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. 2014 Jan 4;105(2):159–167. doi: 10.1111/cas.12332

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© 2013 The Authors. Cancer Science published by Wiley Publishing Asia Pty Ltd on behalf of Japanese Cancer Association.

This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made.

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Fig. 5 — The combination therapy suppresses the accumulation of Tregs in tumors. Thirteen days after the injection of adenoviruses, fresh frozen sections of subcutaneous tumors were processed for immunohistochemistry. Data are representative of three separate experiments with similar results. (a) Immunostaining of CD4⁺ T cells. Rt.: right leg, Lt.: left leg. (b) Immunostaining of CD8⁺ T cells. (c) Immunostaining of Foxp3⁺ cells. (d) Relative ratio of CD4⁺ or CD8⁺ T cells per Foxp3⁺ cells in tumors. The number of CD4⁺ (left panel) and CD8⁺ T cells (right panel) in tumors was compared with that of Foxp3⁺ cells. (e) Proliferative status of CD4⁺ and CD8⁺ T cells infiltrated into Pan02 tumors. Tumors were harvested at 2 weeks after the combination therapy, processed into single-cell suspension, and the frequencies of Ki-67⁺ cells per CD4⁺ or CD8⁺ T cells were analyzed by flow-cytometry with PE-anti mouse Ki-67 antibody (eBioscience, San Diego, CA, USA; n = 3). We gated the lymphocyte regions in fluorescence-activated cell sorting (FACS) plots, and developed spots in two dimensions. A hundred thousand live cells were analyzed.