1. TGFβ1-Induced Inflammation in Premalignant Epidermal Squamous Lesions Requires IL-17 
Javed Mohammed, Andrew Ryscavage, Rolando Perez-Lorenzo, Andrew J. Gunderson, Nicholas Blazanin, Adam B. Glick 
Journal of Investigative Dermatology 
Volume 130, Issue 9, Pages (September 2010)
DOI: /jid
Copyright © 2010 The Society for Investigative Dermatology, Inc Terms and Conditions

2. Figure 1
Induction of TGFβ1 in papillomas causes tumor regression. Squamous papillomas were induced in 7- to 9-week-old K5/rTA × tetOTGFβ1 (DT) and K5/rTA (ST) mice. Tumor-bearing mice were placed on a dox diet (1gkg−1) and analyzed after 48hours or followed up for 10 days. (a) RT–PCR analysis of tetOTGFβ1 transgene expression in papillomas with or without dox for 48hours. (b) Immunohistochemical detection of phosphorylated Smad2 using a p-Smad2-specific antibody. Bars=20μM. (c) Quantitation of nuclear Smad2 in papillomas expressing TGFβ1 (*P=0.02). (d) Tumor cell proliferation in TGFβ1-expressing papillomas after 48hours (*P<0.01). (e) Apoptosis in papillomas overexpressing TGFβ1. Each histogram represents the average percent of TUNEL-positive cells of four tumors (*P=0.028). (f) Changes in tumor volume over 10 days after TGFβ1 induction. At least five tumors were measured for each genotype (*P=0.001), using one-way repeated-measures analysis of variance. Error bars=±SEM.
Journal of Investigative Dermatology  , DOI: ( /jid )
Copyright © 2010 The Society for Investigative Dermatology, Inc Terms and Conditions

3. Figure 2
TGFβ1 induces IL-17 production in CD4+ and γδ+ tumor-infiltrating T cells. Tumor-infiltrating lymphocytes were isolated from papillomas after 2 days of dox treatment and analyzed for cell-surface markers and intracellular cytokines. (a) A representative flow cytometry profile of IL-17+ and IFNγ+ T cells from ST and DT papillomas. (b) Percentage of IL-17+ and IFNγ+ CD4+ T cells from ST (n=11) and DT (n=12) papillomas, and IL-17+ γδ+ T cells from ST (n=4) and DT (n=7) papillomas. (c) Expression of cytokine genes in ST and DT papillomas. For each gene, PCR reactions were performed at the same time for all tumors, and products were run on the same agarose gel. GAPDH was used as loading control. Each column represents the same tumor. Densitometry was performed on gel images for each gene using Kodak Molecular Imaging Software version 4.0, and intensity values for each gene were normalized to the corresponding GAPDH value. Error bars=±SEM.
Journal of Investigative Dermatology  , DOI: ( /jid )
Copyright © 2010 The Society for Investigative Dermatology, Inc Terms and Conditions

4. Figure 3
Autocrine TGFβ1 induces proinflammatory and Th17-polarizing cytokines in v-rasHa-expressing keratinocytes. ST and DT keratinocytes were infected with the v-rasHa retrovirus and treated with dox to induce TGFβ1 expression and RNA or conditioned media isolated after 3 days. (a) Quantitative RT–PCR analysis of cytokine gene expression in v-rasHa-transduced primary keratinocytes. Induction of the tetOTGFβ1 transgene is shown by RT–PCR. (b) IL-23p19 expression in v-rasHa ST and DT keratinocytes treated with dox and in the presence or absence of 1μM SB Data in panels a and b are derived from three independent experiments. Error bars=±SEM. (c) Expression of IL-17A and IL-17F mRNA by T cells activated for 5 days in conditioned media from v-rasHa DT keratinocytes. (d) ELISA for IL-17A/F in supernatants from T cells activated in conditioned media from v-rasHa DT keratinocytes for 5 days, with and without 1μM SB Results in panels c and d are representative of two independent experiments. IL-17 was not detected in the keratinocyte-conditioned media alone. All quantitative PCR and data were normalized to GAPDH mRNA. For panel b, data were log-transformed before statistical analysis.
Journal of Investigative Dermatology  , DOI: ( /jid )
Copyright © 2010 The Society for Investigative Dermatology, Inc Terms and Conditions

5. Figure 4
TGFβ1 overexpression increases inflammatory infiltrate in benign tumors. ST papillomas (a, c) and DT papillomas (b, d–f) were harvested from mice placed on dox for 1 week. Tumors were stained with hematoxylin and eosin-Y (a, b), antiMPO for myeloid cells (c, d), and anti-CD3ε (e) and anti-CD8 (f). Bars=20μM. The arrows indicate infiltrating leukocytes. The photomicrographs are representative of at least three different tumors per genotype. S, stroma; T, tumor. (g–i) Quantitation of tumor-infiltrating T cells from immunohistochemical analysis for CD3+ (g), CD4+ (h), and CD8+ (i) cells. No staining was detected in the absence of primary antibody. Each histogram represents the average from at least four tumors of each genotype, with more than five fields counted per section. Error bars=±SEM.
Journal of Investigative Dermatology  , DOI: ( /jid )
Copyright © 2010 The Society for Investigative Dermatology, Inc Terms and Conditions

6. Figure 5
TGFβ1 increases F4/80+ tumor-infiltrating macrophages and immune activation in skin-draining LNs. Tumor-bearing DT mice were kept on regular or dox chow for 5 days, and tumors were analyzed for CD11b+, CD11c+, and F4/80+ cells and inguinal LNs for CD8+ effectors and IFN+ cells. (a) Representative dot plots of tumor-infiltrating leukocytes stained for CD11b and F4/80 markers from three tumors per group. (b) Quantitation of CD11c−F4/80+ population after gating on CD11b+ tumor-infiltrating myeloid cells (n=3). (c) Fold change in CD45+CD11b+ and CD45+CD11c+ cells in the LNs. (d) Fold change in CD8+ effector T cells (CD44+CD62L−) and IFNγ+ CD8+ T cells in the LN. Data in panels c and d are derived from three different experiments. Error bars=±SEM.
Journal of Investigative Dermatology  , DOI: ( /jid )
Copyright © 2010 The Society for Investigative Dermatology, Inc Terms and Conditions

7. Figure 6
TGFβ1-induced changes in tumor-associated myeloid cell populations are IL-17-dependent and IL-17-independent. Tumor-bearing mice were injected intraperitoneally with either isotype control IgG or neutralizing anti-IL-17 antibody and placed on regular or dox chow for 5 days. (a) Representative dot plots of anti-CD11b- and anti-Ly6G-stained cells from each treatment group within live CD45+ cells. (b) Quantitation of CD11b+ Ly6G− and CD11b+ Ly6G+ populations in tumors from each treatment group (n=3 for DT+IC; n=6 for DT+dox+IC; and n=7 for DT+dox+anti-IL-17). Error bars=±SEM. IC, IgG isotype control.
Journal of Investigative Dermatology  , DOI: ( /jid )
Copyright © 2010 The Society for Investigative Dermatology, Inc Terms and Conditions