Carcinogen induced expansion of atypical B cells and pre-treatment tumor adjacent tertiary lymphoid structures associate with poor response to BCG in non-muscle invasive bladder cancer

Intravesical bacillus Calmette-Guérin (BCG) G immunotherapy is the gold standard treatment for patients with non-muscle invasive bladder cancer (NMIBC). Unfortunately, more than 50% patients experience early recurrence or progression. Increased intra-tumoral immune infiltration in pre-treatment tumors has been shown to associate with poor outcomes post BCG therapy. Specifically, we recently showed that high intra-tumoral B cell density was associated with early recurrence and progression of NMIBC. Here, we address a major knowledge gap in the role of BCG induced expansion of a B cell population called, ‘atypical B cells (ABCs)’ in the context of age and sex, the two major variables in the pathophysiology of NMIBC. We first investigated whether pre-BCG TLSs associate with response to BCG. Using multiplex immunofluorescence and NanoString GeoMx digital spatial profiling-based characterization, we found higher density of tumor adjacent (TA) TLSs in patients categorized as BCG non-responders. Spatial proteomic profiling of 49 immune function and phenotype associated proteins revealed increased expression of immune exhaustion associated proteins in pre-BCG TLSs in tumors from both BCG responders and non-responders. It is established that patient age is a risk factor associated with poor outcomes in NMIBC and that biological aging associates with exhausted populations of immune cells. The expansion of ABCs is known to accompany biological aging and repetitive immunizations. We hypothesized that ABCs are recruited to the bladder mucosa due to repeated BCG instillations in the induction phase and dampen local anti-tumor immunity in patients deemed as BCG non-responders. Using the N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN) carcinogen induced murine model, we investigated the role of B cells in BCG response in aging mice. Bladder local and systemic immune alterations were characterized at post BCG therapy with or without B cell depletion. Since B cells exhibit significant shifts with biological aging in a sex associated manner, we used the four-core genotype mouse model to determine such differences following repeated treatment with BCG. BCG treatment in combination with B-cell depletion led to distinct bladder immune microenvironment states and systemic immune profiles. Overall, this study demonstrates the significant role of ABCs in disease progression and that the B cell infiltrated pre-BCG therapy TA-TLSs reflect a systemically exhausted B cell functional state in patients deemed as BCG non-responders. Findings from this study provide the first evidence suggesting the role of B cells/ABCs in BCG response and will inform future translation to development of therapies along the B cell exhaustion axis. ![Figure][1] ### Competing Interest Statement The authors have declared no competing interest. [1]: pending:yes