Foxp3+ Regulatory T Cells and Natural Killer Cells Distinctly Infiltrate Primary Tumors and Draining Lymph Nodes in Pulmonary Adenocarcinoma Thomas Schneider,

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Foxp3+ Regulatory T Cells and Natural Killer Cells Distinctly Infiltrate Primary Tumors and Draining Lymph Nodes in Pulmonary Adenocarcinoma Thomas Schneider, MD, Silvia Kimpfler, PhD, Arne Warth, MD, Philipp A. Schnabel, MD, PhD, Hendrik Dienemann, MD, PhD, Dirk Schadendorf, MD, PhD, Hans Hoffmann, MD, PhD, Viktor Umansky, MD, PhD Journal of Thoracic Oncology Volume 6, Issue 3, Pages 432-438 (March 2011) DOI: 10.1097/JTO.0b013e31820b80ca Copyright © 2011 International Association for the Study of Lung Cancer Terms and Conditions

FIGURE 1 Regulatory T cell (Treg)-infiltrate primary tumors (A) and metastatic lymph nodes (B) in patients with lung cancer. Consecutive paraffin sections of 1 μm thickness were stained with Foxp3 (brown) monoclonal antibodies (mAbs) and counterstained with hemalaun. Journal of Thoracic Oncology 2011 6, 432-438DOI: (10.1097/JTO.0b013e31820b80ca) Copyright © 2011 International Association for the Study of Lung Cancer Terms and Conditions

FIGURE 2 Analysis of CD4+ T cells in lung tissue of patients with cancer. Single-cell suspensions prepared from healthy lung tissues, tumor centers, and tumor borders were stained with monoclonal antibodies (mAbs) for CD4 and the leukocyte marker CD45 followed by flow cytometry. A, Representative dot plots and gating strategy. B–D, Percentage of CD4+ T cells within CD45+ leukocytes in the lung tissue of all patients (B; n = 30) and of patients with adenocarcinoma (C; n = 16) or squamous cell carcinoma (D; n = 14). *p = 0.02, significant differences between the groups indicated with the lines. Journal of Thoracic Oncology 2011 6, 432-438DOI: (10.1097/JTO.0b013e31820b80ca) Copyright © 2011 International Association for the Study of Lung Cancer Terms and Conditions

FIGURE 3 Distribution of CD4+CD25+Foxp3+ regulatory T cells (Tregs) in lung tissue. Cells from healthy lung tissues, tumor centers, and tumor borders were stained with monoclonal antibodies (mAbs) for CD45, CD4, CD25, and FoxP3, followed by flow cytometry. A, Representative dot plots and gating strategy. B–D, Frequency of CD4+CD25+FoxP3+ Tregs in the lung tissue of all patients (B; n = 30; *p < 0.05) and of patients with adenocarcinoma (C; n = 16; *p < 0.01) or squamous cell carcinoma (D; n = 14; *p < 0.002) expressed as percentage within total CD45+CD4+ T cells. Journal of Thoracic Oncology 2011 6, 432-438DOI: (10.1097/JTO.0b013e31820b80ca) Copyright © 2011 International Association for the Study of Lung Cancer Terms and Conditions

FIGURE 4 Phenotypic analysis of CD56+CD3− natural killer (NK) cells in lung tissue. Cells isolated from healthy lung tissues, tumor centers, and tumor borders were stained with monoclonal antibodies (mAbs) for CD45, CD3, and CD56, followed by FACS analysis. A, Representative dot plots and gating strategy. B–D, Amount of CD56+CD3− NK cells in the lung tissue of all patients (B; n = 30; *p < 0.01) and of patients with adenocarcinoma (C; n = 16; *p < 0.005) or squamous cell carcinoma (D; n = 14; *p < 0.02) expressed as percentages of all CD45+ leukocytes. Journal of Thoracic Oncology 2011 6, 432-438DOI: (10.1097/JTO.0b013e31820b80ca) Copyright © 2011 International Association for the Study of Lung Cancer Terms and Conditions

FIGURE 5 Evaluation of CD4+ T cells in lymph nodes from patients with lung cancer. Cell suspensions prepared from tumor-infiltrated lymph nodes, and lymph nodes lacking tumor infiltration were stained with monoclonal antibodies (mAbs) for CD3 and CD4, followed by flow cytometry. A–C, Percentage of CD3+CD4+ T cells within mononuclear cells (MNC) in lymph nodes of all patients (A; n = 30), of patients with adenocarcinoma (B; n = 16), or squamous cell carcinoma (C; n = 14). *p = 0.01, significant differences between indicated groups. Journal of Thoracic Oncology 2011 6, 432-438DOI: (10.1097/JTO.0b013e31820b80ca) Copyright © 2011 International Association for the Study of Lung Cancer Terms and Conditions

FIGURE 6 Frequency of CD4+CD25+Foxp3+ regulatory T cells (Tregs) in lymph nodes from patients with lung cancer. Single-cell suspensions prepared from tumor-infiltrated lymph nodes and lymph nodes lacking tumor infiltration were stained with monoclonal antibodies (mAbs) for CD3, CD4, CD25, and FoxP3, followed by FACS analysis. A–C, Amount of CD4+CD25+FoxP3+ Tregs in the lung tissue of all patients (A; n = 20) and of patients with adenocarcinoma (B; n = 12) or squamous cell carcinoma (C; n = 8) expressed as percentages of all CD45+CD4+ T cells. *p = 0.01, significant differences between indicated groups. Journal of Thoracic Oncology 2011 6, 432-438DOI: (10.1097/JTO.0b013e31820b80ca) Copyright © 2011 International Association for the Study of Lung Cancer Terms and Conditions