Immunosenescence, the term researchers use for the structural and functional reorganization of immunity over a lifetime, is closely tied to increased vulnerability to infections, weaker vaccine responses, and a heavier burden of chronic disease. While flow cytometric profiling of blood lymphocyte subsets has become a standard tool for characterizing this process, systematic age-related data on natural killer cell subsets and B cell subsets in healthy Chinese adults have remained scarce. Quantitative trends for CD57 and TEMRA cells, widely accepted markers of terminal T cell differentiation, have also been incompletely described in this population. The new study was designed to fill that gap with two complementary 12-color flow cytometry panels applied across a broad age range.

The researchers prospectively enrolled 146 healthy adults attending the hospital’s annual health examination center between 2023 and 2024. Panel 1 included 103 participants aged 20 to 82 years, while Panel 2 included 43 participants aged 25 to 92 years, deliberately enriched for older individuals to strengthen statistical power at the upper end of the age spectrum. Eligibility required no clinically significant abnormalities on routine examination, and the team excluded anyone with autoimmune disease, chronic infections including HIV, hepatitis B, or hepatitis C, hematological disorders, recent immunosuppressive treatment, acute infection within the preceding four weeks, pregnancy, or evidence of malignancy or severe organ dysfunction.

Panel 1 was a single-tube 12-color assay that simultaneously covered T cells, including CD57 expression, the three-way classification of NK cells by CD56 and CD16 intensity, and the four-way classification of B cells by IgD and CD27. Panel 2 was a dual-tube 12-color assay focused on detailed T cell differentiation, incorporating CD45RA and CD62L to resolve naive, central memory, effector memory, and TEMRA subsets, along with CD28, CD31, CD38, and HLA-DR. At least 100,000 lymphocyte events were acquired per tube on a BD LSRFortessa instrument. Statistical analysis employed Spearman rank correlation, Kruskal-Wallis testing, and Mann-Whitney U tests, with multivariate models controlling for sex.

The strongest age-related signals emerged in the CD8-positive T cell compartment. TEMRA accumulation showed a correlation coefficient of +0.532, CD28 loss reached -0.552, and naive cell depletion reached -0.534, all with p values below 0.001. Together these three changes form a coherent triad representing the hallmarks of replicative senescence in CD8 T cells, painting a picture of progressive, co-stimulation-independent terminal differentiation that dominates this compartment in older individuals. Activation and proliferation markers reinforced the pattern: CD8 cells expressing HLA-DR, CD38 together with HLA-DR, and Ki67 all rose significantly with age, indicating that despite their differentiated phenotype, CD8 T cells in older adults remain in a state of chronic activation.

In the CD4 compartment, two significant age-related changes stood out. CD4-positive CD31-positive cells, which serve as a marker of recent thymic emigrants, declined with age at a correlation of -0.361, consistent with thymic involution progressively curtailing the output of new naive T cells. CD4-positive naive cells also showed a significant decline at -0.303. In Panel 1, the two strongest correlations were both CD57-related: CD4-positive CD57-positive cells rose at +0.421 and CD8-positive CD57-positive cells at +0.338, both highly significant, reflecting progressive accumulation of terminally differentiated T cells across both major subsets. When all 146 participants were pooled for shared markers, both CD4-positive and CD8-positive PD-1 expression became significantly positive, suggesting that exhaustion-like checkpoint upregulation accompanies terminal differentiation as a co-occurring feature of immune aging.

The NK cell findings revealed a more nuanced picture. Total NK cell frequency increased with age, yet within that expanding pool the CD56-high CD16-negative regulatory subset contracted significantly, while the cytotoxic CD56-dim CD16-positive fraction showed no significant proportional change. NK cell PD-1 expression also rose with age, a less commonly reported observation suggesting that NK cells, like T cells, may undergo progressive phenotypic exhaustion. In segmented analysis of the combined dataset, the atypical CD56-negative CD16-positive subset showed no significant overall age correlation but became significantly positively associated with age in participants aged 65 and above, reaching a correlation of +0.482. The researchers caution that the formal slope-change test did not reach significance, so this pattern is best described as a late-life tendency requiring replication rather than a confirmed threshold effect.

B cell aging followed a different timeline. In Panel 1, total B cell frequency was stable across the age range, and among the four IgD/CD27 subsets only the IgD-positive CD27-positive marginal zone-like population showed a significant decline at -0.257. This subset represents antigen-experienced but unswitched B cells, and its contraction may reflect reduced T-independent responses with aging. The more dramatic changes emerged in Panel 2, where total B cell frequency declined significantly at -0.381 and switched memory B cells contracted at -0.379. That these changes appeared in an older-enriched cohort with a median age of 70 but not in the broader Panel 1 sample suggests that substantial B cell contraction may be a relatively late phenomenon, becoming apparent mainly in the oldest age ranges.

Sex was a meaningful source of variation throughout the study. Women tended to have higher CD8-positive fractions and lower CD4-to-CD8 ratios than men of the same age, a pattern consistent with established sex differences in immune aging. Both panels showed higher CD31-positive fractions in women, consistent with greater thymic output or slower naive T cell attrition. Men had higher NK frequency in Panel 2. Critically, however, multivariate analysis showed that sex did not explain the primary age effects; all ten significant age correlations in Panel 1 survived adjustment for sex, and the three core CD8 markers remained strongly significant regardless. The researchers note that the male-heavy composition of Panel 1, at 83.5 percent, introduces some interpretive caution and encourage sex-stratified reporting when these data are used as clinical reference ranges.

To integrate the multidimensional age signal into a single summary measure, the team constructed an Immunological Composite Score from nine parameters showing consistent, sex-independent age associations across both panels. Each parameter was z-scored and combined with directional signs aligned to the age correlation direction. Across all 146 participants, the composite score correlated strongly with chronological age at +0.575, and Kruskal-Wallis testing demonstrated highly significant group separation between young, middle-aged, and older cohorts. The older group differed significantly from both the young and middle-aged groups, while the young and middle-aged groups did not differ significantly from each other. The authors emphasize that the score is presented as a proof-of-concept instrument, derived from and validated in the same dataset, and that external prospective validation in an independent cohort with longitudinal follow-up and clinical endpoints such as infection susceptibility or vaccine immunogenicity is required before any clinical application.

Subject of Research: Age-related changes in T cell, NK cell, and B cell subsets measured by multicolor flow cytometry in healthy Chinese adults

Article Title: Age-associated remodeling of NK cell subsets, B cell subsets, and T cell surface markers in peripheral blood of healthy Chinese adults: a prospective multicolor flow cytometry study

Article References: Zheng, L., Cao, W., Wang, Y., Xu, T., Wang, F., Zhang, L., Liu, X., & Li, T. (2026). Age-associated remodeling of NK cell subsets, B cell subsets, and T cell surface markers in peripheral blood of healthy Chinese adults: a prospective multicolor flow cytometry study. Frontiers in Aging, 7, Article 1847033. https://doi.org/10.3389/fragi.2026.1847033

Image Credits: AI Generated

DOI: 10.3389/fragi.2026.1847033

Keywords: immunosenescence, flow cytometry, T cells, NK cells, B cells, TEMRA, CD57, PD-1, aging, healthy Chinese adults, Peking Union Medical College Hospital, immune aging