Liver allografts are well tolerated and other solid organ allografts, when transplanted concurrently with livers, show improved outcomes. However, the mechanisms underlying “hepatic tolerance” have yet to be elucidated. Previous data show that hepatic dendritic cells (DC) have diminished immune functions. Immature plasmacytoid (p)DC have been shown to induce graft prolongation and we have previously shown miR-181a1b1 is increased in pDC as compared to conventional DC. Wild-type (WT) BALB/c mice were transplanted with allogeneic (C57BL/6) vascularized heterotopic heart grafts. Animals received either no treatment (n=3) or were injected i.v. with 0.5×10⁶ hepatic pDC (n=5) or miR-181a1b1^-/- (KO) pDC (n=3) at day (D)−7 from transplant. Recipients with no treatment rejected their allograft by D7 while recipients pre-treated with hepatic pDC had significantly prolonged allograft survival to D15-21 (p<0.01). However, recipients pre-treated with hepatic KO pDC rejected their allografts by D7-9. To determine the mechanism of graft prolongation, splenocytes were obtained at D7 and cytometry by time-of-flight (CyTOF) was utilized to comprehensively characterize the immune response post-transplant. Correlation analyses of molecular features derived from mass cytometry data were performed using Citrus, a method for unsupervised identification of significant cellular populations. Cells were clustered on the basis of the expression of 17 surface and 3 intracellular markers with significant changes in cell frequency inferred with the “glmnet” package in R. CD19⁺B220⁺CD44⁺IL-17⁺ B cells were elevated in the rejecting KO group, while CD4⁺ T cells expressing IL7Ra and CD44 were elevated in group that received WT pDC and had prolonged graft survival. Further analysis of B cell subsets by flow cytometry revealed a significant increase in IgM⁺ and marginal zone B cells in the KO group as compared to WT. There was a trend towards an increase in the Treg (CD4⁺CD25⁺Foxp3⁺), memory CD4⁺, and CD4⁺CD25^–CD69⁺ nontraditional T regulatory subsets in the WT group. These data demonstrate that CyTOF is a powerful new technology to identify immune cell populations post-transplant. Our findings suggest that graft prolongation by miR-181-expressing pDC is associated with an increase in both traditional and non-traditional Treg subsets.

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