Reconstitution of the uterine immune milieu after transplantation (scRNA-seq)

Maintenance of tissue-specific immunity is important for immunological fitness, but its establishment have been difficult to assess in humans. Here, we investigated reconstitution of the human uterine immune system by studying women undergoing uterus solid organ transplantation (UTx) or hematopoietic stem cell transplantation (HSCT). Through single-cell identification based on SNPs and disparate HLA expression using single-cell RNA sequencing or high-parameter flow cytometry, donor vs recipient cell origin was determined, and features of these cells were studied. Endometrial immune cell reconstitution occurred after both UTx and HSCT, at the transcriptomic, phenotypic, and spatial level. This occurred despite tacrolimus-induced calcineurin-mediated NFAT pathway inhibition, which affected de novo induction of tissue-residency features in vitro. Intriguingly, after HSCT, immune cells of male origin could reconstitute the endometrium. Collectively, our results proved insights into tissue immune system persistence and reconstitution capabilities in an organ undergoing continuous regeneration. Overall design: Samples from in total three UTX and two HSCT patients were subjected to single-cell RNA sequencing (scRNAseq, via 10x Genomics platform according to manufacturer's protocol). For the first UTX sample, we performed a targeted fluorescence-activated cell sorting (FACS) to obtain distinct leukocyte subsets from either recipient (B cells) or donor peripheral blood (monocytes), as well as T and NK cells from recipient tissue. Cells were then combined for scRNAseq. The other UTX and HSCT samples were sorted as alive CD45+ mononuclear cells from either tissue or peripheral blood before being subjected to scRNAseq.