Volume 66, Issue 4, Pages 677-688 (October 2014) Bacillus Calmette-Guérin Strain Differences Have an Impact on Clinical Outcome in Bladder Cancer Immunotherapy  Cyrill A. Rentsch, Frédéric D. Birkhäuser, Claire Biot, Joël R. Gsponer, Aurélie Bisiaux, Christian Wetterauer, Micheline Lagranderie, Gilles Marchal, Mickael Orgeur, Christiane Bouchier, Alexander Bachmann, Molly A. Ingersoll, Roland Brosch, Matthew L. Albert, George N. Thalmann  European Urology  Volume 66, Issue 4, Pages 677-688 (October 2014) DOI: 10.1016/j.eururo.2014.02.061 Copyright © 2014 Terms and Conditions

Fig. 1 Consolidated Standards of Reporting Trials flow diagram of the prospective randomized clinical trial. BCG=bacillus Calmette-Guérin. European Urology 2014 66, 677-688DOI: (10.1016/j.eururo.2014.02.061) Copyright © 2014 Terms and Conditions

Fig. 2 Estimated probability of recurrence-free survival and progression-free survival by treatment. Kaplan-Meier survival curves of patients with high-risk non–muscle-invasive bladder cancer treated with either bacillus Calmette-Guérin (BCG) Connaught (orange lines) or BCG Tice (blue lines). (a) Estimated probability of recurrence-free survival. The calculated hazard ratio (log rank) for treatment with BCG Connaught is 0.4 (95% confidence interval, 0.25–0.71). (b) Estimated probability of progression-free survival. European Urology 2014 66, 677-688DOI: (10.1016/j.eururo.2014.02.061) Copyright © 2014 Terms and Conditions

Fig. 3 Comparison of bacillus Calmette-Guérin (BCG) Connaught and BCG Tice in the induction of BCG-specific CD8+ T cells and recruitment of CD8+ T cells to the bladder. (a) The scheme illustrates the experimental design. Animals were instilled intravesically with either phosphate-buffered saline (PBS) or one of two BCG strains, BCG Connaught or BCG Tice, at indicated times (arrows). After four weekly instillations, spleens and lymph nodes were harvested and digested, and the resulting cell suspensions were analyzed by flow cytometry. (b) Representative flow cytometric plots of tetramer staining for H2-Db-Mtb32309–318 positive CD8+ T cells in spleens from mice intravesically treated with PBS, BCG Tice, and BCG Connaught (left to right). Plots represent (b) concatenated data from (c) individual mice. (c) The graph depicts the percentage of H2-Db-Mtb32309–318 positive CD8+ T cells among all CD8+ T cells from the spleen. (d) The graph depicts colony-forming units (CFUs) recovered from regional lymph nodes in mice treated with intravesical BCG. Bars indicate medians. ** p<0.01; Mann-Whitney test. DLN=draining lymph node. European Urology 2014 66, 677-688DOI: (10.1016/j.eururo.2014.02.061) Copyright © 2014 Terms and Conditions

Fig. 4 Recruitment of T cells to the bladder. Animals were instilled intravesically with either phosphate-buffered saline (PBS) or bacillus Calmette-Guérin (BCG) according to the scheme depicted in Figure 3a. (a) Representative flow cytometry plots from bladders treated with BCG Tice and Connaught depicting gating strategies to delineate T, natural killer (NK), and natural killer T (NKT) cells. (b) Absolute numbers of indicated events per bladder of animals treated with phosphate-buffered saline (PBS), BCG Connaught, or BCG Tice. (c) Representative histogram of bladder-derived CD4+ T cells expressing intracellular T-bet transcription factor. For each treatment group, three bladders were pooled for analysis. Yellow and green lines: respective isotype controls for animals treated with BCG Connaught and BCG Tice, respectively; blue line: Tice; orange line: Connaught. (d) Graphs show the frequency and absolute numbers of T-bet positive CD4+ T cells in regional lymph nodes. In graphs, bars indicate medians. * p<0.05; Mann-Whitney test. PBS-treated animals were not included in the statistical analysis. European Urology 2014 66, 677-688DOI: (10.1016/j.eururo.2014.02.061) Copyright © 2014 Terms and Conditions

Fig. 5 Evaluation of bacterial titers and bacillus Calmette-Guérin (BCG) formulation. (a) Titrations of clinical-grade (purchased) BCG freshly reconstituted from vials; (b) laboratory-grade BCG preparations (produced at Institut Pasteur, Paris). (c, d) Mice were immunized subcutaneously with a single dose of BCG. After 14 d, animals were killed and T-cell priming assessed. Graphs depict the frequency of H2-Db-Mtb32309–318 tetramer positive CD8+ T cells in spleens of animals injected with either (c) clinical or (d) laboratory-grade BCG Tice or BCG Connaught. * p<0.05; Mann-Whitney test. Untreated animals were not included in the statistical analysis. CFU=colony-forming unit. European Urology 2014 66, 677-688DOI: (10.1016/j.eururo.2014.02.061) Copyright © 2014 Terms and Conditions