Background Immunosenescence refers to the age-associated decline of the adaptive immune system, which results in increased incidence and severity of infections, cancers, and autoimmunity. The elderly show reduced numbers of naïve T cells, skewed CD4:CD8 ratio, reduced proliferative and functional capabilities, and increased expression of senescence markers. These phenomena have strong repercussion in adoptive immunotherapy.
Notably, the ex vivo manufacturing process of CAR-T cells per se induces senescence extremely quickly; 15 days of T cell expansion age cells 30 years, as measured by telomere length, T cells differentiation and CDKN2a mRNA levels.
To circumvent this problem, we here propose the modulation of USP16, an epigenetic regulator of stem cells and senescence in multiple tissues. Downregulation of USP16 rejuvenates T cells, offering a powerful tool to dramatically improve the efficacy of CAR-T treatments.
Methods During ex vivo CAR-T cell manufacture, cells age very rapidly, strongly decreasing T cell fitness. Importantly, we observed that cellular senescence is an early event that precedes T cell exhaustion upon CD3/CD28 T cell stimulation, making it a very interesting pathway to target. In line with this hypothesis, we demonstrated that reducing cellular senescence increases CAR-T cell functions both in vitro and in vivo.
Results We identified an epigenetic regulator, USP16, whose mRNA levels increase during T cell expansion and correlate with the expression of the aging marker par excellence, CDKN2a. Genetic modulation of USP16 in CD19 and GD2 CAR-expressing T cells not only reduces senescence markers but also expands the naive (CD45RA+CD62L+) population and enhances cell self-renewal, without negative effects on T cell expansion. USP16 modulation also results in increased killing, polyfunctionality, and expansion upon in vitro stimulation with tumor cells. Notably, the delay of cellular senescence induces long-lasting cellular fitness (figure 1) as T cells are less exhausted upon multiple tumor challenges. In vivo, T cells rejuvenated by USP16 modulation, are 60% more efficient in controlling tumor growth in a mouse model of leukemia (NALM-6) and neuroblastoma (CHLA-255).
Conclusions We demonstrated that modulation of USP16 prevents cellular senescence and increases self-renewal in T cells. This approach can significantly improve CAR-T therapy in multiple diseases, including leukemias and solid tumors. Development of small molecules against USP16 could offer a viable solution to improve T cell fitness during manufacturing.
Ethics Approval The study was approved by Institutional Animal Care and Use Committees (IACUC), approval number CR-0104.
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