Heterogeneity of thymic output in the elderly and its association with sex and smoking

Abstract

BACKGROUNDThymic involution with age leads to reduced T cell output and impaired adaptive immunity. However, the extent to which thymic activity persists later in life and how this contributes to immunological aging remains unclear. This study aimed to assess the presence and function of thymic tissue in older adults and identify factors influencing residual thymopoiesis.METHODSPatients aged 50 or older undergoing cardiothoracic surgery were recruited. Thymic structures within mediastinal adipose tissue were evaluated using histology, immunofluorescence, flow cytometry, T cell receptor (TCR) sequencing, and RNA sequencing. Recent thymic emigrants (RTEs) were quantified in peripheral blood and correlated with transcriptomic, epigenetic, and TCR repertoire data. Primary outcomes included thymic tissue identification, RTE frequency, and immune correlates.RESULTSFunctional thymic tissue was identified in mediastinal adipose tissue of older individuals. The frequency of CD31+CD4+ T cells (RTEs) positively correlated with the presence of thymic tissue. Thymic output showed substantial heterogeneity and was influenced by sex and smoking history. Thymic activity was associated with increased TCR repertoire diversity, improved immune protection against infections, and reduced epigenetic aging. Detailed profiling uncovered functional and phenotypic heterogeneity within naive CD4+ T cell subsets shaped by thymic activity.CONCLUSIONThis study demonstrates that thymic function can persist into later life and is modulated by factors such as sex and smoking. These findings suggest that thymic activity during aging is heterogeneous and influenced by more than chronological age alone, with potential implications for immune competence in older adults.

Keywords: Adaptive immunity; Adipose tissue; Cardiology; Immunology.

Figure 1. Characterizing immune cellular landscape and function of thymic structures in aged patients.

(A) Schematic illustrating project workflow. (B) Identification of thymocyte populations via flow cytometry in thymic⁺ (n = 27) and thymic^– (n = 19) samples. Statistical significance was evaluated by 2-tailed Mann-Whitney U test. (C) Representative thymic structures observed through H&E staining. Scale bars: 200 μm. (D) Immunophenotyping single-positive CD4⁺ and CD8⁺ T cells, conventional dendritic cells (cDC1 and cDC2), plasmacytoid dendritic cells (pDCs), and M2 macrophages (M2) in thymic⁺ and thymic^– samples. Statistical significance was evaluated by 2-way ANOVA and Šidák’s test for multiple comparisons. Data in B and D presented as mean ± SD. (E) Volcano plot depicting the average log-fold gene expression changes and Benjamini-Hochberg–corrected P values for pairwise comparisons between thymic^– control adipose tissue (n = 4) and thymic⁺ tissues (n = 3). (F) Shannon diversity index for TCR α (TRA) and β (TRB) chains between thymic⁺ and thymic^– samples. Significance evaluated by unpaired, 2-tailed Student’s t test. Boxes represent the 25^th and 75^th percentiles, lines inside the boxes represent medians, whiskers represent the upper and lower adjacent values. (G) CDR3 length for TRA and TRB chains (thymic⁺ n = 4; thymic^– n = 4).

Figure 2. Thymic output correlates to peripheral T cell subpopulation and associates with sex and smoking in older patients.

(A) Proportion of CD4⁺ and (B) CD8⁺ naive, TCM, TEM, and TEMRA cells in the blood of patients classified as either thymic⁺ or thymic^–. Data presented as mean ± SD. Statistical significance evaluated by 2-way ANOVA with Šídák’s multiple-comparison test. (C) Linear regression analysis comparing RTE% in blood to peripheral T cell pools. (D) Correlative linear regression analysis between chronological age and RTE% in blood for individuals aged <50 years (young; green) (n = 35) and ≥50 years (old; purple) (n = 110). (E) Correlation matrix comparing clinical characteristics to thymic output (relative RTE%) and naive T cell percentage in the blood of patients ≥50 years. Spearman’s coefficients with Bonferroni’s correction P value cutoff <0.002. Significant correlation coefficients are shown as either blue (positive correlation) or red (negative correlation) dots. (F) Graph illustrating RTE% in blood between smokers and nonsmokers and males and females. Truncated violin plots show medians and interquartile ranges, and significance was evaluated by 2-tailed Mann-Whitney U test. The r² values in D and E were calculated using Pearson’s correlation coefficients and significance by 2-sided P value analysis.

Figure 3. Adipose tissue inflammation in old patients is independent of thymic activity.

