Identifying age related changes in the human immune system by using polychromatic flow cytometry

Introduction: Aging is associated with a defective immune response. Both the innate and adaptive arms of the immune system are affected with age, causing an overall decline of immunocompetences. With polychromatic flow cytometry (PFC), the project aimed to look deeper at the immune system to identify age and sex-related changes. Designing Panels: Multicolour panel design for profiling young and old lymphocytes obtained from peripheral blood has been completed. To do this, markers of interest were selected to address the aims of the study and then moved to carefully match the markers with the ideal fluorophores, considering the Flow Cytometer available (BD LSR Fortessa X20 – Blue, Red, Violet, Ultraviolet, Yellow/Green). Antibody titrations, Spreading error assessments and machine standardisation experiments were done. Testing was critical and allowed the review of controls necessary for proper analysis of the data. The analysis of optimisation experiments, tests and cohort runs were done by using FlowJo 10th edition. Results: We show that out of fifty four healthy donors with age have an overall weakened T cell immune compartment in both sexes, and display several alterations mostly involving CD8+ T cell subsets. We also define changes in markers of exhaustion (PD1 and TIGIT) and senescence (CD57) with age. Meanwhile, we define an increase with age amongst a very rare T cell subset that has been recently discovered amongst CD8+ TSCM memory cells (CCR7+, CD45RO-, CD95+, TIGIT+, PD1+). In B cell subsets we assess transcription factors PAX5 and XBP1 and for the first time, we report a decrease in the frequencies of XBP1 with age. We also measure mitochondrial superoxide indicators and show changes with age in T cells, but also in B and NK cells, which specify an impaired redox balance with age. NK cells display a decrease of CD56BRIGHT subset mainly driven my males. Conclusion: Overall, the standardized PFC assays can detect multiple biological phenomena and can provide a better understanding of age-related diseases and immunosenescence, thus offering a powerful target for interventions to improve the health of the elderly.