Human Stem Cell-like Memory T Cells Are Maintained in a State of Dynamic Flux

Abstract

Adaptive immunity requires the generation of memory T cells from naive precursors selected in the thymus. The key intermediaries in this process are stem cell-like memory T (T_SCM) cells, multipotent progenitors that can both self-renew and replenish more differentiated subsets of memory T cells. In theory, antigen specificity within the T_SCM pool may be imprinted statically as a function of largely dormant cells and/or retained dynamically by more transitory subpopulations. To explore the origins of immunological memory, we measured the turnover of T_SCM cells in vivo using stable isotope labeling with heavy water. The data indicate that T_SCM cells in both young and elderly subjects are maintained by ongoing proliferation. In line with this finding, T_SCM cells displayed limited telomere length erosion coupled with high expression levels of active telomerase and Ki67. Collectively, these observations show that T_SCM cells exist in a state of perpetual flux throughout the human lifespan.

Keywords: CD4(+) T cells; CD8(+) T cells; adaptive immunity; in vivo heavy water labeling; memory T cell maintenance; memory T cells; proliferation; stem cell-like memory T cells; telomerase activity; telomere length.

Graphical abstract

Figure 1

Label Incorporation in Naive and Stem Cell-like Memory T Cells (A) Schematic representation of the ²H₂O-labeling protocol and sampling time points. (B) Successive panels depict the flow cytometric gating strategy used to sort CD4⁺ and CD8⁺ T_N and T_SCM cells. Lymphocytes were identified in a forward-scatter versus side-scatter plot, and single cells were resolved in a forward-scatter-height versus forward-scatter-area plot. Boolean gates were drawn for analysis only to exclude fluorochrome aggregates. Live CD3⁺CD14^–CD19^– cells were assigned to the CD4⁺ or CD8⁺ lineage, and potentially naive CD27^brightCD45RO^– cells were separated from memory T cells. Sort gates were then fixed on CCR7⁺CD95^– T_N cells and CCR7⁺CD95⁺ T_SCM cells. Histogram overlays show expression of CD28, CD45RA, CD57, and CD127 in the T_N, T_SCM, and memory subsets. (C) Schematic representation of the mathematical models applied to the labeling data. In the depicted variation, a precursor compartment replenishes T_N cells, which do not proliferate. Two further variations were considered, one eliminating the precursor compartment, and the other assuming T_N cell proliferation. Similar results were obtained with all three variations. (D) Experimental labeling data (black filled circles) and modeled curve fits for subject DW01 (young adult). The curve fits for model 1 overlie the curve fits for model 2.

Figure 2

Comparative Label Enrichment in Naive, Stem Cell-like Memory and Other Memory T Cells Experimental labeling data for T_N, T_SCM, CD45RA^– memory, and CD45RA⁺CD45RO⁺ transitional memory T cells from subjects DW01, DW09, DW10, and DW11 (young adults), and DW04, DW03, and DW02 (elderly).

Figure 3

Ki67 Expression in Naive, Stem Cell-like Memory, and Other Memory T Cells (A) Intracellular Ki67 expression in the depicted T cell subsets from subject DW01 (young adult). Live CD3⁺CD14^–CD19^– lymphocytes within the CD4⁺ and CD8⁺ lineages were identified as shown in Figure 1B. Conservative gates were placed around CCR7⁺CD95^– T_N cells and CCR7⁺CD95⁺ T_SCM cells within a naive-like phenotype (CD45RA^brightCCR7⁺). (B) Intracellular Ki67 expression in CD4⁺ (left) and CD8⁺ (right) T cell subsets from healthy adult volunteers and subject DW01 (young adult). Peripheral blood mononuclear cells were stained in triplicate directly ex vivo. Horizontal bars represent mean values with SEs. T_CM (CD45RA^–CCR7⁺); T_EMRA (CD45RA⁺CCR7^–). Significance was assessed using a two-tailed Mann-Whitney test. Asterisks indicate p < 0.001 for all comparisons.

Figure 4

Telomere Lengths in Naive and Stem Cell-like Memory T Cells (A) Representative single telomere length analysis data from subjects DW02 (elderly), DW01 (young adult), and DW04 (elderly). Single telomere length analysis was conducted at the XpYp telomere for CD4⁺ and CD8⁺ T_N and T_SCM cells. Mean values and telomere length differentials are shown (bottom). (B) XpYp telomere length distributions as scatterplots. Significance was assessed using a two-tailed Mann-Whitney test.