Volume 74, Issue 6, Pages (December 2018)

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Volume 74, Issue 6, Pages 688-692 (December 2018) Neoadjuvant Chemotherapy Reinforces Antitumour T cell Response in Urothelial Urinary Bladder Cancer David Krantz, Ciputra Adijaya Hartana, Malin E. Winerdal, Markus Johansson, Farhood Alamdari, Tomasz Jakubczyk, Ylva Huge, Firas Aljabery, Karin Palmqvist, A. Ali Zirakzadeh, Benny Holmström, Katrine Riklund, Amir Sherif, Ola Winqvist European Urology Volume 74, Issue 6, Pages 688-692 (December 2018) DOI: 10.1016/j.eururo.2018.06.048 Copyright © 2018 Terms and Conditions

Fig. 1 Sentinel node (SN) CD8+ and CD4+ effector T cells are less exhausted with increased effector capacity after NAC. Phenotypic, epigenetic, and functional analysis of the impact of NAC on CD8+ and CD4+ effector T cells acquired from SNs of MIBC patients. (A and B) Expression of the checkpoint receptor PD-1 on CD8+ T cells and CD4+ effector T cells was assessed by FACS. The mean percentage of PD-1+ CD8+ T cells and PD-1+ CD4+ effector T cells are displayed (n = 10). (C and D) The expression of the cytotoxic markers T-bet, granzyme B, and perforin in CD8+ T cells was compared in patients receiving two or three cycles of NAC (n = 13), and the mean percentage of CD8+ T cells expressing these markers is displayed. (E and F) DNA methylation status of PRF1 and IFNG loci in CD8+ T cells from patient lymph nodes was measured by pyrosequencing. DNA methylation percentage was compared between non–complete responders (nonresponders and partial responders) versus complete responders (n = 7). (G) SN-derived lymphocytes were cultured for 7 d in the presence or absence of autologous tumour tissue homogenate. Dot plots display blasting T cell populations (FSC-A vs SSC-A) in representative patients from the control and stimulated groups (NAC naïve and NAC patients). (H) Average stimulation index representing CD4+ T cell blast percentage of total CD4+ T cells in tumour- or non–tumour-stimulated SN-derived lymphocytes from NAC-naïve or NAC patients (n = 18). The lines in all scatter plots and bar graphs represent means, with error bars indicating SEM. Statistical methods used were independent t test (A, B, D, and E) and Mann-Whitney test (D, F, and H). FACS = fluorescence-activated cell sorting; NAC = neoadjuvant chemotherapy; SEM = standard error of the mean. * p < 0.05. ** p < 0.01. *** p < 0.001. **** p < 0.0001. European Urology 2018 74, 688-692DOI: (10.1016/j.eururo.2018.06.048) Copyright © 2018 Terms and Conditions

Fig. 2 Treg frequency and suppressive function are reduced after NAC and correlate to a better NAC response. Comparison of Treg frequency and expression of Treg activation and effector markers in NAC-naïve and NAC patients after phenotypical analysis by FACS. Frequency of Tregs (CD4+ FOXP3+) was measured and displayed in (A) density plots of data from representative NAC-naïve and NAC patients and (B) a scatter plot including mean percentage of Tregs among these groups (n = 27). (C) Treg frequency was compared in patients receiving two and three or more cycles of NAC (n = 18). (D–F) The fraction of Tregs expressing CD39, HLA-DR, and PD-1 was compared between NAC-naïve and NAC-treated patients (n = 17). Histograms overlays of the investigated markers in CD4+FOXP3+ T cells from SNs of representative NAC-naïve and NAC -patients, as indicated. (G) The ratio of CD4+ Teff frequency to bulk Treg frequency in nonresponders and complete responders to NAC (n = 13). Corresponding to Figure 2G, the ratio of CD4+ Teff frequency to the frequency of Tregs expressing (H) the effector molecule CD39+ (n = 6) or (I) the activation marker CD69+ (n = 6). The FOXP3 gates and all gates for Treg markers were based on isotype controls. In the scatter plots, the lines represent means, with error bars indicating SEM. Statistical methods used were independent t test (F) and Mann-Whitney test (B–E). NAC = neoadjuvant chemotherapy; SEM = standard error of the mean; SN = sentinel node; Teff = effector T cell; Treg = regulatory T cell. * p < 0.05. ** p < 0.01. *** p < 0.001. **** p < 0.0001. European Urology 2018 74, 688-692DOI: (10.1016/j.eururo.2018.06.048) Copyright © 2018 Terms and Conditions