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Blockade of inhibitory receptors (IR), overexpressed by T-cells, can activate anti-tumor 30immune responses resulting in the most promising therapeutic approaches, particularly in 31 bladder cancer, currently able to extend patient survival. Thanks to their ability to cross-32 present antigens to T cells, dendritic cells (DC) are an immune cell population playing a 33 central role in the generation of effective anti-tumor T-cell responses. While function and 34 expression of IRs have been mostly investigated in T cells, very few data are available for 35 DC. Therefore, we analyzed whether DC may express IRs able to decrease their functions. 36For that purpose, we investigated several IR: PD-1, CTLA-4, BTLA, TIM-3 and CD160, in 37 patients. pDC and conventional CD1c + and CD141 + DC were identified using a combination 89 of phenotypic markers ( Supplementary Fig. 1) and expression of IR was determined. PD-1, 90CTLA-4 and CD160 were not expressed by any subtype of DC from HD or UCa patients 91 (data not shown). In contrast, BTLA was observed in all DC subsets, albeit at a very low level 92 in CD1c + DC and TIM-3 was only expressed by CD1c + and CD141 + DC, in HD. Comparison 93 to UCa patients showed that BTLA was significantly overexpressed by CD141 + DC and pDC, 94Chevalier et al. European Urology -Revised manuscript whereas only a slight increase of TIM-3 expression was observed in CD141 + DC (Fig. 1A). 95This result suggests that bladder tumor microenvironment may increase BTLA and TIM-3 96 expression on DC. In order to have more insights into BTLA and TIM-3 expression by DC, 97 we segregated the data from UCa patients in two groups, according to the stage of the disease 98 (Supplementary Table 1): non-muscle invasive bladder cancer (NMIBC) and MIBC patients. 99A significantly higher expression of TIM-3 was only found in CD141 + DC from MIBC 100 patients as compared to HD, suggesting that UCa-mediated overexpression of TIM-3 is later 101 than for BTLA, which was overexpressed in CD141 + DC and pDC from both types of patients 102 (Supplementary Fig. 2). 103Seeking further evidence that BTLA and TIM-3 expression may be altered by the bladder 104 tumor microenvironment, we analyzed their expression on tissue-infiltrating DC 105 subpopulations from bladder tumor and paired non-tumoral adjacent tissue from surgical 106 specimen recovered after cystectomy. Since DC subtypes from tissue are phenotypically 107 different than circulating DC, we focused on CD14 + CD11c + and CD14 neg CD11c + tissue-108 infiltrating DC [7, 8] (Fig. 1B). Notably, we observed a significant overexpression of TIM-3 109 in both types of bladder DC and a higher frequency of BTLA + CD14 neg CD11c + bladder DC 110 within tumor tissue (Fig. 1C). Similar results were obtained when comparing non-paired 111 tissue samples (Supplementary Fig. 3). 112We next sought to determine whether BTLA and TIM-3 expressed on DC are functional 113 and could lead to an inhibition of DC function, as monitored by cytokine (IL-12, IL-1β and 114 TNF-α...
