Intravesical instillation of bacillus Calmette-Guérin (BCG) for non-muscle invasive bladder cancer is one of the few examples of successful immunotherapy in the clinic, with 50-70% treatment response. While success of therapy is known to rely on repeated instillations of live BCG, administered as adjuvant therapy shortly after tumor resection, its precise mechanisms of action remain unclear. I established an experimental mouse model to study the dynamics of the immune response following intravesical BCG regimen. Based on experimental work in humans, I focused my attention on the establishment of a robust acute inflammatory response, together with the activation and recruitment of T lymphocytes. I demonstrated that BCG dissemination to bladder draining lymph nodes and priming of interferon-!-producing T cells could occur following a single instillation. However, repeated instillations with live BCG were necessary for a robust T cell infiltration into the bladder. Interestingly, subcutaneous immunization with BCG prior to instillation overcame this requirement, triggering a more robust acute inflammatory process following the first intravesical instillation and accelerating T cell entry into the bladder, as compared to the standard protocol. Moreover, subcutaneous immunization with BCG prior to intravesical treatment of an orthotopic tumor dramatically improved response to therapy. These data prompted analysis of clinical data, which showed a significant difference in recurrence-free survival, favoring those patients with sustained pre-existing immunity to BCG. In parallel, using clinical and in vitro experimental data, I contributed to the construction and parameterization of a stochastic mathematical model describing the interactions between BCG, the immune system, the bladder mucosa and tumor cells. First, we could show that tumor extinction mediated by the innate immune system acting on its own is an unlikely occurrence, as it would require a bystander killing capacity per innate effector cell that is much higher than suggested by experimental data. Second, we refined our mathematical model to take into account the adaptive immune response, and evaluated optimal clinical parameters of BCG induction therapy, including (i) duration between resection and the first instillation, (ii) BCG dose, (iii) indwelling time, and (iv) treatment interval of induction therapy, which all had an impact on the probability of tumor extinction. A remarkable finding is that an inter-instillation interval two times longer than the seven-day interval used in the current standard of care would substantially improve treatment outcome. Together these data provide new insights into a long-standing clinically effective immunotherapeutic regimen and predict strategies that may improve patient management. Most importantly, I suggest that monitoring patients' response to purified protein derivative (PPD) test, and, if negative, boosting BCG responses by parenteral exposure prior to intravesical treatment initiation, may be a safe and effective means of improving intravesical BCG-induced clinical responses.
