Site-specific development and progressive maturation of human tissue-resident memory T cells over infancy and childhood

Abstract

Infancy and childhood are critical life stages for generating immune memory to protect against pathogens; however, the timing, location, and pathways for memory development in humans remain elusive. Here, we investigated T cells in mucosal sites, lymphoid tissues, and blood from 96 pediatric donors aged 0-10 years using phenotypic, functional, and transcriptomic profiling. Our results revealed that memory T cells preferentially localized in the intestines and lungs during infancy and accumulated more rapidly in mucosal sites compared with blood and lymphoid organs, consistent with site-specific antigen exposure. Early life mucosal memory T cells exhibit distinct functional capacities and stem-like transcriptional profiles. In later childhood, they progressively adopt proinflammatory functions and tissue-resident signatures, coincident with increased T cell receptor (TCR) clonal expansion in mucosal and lymphoid sites. Together, our findings identify staged development of memory T cells targeted to tissues during the formative years, informing how we might promote and monitor immunity in children.

Keywords: T cells; developmental immunity; human immunology; infant immunity; mucosal immunity; tissue-resident memory T cells.

Figure 1.. Pediatric samples for examining T cell differentiation across blood and tissues over childhood and accumulation in mucosal sites.

(A) Donor and subjects included in this study organized by age (left; organ donors: n=68, outpatients: n=28) and samples (right) used in this study. (B) Donor and subject demographics. Pie charts show stratification of pediatric donors and subjects combined by reported ethnicity and race. (C) T cell accumulation in mucosal sites over early life and childhood. Immunofluorescence imaging on sections obtained from lungs and intestines of organ donors (n=9). Representative staining with antibodies to T cell receptor (TCR) β, TCR γδ, and DAPI on lung (left) and jejunum (right) sections from 5 organ donors paired by row. CD3⁺ cells are shown based on their expression of TCR β (green) and TCR γδ (red). Sections shown at 40x magnification (with line demonstrating 100μm distance) with dotted square box shown as inset at 80x (line depicting 50μm distance). (D) Compiled analysis of αβ and γδ T cells by area (y-axis, cells/mm²) and age (x-axis, years) from lung (left) and jejunum (right). Data points represent mean cell/mm² of sections from each individual donor (n=3–6 sections/donor) with shading depicting one standard deviation from the mean. Statistical testing was performed by Pearson correlation demonstrated correlation between age and cells/mm²: lung αβ; p=0.0025, jejunum αβ; p = 0.0292, and jejunum γδ; p = 0.0026. Line depicts best fit as determined by modeling (see methods). T cell subsets displayed as lung αβ (blue circles), lung γδ (purple diamonds), jejunum αβ (orange squares), jejunum γδ (green diamonds). See also Figures S1–3 and Table S1.

Figure 2.. Memory T cells develop rapidly in mucosal sites with slower accumulation in blood and lymphoid organs.

High-dimensional flow cytometry analysis was performed to identify major T cell subsets by tissue and age. (A) T cell subsets were defined by expression of CCR7 and CD45RA: Naïve (CCR7⁺CD45RA⁺, blue), Central Memory (T_CM cells; CCR7⁺CD45RA⁻, light gray), Effector Memory (T_EM cells; CCR7⁻CD45RA⁻, red), and Effector Memory cells expressing CD45RA (T_EMRA cells; CCR7⁻CD45RA⁺, dark gray). Piecharts showing the proportion of CD4⁺ (left) and CD8⁺ (right) memory subsets within each tissue (row) for representative donors (column) of indicated ages over the first 10 years of life. (B) Compiled data from all donors showing relationship of total memory cells (T_EM + T_CM + T_EMRA) (green) to naïve (blue) T cells vs age over the first 10 years of life, organized by tissue with lymphoid (left; blood n = 31, spleen n = 36, lung lymph node (LLN) n = 33, gut lymph node (GLN) n = 35) and mucosal (right; lung n = 36, jejunum n = 41, ileum n = 29, and colon n= 25). Lines depict best fitting model (see methods) logit-linear (lymphoid tissues) or nonlinear (mucosal tissues) with shading indicating 95% confidence intervals. C) Graph depicts log scaled maximal % accumulation rate per year (x-axis) of CD4⁺ and CD8⁺ memory T cells by tissue site (y-axis) with data organized by highest to lowest rate. Dots represent mean and bars represent 95% confidence intervals. See also Figure S2–5 and Table S1.

Figure 3.. Functional maturation of effector-memory T cells over childhood.

T cells from lymphoid and mucosal sites of organ donors aged 0–10 years were stimulated with PMA/ionomycin and effector cytokine production (TNF-α, IFN-γ) from T_EM cells was assessed by intracellular cytokine staining (ICS). (A) Representative cytokine production by CD4⁺ (top) and CD8⁺ (bottom) T_EM cells from indicated tissues obtained from an infant (7 months, left) and older pediatric (8 years, right) organ donor, showing frequency of IFN-γ⁺, TNF-α⁺ or IFN-γ/TNF-α⁺ cells in each site. Quadrants delineated based on unstimulated controls. (B) Effector cytokine production stratified by tissue site. Plots show cytokine production (TNFα⁺ (top), IFNγ⁺ (middle), and TNFα⁺/IFNγ⁺ (bottom)) by CD4⁺ (left) and CD8⁺ (right) T_EM cells for each site for each individual pediatric donor. Lines depict mean with standard deviation. Statistical testing by Kruskal-Wallis Anova with Dunn’s correction for multiple comparisons. (C) Progressive acquisition of effector function over childhood. Cytokine production by CD4⁺ (left) and CD8⁺ (right) T_EM cells in indicated sites over age showing frequency of cells expressing either TNFα⁺ or IFNγ⁺ (blue circles) and or both TNFα⁺/IFNγ⁺ (green squares). Lines depict best fitting linear or nonlinear model. Statistical testing using Spearman correlation. Donors; lung lymph node n= 21, gut lymph node n = 24, spleen n = 17, lung n = 22, and jejunum n=20. ns = p>0.05, * = p<0.05, **= p<0.01, ***=p<0.001, ****=p<0.0001. See also Figures S2,S6 and Table S1.

