It is unknown how intestinal B cell populations and B cell receptor (BCR) repertoires are established and maintained over time in humans. Following intestinal transplantation (ITx), surveillance ileal mucosal biopsies provide a unique opportunity to map the dynamic establishment of gut lymphocyte populations. Using polychromatic flow cytometry that includes HLA allele group-specific mAbs distinguishing donor from recipient cells along with high throughput BCR sequencing, we tracked the establishment of recipient B cell populations and BCR repertoire in the allograft mucosa of ITx recipients. We confirm the early presence of naive donor B cells in the circulation and, for the first time, document the establishment of recipient B cell populations, including B resident memory cells, in the intestinal allograft mucosa. Recipient B cell repopulation of the allograft was most rapid in infant (<1 year old)-derived allografts and, unlike T cell repopulation, did not correlate with rejection rates. While recipient memory B cell populations were increased in graft mucosa compared to circulation, naive recipient B cells remained detectable in the graft mucosa for years. Comparisons of peripheral and intra-mucosal B cell repertoires in the absence of rejection revealed increased BCR mutation rates and clonal expansion in graft mucosa compared to circulating B cells, but these parameters did not increase markedly after the first year post-transplant. Furthermore, clonal mixing between the allograft mucosa and the circulation was significantly greater in ITx recipients, even years after transplantation, than in healthy control adults. Collectively, our data demonstrate intestinal mucosal B cell repertoire establishment from a circulating pool, a process that continues for years without evidence of establishment of a stable mucosal B cell repertoire.

The authors have declared no competing interest.

The study is part of Program Project Grant (PPG) P01 AI106697 funded by the National Institute of Allergy and Infectious Diseases (NIAID). J.F. was supported by Nelson Faculty Development Awards from the Nelson Family Transplantation Innovation Award Program at Columbia University Irving Medical Center. E.W. was supported by NIAID training grant T32AI148099. T.H., A.S. and U.H. were also supported by the European Union's Horizon 2020 Research and Innovation Program under grant agreement 825821 and by Israel Science Foundation (ISF) grant 1327/22. Research reported here was performed in the CCTI Flow Cytometry Core, supported in part by the Office of the Director, National Institutes of Health (NIH) awards S10RR027050 and S10OD020056 and in the Human Immunology Core at the University of Pennsylvania (RRID: SCR 022380), supported in part by NIH awards P30-AI045008 and P30-CA016520.

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