Increased PD-1+ NK Cell Subset in the Older Population

Abstract

Background: The aging of the immune system is associated with various diseases. It is worth exploring the changes of the immune system in aging. Previous studies have shown that aged T cells have enhanced expression of co-inhibitory molecules. However, it remains unclear whether aged NK cells exhibit similar characteristics to aged T cells. The objective of our research was to clarify this aspect.

Patients and methods: This study included 98 adults aged 24-90 years (50 males and 48 females). We detected the subset of peripheral blood NK cells and the expression of various receptors on NK cells among donors of different age groups by flow cytometry. Immune subsets were initially defined by forward and side-scatter characteristics and then staining with the appropriate marker.

Results: The absolute number and subset distribution of NK cells were not associated with age. However, CD57 expression and CD69 expression were correlated with age. Furthermore, we found that PD-1 was up-regulated on NK cells in older people, associated with aging, while no such change was observed in other co-inhibitory molecules, including 2B4, CTLA-4, TIM-3, BTLA, CD70, CD39, CD160, and TIGIT. PD-1⁺ NK cells expressed high levels of CD57 and CD69, indicating PD-1⁺ NK cells displayed a phenotype of over-activation and aging.

Discussion: This study indicated that PD-1⁺ NK cells were one of the characteristics of NK cells in older people.

Conclusion: This study indicated that PD-1⁺ NK cells were one of the characteristics of NK cells in older people. Those findings provided new ideas to explore the underlying drivers of NK aging.

Keywords: NK cell; PD-1; aging; co-inhibitory molecules.

Figure 1

Changes of absolute number, subset distribution and phenotype on NK cells with age. Flow cytometry analysis of NK cell absolute number, subset distribution and phenotype was performed on PBMCs collected from donors of different ages. (A–D) Box plots of the absolute number of NK cells (A, left), CD56^brightCD16⁻ NK cells (B, left), CD56^dimCD16⁺ NK cells (C, left) and CD56^negCD16⁺ NK cells (D, left) from donors in different age groups (n = 14–22 each group). Correlation analysis of the absolute number of NK cells (A, right), CD56^brightCD16⁻ NK cells (B, right), CD56^dimCD16⁺ NK cells (C, right) and CD56^negCD16⁺ NK cells (D, right) with age from all donors. (E–G) Box plots (left) of the percentage of NK cells subset from donors in different age groups (n = 14–22 each group) and correlation analysis (right) of the percentage of NK cells subset with age from all donors, including CD56^brightCD16⁻ NK cells (E), CD56^dimCD16⁺ NK cells (F) and CD56^negCD16⁺ NK cells (G). (H and I) Box plots of the percentage of CD57⁺ (E, left) and CD69⁺ (F, left) NK cells from donors in different age groups (n = 14–22 for each group). Correlation analysis of CD57 expression (E, right) or CD69 expression (F, right) with age on NK cells from all donors. P values were obtained by the Kruskal–Wallis test followed by Dunn’s multiple comparisons test. Spearman’s non-parametric test was used to test for correlations. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Figure 2

Expression of co-inhibitory molecules on NK cells in different age groups. Flow cytometry analysis of expression of 2B4 (A), CTLA-4 (B), TIM-3 (C), BTLA (D), CD70 (E), CD39 (F), CD160 (G) and TIGIT (H). Box plots (left) of the positive percentage of the above receptors on NK cells from donors in different age groups (n = 14–22 for each group). Correlation analysis (right) of the expression of the above receptor with age on NK cells from all donors. P values were obtained by the Kruskal–Wallis test followed by Dunn’s multiple comparisons test. Spearman’s non-parametric test was used to test for correlations. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 3

PD-1-positive NK cells accumulated in elder populations. (A) Representative flow data show the expression of PD-1 gated on NK cells from two donors in two age groups. (B and C) Box plots of the percentage of PD-1⁺ cells among NK cells (B, left), CD56^dimCD16⁺ NK cells (C, left) and CD56^brightCD16⁻ NK cells (D, left) from donors in different age groups (n = 14–22 each group). Correlation analysis of the percentage of PD-1⁺ cells among NK cells (B, right), CD56^dimCD16⁺ NK cells (C, right) and CD56^brightCD16⁻ NK cells (D, right) with age on NK cells from all donors. P values were obtained by the Kruskal–Wallis test followed by Dunn’s multiple comparisons test. Spearman’s non-parametric test was used to test for correlations. *P < 0.05, **P < 0.01.

Figure 4

Other receptors on PD-1⁺ and PD-1⁻ NK cells. Flow cytometry analysis of CD57 (A), CD69 (B), CD160 (C) and 2B4 (D), TIGIT (E) and CD70 (F) expression on PD-1⁻ vs PD-1⁺ NK cells from the elderly (60–90 years, n = 26). Representative histograms (left) and plots (right) displayed the expression of the above receptors PD-1⁻ vs PD-1⁺ NK cells from the same individual. The p-values were obtained by paired t-test (CD57, CD69, CD160, TIGIT) or Wilcoxon matched-pairs signed rank test (2B4 and CD70).

Figure 5

Correlation of PD-1 expression and other receptors expression on NK cells. (A–F) Correlation analysis of 2B4 (A), CD57 (B), CD69 (C), CD160 (D), TIGIT (E) and CD70 (F) expression with PD-1 expression on NK cells from the elderly (60–90 years, n = 26). Spearman’s non-parametric test was used for correlation analysis.