*Purpose: Tim-1 signaling has an important role in maintaining the function of Regulatory B cells. Our previous study suggested Tim-1+ Bregs can prolong the survival of islets significantly in an allo-transplantation model (Balb/c to B6). In this study, we analyzed transcriptional data to determine potential pathways of tolerance induction by TIM-1+ Bregs.

*Methods: C57BL/6 mice were given anti-TIM-1, anti-CD45RB, and irradiated BALB/c splenocytes. On day 14, TIM-1+ and TIM-1- B cells were sorted, and RNA was extracted for RNA sequencing. Naïve C57BL/6 B cell RNA was used as a control. The data were analyzed using R (version: 3.6.1).

*Results: In comparison with the transcripts of the naïve B cell group, the Tim-1- B cell group (n=3) contained 139 differentially expressed genes (DEGs, Log2 |FC| > 1 and P < 0.05), among which 68 transcripts were up-regulated and 71 transcripts were down-regulated. In contrast, the Tim-1+ Breg group (n=3) contained 900 DEGs (759 up-regulated and 141 down-regulated). Between the Tim-1- and Tim-1+ B cell groups, there were 972 transcripts up-regulated and 53 transcripts down-regulated in the Tim-1+ group. Pearson correlation analysis (PCA) found the transcripts of Tim-1- B cells and Tim-1+ Bregs to be significantly different (P < 0.05, Fig. A). Ten of the most different DEGs (according to the contributed score) were: CDC6, Pparg, Rxra, Tyms, Slc11a1, Emr1, Clec1b, Tlr8, Ckap2, and C2 (Fig. B). GSEA enrichment analysis displayed up-regulated DEGs as mainly enriched in 28 different pathways (|NES|>1, NOM p-val<0.05, FDR q-val<0.25). Notable pathways include: angiogenesis, IL6-Jak-Stats signaling, IL-2-Stat5 signaling and TGF-beta signaling (Fig. C).

*Conclusions: Significant transcriptional differences were found between naïve B cells, Tim-1-B cells, and Tim-1+ Bregs. These differences indicate potential mechanistic pathways responsible for suppression of immune response and prolongation of graft survival by Tim-1+ Bregs. Further research on these DEGs is ongoing in vitro.

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