Background: Immune checkpoint inhibitors (ICIs), such as anti-PD-1/PD-L1 antibodies, have emerged as a successful immunotherapeutic strategy for advanced and metastatic urothelial cancer (UC). Therapeutic blockade of PD-1 or PD-L1 with monoclonal antibodies leads to durable tumor regressions in up to 25% metastatic muscle invasive UC (MIBC). Neoadjuvant use of ICI also showed remarkable efficacy and represents a unique opportunity to study immunodynamics during PD-1 blockade to decipher functional predictors of response and resistance. Methods: Patients diagnosed with T2-T4aN0M0 MIBC were treated with 3 cycles of neoadjuvant pembrolizumab before cystectomy in the PANDORE trial (NCT03212651). The primary endpoint was pathologic complete response (ypT0N0). Secondary endpoints focused on safety, progression-free survival (PFS) and biomarker analysis. We performed longitudinal analysis of peripheral and tumor infiltrating lymphocytes, tumor microbiome as well as soluble factors using high dimensionnal immune phenotyping by mass cytometry, immuno-fluorescence and -histochemistry and multiplex immunoassays. Humoral and cellular recall immune memory against urinary tract commensals were studied. Results: Thirty-nine patients were enrolled from October 2017 to December 2019. All but 5 (n = 34 patients (87.2%)) proceeded with cystectomy. Ten patients presented with ypT0N0 (29.4%; 95% CI: 15.1 %-47.5 %). Multidimensional biomarkers analysis showed that baseline follicular T helper (Tfh) and post-pembrolizumab tertiary lymphoid structure (TLS) and activated B cells were associated with outcome (p= 0.005, p= 0.01 and p= 0.04, respectively). Plasma CXCL13 (the prototypic chemokine secreted by Tfh and involved in TLS functions) increased after 1 cycle of PD-1 blockade in responders and patients without progression at 24 months (p= 0.002 and p= 0.0001, respectively). Focusing on MIBC tumor microbiome, we showed that intracellular Gram negative bacteria and other commensals were more frequent in tumoral than in normal urothelium (p= 0.04). Interestingly, basal CXCL13-secreting CD4⁺ T cells and IgG directed against urinary pathobionts such as Escherichia coli predicted prolonged PFS (p= 0.01 and p= 0.001, respectively). Conclusions: Our results suggest that urothelial commensals could induce specific Tfh and B cell responses that were re-invigorated by PD-1 blockade and associated with clinical benefit to pembrolizumab. Further analyses are needed to validate the predictive value of commensal-specific Tfh in UC and other epithelial cancers that are directly or indirectly exposed to bacteria. Clinical trial information: NCT03212651.