Ret mouse very large tumors (VLTs) display altered ratios of infiltrating memory to naive T cells: Roles in tumor expansion Mohammad W. Khan, Viktor.

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Ret mouse very large tumors (VLTs) display altered ratios of infiltrating memory to naive T cells: Roles in tumor expansion Mohammad W. Khan, Viktor Umansky Pathophysiology Volume 23, Issue 3, Pages 211-220 (September 2016) DOI: 10.1016/j.pathophys.2016.06.001 Copyright © 2016 Elsevier B.V. Terms and Conditions

Fig. 1 Immunohistochemistry-based staining of CD4+ or CD8+ T cells in spleen from C57B6L/J mice (A) CD4+ T cells or (B) CD8+ T cells were not detected in formalin fixed spleen even with heat-induced antigen-retrieval. Negative control-C) no CD4-primary antibody used or (D) no CD8-primary antibody were used in the immunohistochemistry-based staining on zinc-fixed spleen without antigen-retrieval, indicating specificity of the method. Immunohistochemistry-based detection of (E) CD4+ T cells or (F) CD8+ T cells (note red colored cell-membrane bound staining) in spleens fixed with zinc fixative, without any antigen-retrieval method, indicated by black arrowheads. Each experiment was repeated 3 times. Images acquired at 400X magnification. Scale bar=50μm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.). Pathophysiology 2016 23, 211-220DOI: (10.1016/j.pathophys.2016.06.001) Copyright © 2016 Elsevier B.V. Terms and Conditions

Fig. 2 Immunohistochemistry reveals the presence of melanoma-associated antigens Tyrosinase, TRP-1, TRP-2 and gp-100 in VLTs from ret transgenic mice fixed with zinc-based fixative. A) Negative control (no primary antibody used) staining on zinc-fixed melanoma tissue without antigen-retrieval, indicating specificity of the method. Intra-cytoplasmic staining of B) Tyrosinase+ C) TRP-1+ D) TRP-2+ and E) gp-100+ expressing cells in melanoma without antigen-retrieval. Images acquired at 400X magnification. Scale bar=50μm. Pathophysiology 2016 23, 211-220DOI: (10.1016/j.pathophys.2016.06.001) Copyright © 2016 Elsevier B.V. Terms and Conditions

Fig. 3 Double-immunohistochemistry based detection of TILs in VLTs from ret transgenic mice fixed using zinc-based fixative. A). CD4+ T cell (red cells, indicated by black arrows) detected in melanoma. B) CD8+ T cell (red colored cells, indicated by black arrows) detected in melanoma. C) Cell membrane staining of CD4 (using Vector Red) and nuclear staining of Foxp3 (using Vector Black) in melanoma from ret transgenic mice, note colocalization with black arrows arrowheads. D) Negative control (no primary antibody) staining in melanoma using Vector Red and Vector Black substrate, indicating specificity of the method. Images acquired at 400X magnification. Scale bar=50μm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) Pathophysiology 2016 23, 211-220DOI: (10.1016/j.pathophys.2016.06.001) Copyright © 2016 Elsevier B.V. Terms and Conditions

Fig. 4 Analyses of tumor infiltrating CD3+CD4+ T cells population in VLTs from ret transgenic mice using flow cytometry. A) Representation of the gating strategy for the CD3 isotype control (Rat IgG2b). B) Representation of the gating strategy for the characterization of the CD3+, CD3+CD4+, CD3+CD4+CD45RB+CD62L+ (naive), CD3+CD4+CD45RB−CD62L+ (central memory) and CD3+CD4+CD45RB−CD62L− (effector memory). Also, quantification of naive, central and effector memory cell infiltration in melanoma from ret transgenic mice expressed as% mean±S.D per total C) CD3+CD4+ T cells and D) CD3+ T cells is shown in bar graphs. Black arrows indicate flow of gating strategy. * P<0.01 and ** P<0.001 represents significance and the results of One-way ANOVA. Pathophysiology 2016 23, 211-220DOI: (10.1016/j.pathophys.2016.06.001) Copyright © 2016 Elsevier B.V. Terms and Conditions

Fig. 5 Analyses of tumor infiltrating CD3+CD8+ T cells population in VLTs from ret transgenic mice using flow cytometry. A) Representation of the gating strategy for the characterization of the CD3+, CD3+CD8+, CD3+CD8+CD44highCD62L+ (naive), CD3+CD8+CD44lowCD62L+ (central memory) and CD3+CD8+CD44lowCD62L− (effector memory). Also, quantification of naive, central and effector memory cell infiltration in melanoma from ret transgenic mice expressed as% mean±S.D per total B) CD3+CD8+ T cells and C) CD3+ T cells is shown in bar graphs. Black arrows indicate flow of gating strategy. ***P < 0.0001 represents significance and the results of One-way ANOVA. Pathophysiology 2016 23, 211-220DOI: (10.1016/j.pathophys.2016.06.001) Copyright © 2016 Elsevier B.V. Terms and Conditions