Early-life compartmentalization of human T cell differentiation and regulatory function in mucosal and lymphoid tissues

Abstract

It is unclear how the immune response in early life becomes appropriately stimulated to provide protection while also avoiding excessive activation as a result of diverse new antigens. T cells are integral to adaptive immunity; mouse studies indicate that tissue localization of T cell subsets is important for both protective immunity and immunoregulation. In humans, however, the early development and function of T cells in tissues remain unexplored. We present here an analysis of lymphoid and mucosal tissue T cells derived from pediatric organ donors in the first two years of life, as compared to adult organ donors, revealing early compartmentalization of T cell differentiation and regulation. Whereas adult tissues contain a predominance of memory T cells, in pediatric blood and tissues the main subset consists of naive recent thymic emigrants, with effector memory T cells (T(EM)) found only in the lungs and small intestine. Additionally, regulatory T (T(reg)) cells comprise a high proportion (30-40%) of CD4(+) T cells in pediatric tissues but are present at much lower frequencies (1-10%) in adult tissues. Pediatric tissue T(reg) cells suppress endogenous T cell activation, and early T cell functionality is confined to the mucosal sites that have the lowest T(reg):T(EM) cell ratios, which suggests control in situ of immune responses in early life.

Figure. 1. Naïve T cells predominate in infant tissues with T_EM cells confined to mucosal sites

(a) Flow cytometry plots of CD45RA/CCR7 expression by CD4⁺ and CD8⁺ T cells in pediatric blood and tissues from a representative infant (left, donor 118, 2 months) and older baby (right, donor 213, 2 yrs). (b) Mean frequency ± SEM of each T cell subset (naïve, T_CM, T_EM, T_EMRA) expressed as a percent of CD4⁺ (top) or CD8⁺ (bottom) T cells in circulation, lymphoid, and mucosal tissue (ordered from circulation, red; to mucosal sites, green) separated by age group (pediatric (0–2 years) 15 donors, red; young adult (15–25 years), 9 donors, black). (c) Graph showing mean frequency ± SEM of CD31 expression by total CD4 T cells (top) and naive CD4⁺ T cells (bottom) from 8 pediatric and 9 young adult donors as in (b). (d) Representative staining of CD4⁺ and CD8⁺ T cell populations in thymic tissue of an infant (top, donor 107, 4 months) and young adult (bottom, donor 102, 22 yrs) and compiled frequencies of CD4⁺CD8⁺ (double positive, DP) cells in thymii of 6 pediatric and 5 young adult donors. Statistical significance represents comparisons between the indicated frequencies in pediatric and young adult donors in the same tissue sites measured by multiple t tests adjusted for multiple comparisons indicated as *P < 0.05; **P < 0.001. Individual donors used, descriptive statistics and individual p-values are shown in (Supplementary Tables 2, 3 and 4).

Figure. 2. Distinct properties of early tissue memory T cells in lymphoid and mucosal sites

(a) Representative histograms of CD69 expression by naïve (red) and T_EM (blue) CD4⁺ (top) and CD8⁺ (bottom) T cells from an infant donor (donor 127, 16 months). (b) Mean frequencies ± SEM of CD69 CD4⁺ (top) or CD8⁺ (bottom) T_EM cells from 15 pediatric (0–2 years, red) or 9 young adults (15–25 years, black). (c) Representative flow cytometry plots of CD69 and CD103 expression on CD8⁺ T_EM cells from a pediatric donor (top, donor 63, 26 months) and a young adult donor (bottom, donor 105, 20 years). (d) Mean frequencies ± SEM of CD69⁺CD103⁺ CD8⁺ (left) and CD69⁺CD103⁻ (right) T_EM in 9 pediatric (white) and 8 young adult (black) donors. Statistical significance for each site indicated as *P < 0.05; ** P < 0.001. (e) Cytokine content in culture supernatants (pg/ml, mean ±SEM) of infant (n=4, white) and adult (n=3, black) MLN T cells following stimulation with anti-CD2/CD3/CD28-coupled beads for four days. Unstimulated cells produced <1pg/ml cytokines (data not shown). Statistical significance indicated as *P < 0.05. (f) Average IFN-γ production from indicated tissues of 5 pediatric donors and 3 adult donors stimulated with PMA/ionomycin for 4 h. (g) IFN-γ production versus CD45RA expression from CD3⁺ T cells of donor 213 (2 years) stimulated with PMA/ionomycin for 4 hours. Individual donors used, descriptive statistics and individual P values are shown in (Supplementary Tables 2 and 3).