Figure 4.. Site- and age-regulated expression of tissue resident markers by mucosal memory T cells.

Flow cytometric analysis of expression of CD69 on effector memory (T_EM) T cells. (A) Compiled data of all CD4⁺ (top) and CD8⁺ (bottom) % CD69⁺ T_EM cells over age based on tissue site. Data organized by left (lymph nodes (LN); lung, gut), middle (spleen), right (mucosal; lung and jejunum). Statistical testing via Pearson or Spearman correlation with lines depicting best fitting model (linear vs nonlinear). Donors; spleen n = 35, LLN n = 31, GLN n = 36, lung n = 33, jejunum n = 39. (B) Flow cytometry histograms of core T_RM marker expression by CD4⁺ (top) and CD8⁺ (bottom) CD69^hi T_EM cells from the jejunum of representative donors displayed by age grouping; <1 year, 2–4 years, and 5–10 years. Histograms normalized to mode. (C) Expression of T_RM cell core markers (CD103, CD101, CXCR6, and CD49a) in the lung and jejunum over age shown in compiled data from infant (n=13–16) and adult organ donors (n=4 per graph, 8 total donors) by CD4⁺ (left) and CD8⁺ (right) CD69⁺ T_EM cells. Values from adult donors (aged 20–50 years) are shown in each plot. Statistical testing performed using Pearson or Spearman correlation. * = p<0.05, ** p<0.01. See also Figures S2,S7 and Tables S1.

Figure 5.. Transcriptional maturation of mucosal memory T cells over childhood.

CD8⁺CD69⁺T_EM cells were sorted from lungs and intestines from donors of indicated ages for whole transcriptome profiling by RNAseq (See methods). (A) Principal component analysis (PCA) of samples from the lung (circles; n = 10) and intestine squares; jejunum n=10, and triangle; ileum n=9) from n=11 organ donors (aged 3 months – 8 years) colored based on donor age. Circles represent one standard deviation based on tissue site (lung or intestine). (B) Differentially expressed (DE) genes from pediatric organ donors based on age groups (1–3 years, 4–6 years, and >7 years) relative to the youngest donors (<1 year). Venn diagrams (left) depicting DE genes (log2fold > ±0.5, p<0.05) within and across donor groups compared to the youngest donors. Line graph depicting unique DE genes in each age group compared to youngest donor group (lungs; blue squares and intestines; orange squares). (C) Heatmaps depicting row normalized gene expression of key genes differentially expressed over age by memory T cells isolated from lungs (left) and intestines (right) with donors (columns) arranged in increasing age. Statistical testing by Spearman or Pearson correlation analysis. * = p<0.05. See also Figure S2,S8 and Table S1–4.

Figure 6:. Dynamic shifts in functional capacity for memory CD4⁺ T cells over childhood.

(A) Heatmaps depicting row normalized cytokine gene expression from CD69⁺ CD4⁺ T_EM cells within mucosal sites (lung; blue circles, n = 10 and intestine (jejunum and ileum); orange squares, n = 19) from n = 11 organ donors over age (0.3 – 8 years)). Statistical testing by Pearson or Spearman correlation. (B) Expression of indicated chemokine receptors denoting T-helper cell differentiation states by memory CD4⁺ T cells in lung (top; n=7) and jejunum (bottom; n=8) from organs donors aged 0.02–7 years shown as representative histograms (normalized to mode) from infant (<2 years; purple) and older child (>4 years; green) and compiled data from multiple donors. Statistical testing done with Mann-Whitney T test, lines depict mean and standard deviation. * = p <0.05, ** = p <0.01, *** = p<0.001. LLN = lung lymph node, GLN = gut lymph node. See also Figure S2 and Tables S1,S2.

Figure 7.. T cell receptor analysis reveals loss of diversity and differential overlap between sites over age.

T cell receptor (TCR) analysis performed on population RNA sequencing of CD69⁺ T_EM cells as in Fig. 5. (A) Clonotype analysis organized by tissue site (columns) and CD4⁺ (top) and CD8⁺ (bottom) with proportion of repertoire space (y-axis) occupied by clonotypes vs individual donors (x-axis; stacks) organized by increasing age. Gray bars depict donors/tissues were no sample was available. (B) R20 (top) and R50 (bottom) over age for CD4⁺ (blue circles) and CD8⁺ (red squares) T cells by tissue site. Statistical testing performed by Wald test. Line depicts best fitting linear or non-linear model. (C) Clonal overlap matrices for individual donors with CD4⁺ (top) and CD8⁺ (bottom) organized by individual donors (columns) by increasing age. Box color depicts % overlap. Gray boxes depict tissues were no sample was available. (D) Dotplots showing clonal overlap over age (years) for CD4⁺ (blue circles) and CD8⁺ (red squares) T cells. Line depicts best fit with 95% confidence interval. Statistical testing with Wald test. ns = p>0.05, * = p<0.05, ** = p<0.01, *** = p<0.001, **** = p<0.0001. Abbreviations; Ile- Ileum, Jej- Jejunum, Lg- Lung. See also Tables S1,S5.