Characterization of Ty21a immunostimulatory effects in the mouse bladder

Intravesical treatment with Salmonella enterica Ty21a, an oral typhoid-fever vaccine, has shown therapeutic potential against bladder tumors mainly through local immune-cell recruitment, particularly CD8⁺ T-cells. However, the mechanisms underlying its efficacy and the impact of bacterial formulation remain unclear. Here, we show that increased immune-cell infiltration was neither associated with modification in blood vessel density nor the generation of high endothelial venules, but rather with a transient increase in local vessel permeability, requiring live bacteria. Giving prior evidence that freshly harvested bacteria (Ty21a^FR) were more efficient than lyophilized bacteria (Ty21a^LYO), we tested both formulations intravesically in mice. Although, both similarly increased vascular permeability, Ty21a^FR induced significantly greater immune-cell recruitment locally and more effective tumor regression in the orthotopic MB49 bladder cancer model. Chemokine analysis showed higher levels of C5a, CXCL2 and CXCL5 in Ty21a^FR-treated bladders, however their receptors (C5aR, CXCR2) were barely detected on infiltrating T cells, precluding their direct involvement in T-cell recruitment. Instead, Ty21a^FR increased C5aR⁺ and C5aR^-CD11b^high myeloid cells, suggesting their indirect influence on T-cell recruitment. We hypothesized that LPS, a TLR4 agonist, from Salmonella, might be involved. Indeed, CD8⁺ T-cell infiltration following Ty21a^FR was significantly decreased in TLR4- and MyD88-KO mice. In contrast, myeloid-cell recruitment was only reduced in MyD88-KO mice, suggesting the involvement of TLR4-independent pathways in that process. This study is the first to identify Ty21a formulation-driven immunostimulatory differences in bladder cancer. Altogether, our data provide new insights into Ty21a's immunostimulatory mechanisms and highlight the importance of bacterial formulation for optimizing bladder cancer treatment.