Single-cell RNAseq of murine Peyer's patches after one dose of anti-α4β7 antibody or PBS. Single-cell RNAseq of murine Peyer's patches after one dose of anti-α4β7 antibody or PBS

Targeting the α4β7-MAdCAM-1 axis with vedolizumab (VDZ) is a first-line treatment in ulcerative colitis (UC). However, mechanism(s) of action (MOA) of VDZ remain relatively undefined. Here, we examined five distinct cohorts of patients with UC (n=83, n=60, n=21, n=31, n=401), to determine the effect of VDZ on the mucosal and peripheral immune system. We found a significant decrease in colonic and ileal naïve B and T cells and circulating gut- homing plasmablasts (β7+) in VDZ-treated patients, pointing to gut-associated lymphoid tissue (GALT) targeting by VDZ. Murine Peyer’s patches (PP) demonstrated a significant loss cellularity associated with reduction in follicular B cells, including a unique population of epithelium-associated B cells, following anti-α4β7 antibody (mAb) administration. Photoconvertible (KikGR) mice demonstrated impaired cellular entry into PPs in anti-α4β7 mAb treated mice. In VDZ-treated, but not anti-tumor necrosis factor-treated UC patients, lymphoid aggregate size was significantly reduced in treatment responders compared to non- responders, with an independent validation cohort further confirming these data. Response to VDZ was associated with a significant decrease of naïve B cells and a reduction in B cell follicle organization in the GALT. Responders had a significant reduction of β7+IgG+ plasmablasts in circulation, as well as IgG+ plasma cells and FcgR-dependent signaling in the intestine. GALT targeting represents a previously unappreciated MOA of α4β7-targeted therapies, with major implications for this therapeutic paradigm in UC, and for the development of new therapeutic strategies. Overall design: Peyer's patches were isolated from the small inthestine of treated mice and incubated with collagenase D, before mechanical homogenization to obtain a single-cell suspension.