CD4+ T Cells in Lymph Nodes of UVB-Irradiated Mice Suppress Immune Responses to New Antigens Both In Vitro and In Vivo  Shelley Gorman, Jamie W.-Y. Tan, Stephanie T. Yerkovich, John J. Finlay-Jones, Prue H. Hart  Journal of Investigative Dermatology  Volume 127, Issue 4, Pages 915-924 (April 2007) DOI: 10.1038/sj.jid.5700600 Copyright © 2007 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Reduced proliferation of CD4+ T cells from the SDLNs of UVB-irradiated mice. (a) CD4+ T cells were purified from the SDLNs of OVA-TCR transgenic mice irradiated with 8kJ/m2 UVB (shaded) and control (unshaded) mice, labeled with CFSE and cultured with APC (at a ratio of 100:1) and OVA323–339 peptide for up to 120hours. Proliferation is shown by dilution of the CFSE label. (b) IL-2 levels in supernatants for three replicates per treatment (*P<0.05). In (c), results from the 96hours time point from (a) are re-graphed with the number of cells on the y axis and the UVB-treated group now shown by a broken line. (d) Reduced IL-2, but not IFN-γ and IL-10 in supernatants of CD4+ T cells from the SDLNs of UVB-irradiated mice. Supernatants were harvested after 96hours culture (n=8 experiments, mean±SEM, with three replicates for each treatment per experiment). Journal of Investigative Dermatology 2007 127, 915-924DOI: (10.1038/sj.jid.5700600) Copyright © 2007 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Reduced proliferation of CD4+ T cells from the SDLNs of irradiated mice is dependent upon the timing and dose of UVB. CD4+ T cells were purified from the SDLNs of OVA-TCR transgenic mice (a) 2, 4, 7, or 14 days after irradiation with 8kJ/m2 UVB, (b) 4 days after irradiation with 2, 4, or 8kJ/m2 UVB, or (c) 4 days after the last of 4 weekly doses of 4kJ/m2. For (a–c), CD4+ T cells were also purified from the SDLNs of control mice. CD4+ T cells were labeled with CFSE and cultured with APC (at a ratio of 100:1) and OVA323–339 peptide for 96hours. Data are shown as mean±SEM, n=3. Journal of Investigative Dermatology 2007 127, 915-924DOI: (10.1038/sj.jid.5700600) Copyright © 2007 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Adoptively transferred CD4+ T cells purified from the SDLNs of UVB-irradiated mice suppress CHS responses in vivo. CD4+ cells were isolated from donor mice 4 days following irradiation with 8kJ/m2 UVB (shaded) or sham treatment (unshaded). Purified CD4+ T cells (1.5 × 107) from the SDLNs of OVA-TCR transgenic mice were adoptively transferred into naive BALB/c mice. Non-irradiated (“Control”) and UVB-irradiated (“UVB”) mice provided controls for these adoptive transfers. For the sensitization of all mice, the shaved abdomens were painted with DNFB 24hours after adoptive transfer of cells. After 5 days, the pinnae of each ear were painted with DNFB. Ear-swelling responses were measured 24hours after ear challenge. Significantly different (P<0.05) ear-swelling responses are denoted by an asterisk, where results from sham-irradiated mice were compared with those from irradiated mice, and results from mice that received control CD4+ cells (“control CD4+”) compared with those that received CD4+ cells from UVB-irradiated mice (“UVB CD4+”). Data are shown as mean±SEM, n=10. Journal of Investigative Dermatology 2007 127, 915-924DOI: (10.1038/sj.jid.5700600) Copyright © 2007 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Reduced in vitro proliferation of CD4+ T cells purified from the SDLNs 4 days after irradiation with 8kJ/m2 UVB is due to CD4+CD25+ regulatory T cells. (a) Purified CD4+ T cells from control (unshaded) and UVB-irradiated (shaded) OVA-TCR transgenic mice were depleted of CD25+ cells. CD4+ and CD4+CD25- cells were then cultured for 96hours with APC (at a ratio of 100:1) and OVA323–339 peptide. Proliferation by dilution of the CFSE label is shown in (a) and IL-2 levels in supernatants in (b). For (b), results were pooled for two experiments for three replicates per treatment (*P<0.002, **P<0.003 when compared with non-CD25+-depleted controls; NS=no significant difference). (c) Addition of IL-2 removed the effect of UVB on CD4+ T-cell proliferation in vitro. CD4+ T cells from the SDLN of UVB-irradiated (shaded) and control (unshaded) mice were cultured with IL-2. (d) CD4+ T cells from the SDLNs of UVB-irradiated and control mice were cultured with rIL-12. In order to calculate the percent reduction in proliferation, the number of divided CD4+ T cells in control treatments was compared with those in UVB treatments, where IL-12 was (UVB+IL-12) or was not (UVB) also added to control cultures. For (b) and (d), data are shown as mean±SEM, n=3. Journal of Investigative Dermatology 2007 127, 915-924DOI: (10.1038/sj.jid.5700600) Copyright © 2007 The Society for Investigative Dermatology, Inc Terms and Conditions