Development of a humanized mouse model of graft-versus-host disease to assess human regulatory T cell function

We developed a humanized mouse model of graft-versus-host disease in which Treg injected two days after PBMC persist in the blood of infused mice and can be characterized. This model provides in vivo assessment of manipulated Treg. We used that model to evaluate the impact of ex vivo priming of Treg with TNF-alpha on their ability to prevent xenogeneic GVHD. Indeed, it had been demonstrated that Treg function can be enhanced through stimulation of TNFR2. scRNAseq analysis was performed to assess the impact of TNF priming on Treg transcriptomics. Peripheral blood mononuclear cells were isolated from the blood of three healthy donors. Treg were isolated through immuno-magnetic selection then cultured for 24 hours with IL-2 at 10 ng/mL with or without TNF at 100 ng/mL. After 24 hours, cells were collected and washed. We performed cell multiplexing to label samples individually, then pooled them for single cell RNA sequencing. For each donor (named donors 3, 4 and 7), half cells were in the control group (no TNF) and half cells were in the experimental group (with TNF). 10x Genomics 3' Single Cell Gene Expression with CellPlex multiplexing (Set A). Cells were labeled with Cell Multiplexing Oligos prior to pooling and library preparation.