Transcriptomic profiling of mouse spleen mascrophages following adjuvant vaccination

Trained immunity is the phenomenon whereby innate immune cells such as monocytes or macrophages undergo functional reprogramming after exposure to certain microbial components, altering their responses to future exposures. Intravesical instillation with Bacillus Calmette-Guérin (BCG) is the most effective adjuvant therapy in patients with high-risk non-muscle invasive bladder cancer (HR-NMIBC). HR-NMIBC is associated with a high risk of tumor recurrence and progression to muscle-invasive bladder cancer. The precise immunological mechanisms through which BCG mediates anti-tumor immunity are still unclear. The aim of our study was to investigate the (long-term) induction of trained immunity by repeated BCG instillations in NMIBC patients and elucidate the immunological and epigenetic mechanisms that are involved. Another aim was to assess the relationship between trained immunity response and clinical response of NMIBC patients, in terms of recurrence free survival and progression free survival. To determine whether BCG instillations induce trained immunity in peripheral blood mononuclear cells (PBMCs) we performed a prospective cohort study (‘Tribute’) and isolated PBMCs collected before BCG therapy and at 8 time points during BCG therapy. A total of 17 BCG-naïve HR-NMIBC patients were included. After isolation of PBMCs, monocytes were further purified using an isolation procotol with a percoll gradient. RNA was isolated from these purified monocytes were and used as input for RNA-seq analysis. Blood was collected using venapuncture and PBMCs were isolated at three time points during the BCG induction cycle: pre-BCG1, BCG2 and BCG6; and two time points during each subsequent BCG maintenance cycle: pre-BCG7, BCG9, pre-BCG10, BCG12, pre-BCG13 and BCG15. For each donor one replicate for each time point was included. RNA from purified monocytes was isolated for the time points pre-BCG1, BCG6, and pre-BCG7 and the RNA-seq data was analysed. For some patients RNA-seq data for the pre-BCG10 time point was also available, but this time point was not included in the analysis because not every patient had data available for this time point. Comparisons for differentially expressed genes were made using the pre-BCG1 time point as a reference.