Investigating the heterogeneity of the human immune memory compartment

Abstract

Adaptive immunity is defined by the generation and maintenance of memory cells. T-stem cell memory (TSCM) cells are a minimally differentiated subset considered to be the apex of long-term immune memory (Gattinoni et al., 2011). Stable isotope labelling of human TSCM cells revealed kinetic heterogeneity within the compartment, exhibited by at least two subpopulations: A short-lived population with high turnover and a long-lived population with slower turnover and longer half-life (Costa Del Amo et al., 2018). To identify the "true" TSCM subset, we sought to identify and characterize the two subpopulations in vitro. Flow cytometry analysis and cell sorting were used for immunophenotyping of the TSCM compartment. To uncover the multipotency and self-renewal of the two subsets, cells were cultured in vitro under different stimuli. Transcriptional differences were assessed via bulk and single cell RNAseq along with qPCR analysis. Finally, mathematical modelling of samples from healthy individuals labelled with 2H20 was applied to identify the kinetics of the two subpopulations in vivo. We show that the two subpopulations can be distinguished by CD95 expression levels. CD95hi TSCM cells show increased proliferation compared to CD95int TSCM cells by Ki67 staining and measurement of TRECs. Furthermore, CD95hi cells demonstrated the highest self-renewal potential compared to CD95int under stimulation with homeostatic cytokines. qPCR analysis highlighted transcriptional differences with CD95int cells resembling a naive-like state whereas CD95hi cells were closer transcriptionally to central memory (TCM) cells. Finally, CD95hi cells incorporated more deuterium label, suggesting increased division in vivo compared to CD95int subset. In conclusion, we show that CD95 expression can distinguish the two proposed subpopulations of the TSCM compartment, and although the subsets differ in function both play a role in memory maintenance. We propose that CD95hi cells exhibit self-renewal under homeostasis generating other memory subsets while CD95int cells divide less and replenish the CD95hi population.