Human NK cells proliferate and die in vivo more rapidly than T cells in healthy young and elderly adults

Abstract

NK cells are essential for health, yet little is known about human NK turnover in vivo. In both young and elderly women, all NK subsets proliferated and died more rapidly than T cells. CD56(bright) NK cells proliferated rapidly but died relatively slowly, suggesting that proliferating CD56(bright) cells differentiate into CD56(dim) NK cells in vivo. The relationship between CD56(dim) and CD56(bright) proliferating cells indicates that proliferating CD56(dim) cells both self-renew and are derived from proliferating CD56(bright) NK cells. Our data suggest that some dying CD56(dim) cells become CD16(+)CD56(-) NK cells and that CD16(-)CD56(low) NK cells respond rapidly to cellular and cytokine stimulation. We propose a model in which all NK cell subsets are in dynamic flux. About half of CD56(dim) NK cells expressed CD57, which was weakly associated with low proliferation. Surprisingly, CD57 expression was associated with higher proliferation rates in both CD8(+) and CD8(-) T cells. Therefore, CD57 is not a reliable marker of senescent, nonproliferative T cells in vivo. NKG2A expression declined with age on both NK cells and T cells. Killer cell Ig-like receptor expression increased with age on T cells but not on NK cells. Although the percentage of CD56(bright) NK cells declined with age and the percentage of CD56(dim) NK cells increased with age, there were no significant age-related proliferation or apoptosis differences for these two populations or for total NK cells. In vivo human NK cell turnover is rapid in both young and elderly adults.

Figure 1

Flow cytometry gating and representative results. Shown are sample gating and Ki67 data for CD56^bright and CD56^dim NK cells from (A) a 24 year old subject and (B) a 77 year old subject. Cells were gated using DNA content (DAPI area) and pulse width (DAPI width) to exclude cell aggregates and debris (panel 1). Forward and side scatter were used to select lymphocytes (panel 2). After selection of CD3⁻ cells (panel 3), NK cells were selected as cells that expressed either CD16 or CD56 or both, in the four boxed areas, excluding the left lower quadrant (panel 4). The CD56^bright and CD56^dim NK subpopulations and the percentage among total NK cells are denoted. Also shown in panel 4 are CD16⁻CD56^low cells (dashed line box) and CD16⁺CD56⁻ cells (dotted line box). Ki67 results are show for CD56^bright and CD56^dim populations (panels 5 and 6). Identical Ki67 gating was used for all NK and T cell populations.

Figure 2

NK cells have high proliferation and apoptosis rates. PBMC were mAb stained and then permeabilized and stained for Ki67 expression (A) or treated with TUNEL reagents (B). Alternatively, viable and nonviable cells were assessed by forward and side scatter characteristics (C). Shown are means and 95% confidence limits for combined results with young and elderly subjects. Significance of differences between selected groups is indicated, *, p = 0.0003; **, p < 0.0001.

Figure 3

CD16⁻CD56^low NK cells rapidly respond to cellular and cytokine stimulation. NK cells were stimulated for 2 h as indicated, and assayed for cell surface CD107a (A, B), intracellular IFN-γ (C), and cell surface CD69 (D). Results are presented as % positive cells in each indicated NK subset (A, C, D) or as geometric mean fluorescence intensity on the positive cells (B). Results shown from 1 subject are representative of 3 subjects, each analyzed in a separate experiment.

Figure 4

The number of proliferating CD56^dim cells is directly related to the number of proliferating CD56^bright NK cells. The relative number of proliferating CD56^bright and CD56^dim cells was calculated as (% of cells in subset × % Ki67⁺)/100. The least squares line, Spearman correlation coefficient (r_s), and significance of association (p) are indicated. r_s = 1.0 or −1.0 denote perfect correlation; r_s = 0 denotes no correlation. Young (circles) and elderly (diamonds) analyzed separately showed similar correlations (p< 0.0001).

Figure 5

CD56^dim proliferation, but not CD56^bright proliferation, correlates with percentage CD56^bright NK cells. Ki67 expression in the CD56^dim subset (A) and the CD56^bright subset (B) are plotted against CD56^bright cells as a % of NK cells. A) The Spearman correlation coefficient (r_s) and significance of association are indicated; young (circles) and elderly (diamonds) analyzed separately showed similar correlations and were significant. B) No significant correlations were obtained when young (circles) and elderly (diamonds) were analyzed together or separately.

Figure 6

CD57⁺ T and NK cells proliferate in vivo. The ratio of % Ki67 expression in CD57⁺ cells and CD57⁻ cells within each indicated lymphocyte subset is shown as mean + 95% confidence limit. Differences from an equal ratio are indicated (* p = 0.001; ** p = 0.0005).

Figure 7

Proliferating CD57⁺ T cells do not have low density CD57 expression. For each T cell subset (CD8⁺ top panel, CD8⁻ bottom panel), we defined CD57^hi and CD57^low expression as above and below median CD57 surface staining intensity, respectively. Based on this median, we calculated a ratio of CD57^hi to CD57^low cells among the Ki67⁺ proliferating population. A ratio > 1 indicates that more proliferating cells were CD57^hi than CD57^low.

Figure 8

Model of human NK turnover. CD56^dim cells are maintained by cell division (represented by curved arrow above the oval; arrow thickness is proportional % Ki67⁺). CD56^dim cells also are maintained by input from CD56^bright cells and some input from immature CD16⁺CD56⁻ NK cells (represented by thin arrows between populations). CD56^dim cells may temporarily lose CD16 expression following stimulation. At steady state, the number of new CD56^dim cells is balanced by change of surface phenotype (e.g., loss of CD56 expression) and by death (represented by skull and crossbones; arrow thickness is proportional to % TUNEL⁺). Some Ki67⁺ CD56^dim cells are derived from dividing CD56^bright cells; some dying CD16⁺CD56⁻ cells are derived from apoptotic CD56^dim cells.