Regulatory T Cells Control VEGF-Dependent Skin Inflammation Ingrid Teige, Henning Hvid, Lars Svensson, Peter Helding Kvist, Kåre Kemp  Journal of Investigative Dermatology  Volume 129, Issue 6, Pages 1437-1445 (June 2009) DOI: 10.1038/jid.2008.375 Copyright © 2009 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Skin-infiltrating cells are highly activated. Large numbers of regulatory T cells present. Ears were collected from seven TPA-treated mice at day 17 and infiltrating cells were isolated by a combination of mechanic and enzymatic methods. Compared to cells isolated from the blood of corresponding mice, the levels of activation markers are very high in cells isolated from ears. (a) CD25 expression on CD4+ T cells and (b) MCH II expression on macrophages and dendritic cells are depicted as examples. This was also true for CD80 and CD86 on macrophages and dendritic cells (data not shown). (c) Activation markers, CD25, CD69, and CD44 were all increased on T cells isolated from ears. Interestingly, numbers of cells with a regulatory phenotype (CD4+CD25+CD127−/low) were also clearly augmented. Figure shows mean values±SD, n=7 mice per group, ***P-value <0.001. Journal of Investigative Dermatology 2009 129, 1437-1445DOI: (10.1038/jid.2008.375) Copyright © 2009 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Depletion of CD4+ T cells strongly enhances skin inflammation. Mice were treated with the CD4-depleting antibody GK1.5 day -3, day 0, and day 8. Skin inflammation was induced as before and disease development was studied for 17 days. (a) Treatment with anti-CD4 antibody strongly reduces the number of circulating CD4+ cells measured in peripheral blood day 16, n=8 per group. (b) Depletion of CD4+ T cells enhances skin inflammation as measured by cumulative ear thickness. Isotype control does not affect disease development. The figure shows the mean area under the curve (AUC) for the increased ear thickness (compared to day 0)±SD from day 0 to day 16, n=8 mice per group, ***P-value <0.001. Journal of Investigative Dermatology 2009 129, 1437-1445DOI: (10.1038/jid.2008.375) Copyright © 2009 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Depletion of CD4+ T cells increases cytokine production. Depletion of CD4+ T cells severely enhances local proinflammatory cytokine production within the skin measured at the day of termination. Isotype control treatment does not affect this. *P-value <0.05, **<0.01 when calculating anti-CD4 versus both vehicle and isotype control, n=7 per group, figure shows mean values±SD. Journal of Investigative Dermatology 2009 129, 1437-1445DOI: (10.1038/jid.2008.375) Copyright © 2009 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Sorted CD4+CD25+ T cells inhibit IL-2 production in a mixed lymphocyte reaction. Mitomycin-treated Balb/c splenocytes served as stimulator cells for sorted CD4+CD25− responder T cells from K14/VEGF mice (FVB background). To this mixed lymphocyte cultures, sorted regulatory CD4+CD25+ suppressor T cells were added in different ratios. These regulatory T cells efficiently suppressed IL-2 production in a concentration-dependent manner, max suppression was maintained even at a 1:0.25 ratio. The 1:0 value is the positive control without any CD4+CD25+ cells present and the 0:0 value represents cultures with stimulator splenocytes only. Journal of Investigative Dermatology 2009 129, 1437-1445DOI: (10.1038/jid.2008.375) Copyright © 2009 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 Transfer of CD4+CD25+ T cells protects the mice from severe skin inflammation. Transfer of as little as 1 × 106 CD4+CD25+ T cells (sorted from the spleen of naive VEGF transgenic mice) 1 day before the first TPA application significantly protected the mice from VEGF-driven skin inflammation. (a) Ear thickness development is shown in where arrows indicate TPA applications, n=7 per group. Endpoints at termination day 18 were measured as: (b) reduced mean AUC±SD for increased ear thickness and (c) reduced ear biopsy weight±SD; n=7 per group, **P-value <0.01. Journal of Investigative Dermatology 2009 129, 1437-1445DOI: (10.1038/jid.2008.375) Copyright © 2009 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 6 CD4+CD25+ T-cell transfer significantly reduces cytokine production. Transfer of 1 × 106 CD4+CD25+ T cells 1 day before the first TPA application diminishes proinflammatory cytokine production in the ear. (a) Local TNF-α and (b) local IL-12p40 levels. (c) Regulatory T-cell transfer also decrease systemic IL-12p40 levels; n=7 per group, *P-value <0.05, figures show mean values±SD. Journal of Investigative Dermatology 2009 129, 1437-1445DOI: (10.1038/jid.2008.375) Copyright © 2009 The Society for Investigative Dermatology, Inc Terms and Conditions