Volume 49, Issue 1, Pages 59-70 (January 2006) Detection of Immune Responses Against Urinary Bladder Cancer in Sentinel Lymph Nodes  Per Marits, Mona Karlsson, Amir Sherif, Ulrike Garske, Magnus Thörn, Ola Winqvist  European Urology  Volume 49, Issue 1, Pages 59-70 (January 2006) DOI: 10.1016/j.eururo.2005.09.010 Copyright © 2005 Elsevier B.V. Terms and Conditions

Fig. 1 Location and histology of sentinel nodes. The anatomical locations of nonmetastatic sentinel nodes (blue circles) from patient 3 (A, left panel) are shown. In patient 9 (A, right panel), two metastatic lymph nodes, which were not detected as sentinel nodes, i.e. false negative (FN), are also shown (yellow circles) and detected metastatic sentinel nodes are indicated with light red circles. Bold arrows indicate the sentinel nodes subjected to immunologic analyses. Hematoxylin and eosin stained sections from patients 3 and 9 are shown (B). Top panel (B) are sections from tumours (T) at 20× magnification. The scale bar represents 50μm. Bottom panel (B) show stained sentinel nodes (SN) from patients 3 and 9, respectively, at 10× magnification. The scale bar represents 100μm. European Urology 2006 49, 59-70DOI: (10.1016/j.eururo.2005.09.010) Copyright © 2005 Elsevier B.V. Terms and Conditions

Fig. 2 Anatomical location of sentinel nodes in the 14 patients. Detected nonmetastatic sentinel nodes (blue circles), false negative sentinel nodes, i.e. not detected metastatic nodes (yellow circles) are shown. Detected metastatic sentinel nodes are indicated with light red circles. The black arrows in patients 6 and 9 indicate dissection of specified nodes in the triangle of Marceille. European Urology 2006 49, 59-70DOI: (10.1016/j.eururo.2005.09.010) Copyright © 2005 Elsevier B.V. Terms and Conditions

Fig. 3 Flow cytometric analysis of lymph node cells. Sentinel node derived single cells from patient 3 stained with antibodies against the surface molecules CD4, CD8 and CD69 are shown (A). Lymphocytes were gated on the basis of forward and side scatter characteristics. Cells were further gated on the presence of CD4+ (B left panel) or CD8+ (B right panel), respectively, and investigated with the activation markers CD69 and CD62L. European Urology 2006 49, 59-70DOI: (10.1016/j.eururo.2005.09.010) Copyright © 2005 Elsevier B.V. Terms and Conditions

Fig. 4 Detection of tumour cells with flow cytometry. Cell suspensions from the tumour (A), sentinel node (B), a false negative sentinel node (C) and peripheral blood leukocytes (D), from patient 9, were stained intracellularly with antibodies against cytokeratin-20, followed by a FITC labelled secondary antibody. The side scatter characteristics of the cells are plotted against the FL-1 (green) fluorescence. Right panels are the negative controls, stained with only the secondary antibody. European Urology 2006 49, 59-70DOI: (10.1016/j.eururo.2005.09.010) Copyright © 2005 Elsevier B.V. Terms and Conditions

Fig. 5 Proliferative response of lymph node cells to autologous tumour extract. Proliferation assays from patients 3 (A) and 11 (B) are shown. Sentinel node(s) (filled or shaded circles) and non-sentinel node lymphocytes (open circles) were incubated with increasing amounts autologous homogenized tumour extract. Left panels show the dose-response on day 5 (A) and 6 (B), respectively. Time courses are shown for the highest amount of antigen extract used (Right panels). The proliferation of TIL is shown for patient 11 (B) (filled triangles). Proliferation is expressed as mean cpm of triplicate samples +/− 1 SD. European Urology 2006 49, 59-70DOI: (10.1016/j.eururo.2005.09.010) Copyright © 2005 Elsevier B.V. Terms and Conditions

Fig. 6 Proliferative responses to the mitogen Con A. To evaluate general T cell responsiveness in sentinel nodes, cells were cultured with Con A and pulsed with [3H]-thymidine 18hours prior to harvesting on day 3. The Con A stimulated proliferation of lymphocytes derived from sentinel (SN) and non-sentinel nodes (non-SN) is shown (grey bars). Black bars represent negative controls cultured in medium alone. Patient 3, with a non-metastatic sentinel node (A) and patient 9 with a metastatic sentinel node (B) are shown. In addition, the ConA stimulation of TIL is shown for patient 9 (B). Proliferation is expressed as mean cpm of triplicate samples. Error bars indicate 1 SD. European Urology 2006 49, 59-70DOI: (10.1016/j.eururo.2005.09.010) Copyright © 2005 Elsevier B.V. Terms and Conditions

Fig. 7 Expansion of T cells from the sentinel node. Cells from the sentinel node of patient 8 were cultured in vitro for several weeks. Single cells were stained for the T cell markers CD4 and CD8, and the activation markers CD69 (A) and CD62L (B) at the start (black line) of the expansion and after 17 days (solid grey). The ratio of the number of CD4+/CD8+ cells are plotted at the start of the expansion and at days 11 and 17 (C). European Urology 2006 49, 59-70DOI: (10.1016/j.eururo.2005.09.010) Copyright © 2005 Elsevier B.V. Terms and Conditions