Transient CD44 Variant Isoform Expression and Reduction in CD4+/CD25+ Regulatory T Cells in C3H/HeJ Mice with Alopecia Areata Margot Zöller, Kevin J.

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Transient CD44 Variant Isoform Expression and Reduction in CD4+/CD25+ Regulatory T Cells in C3H/HeJ Mice with Alopecia Areata Margot Zöller, Kevin J. McElwee, Peter Engel, Rolf Hoffmann Journal of Investigative Dermatology Volume 118, Issue 6, Pages 983-992 (June 2002) DOI: 10.1046/j.1523-1747.2002.01745.x Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Enrichment of CD4+ and CD8+ cells in AA-affected skin. Skin containing anagen stage hair follicles from mice with chronic AA (A, G), normal-haired mice (B, H), mice 2 wk after receiving a transplant of AA-affected skin (C, D, I, J), and sham-grafted mice 2 wk after surgery (E, F, K, L) were labeled with anti-CD4 (A–F) and anti-CD8 (G–L) MoAb. The skin of normal-haired mice (B), sham-grafted mice within the graft (E) and host skin immediately adjacent to the graft site (F) contained isolated CD4+ cells. Similarly, the skin of normal-haired mice (H) and host skin immediately adjacent to the graft site of sham-grafted mice (L) were virtually devoid of CD8+ cells, whereas within the graft of sham-grafted mice (K) isolated CD8+ cells were apparent. In contrast, chronic AA-affected hair follicles (A)and hair follicles within the AA-affected skin transplant (C) exhibited an increased number of perifollicular CD4+ cells. CD8+ cells were observed in both perifollicular and intrafollicular locations in chronic AA-affected skin (G) and within the AA-affected skin graft (I). In addition, host skin immediately adjacent to the AA-affected skin graft site exhibited isolated CD4+(D) and CD8+ cells (J). Scale bar: 100 µm. Journal of Investigative Dermatology 2002 118, 983-992DOI: (10.1046/j.1523-1747.2002.01745.x) Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Leukocyte subset distribution in the skin of AA-affected mice. SkIL were prepared from mice (three per group) with chronic AA and normal-haired mice as well as of mice receiving a transplant of AA-affected skin or of normal-haired skin at 2 and 6 wk after transplantation. Isolated leukocytes were labeled with anti-CD4, anti-CD8, anti-surface IgM, anti-CD11b MoAb and an antibody specific for dendritic cells. Live cells were gated and the percentage of positive cells was evaluated. Mean ± SD of three experiments are shown. Significant differences (p ≤ 0.01) as compared with non-AA-affected mice are indicated by an asterisk. Journal of Investigative Dermatology 2002 118, 983-992DOI: (10.1046/j.1523-1747.2002.01745.x) Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Expression of costimulatory molecules and their ligands in SkIL of AA-affected mice. SkIL of the mice described in Figure 2 were labeled with anti-CD25, anti-CD28, anti-CD40, anti-CD80, and anti-CD86 MoAb. Live cells were gated and the percentage of positive cells was evaluated. Mean ± SD of three experiments are shown. Significant differences (p ≤ 0.01) as compared with non-AA-affected mice are indicated by an asterisk. Journal of Investigative Dermatology 2002 118, 983-992DOI: (10.1046/j.1523-1747.2002.01745.x) Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Decrease in CD4+/CD25+ lymphocytes in skin, lymph nodes, and spleen of AA-affected mice. SkIL, draining LNC, and spleen cells from normal-haired mice, mice with chronic AA, sham-grafted mice, and mice receiving a transplant of AA-affected skin; the latter two mice at 2 wk after transplantation, were double labeled with PE-conjugated anti-CD4 and fluorescein isothiocyanate-conjugated anti-CD25 MoAb. The population of lymphoblasts was gated. The negative control and the percentage of CD4-, CD25- and CD4/CD25 double positive cells in the gated population are shown. Reduced numbers of CD4+/CD25+ cells were recovered from SkIL, draining LNC, and spleen cells of mice with chronic AA and from mice transplanted with AA-affected skin. Journal of Investigative Dermatology 2002 118, 983-992DOI: (10.1046/j.1523-1747.2002.01745.x) Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 Cytokine expression in SkIL of AA-affected mice. SkIL of the mice described in Figure 2 were fixed and permeabilized and labeled with anti-IL-2, anti-IL-4, anti-IL-10, anti-IL-12, anti-IFN-γ, and anti-TNF MoAb. The percentage of positive cells was evaluated. Mean ± SD of three experiments are shown. Significant differences (p ≤ 0.01) as compared with non-AA-affected mice are indicated by an asterisk. Journal of Investigative Dermatology 2002 118, 983-992DOI: (10.1046/j.1523-1747.2002.01745.x) Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 6 Expression of CD44 isoforms in SkIL and draining LNC of AA-affected mice. (A) Skin containing anagen stage hair follicles from normal-haired mice, mice with chronic AA, mock transplanted mice, and mice receiving a transplant of AA-affected skin; the latter two mice at 2 wk after transplantation, were labeled with biotinylated anti-panCD44, anti-CD44v3, anti-CD44v6, anti-CD44v7, and anti-CD44v10 MoAb, and were counter-labeled with biotinylated goat anti-rat IgG or goat anti-mouse IgG and alkaline phosphatase conjugated avidin–biotin complex solutions in sequence. Only the skin of mice receiving an AA-affected transplant contains a high number of CD44v3+ and of CD44v10+ leukocytes (scale bar: 100 µm). The finding was confirmed by flow cytometry. (B) The population of lymphoblasts was gated in draining LNC and SkIL. The gated populations, and the histogram of negative control of the gated population as well as the histograms of the gated population stained with anti-CD44v3, anti-CD44v6, anti-CD44v7, and anti-CD44v10 in draining LNC and SkIL of normal-haired mice, sham-grafted, and AA-grafted mice at 2 wk after surgery are shown. The cut-off level in the negative controls (M1) is also shown in the anti-CD44 stained probes. (C, D) Staining of the total leukocyte population (excluding the keratinocytes in SkIL) with anti-panCD44, anti-CD44v3, anti-CD44v6, anti-CD44v7, and anti-CD44v10 MoAb is presented. Mean ± SD of three experiments are shown. Significant differences (p ≤ 0.01) as compared with non-AA-affected mice are indicated by an asterisk. Journal of Investigative Dermatology 2002 118, 983-992DOI: (10.1046/j.1523-1747.2002.01745.x) Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 6 Expression of CD44 isoforms in SkIL and draining LNC of AA-affected mice. (A) Skin containing anagen stage hair follicles from normal-haired mice, mice with chronic AA, mock transplanted mice, and mice receiving a transplant of AA-affected skin; the latter two mice at 2 wk after transplantation, were labeled with biotinylated anti-panCD44, anti-CD44v3, anti-CD44v6, anti-CD44v7, and anti-CD44v10 MoAb, and were counter-labeled with biotinylated goat anti-rat IgG or goat anti-mouse IgG and alkaline phosphatase conjugated avidin–biotin complex solutions in sequence. Only the skin of mice receiving an AA-affected transplant contains a high number of CD44v3+ and of CD44v10+ leukocytes (scale bar: 100 µm). The finding was confirmed by flow cytometry. (B) The population of lymphoblasts was gated in draining LNC and SkIL. The gated populations, and the histogram of negative control of the gated population as well as the histograms of the gated population stained with anti-CD44v3, anti-CD44v6, anti-CD44v7, and anti-CD44v10 in draining LNC and SkIL of normal-haired mice, sham-grafted, and AA-grafted mice at 2 wk after surgery are shown. The cut-off level in the negative controls (M1) is also shown in the anti-CD44 stained probes. (C, D) Staining of the total leukocyte population (excluding the keratinocytes in SkIL) with anti-panCD44, anti-CD44v3, anti-CD44v6, anti-CD44v7, and anti-CD44v10 MoAb is presented. Mean ± SD of three experiments are shown. Significant differences (p ≤ 0.01) as compared with non-AA-affected mice are indicated by an asterisk. Journal of Investigative Dermatology 2002 118, 983-992DOI: (10.1046/j.1523-1747.2002.01745.x) Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions