An updated immunosenescence exploration in healthy Chinese donors: circular elevated PD-1 on T cell and increased Ki67 on CD8+ T cell towards aging

Materials and Methods

Subjects

A cross-sectional study was conducted between June and September 2022 among 150 healthy adults, including 90 males and 60 females, aged over 18 years. Subjects testing positive to HIV, systemic infection, connective tissue disease, abnormal tumor marker or cancer were excluded. According to the defined criteria from the SENIEUR protocol guideline [14], subjects were classified as older (≥65 years), middle-aged (45–64 years), and younger (18–44 years). All subjects received informed consent, and the ethics committee of Peking Union Medical College Hospital approved the study (Ethics number: I-23PJ463).

Lymphocyte count and phenotyping analysis

Fresh EDTA-anticoagulated whole blood was collected and treated by flow cytometry within 6 h. Eighteen color flow cytometry (LSRFortessa and trade; BD Biosciences, La Jolla, CA, USA) analyzed peripheral blood lymphocyte subsets. Isolated PBMC from whole blood was incubated and tested with a panel of monoclonal antibodies against: anti-CD56-PE/anti-PerCP-cy5.5-HLA-DR/anti-CD38-APC/anti-CD3-PE-cy7/anti-CD8-APC-cy7/anti-CD4-AF700/anti-CD19-V450/anti-CD45-V500C/anti-PD-1 BV605/anti-CD45RA-FITC/anti-CD62L-PE/anti-CD28-PerCP-cy5.5/anti-CD25-APC/anti-CD127-V450/anti-CD31-BV421. Antibodies for this study were purchased from BD Pharmingen (San Diego, CA, USA). The samples were analyzed using FACSDiva software (Becton Dickens, Franklin Lakes, NJ, USA). The gating strategy for lymphocyte subsets is shown in Figure 1. Cell counts for lymphocyte subsets were calculated using a two-platform approach, where white blood cell counts and lymphocyte differentials were obtained from routine blood tests of the same specimen.

Figure 1. Gating strategy. Cells were first gated for lymphocytes (SSC-A vs. CD45), then CD3+ T cells, CD4+ T cells, CD8+ T cells, CD56+ NK cells and CD19+ B cells were positively determined. Next, the subtle subsets underlying each type of cell were further identified and differentiated.

Statistical analysis

All data parameters were subject to normality testing using the Shapiro–Wilk normality test. The continuous variables were described as mean or median and compared by Kruskal–Wallis Rank Sum Test or Mann–Whitney U-test when data did not conform to a normal distribution. Probability value was obtained from two-sided tests and P < 0.05 were considered significant. Statistical analysis was performed with SPSS software (SPSS^® for Windows™ version 21.0, SPSS Inc, Chicago, IL, USA) and GraphPad Prism software (GraphPad Software^® for Windows™ version 9.0, Boston, MA, USA).

Results

The characteristics of study population

A total of 150 healthy Chinese adults were recruited in this study, including 90 men (60.0%) and 60 women (40.0%) with age spanning from 19 to 83. In this study, the 95% confidence interval recommended by the International Committee for Clinical Laboratory Standardization was used to determine the detailed distributions of lymphocyte subsets by the combination of normal distribution method and percentile method, as shown in Table 1.

Table 1. Peripheral blood lymphocyte subset reference values.

Parameters		Mean	Median	Std. deviation	Range	Min–Max
Lymphocyte	(%)	26.2	29.7	5.4	20.6–35.8	20.6–52.7
	(cells/μl)	1936	1901	466	1551–2321	950–2947
CD19+ B	(%)	9.96	10.00	3.8	7.0–12.0	2.2–22.7
	(cells/μl)	190	186	80	131–247	18–434
CD16+CD56+ NK	(%)	16.6	15.0	6.8	12.0–21.2	2.1–37.0
	(cells/μl)	324	301	162	205–403	36–789
CD3+ T	(%)	69.1	70.8	8.5	62.3–75.5	44.9–86.5
	(cells/μl)	1338	1292	362	1091–1553	509–2260
CD3+CD4+/CD3+	(%)	52.5	51.9	10.5	44.3–60.1	27.7–77.5
	(cells/μl)	702	644	250	525–842	233–1506
CD3+CD8+/CD8+	(%)	37.9	37.5	9.6	32.3–45.0	16.0–65.3
	(cells/μl)	506	485	187	358–609	95–1013
CD4+/CD8+	(%)	1.6	1.4	0.8	1.00–1.80	0.42–4.49
CD4+CD25+CD127-/CD4+	(%)	6.3	6.2	2.0	5.9–6.7	1.2–11.0
	(cells/μl)	42	39	18.0	34–43	10–103
CD4+CD45RA-/CD4+	(%)	66.0	65.3	13.6	56.5–75.0	29.5–98.3
	(cells/μl)	456	439	171	333–549	194–1141
CD4+CD45RA+/CD4+	(%)	37.7	36.0	13.8	25.0–43.5	1.7–70.5
	(cells/μl)	455	439	176.9	291–465	20–1141
CD4+CD45RA+CD62L+/CD4+	(%)	34.2	35.0	13.6	25.0–43.8	1.9–69.2
	(cells/μl)	247	228	155	141–324	14–1037
CD4+CD28+/CD4+	(%)	91.6	94.8	9.9	87.4–97.9	48.4–99.9
	(cells/μl)	635	606	226	477–770	175–1478
CD4+CD38+/CD4+	(%)	10.6	8.8	4.1	5.2–14.3	1.0–29.7
	(cells/μl)	75	59	35.1	37–98	4–286
CD4+HLA-DR+/CD4+	(%)	7.3	6.1	4.0	4.2–9.6	1.3–18.0
	(cells/μl)	51	49	30	25–67	8–229
CD4+CD38+HLA-DR+/CD4+	(%)	1.0	0.7	0.3	0.5–1.1	0.1–10.0
	(cells/μl)	6	5	2	3–8	0–40
CD4+PD-1+/CD4+	(%)	14.5	13.5	5.2	10.5–18.1	2.5–28.9
	(cells/μl)	99	93	38	63–129	14–257
CD4+Ki-67+/CD4+	(%)	0.8	0.7	0.5	0.3–1.2	0.1–2.3
	(cells/μl)	6	5	3	2–8	0–19
CD4+CD45RA+CD31+/CD4+	(%)	23.5	24.2	11.9	14.2–31.3	0.6–59.8
	(cells/μl)	170	157	120	83–229	4–896
CD8+CD45RA-/CD8+	(%)	48.7	47.3	15.4	35.8–60.8	19.4–85.3
	(cells/μl)	244	219	125	161–313	40–781
CD8+CD45RA+CD62L+/CD8+	(%)	27.4	24.7	16.4	13.8–39.6	1.6–71.2
	(cells/μl)	137	113	96	62–204	6–464
CD8+CD28+/CD8+	(%)	52.5	53.8	20.2	36.2–67.2	13.6–90.0
	(cells/μl)	254	240	122	165–320	45–789
CD8+CD38+/CD8+	(%)	4.6	4.0	3.6	2.0–5.7	0.3–19.0
	(cells/μl)	23	17	20	9–30	2–111
CD8+HLA-DR+/CD8+	(%)	12.9	10.7	7.9	6.8–18.0	2.2–33.1
	(cells/μl)	66	50	42	27–96	8–308
CD8+CD38+HLA-DR+/CD8+	(%)	1.9	1.7	1.2	0.9–2.5	0.1–6.0
	(cells/μl)	10	8	6	4–13	1–45
CD8+PD-1+/CD8+	(%)	13.7	14.1	5.5	9.5–16.5	3.6–30.5
	(cells/μl)	69	61	37	41–88	9–221
CD8+Ki-67+/CD8+	(%)	0.7	0.5	0.3	0.3–0.8	0.1–2.1
	(cells/μl)	3	2	1	1–4	1–11
CD19+CD38+/CD19+	(%)	17.8	15.7	10.5	9.3–24.0	2.5–49.9
	(cells/μl)	34	27	26	15–48	2–200
CD19+PD-1+/CD19+	(%)	2.3	1.7	1.5	0.9–3.1	0.2–6.4
	(cells/μl)	3	2	1	2–5	1–14
CD19+Ki-67+/CD19+	(%)	1.2	0.9	0.6	0.6–1.3	0.1–3.8
	(cells/μl)	2	2	1	1–3	1–7
CD16+CD56+CD38+/CD16+CD56+	(%)	42.9	43.5	19.3	28.4–58.7	8.6–82.1
	(cells/μl)	131	111	90	75–161	14–631
CD16+CD56+HLA-DR+/CD16+CD56+	(%)	6.3	4.5	3.5	2.1–9.0	0.8–20.9
	(cells/μl)	21	13	13	6–31	1–112
CD16+CD56+PD-1+/CD16+CD56+	(%)	1.6	0.8	0.7	0.3–1.7	0.1–7.5
	(cells/μl)	5	3	3	1–5	1–35
CD16+CD56+Ki-67+/CD16+CD56+	(%)	1.1	0.9	0.7	0.4–1.6	0.1–3.8
	(cells/μl)	3	2	3	1–4	1–20

The relationship between lymphocyte subsets and aging

To investigate the influence of the age on the lymphocyte subsets, we divided the subjects into three age groups. A total of 55 (36.6%) were in young adult group (19–44 years old, 34 males, 21 females, mean age 29.2 years), 46 (30.7%) belonged to the middle-aged adult group (45–64 years old, 24 males, 22 females, mean age 52.6 years) and 49 (32.7%) belonged to the elderly (over 65 years, 31 males, 18 females, mean age 68.0 years). The Chi-square tests showed that there were differences between the gender in different age groups. (P = 0.045). We then observed associations between a set of parameters and age (results shown in Figure 2). Most parameters varied with age except for CD19+ B cells (p = 0.304), CD16+CD56+ NK cells (p = 0.727), CD3+ T cells (p = 0.966), CD3+CD4+ T cells (p = 0.162) and CD3+CD8+T cells (p = 0.204), especially a clear association was shown in the CD4 and CD8 T cell subsets combinations. Gender impacts the fluctuation of many physiological parameters, furtherly, the lymphocyte subsets distributions correlated with age in males and females at different stages were evaluated and regression analysis was performed.

Figure 2. Correlation and regression analysis of different T cell subsets and ages were calculated. The left represents the frequency, and the right represents the absolute number. The red points and bars represent the R-value and 95% confidence interval of the regression equation, and the P-value to the right of the figure indicates the statistical significance of each subset.

T cell subsets decreased with older ages in male and female populations

The results of subgroup distribution of males and females showed that CD4/CD8 percentage and the percentage of CD4+CD25+CD127-T cells had slightly increased or decreased trend with age (Figure 3A, 3F), whereas there was no significant difference among further groups (Figure 4A, 4F). Data showed a moderate decreased expression level of CD4+CD45RA+ T cells percentage (r = −0.303, p = 0.002, Figure 3C) and CD4+CD45RA+CD62L+ T cells percentage (r = −0.326, p < 0.001, Figure 3D) with age growing in males (Figure 4C, 4D). Meanwhile, it was found that aging did not seem to have a significant effect on women, but still showed an obvious downward trend. Significant differences existed in CD8+CD45RA+CD62L+ T cells and CD4+CD45RA+CD31+ T cells between the gender in the whole age stages and the difference mainly lay in males (Figure 4H, 4E). Figure 3E, 3H shows the strong negative correlation between these two types of cells with age (r = −0.598, P < 0.001 and r = −0.3379, p < 0.001). A trend of decrease in CD4+CD38+ T cells and CD8+CD38+ T cells were also observed with age (r = −0.446, P < 0.001 and r = −0.200, P < 0.001, Figure 3I, 3M), but there was only obvious difference with age growing in the males (Figure 4I). Differences in the percentage of CD8+CD38+ T cells mainly lay in the older group, and women were higher than men (Figure 4M). The same changes can also be observed in CD8+CD28+ T cells (Figure 4L), with a strong negative correlation in the CD8+CD28+ T percentage (r = −0.456, P < 0.001, Figure 3L), particularly evident in females.

Figure 3. Relationship between age and percentages of lymphocyte subsets in the male or female population. (A) CD4+/CD8+ percentage, (B) CD4+CD45RA- T cell percentage, (C) CD4+CD45RA+ T cell percentage, (D) CD4+Naïve T cell percentage, (E) CD4+CD45RA+CD31+T cell percentage, (F) CD4+CD25+CD 127- T cell percentage, (G)CD8+CD45RA- T cell percentage, (H) CD8+Naive T cell percentage, (I) CD4+CD38+ T cell percentage, (J) CD4+HLA-DR+ T cell percentage, (K) CD8+HLA-DR+ T cell percentage, (L) CD8+CD28+ T cell percentage, (M) CD8+CD38+ T cell percentage, (N) CD4+PD-1+ T cell percentage, (O) CD8+Ki67+ T cell percentage, (P) CD8+PD-1+ T cell percentage. Correlations between two variables were done using Spearman’s correlation and linear regression was used to plot graph.

Figure 4. Comparison of lymphocyte subsets in different age groups of male or female populations with significant differences. (A) CD4+/CD8+percentage (%), (B) CD4+CD45RA- T cell (% and cells/μl), (C) CD4+CD45RA+ T cell (% and cells/μl), (D) CD4+ Naïve T cell (% and cells/μl), (E) CD4+CD45RA+CD31+T cell (% and cells/μl), (F) CD4+CD25+CD127- T cell percentage (%), (G) CD8+CD45RA- T cell (% and cells/μl), (H) CD8+Naive T cell (% and cells/μl), (I) CD4+CD38+ T cell (% and cells/μl), (J) CD4+HLA- DR+ T cell (% and cells/μl), (K) CD8+HLA-DR+ T cell (% and cells/μl), (L) CD8+CD28+ T cell (% and cells/μl), (M) CD8+CD38+ T cell (% and cells/μl), (N) CD4+PD-1+ T cell (% and cells/μl), (O) CD8+Ki67+ T cell (% and cells/μl), (P) CD8+PD-1+ T percentage (%). (^*p < 0.05, ^**p < 0.01, ^***p < 0.001).

T cell subsets increased significantly with age between male and female individuals

Compared with CD45RA+ T cells, the reverse trend between CD4+CD45RA-T cells (r = 0.251, p = 0.001, Figure 3B) and CD8+CD45RA-T cells (r = 0.258, p = 0.018, Figure 3G) were also shown in our study which gradually increased with age (Figure 4B, 4G). Significant gender difference was found among three cohorts respectively in memory CD8+ T cells and were higher in women (Figure 4G). Data showed an increased expression level of CD4+HLA-DR+ T cells (r = −0.254, p = 0.002, Figure 3J) and the percentage of CD8+HLA-DR+ T cells (r = −0.226, p < 0.001, Figure 3K) with age growing. Meanwhile, it was found that age seem to have a significant effect on men between the older and the younger, and that men were significantly higher than women in the older group (Figure 4J). The same changes could be seen in CD8+HLA-DR+ percentage (Figure 4K). More interestingly, we found a weak positive correlation between age and CD4+PD-1+ T cells (r = 0.218, p = 0.016, Figure 3N), as well as CD8+PD-1+ percentage (r = 0.241, p < 0.001, Figure 3P). Age-related variation was obvious with CD4+PD-1+ T cells in the males (Figure 4N). Similarly, the percentage of CD8+PD-1+showed increased expression level with age growing in the males, and the gender difference was significant in the older group (Figure 4P). An increased trend of CD8+ki-67+ percentage was also observed with age (r = 0.201, p < 0.001, Figure 3O), and the obvious difference existed in men versus women towards the younger and the other older groups (Figure 4O).

Author Contributions

YC contributed to the design, and interpretation of the results and drafts of the manuscript. Other authors were involved in the supervision, funding acquisition, and manuscript review. All authors contributed to the article and approved the submitted version.

Conflicts of Interest

The authors declare no conflicts of interest related to this study.

Ethical Statement and Consent

All subjects received informed consent, and the ethics committee of Peking Union Medical College Hospital approved the study (Ethics number: I-23PJ463).

Funding

This work was funded by Special Research Fund for the Chinese Academy of Medical Sciences (CAMS) Innovation Fund for Medical Sciences (Grant Number: 2021-I2M-1-037), and Central High-level Hospitals of Peking Union Medical College Hospital (Grant Number: 2022-PUMCH-D-008), and Central High-level Hospitals of Peking Union Medical College Hospital (Grant Number: 2022-PUMCH-B-117).

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