O27 - Assessing the Intersection of O-glycosylation and Aging on Immune Cells

Introduction: : As we age, we experience a series of changes in functionality in multiple organ systems, such as the lymphatic system. Lymphatic vasculature has been shown to have increased permeability and decreased vessel size, which affects the transport of fluids and cells to the organs within this system. T cells in particular are impacted by this, experiencing a decrease in proliferation and presence in the circulation resulting in a reduced ability to combat cancers and fight infections. Lymphoid tissues, such as the spleen, play an integral role in immune cell maturation and differentiation as well as experience structural changes with age. However, these changes and their effect on cellular behaviors have not been fully investigated. The glycocalyx, a sugar-rich coating on all mammalian cells, has been established in these immune cell-pathogen and microenvironment interactions. Therefore, we are investigating the impact of age on glycocalyx expression, motility, and T cell-mediated cytotoxicity of cancer cells, and T cell maturation in the spleen. Overall, our findings suggest that aging does impact glycocalyx expression, T cell-mediated cytotoxicity, and motility. Future work will further investigate the development of a spleen tissue-engineered model to further probe T cell-lymphatic crosstalk behavior.

Materials and

Methods: : Blood samples were collected from young and aged patients. Using the EasySep Human T Cell Isolation Kit, T cells were isolated and cultured with 25 ug/mL gentamycin, IL-2, and T Cell Activators for expansion and activation. Leukemia cell lines were cultured in RPMI 1640 supplemented with 10% FBS. T cell killing assays were conducted at a 10:1 and 5:1 effector to target cell ratio, and motility was assessed via live imaging over 24 hours. O-glycosylation was analyzed using lectins for Tn, sTn, and T antigens on healthy peripheral mononuclear cells, Jurkat, Nalm7, CEM, REH, and young and aged T cells. Mouse spleens from different-aged C57/BL6 mice were decellularized, cryosectioned, and stained for analysis.

Results, Conclusions, and Discussions:: Glycocalyx expression was altered between healthy peripheral mononuclear cell and malignant cell lines, particularly in Jurkats. The glycocalyx expression of aged T cells was on was found to have less expression than the young T cells. We expected to see this as this could indicate a decrease in interactions in vasculature, different microenvironments, and pathogens within the lymphatic system. Breast cancer cells exposed to aged T cells had lower cell death as compared to those exposed to young T cells at all three measured time points. However, the aged T cells had a higher percentage of dead cells when exposed to breast cancer cells at all three time points. This suggests that the older T cells may be experiencing T cell exhaustion. The successful decellularization of mouse spleen tissues will further inform the development of our tissue-engineered model. Ongoing work is being done to assess cell motility between the aged and young samples. Overall, these findings will better inform the mechanisms that modulate T cell behavior in aging disease pathologies on a cellular and tissue-based level.

Acknowledgements and/or References (Optional)::