Single cell analysis revealed that two distinct, unique CD4+ T cell subsets were increased in the small intestinal intraepithelial lymphocytes of aged mice
Recent advances in research suggest that aging has a controllable chronic inflammatory disease aspect. Aging systemic T cells, which secrete pro-inflammatory factors, affect surrounding somatic cells, and accelerate the aging process through chronic inflammation, have attracted attention as potential therapeutic targets in aging. On the other hand, there are few reports on the aging of the intestinal immune system, which differs from the systemic immune system in many ways. In the current study, we investigated the age-related changes in the intestinal immune system, particularly in T cells. The most significant changes were observed in the CD4+ T cells in the small intestinal IEL, with a marked increase in the proportion of this fraction in old mice and reduced expression of the co-stimulatory molecules CD27 and CD28, which are characteristic of aging systemic T cells. The proliferative capacity of aging IEL CD4+ T cells was significantly more reduced than that of aging systemic T cells. Comprehensive transcriptome analysis showed that the expression of inflammatory cytokines such as TNF was not upregulated, whereas Cd8a, NK receptors, and Grazymes were upregulated in aging IEL CD4+ T cells. Functional analysis showed that aging IEL T cells had a higher cytotoxic function against intestinal tumor organoids in vitro than young IEL T cells. scRNAseq revealed that splenic T cells show a transition from naïve to memory T cells, whereas intestinal T cells show the emergence of a CD8aa+CD4+ T cell fraction in aged mice, which is rarely seen in young cells. Further analysis of the aging IEL CD4+ T cells showed that two unique subsets are increased, which are distinct from the systemic CD4+ T cells. Subset 1 has a pro-inflammatory component, with expression of IFN? and upregulation of NFkB signaling pathways. Subset 2 does not express IFN?, but upregulates inhibitory molecules and nIEL markers. Expression of granzymes and CD8a was common to both. These two fractions were in opposite positions in the clustering by UMAP and indeed had different TCR repertoires. They may be involved in the suppression of intestinal aging and longevity through anti-tumor immunity, elimination of senescent cells and stressed cells in the aging environment. This finding could be a breakthrough in aging research. Overall design: SI-IELs or SP cells were collected and pooled from young (6-week-old) or old C57BL/6 mice (80-week-old), the cells from 3 young mice and 3 old mice were mixed and pooled as young SP, young IEL, old SP and old IEL. CD45+ cells were enriched from each group using CD45 microbeads (Miltenyi Biotec), followed by cell hashing with TotalSeq-C anti-mouse hashtag antibodies (hashtag1-young IEL, hashtag2-old IEL, hashtag3-young SP, hashtag4-old SP). After the Fc blocking, cells were stained with anti-CD3e-FITC mAbs and anti-CD4-APC mAbs, and the CD3+ cells and CD3+CD4+ cells were sorted from each group by FACSMelody. Dead cells removal was performed with 7-AAD. To enrich CD4+ cells, which are rare population in young IEL, CD3+ cells and CD3+CD4+ cells were mixed as 1:1 in young and old IEL.