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Supplementary Appendix 4. Activation of Immune Mechanisms in Association with Intratumoral T Cells.
In Panel A, double immunofluorescence with a monoclonal antibody against CD83 (Texas red; red, arrows) and with a polyclonal antibody against CD3 (fluorescein; green) demonstrates clustering of T cells around a CD83+ antigen-presenting cell. The inset shows immunohistochemical detection of a CD83+ cell with morphologic features of a dendritic cell, surrounded by small mononuclear cells with scant cytoplasm, identifying them as lymphocytes. Panel B shows quantification by image analysis of CD83+ cells detected by immunohistochemical analysis in tumors with T cells and those without T cells. There is a significantly higher number of CD83+ cells in tumors harboring T cells than in tumors without T cells. In Panel C, double immunofluorescence with a monoclonal antibody against CD45RO (Texas red; red) and a polyclonal antibody against CD3 (fluorescein; green) shows memory CD45RO+CD3+ T cells in a tumor containing T cells. Double-positive cells appear yellow due to colocalization of both CD45RO and CD3 on the cell surface. Panel D shows quantification by image analysis of CD45RO+ (memory) T cells detected by immunohistochemical analysis in the stroma of tumors with or without T cells. There are significantly more memory T cells in the stroma of tumors containing T cells than in the stroma of tumors without T cells. In Panel E, double immunofluorescence with a monoclonal antibody against CD3 (fluorescein; green) and a polyclonal antibody against Ki-67 (Texas red; red) reveals activated Ki-67+CD3+ T cells in a tumor containing T cells. Panel F shows the results of real-time quantitative PCR analysis of the levels of interleukin-2 and interferon- messenger RNA (mRNA) in 16 ovarian carcinomas harboring intratumoral T cells and 10 ovarian carcinomas without intratumoral T cells. Higher levels of interleukin-2 mRNA (P=0.09) and interferon- mRNA (P=0.02) were found in tumors containing T cells than in tumors without T cells. The presence of both cytokines indicates antigen-dependent T-cell activation and type 1 polarization. The y axis represents the relative expression of interferon- and interleukin-2 in relation to glyceraldehyde-3-phosphate dehydrogenase.
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