Jindong Zhao ^* Zhaohui Fang ^*

• First Affiliated Hospital of Anhui University of Traditional Chinese Medicine, Hefei, China

Type 2 diabetes mellitus (T2DM) is a disease that involves autoimmunity. However, how immune cells function in the peripheral blood remains unclear. Exploring T2DM biomarkers via single-cell RNA sequencing (scRNA-seq) could provide new insights into the underlying molecular mechanisms. In this study, we included three healthy participants and three T2DM patients. The observed clinical indicators included weight and fasting blood glucose (FBG), glycosylated haemoglobin A1c (HbA1c) and fasting insulin levels. Direct separation and purification of peripheral blood mononuclear cells (PBMCs) were performed via the Ficoll density gradient centrifugation method. Immune cell types were identified via scRNA-seq. The differentially expressed genes, biological functions, cell cycle dynamics, and correlations between blood glucose indicators and genes in different cell types were analysed. There were differences between the healthy and T2DM groups in terms of FBG and HbA1c (p<0.05 or p<0.01). We profiled 13,591 cells and 3188 marker genes from PBMCs. B cells, T cells, monocytes, and NK cells were grouped into 4 subclusters from PBMCs. CD4+ T cells are mainly in the memory activation stage, and CD8+ T cells are effectors. Monocytes include mainly CD14+ monocytes and FCGR3A+ monocytes. There were 119 differentially expressed genes in T cells and 175 differentially expressed genes in monocytes. Gene set enrichment analysis revealed that the marker genes were enriched in HALLMARK_INTERFERON_GAMMA_RESPONSE and HALLMARK_TNFA_SIGNALING_VIA_NFKB. Moreover, TNFRSF1A was identified as the core gene involved in network interactions in T cells. Our study provides a transcriptional map of immune cells from PBMCs and provides a framework for understanding the immune status and potential immune mechanisms of T2DM patients via scRNA-seq.