Abstract
Purpose :
Ins2Akita (Akita) mice spontaneously develop diabetes, and induce early pathophysiological changes by diabetic complications. On the other hand, interferon-γ knock out (GKO) mice exhibit high differentiation and activation of Th2 and Th17 cells as a result of Th1 cell deficiency. Recently, we demonstrated that in Akita mice back crossed with GKO mice (Akita-GKO mice), immune responses shifted to Th17 cells, and development of diabetic retinopathy (DR) was promoted compared with Akita mice. In addition, T cells producing osteopontin (OPN) increased in Akita-GKO mice. In this study, we analyzed peripheral OPN- and IL-17-producing T cells in Akita mice by single-cell RNA sequencing.
Methods :
Spleens were harvested form 6 Akita mice and 6 C57BL/6 (WT) mice. T cells were purified by magnetic negative selection using MACS cell separation, and pooled in WT mice or Akita mice individually. The number of purified T cells applied to scRNAseq analysis was 2888 cells in WT mice and 3637 cells in Akita mice, but the number of filtered cells by quality control was 2192 cells in WT mice and 2807 cells in Akita mice. Cluster analysis was performed on these cells and the expression levels of approximately 6,000-7,000 genes were compared by differential expressed genes (DEGs) analysis.
Results :
When compared the expression of spp1 (OPN gene) in whole T cell population of Akita mice and wild-type mice, spp1-expressing T cells were detected only in Akita mice, but not in WT mice (Mean of normalized value of spp1 expression, Akita vs WT; 0.011 vs 0, p=0.0012). The ratio of cells in the Th17 cluster was Akita:WT = 65:35, and Th17 population was increased in Akita mice compared to WT. However, cluster analysis confirmed that spp1 is mostly expressed in clusters of follicular helper T cells (Tfh), but not in Th17 cell cluster. DEGs analysis of Akita vs. WT in the Tfh cluster also confirmed spp1 at the top. Additionally, spp1 expression was significantly higher in senescence-associated T cells with sostdc1 of Tfh cluster (p=0.0374).
Conclusions :
These results indicated that OPN-producing T cells and IL-17-producing cells were different cell populations and OPN was mainly produced by senescence-associated T cells. Both populations increased in Akita mice compared with WT mice, suggesting that these T cells would be synergically involved in development of DR in Akita mice.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.