transcription PCR was performed with primers specific for γc, its co-receptors, and GAPDH. (b) KC, NIKS, CD8+ T cells, and ED-7R cell lines were stained with anti-γc antibody and analyzed by flow cytometry. (c) NIKS and KCs were lysed and analyzed for their expression of IL-7Rα and IL-2Rβ by Western blot. (d) NIKS were stained with antibodies for γc–co-receptors and analyzed by flow cytometry. (e) NIKS were cultured in serum-free medium overnight, stimulated with 100 ng/ml of the indicated cytokines for 10 minutes, and analyzed by immunoblot for the expression of pAKT. γc, common gamma chain; APC, allophycocyanin; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; KC, keratinocyte; NIKS, normal immortalized keratinocyte cell line; pAKT, phosphorylated protein kinase B. Journal of Investigative Dermatology 2017 137, 2120-2130DOI: (10.1016/j.jid.2017.05.024) Copyright © 2017 The Authors Terms and Conditions

Figure 2 Generation of a γc-knockdown cell line. NIKS were transduced with short hairpin RNA against γc or with scrambled control (scr) short hairpin RNA. (a) RNA was isolated from scr and γc-knockdown (KD) NIKS cell lines, and reverse transcription PCR was performed using γc- and GAPDH-specific primers; bar charts showing the band intensities compared with untransduced, mean ± standard error of the mean. ∗P < 0.02, n = 4. (b) γc-expression in scr and KD cells was analyzed by flow cytometry. (c) NIKS cell lines were cultured in serum-free medium overnight, stimulated with 10 ng/ml IL-15 for 10 minutes, lysed, and analyzed for the expression of pAKT. Bar chart shows comparison of intensities of the Western blot bands, mean ± standard error of the mean. ∗P < 0.05, n = 5. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; APC, allophycocyanin; KD, γc-knockdown; NIKS, normal immortalized keratinocyte cell line; pAKT, phosphorylated protein kinase B; scr, scrambled control. Journal of Investigative Dermatology 2017 137, 2120-2130DOI: (10.1016/j.jid.2017.05.024) Copyright © 2017 The Authors Terms and Conditions

Figure 3 Secretion of chemokines after IL-15 stimulation. Scrambled control (scr) and γc-knock-down (KD) NIKS were serum-depleted for 6 hours and then stimulated for 24 hours with 100 ng/ml IL-15 at 37 °C. Supernatants were collected and analyzed for secretion of (a) CXCL1, (b) CXCL8, (c) CCL20, (d) CCL5, (e) IL-1α, and (f) CXCL10 using Luminex bead assays, mean ± standard error of the mean. (a–c) n = 6, ∗P < 0.05. (d–f) n = 3. KD, γc-knockdown; NIKS, normal immortalized keratinocyte cell line; scr, scrambled control. Journal of Investigative Dermatology 2017 137, 2120-2130DOI: (10.1016/j.jid.2017.05.024) Copyright © 2017 The Authors Terms and Conditions

Figure 4 Migration of neutrophils. Neutrophils were isolated from whole blood and used for migration experiments using Dunn chambers, with the cells imaged every minute for 1 hour. (a) Cytospin of neutrophils before plating them on coverslips for migration. Scale bar = 25 μm. (b, c) Quantification for migration of one representative donor, where each dot represents one tracked cell. (d, e) Quantification for multiple migration experiments, where each dot represents the mean value for all cells analyzed from one donor. (b–e) “None” contains no chemoattractant; “chemokine cocktail” represents a cocktail of 10 ng/ml CXCL1, 3 ng/ml CXCL8, and 1 ng/ml CCL20. fMLP at 100 nmol/L was used as a positive control. Mean ± standard error of the mean; ∗P < 0.05, ∗∗∗P < 0.001; n = 4. Shown are (b, d) migratory speed and (c, d) directionality. fMLP, N-formylmethionyl-leucyl-phenylalanine; ns, not significant. Journal of Investigative Dermatology 2017 137, 2120-2130DOI: (10.1016/j.jid.2017.05.024) Copyright © 2017 The Authors Terms and Conditions

Figure 5 Migration of dendritic cells and CD4+ T cells. (a–c) CD14+-derived dendritic cells (DCs) were cultured on μ-Slides Chemotaxis 3D (Ibidi, Martinsreid, Germany; collagen IV coated) for migration assays. (a) Microscopic image of adherent DCs. Scale bar = 10 μm. (b) Quantification of an individual migration experiment, where each dot represents one cell tracked. (c) Quantification for multiple experiments, where each dot represents the mean value for all cells analyzed from one donor. (d, e) CD4+ T cells were isolated from whole blood and used for Transwell migration assays; values were normalized to the sample containing no chemoattractant. Assays were run in triplicate. (a–d) “None” contains no chemoattractant; “scr + IL-15” is a cocktail of 10 ng/ml CXCL1, 3 ng/ml CXCL8, and 1 ng/ml CCL20; and “KD + IL-15” the same chemokines at half the concentration. (e) Migration toward CX3CL1 at 40 ng/ml, n = 4, mean ± standard error of the mean. ∗P < 0.05. KD, γc-knockdown; min, minute; ns, not significant; scr, scrambled control. Journal of Investigative Dermatology 2017 137, 2120-2130DOI: (10.1016/j.jid.2017.05.024) Copyright © 2017 The Authors Terms and Conditions

Figure 6 Chemokine secretion from HPV18-positive cells. (a) Scrambled control (scr) and γc-knockdown (KD) NIKS were transfected with HPV18 plasmid. DNA was isolated and used for PCR using HPV18-specific primers. (b) CD4+ T cells were isolated from whole blood and used for Transwell migration assays; migration was measured toward supernatants harvested from scr control and KD NIKS with and without HPV18. (c) CD8+ T cells were isolated from whole blood and used for Transwell migration assays as with CD4+ T cells. (b, c) Values shown represent the number of migrating cells normalized to experiments using supernatant from HPV18-negative samples; each dot represents the mean of duplicate wells analyzed for a single supernatant. Four different sets of supernatants and two different blood donors were used; mean ± standard error of the mean are shown. *P < 0.05. HPV, human papilloma virus; NIKS, normal immortalized keratinocyte cell line; scr, scrambled control. Journal of Investigative Dermatology 2017 137, 2120-2130DOI: (10.1016/j.jid.2017.05.024) Copyright © 2017 The Authors Terms and Conditions