Figure. 3. Elevated frequency and broad tissue distribution of T_reg cells in pediatric as compared to adult tissues

(a) Frequency of CD25⁺FOXP3⁺CD4⁺ T cells in tissues from an infant (donor 118, 2 months) and adult (donor 105, 20 years). (b) Gating strategy for T_reg cells from pediatric (top, donor 68, 6 months) and adult (bottom, donor 110, 40 yrs) LLN showing and initial CD25⁺CD127⁻ gate (left) subsequently gated on FOXP3 expression, versus non-T_reg (CD25⁻CD127⁺) cells as a control. (c) T_reg cell frequency (mean±SEM) in tissues from 13 pediatric (red) and 24 adult (black) donors expressed as percent CD25⁺CD127⁻FOXP3⁺ cells from total CD4⁺ T cells (left). Statistical significance indicated as *P < 0.05; **P < 0.001. (d) T_reg cell tissue distribution in pediatric and adult donors. Graphs show Treg cell frequency tissues from pediatric (top row, 2 months, 4 months, 1 year) and adult (lower, 20 years, 38 years, 51 years) donors. (e) CD45RA expression by T_reg cells from 13 pediatric (red) and 24 adult (black) donors, with * P < 0.05. (f) CD69 and CD103 expression on T_reg cells shown as representative flow cytometry plots(left) and compiled percentages (mean±SEM) of CD69⁺CD103⁻ (white) and CD69⁺CD103⁺ (grey) populations from 16 donors (right). (g) Percent Ki67 expression (mean±SEM) by T_reg (purple) and non-T_reg (CD4⁺CD25⁻CD127⁺) cells (black) in blood and tissues from 12 donors. Statistical significance indicated as *P < 0.05; **P < 0.001. Individual donors used, descriptive statistics and P values are shown in (Supplementary Tables 2, 4 and 5).

Figure. 4. Pediatric tissue T_reg cells suppress endogenous T cell proliferation and function

(a) FOXP3 expression by pediatric and adult T_reg cells. Representative FOXP3 expression by MLN CD4⁺CD25⁺CD127⁻ cells (red) compared to CD25⁻CD127⁺FOXP3⁻ cells (black dashed) from a pediatric (16 months, left) and adult (22 yrs, right) donor. ΔMFI = MFI(T_reg cells)–MFI(control non-T_reg cells). (b) Normalized FOXP3 MFI as in (a) of T_reg cells from blood and lymphoid tissues of 10 pediatric (white) and 17 adult (black) donors. *P < 0.05; **P < 0.001; n.s.: not significant. (c) Proliferation of unfractionated T cells (red dotted) or CD25-depleted T cells (black), isolated from pediatric and adult MLN of indicated ages following anti-CD3/CD28 stimulation. Left: CFSE dilution of activated compared to unstimulated (grey shaded) CD4⁺ (top) and CD8⁺ (bottom) T cells. Right: Percent of CFSE⁺ (undivided) CD4⁺ (top) or CD8⁺ (bottom) T cells stimulated with or without CD25-depletion for each individual infant and adult donor. (d) Cytokine production in the presence or absence of T_reg cells. Spleen and MLN T cells from pediatric and adult tissues were activated as in (c) with or without CD25 depletion and IFN-γ and IL-2 in supernatants was measured after 48 hours Graph shows fold change of cytokine production by the CD25-depleted versus unfractionated pediatric (white, n=4) and adult (black, n=4) T cells, with *P < 0.05; **P < 0.001. (e) Average T_reg:T_EM cell ratios in tissues from pediatric donors (0–2 yrs, n=17). Red dashed line indicates the maximum T_reg:T_EM cell ratio in adult tissues.