(A) Bubble plot showing linear regression analysis comparing T cell percentage in EAT to blood T cell populations and clinical characteristics. Significant correlations (P < 0.05) are shown as red (positive correlation) or blue (negative correlation) dots. (B–D) Proportion of RTE, CD8⁺ TEMRA, and CD4⁺ TEMRA in the blood of CMV⁺ (n = 55) and CMV^– (n = 52) patients. (E) Comparison of chronological age between CMV⁺ and CMV^– patients. Violin plots show medians and interquartile ranges, and significance was evaluated by 2-sided Mann-Whitney U test. (F) Linear regression analysis between chronological age and proportion of RTE, (G) CD8⁺ TEMRA, and (H) CD4⁺ TEMRA in patients ≥50 years. The r² values in A and F–H were calculated using Pearson’s correlation coefficients and significance by 2-sided P value analysis.

Figure 4. Evaluating transcriptional changes associated with thymic output in old age.

(A) Volcano plot depicting differentially expressed genes between RTE-high and RTE-low patients. (B) Dendrogram with heatmap showing expression (z score) of the 30 statistically significant genes identified from RTE-high versus -low analysis applied to all patients (n = 94). Red and blue markers represent samples initially classified as RTE-high and RTE-low, respectively. (C) GSEA between patients grouped as RTE-low (RTE% ≤ 10) (n = 28) and RTE-high (RTE% ≥ 20) (n = 22). (D) Gene expression of MMP28, SLC16A10, CTLA4, IL2RB, and TGFBR3 in human CD31⁺ (RTE) and CD31^– (MN) naive CD4⁺ T cells (n = 20). FDR < 0.05. Data presented as mean ± SD. Statistical significance evaluated by unpaired, 2-tailed Student’s t test.

Figure 5. Preserved thymic activity in old patients promotes TCR repertoire diversity.

(A) Graphs showing CD69⁺PD-1^– and CD69^–PD-1⁺ T cells 5 days after stimulation. Statistical significance evaluated by paired, 2-tailed Student’s t test (n = 5). (B) Proliferation index calculated via CFSE fluorescence cocultures with or without IL-7. (C) Graph showing the proportion of IFN-γ⁺ T cells cocultured with or without IL-7. Data in A–C presented as mean ± SD; significance between groups evaluated by 2-way ANOVA with Tukey’s test for multiple comparisons (n = 3). (D) Shannon diversity index (SDI) values for α and (E) β TCR chains correlated to chronological age and RTE% (n = 25). The r² values in D and E were calculated using univariate Pearson’s correlation coefficients and significance by 2-sided P value analysis. (F) Graph showing influenza A–specific IgG antibody levels in patients 1 (n = 7–8), 3 (n = 12–11), 6 (n = 8–9), and 9 months (n = 11–12) after immunization. Data presented as mean ± SD. Statistical significance evaluated by 2-way ANOVA with Šídák’s multiple-comparison test.

Figure 6. Reduced thymic activity associates with epigenetic age acceleration in old patients.

(A) GrimAge age acceleration (AgeAccelGrim) against RTE% in blood (n = 32). (B) AgeAccelGrim values compared between male nonsmokers (n = 11) and smokers (n = 10). Truncated violin plots show medians and interquartile ranges, and significance was evaluated by unpaired, 2-tailed Student’s t test. (C) Correlation between RTE% in blood and DNAm surrogates for cystatin C, (D) plasminogen activator inhibitor 1 (PAI-1), (E) smoking (PackYears), and (F) mitotic age (epiTOC2). (G) Significant (orange) and nonsignificant (gray) smoking-associated CpGs correlating with RTE%. Model 1: No covariates; Model 2: age, sex, and major blood cell type DNAm estimates (monocytes, B cells, neutrophils, CD4⁺ T cells, NK cells). (H) Linear regression between RTE% and GPR15 gene expression from whole blood microarray data. (I) GRP15 gene expression from whole blood between patient subgroups defined by sex and smoking status. Violin plots show medians and interquartile ranges, and significance was evaluated by 1-way ANOVA with Tukey’s test for multiple comparisons. Only significant values are shown. S, smoker; NS, nonsmoker. (J) GPR15 gene expression (n = 20) and (K) geometric mean fluorescence intensity (gMFI) of GPR15 protein expression from CD31⁺ and CD31^– naive CD4⁺ T cells (n = 37). Data presented as mean ± SD; significance by paired, 2-tailed Student’s t test. The r² values in A, C–F, and H were calculated using Pearson’s correlation coefficients and significance by 2-sided P value analysis.