Analysis of T cell response in bladder cancer patients treated with Bacillus of Calmette-Guérin (BCG)

Non-Muscle Invasive Bladder Cancer (NMIBC) is the second most common urological cancer in western countries. Intravesical Bacillus Calmette-Guérin (BCG) immunotherapy is the gold standard for NMIBC treatment, but patients respond variably. Immune response induced and/or enhanced by BCG treatment is thought to be critical for therapeutical success, i.e., in limiting disease recurrence and progression. Nevertheless, correlations between BCG-elicited immune response and clinical outcome are poorly understood. More knowledge is needed about the roles exerted by T cells in BCG-treatment of NMIBC patients. The aim of this study is the analysis of circulating T cell immuno-profile, cell cycle, and BCG antigen-specific cytokine production following BCG immunotherapy of NMIBC patients, and the correlations of possible changes of these parameters to clinical response. T cell response has been studied in a cohort of 15 NMIBC patients at three different timepoints during BCG treatment induction by weekly BCG instillation: baseline (t0), immediately before the start of BCG therapy, at week 1 (t1) and 2 (t2) after the start of BCG therapy. Immunophenotype analysis by flow cytometry resulted in no treatment-related changes or abnormalities in frequencies of CD4+/CD8+, Tregs, and γδ T cells in NMIBC patients. Cell cycle analysis by flow cytometry showed higher percentages of proliferating cells among CD4+ and CD8+ effector memory T cells compared to the other naïve/memory T cell subsets, in NMIBC patients at each timepoint, and in HD used as control group. Flow cytometric Intracellular Cytokine Staining (ICS) assay upon in vitro stimulation with a peptide pool derived from the BCG antigen Ag85b resulted in extremely low percentages of IFNγ-, TNFα-, IL-2-producing cells among CD4+ and CD8+ T cells from PBMCs of most of the NMIBC patients at each timepoint. We identified 3 NMIBC patients having Ag85b-specific cytokine producing T cells above background (so-called ICS-assay-positive). Almost no IL-4 production was observed. No remarkable correlations between ICS results and clinical outcome were found. The strength of our study is based on the analysis of both CD4+ and CD8+ T cell cytokine profiles at different timepoints during BCG therapy, and upon two different times of stimulation. Our data represent a starting point to understand the role of CD4+ and CD8+ T cell responses in NMIBC patients at different times of BCG immunotherapy. In the future, TCRseq analysis from a larger group of NMIBC patients will be performed with the help of our collaborators of Francis Crick Institute and King’s College in London. Documentation for the approval has been submitted.