Single-Cell Transcriptional Analysis of Murine Mesenteric Lymph Nodes Following Oral Lyso-phosphatidylserine Nanoparticle Administration Reveals Cellular Heterogeneity in Tolerance Features

Unwanted immune responses against self-proteins or exogenous therapeutic proteins can trigger autoimmune diseases or compromise the safety and efficacy of life-saving therapeutic biologics. To address this issue, we utilized the ability of phosphatidylserine (PS) to convert an immunogen into a tolerogen and rationally developed a LysoPS-containing tolerogenic nanoparticle platform to prevent and mitigate unwanted immune responses. We demonstrated that prophylactic oral treatment with LysoPS-containing proteins prevents unwanted immune responses by inducing tolerance. However, the biological processes and cellular communication involved in LysoPS-mediated oral tolerance remain unclear. Therefore, this study aimed to characterize immune cell interactions and the potential tolerogenic mechanism in mice treated with LysoPS using Single-cell RNA sequencing (scRNA-seq). Our data showed that LysoPS nanoparticles increased the expression of RNAs associated with tolerogenic features in B cells, T cells, and NK cells, primarily through TGF-β responses. Animals were continuously fed Buffer, Free OVA, and LysoPS-OVA for four weeks via oral administration. Mesenteric lymph nodes (MLNs) were collected, enzymatically digested, and purified into a single live cell suspension before scRNA-seq. Six animal per group were used in this study and animal from the same group were pooled together for scRNA