Scientists have demonstrated that CAR T cells can be employed against senescent intestinal stem cells, improving regeneration and ameliorating age-related symptoms such as “leaky gut” [1].
Cell-on-cell warfare
Cellular senescence is a heterogeneous phenomenon in which cells stop dividing and malfunction, driving up inflammation. While senescent cells play a positive role in certain contexts, such as wound healing [2] and embryonic development, their excessive accumulation with age is widely viewed as detrimental [3]. It happens in multiple, if not all, organs and tissues, including the gut. However, intestinal cellular senescence has not been studied enough.
The gut in itself is important in aging. The intestinal epithelium has one of the highest renewal rates in the body. To maintain gut function, intestinal stem cells (ISCs) actively proliferate, churning out a lot of short-lived epithelial cells that do the hard work of absorbing nutrients. Aging affects ISCs, probably more than many other types of stem cells, diminishing their regenerative capacity and harming gut performance.
The authors of this new study, currently published as a preprint, wanted to see what will happen to ISCs in response to a treatment that eliminates senescent cells (a senolytic). Previously, this group developed an unusual weapon against senescent cells: CAR T cells [4]. CAR stands for “chimeric antigen receptor” and basically means that cytotoxic T cells were genetically modified to express receptors that recognize a specific cell type. When injected into the body, the modified T cells go after the target cells.
CAR T cells are most associated with modern anti-cancer immunotherapies, but this is just one possible use. T cells are a universal weapon that can be deployed against any misbehaving cell subset for people who know how to target it.
Finding the target
First, the researchers characterized cellular senescence in the gut. They found that the most popular senescence marker, senescence-associated beta-galactosidase (SA-β-gal), was greatly elevated in the small intestines of 20-month-old mice compared to 3-month-old mice.
The scientists then found several more markers that correlated with senescence, including the surface protein uPAR. Most of the cells expressing uPAR were of epithelial origin and also expressed SA-β-gal. By performing single-cell RNA sequencing on thousands of cells, the researchers showed that uPAR-expressing cells were comprised mostly of stem cells, enterocytes, and macrophages and significantly overlapped the senescent cell population.
Analysis of human samples from young and old individuals produced a similar picture: age-related elevation in uPAR that was correlated with an increase in senescence signatures. The number of cells co-expressing uPAR and other senescence markers also increased with age. Going back to mice, the researchers found that senescent cell burden was correlated with diminished intestinal function, increased intestinal permeability, and altered microbiome composition.
Unleash the CAR T cells!
Next, the scientists prepared uPAR-targeting CAR T cells and administered them to young and old mice at a dose that was previously found to be optimal in terms of senolytic potential and safety. The CAR T cells promptly got activated, showing that they can recognize uPAR-expressing cells. The treatment led to a significant reduction in SA-β-gal-positive cells and, more importantly, to phenotypic improvements. In particular, it rescued age-related gut permeability (“leaky gut”), which is thought to be a major cause of inflammation. The number of proliferating epithelial cells increased.
CAR T cell treatment also significantly reduced damage in two different mouse models of intestinal injury. “Taken together,” the researchers wrote, “these results show that the accumulation of uPAR positive senescent cells in aged and injured intestines contributes to decreased epithelial integrity and reduced regenerative capacity.”
To elucidate the mechanisms behind this effect, the researchers again employed single-cell RNA sequencing. Aged mouse intestines contained much fewer ISCs, and even those stem cells were more exhausted than in young animals. The CAR T cell treatment reversed both those trends, increasing the abundance of ISCs as well as their fitness according to their RNA profiles. ISCs from the treated intestines were better at forming organoids, showing improved regenerative capacity. Interestingly, differentiated epithelial cells in the treated intestines also performed better.
Herein we identify for the first time the accumulation of intestinal senescent cells during physiological aging and validate uPAR as a reliable marker of senescence in this setting. Harnessing uPAR-targeting CAR T cells we show that in vivo elimination of senescent cells in aged animals significantly improves epithelial integrity and overall intestinal homeostasis. These results suggest that in the context of the aging intestinal stem cell niche and epithelium, senescent cells significantly impair regenerative capacity. Indeed, we identify uPAR expression as a marker of dysfunctional ISCs whose in vitro elimination is sufficient to rejuvenate the regenerative potential of aged intestinal crypts.
Literature
[1] Eskiocak, O., Chowdhury, S., Shah, V., Nnuji-John, E., Chung, C., Boyer, J. A., … & Amor, C. (2024). Senolytic CAR T cells reverse aging-associated defects in intestinal regeneration and fitness. bioRxiv, 2024-03.
[2] Andrade, A. M., Sun, M., Gasek, N. S., Hargis, G. R., Sharafieh, R., & Xu, M. (2022). Role of senescent cells in cutaneous wound healing. Biology, 11(12), 1731.
[3] Di Micco, R., Krizhanovsky, V., Baker, D., & d’Adda di Fagagna, F. (2021). Cellular senescence in ageing: from mechanisms to therapeutic opportunities. Nature reviews Molecular cell biology, 22(2), 75-95.
[4] Amor, C., Feucht, J., Leibold, J., Ho, Y. J., Zhu, C., Alonso-Curbelo, D., … & Lowe, S. W. (2020). Senolytic CAR T cells reverse senescence-associated pathologies. Nature, 583(7814), 127-132.
