(P21-016-25) Single-Cell Analysis of Cellular Heterogeneity and Immune Interactions in the Intestine of BCO2 Deficient Mice

Objectives: To investigate the effects of beta-carotene oxygenase 2 (BCO2) gene on the cellular profiles and communications of intestinal epithelial and immune cells at the single-cell level.

Methods: Ileum tissues were harvested from eight-week-old BCO2 knockout (KO) and its parental strain, C57BL/6J (WT) mice. Following the removal of Peyer's patches, tissues were pre-digested with EDTA-DTT buffer, then proceeded with collagenase digestion and cell strainer filtration. Upon Percoll gradients purification, samples with cell viability greater than 80% were further processed with cell fixation, library construction, and single-cell RNA sequencing (scRNA-seq). Bioinformatics analyses and results visualization were performed using Cellranger (10X Genomics) and R software.

Results: The scRNA-seq analysis profiled 61,929 cells obtained from four biological replicates of KO and WT mice, detecting on average 3,734 genes per cell. Unsupervised graph-based clustering and annotation based on known marker genes revealed the presence of all intestinal epithelial cell lineages, including enterocytes, goblets, stem cells, enteroendocrine cells, Paneth cells, and tuft cells, consistent across both conditions. Differential expressed gene analysis showed increased expression of genes involved in antigen presentation pathways, like CD74, H2-Ab1, and H2-Eb1, across most detected cell types in KO intestines, leading to enrichment in epithelial cell antigen presentation pathways. Consequently, KO intestines exhibited a higher proportion of activated lymphocytes than WTs, including effector CD8+ T cells and plasma B cells, despite a lower overall proportion of immune cells. Interestingly, BCO2 gene deficiency resulted in the accumulation of a rare cell subset of CD4+CD8+ double positive T lymphocytes, thus inducing significantly enhanced MHC-I signaling interactions compared to that in the WT intestine.

Conclusions: The results revealed novel communications hubs between epithelial cells and immune cells in BCO2 KO intestines, suggesting a potential role the BCO2 gene plays in antigen presentation within the intestinal mucosal immune response.

Funding Sources: USDA and NIH