1. Elimination of CD4+ T Cells Enhances Anti-Tumor Effect of Locally Secreted Interleukin- 12 on B16 Mouse Melanoma and Induces Vitiligo-Like Coat Color Alteration 
Hiroshi Nagai, Tatsuya Horikawa, Masahiro Oka, Masamitsu Ichihashi 
Journal of Investigative Dermatology 
Volume 115, Issue 6, Pages (December 2000)
DOI: /j x
Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

2. Figure 1
IL-12-transfected B16 melanoma showed retarded tumor growth in syngeneic mice. C57/BL6 mice were challenged with B16 parental melanoma (B16/P), control vector-transfected cell line (B16/C) or clones of B16 melanoma transduced with IL-12 (B16/IL-12 no. 1, B16/IL-12 no. 2). Mice were subcutaneously injected with 106 cells in the right flank on day 0. Tumor size was measured as the product of the longest diameter × perpendicular diameter. Data are presented as mean values of tumor size of five mice per group. Error bars represent SD. ▴, B16/P; ♦, B16/C; •, B16/IL-12 no. 1; ○, B16/IL-12 no. 2. *p <;0.01, **p <;0.01 (Student's t test).
Journal of Investigative Dermatology  , DOI: ( /j x)
Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

3. Figure 2
CD4+ T cell depletion led to regression of B16 melanoma transduced with IL-12. B16 melanoma transduced with IL-12 (B16/IL-12) were challenged in C57/BL6 mice depleted of natural killer cells, CD8+ T cells, and CD4+ T cells. Depletion of natural killer cells, CD8+ T cells, and CD4+ T cells was performed as described in Materials and Methods. Mice were subcutaneously injected with 106 cells in the right flank on day 0. Tumor measurements were performed as described in Figure 1 and in Materials and Methods. Data are presented as mean values of tumor size of six to eight mice per group. Error bars represent SD. ▴, B16/P in C57/BL6 mice; •, B16/IL-12 in C57/BL6 mice; □, B16/IL-12 in natural killer cell-depleted C57/BL6 mice; ▵, B16/IL-12 in CD8+ T cell-depleted C57/BL6 mice; ▿, B16/IL-12 in CD4+ T cell-depleted C57/BL6 mice. *p <;0.05, **p <;0.05, ***p <;0.01 (Student's t test).
Journal of Investigative Dermatology  , DOI: ( /j x)
Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

4. Figure 3
Alteration of coat color in CD4+ T cell-depleted C57BL/6 mice at 60 d after B16/IL-12 injection. Coat color alteration began at 30–50 d after tumor injection and thereafter increased gradually. Two representative mice showing coat color alteration are presented. B16/IL-12 tumors were subcutaneously injected into the right flank of mice. Note that depigmentation is observed not only in the tumor inoculation site, but also in almost the entire body.
Journal of Investigative Dermatology  , DOI: ( /j x)
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5. Figure 4
Strong accumulation of CD8+ T cells in B16/IL-12 tumors was induced by CD4+ T cell depletion. Immunohistochemical staining with anti-CD8 antibodies of B16/IL-12 tumors 7 d after tumor injection. (a) Staining of B16/IL-12 in mice undepleted of CD4+ T cells, showing a few CD8+ T cells at the periphery of the tumor. (b) Staining of B16/IL-12 in mice depleted of CD4+ T cells, showing strong accumulation of CD8+ T cells (arrows) in close vicinity to B16/IL-12. Scale bar: 100 μm.
Journal of Investigative Dermatology  , DOI: ( /j x)
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6. Figure 5
CD8+ T lymphocytes accumulated around the lower portion of follicles in mice with coat color alteration. (a) Hematoxylin–eosin staining of follicles in mice with coat color alteration, showing that lymphocytes (arrows) accumulate around the lower portion of follicles, but only scarcely around the upper portion. (b) Hematoxylin–eosin staining of follicles in normal mice, showing no lymphocytes around the follicles. (c) Immunohistochemical staining with anti-CD8 antibodies of follicles in mice with coat color alteration, showing that most of the infiltrating cells around the lower follicles were CD8+ T cells (arrows). Scale bar: 50 μm.
Journal of Investigative Dermatology  , DOI: ( /j x)
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7. Figure 6
Th2 cytokine gene expressions, but not Th1, were abolished in the TDLN of B16/IL-12-bearing mice by CD4+ T cell depletion. We used the reverse transcription–Multiplex PCR system, by which multiple amplified targets can be detected under the same conditions. M = PCR markers; P = positive control; lanes 1 and 2 = TDLN of B16/IL-12-bearing mice with (lane 2) or without (lane 1) CD4+ T cell depletion on day 7 after tumor injection; lanes 3 and 4 = TDLN of B16/IL-12-bearing mice with (lane 4) or without (lane 3) CD4+ T cell depletion on day 21 after tumor injection.
Journal of Investigative Dermatology  , DOI: ( /j x)
Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions