Characterizing age induced dysfunction in natural killer cells
Introduction
Cellular aging has become a prominent topic within immune cells particularly when discussing cellular dysfunction. In many cell types including natural killer (NK) cells, it has been observed that as the cells age, dysfunction sets in and cells become senescent. Cellular senescence is defined as irreversible dysfunction in the form of cell cycle arrest that includes changes to gene and transcription factor expression as well as shortening of telomeres. While dysfunction and cellular senescence are very well defined in aging T cells, age induced NK cell dysfunction and senescence are not well defined.
Methods
NK cells are isolated from cord blood and expanded in vitro weekly with irradiated K562 based feeder cells and Interleukin (IL)-2. Each week, an aliquot of cells was frozen at their current expansion age while another aliquot was stimulated and expanded again to continue the aging process. At each collection cells were counted and checked for viability prior to freezing or continued expansion. Once cells were expanded numerous times all the frozen aliquots were then used for multiple assays. Cytoxicity was observed using Incucyte in a tumor rechallenge assay using either Raji cells or MOLM14 cells as target tumor cells, telomere length was analyzed using qPCR, and bulk RNA sequencing and ATAC-sequencing were performed on the aging samples as well.
Results
We show in these studies that as the NK cells age, they become less cytotoxic, which is a problem seen in many age-related diseases such as cancer. We see a significant decrease in the aging NK cells’ ability to kill tumor cells as shown in multiple Incucyte assays. We also see a significant decrease in cell proliferation over time. The telomere length of the aged cells trended downwards with three of the four cords having significantly shorter telomeres at the fourth stimulation compared to the second stimulation. Also in these studies, bulk RNA and ATAC-sequencing were also performed on the aging NK cells to determine potential genes, transcription factors or pathways to exploit. From the bulk RNA sequencing we are able to see significant changes in senescence related genes as the NK cells age through in vitro cellular stimulations. Some of these genes have been thoroughly studied in the context of T cell aging such as FOS, JUN, KLRG1, CCL1, SESN1, SESN2, SESN3, among others. The ATAC-sequencing shows us transcription factors that are upregulated at each different age distinction with a different subset being upregulated at each stimulation time. Some of these changes in transcription factors have also been seen in T cell aging such as a decrease in EOMES expression as the cells age through the in vitro stimulation methods used.
Conclusion
Age induced NK cell dysfunction is a problem that has not been thoroughly addressed or characterized. This dysfunction could have a negative impact on NK cell function and anti-tumor potency, limiting their efficacy as adoptive cell therapy. One way to potentially increase the efficacy of NK cell therapy is to increase the longevity of the cells as well as decrease the incidence of age induced dysfunction such as senescence. We plan to use the initial results from these studies to exploit the differences found within the gene expressions to increase the longevity and cytotoxicity of NK cells allowing for a more potent NK cell therapy.