Single-cell RNA sequencing revealed the role of the Th17 pathway in the development of anti HLA antibodies in a highly sensitized mouse model

The aim of this study is to investigate the specific pathway involved in HLA sensitization using single-cell RNA sequencing analysis and an allo-sensitized mouse model developed with an HLA.A2 transgenic mouse. For sensitization, wild-type C57BL/6 mouse received two skin grafts from C57BL/6-Tg (HLA-A2.1)1Enge/J mouse (allogeneic mouse (ALLO)). For syngeneic control (SYN), skin grafts were transferred from C57BL/6 to C57BL/6. We performed single-cell RNA sequencing analysis on splenocytes isolated from ALLO and SYN mice and compared the gene expression between them. We generated 9,190 and 8,890 single-cell transcriptomes from ALLO and SYN control mice, respectively. Five major cell types (B-cells, T-cells, NK cells, macrophages, and neutrophils) and their transcriptome data were annotated according to the representative differentially expressed genes of each cell cluster. The percentage of B-cells was higher in allogeneic mouse than it was in syngeneic control mouse. KEGG enrichment analyses indicated that the highly expressed genes in the B-cells from ALLO mouse were mainly associated with antigen processing and presentation pathways, allograft rejection, and the Th17 cell differentiation pathway. Upregulated genes in the T-cells of ALLO mouse were involved in the IL-17 signaling pathway. The ratio of Th17 cluster and Treg cluster was increased in the ALLO mouse. On flow cytometry, the percentage of Th17 (IL-17+/CD4+ T) cells was higher and regulatory T cells (FOXP3+/CD4+ T) was lower in the ALLO mouse compared to those in the SYN mouse. Our results indicate that not only the B cell lineage, but also the Th17 cells and their cytokine (IL-17) are involved in the sensitization to HLA. For sensitization (Allogeneic (ALLO) mouse), wild-type C57BL/6 mouse was sensitized twice with skin allografts from C57BL/6-Tg(HLAA2.1)1Enge/J mouse (0 and 5 weeks). Syngeneic (SYN) mouse received skin transplants from wild-type C57BL/6 mouse using the same procedure. Both the donor and recipient mice were anesthetized with intraperitoneal (i.p.) injections of Zoletil®50 (Tiletamine and Zolazepam) 30 mg/kg and Rompun® (Xylazine) 10 mg/kg. Tail skin, segmented into 8 × 8 −10 mm2 sizes, was obtained from the donor mice and grafted onto the dorsal area of the recipient mice. The mice were sacrificed using a CO2 chamber at four weeks after the second skin transplant, and mice spleens were harvested for scRNA-seq analysis.