Volume 61, Issue 2, Pages 385-394 (February 2012) T Lymphocyte Recruitment into Renal Cell Carcinoma Tissue: A Role for Chemokine Receptors CXCR3, CXCR6, CCR5, and CCR6  Kimberley A. Oldham, Greg Parsonage, Rupesh I. Bhatt, D. Michael A. Wallace, Nayneeta Deshmukh, Shalini Chaudhri, David H. Adams, Steven P. Lee  European Urology  Volume 61, Issue 2, Pages 385-394 (February 2012) DOI: 10.1016/j.eururo.2011.10.035 Copyright © 2011 European Association of Urology Terms and Conditions

Fig. 1 Homing phenotype of T cells in renal cell carcinoma (RCC) tumour-infiltrating lymphocytes (TILs) and matched peripheral blood lymphocytes (PBLs). Paired TIL and PBL samples were stained with antibodies specific for (A) 19 chemokine receptors and (B) 7 adhesion molecules. The number of RCC cases stained was as follows: CCR5, CXCR3, and CXCR6: n=16; CCR3, CCR4, CCR6, CCR7, and CXCR4: n=10; CCR8 and CX3CR1: n=8; CCR1, CCR2, CCR9, CCR10, CXCR2, CXCR5, CXCR7, XCR1, and CD11b: n=7; CXCR1, CD62L, and PSGL-1: n=6; and LFA-1, CLA, VLA-4, and BLT-1: n=5. Analysis was conducted by flow cytometry, gating on viable CD3+ CD45RA− cells. Results show the median fluorescence intensity (plus standard error) for each marker. (C) Paired analysis for three chemokine receptors in which expression on viable CD3+ CD45RA− T cells in TILs was significantly greater than the corresponding cells in matched PBLs (measured using the Wilcoxon matched pairs test). European Urology 2012 61, 385-394DOI: (10.1016/j.eururo.2011.10.035) Copyright © 2011 European Association of Urology Terms and Conditions

Fig. 2 Coexpression of CCR5, CXCR3, and CXCR6 on T cells from tumour-infiltrating lymphocytes (TILs) and matched peripheral blood lymphocytes (PBLs). The relative proportions of cells expressing none, one, two, or all three of the chemokine receptors were determined using flow cytometry. Results show the mean of data from six cases (plus standard error). European Urology 2012 61, 385-394DOI: (10.1016/j.eururo.2011.10.035) Copyright © 2011 European Association of Urology Terms and Conditions

Fig. 3 Expression of selected chemokine receptors and their corresponding chemokine ligands in renal cell carcinoma (RCC) tissue. Each of the nine pictures shows immunohistochemical staining for the (A) chemokine receptor or (B) chemokine ligand indicated in the top left corner. Isotype control staining is shown in the top right corner. Every picture is representative of 10 RCC cases. European Urology 2012 61, 385-394DOI: (10.1016/j.eururo.2011.10.035) Copyright © 2011 European Association of Urology Terms and Conditions

Fig. 4 Functional test of CCR5, CXCR3, and CXCR6 on freshly isolated renal cell carcinoma (RCC) tumour-infiltrating lymphocytes (TILs). Infiltrating lymphocytes isolated from RCC tissue were placed in the top chamber of a Transwell filter (3-μm pore size), with the indicated chemokine(s) or medium alone placed in the bottom chamber. All chemokines were used at pretitred concentrations to achieve maximal migration of lymphocytes (ie, 10 ng/ml, except CXCL16, which was used at 20 ng/ml). Numbers of CD3+ T cells that migrated through the transwell were counted and a migration index measured by dividing the number of cells migrating in response to chemokine by the number that migrated in response to medium alone. Results shown represent the mean migration index (plus standard error) using TILs from multiple RCC cases as indicated. The p values were calculated using the Wilcoxon signed rank test and indicate a significant increase in migration activity in response to the chemokine indicated compared with media alone. European Urology 2012 61, 385-394DOI: (10.1016/j.eururo.2011.10.035) Copyright © 2011 European Association of Urology Terms and Conditions

Fig. 5 Defining the regulatory T cell (Treg) population that is enriched within renal cell carcinoma (RCC) tissue. (A) Foxp3+ cells were visualised by immunohistochemistry. The staining shown is representative of 10 RCC cases studied. (B) Relative proportions of Foxp3+ CD3+ CD4+ T cells within paired samples of tumour-infiltrating lymphocytes (TILs) and matched peripheral blood lymphocytes (PBLs) from 25 RCC cases. Horizontal lines indicate mean percentage values. (C) Levels of putative Tregs in TILs and PBLs were examined by flow cytometry for coexpression of CD127 and CD25. Data shown are representative of 25 RCC cases. (D) Intracellular staining for cytokines interferon γ (IFN-γ), interleukin 2 (IL-2), and tumour necrosis factor α (TNF-α) in Foxp3+ T cells from RCC TILs or PBLs from a healthy donor following stimulation with phorbol myristate acetate/ionomycin. Percentage values shown indicate the proportion of Foxp3+ or Foxp3− cells that expressed the cytokine. Data shown are representative of 18 RCC cases. (E) Percentage of CD4+ Foxp3− (non-Tregs) and CD4+ Foxp3+ (Tregs) in TILs that express CTLA4 (data from nine RCC cases). Horizontal lines indicate mean percentage values. All p values shown were measured using the Wilcoxon matched pairs test. European Urology 2012 61, 385-394DOI: (10.1016/j.eururo.2011.10.035) Copyright © 2011 European Association of Urology Terms and Conditions

Fig. 6 Homing phenotype of Treg cells in renal cell carcinoma (RCC) tumour-infiltrating lymphocytes (TILs) and matched peripheral blood lymphocytes (PBLs). Tregs in paired TIL and PBL samples were stained with antibodies specific for (A) 19 chemokine receptors and (B) 7 adhesion molecules. The number of RCC TIL samples stained was as follows: CCR3–7, CXCR3, CXCR4, and CXCR6: n=10; CX3CR1: n=8; CCR1, CCR2, CCR8–10, CXCR1, CXCR2, CXCR5, CXCR7, XCR1, and CD11B: n=7; CD62L and PSGL-1: n=6; and LFA-1, CLA, VLA-4, and BLT-1: n=5. Ten PBL samples were stained for all markers. Analysis was conducted by flow cytometry, gating on viable CD3+ CD45RA− CD4+ Foxp3+ cells. Results show the median fluorescence intensity (plus standard error) for each marker. (C) Paired analysis for four chemokine receptors in which expression on Tregs in TILs was significantly greater than the corresponding cells in matched PBLs (measured using the Wilcoxon matched pairs test). (D) Expression of CCR6 and CCL20 in RCC tissue; isotype control staining is shown inset. CCR6 staining is representative of 10 RCC cases; CCL20 is representative of 3 of 11 cases, with weaker staining detected in another 5 of these cases. European Urology 2012 61, 385-394DOI: (10.1016/j.eururo.2011.10.035) Copyright © 2011 European Association of Urology Terms and Conditions