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letters Early-life compartmentalization of human T cell differentiation and regulatory function in mucosal and lymphoid tissues © 2015 Nature America, Inc. All rights reserved. Joseph J C Thome1,2, Kara L Bickham1, Yoshiaki Ohmura3, Masaru Kubota3, Nobuhide Matsuoka3, Claire Gordon1,4, Tomer Granot1, Adam Griesemer1, Harvey Lerner5, Tomoaki Kato3 & Donna L Farber1–3 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 immunity1–4 and immunoregulation5,6. 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 cells7,8, in pediatric blood and tissues the main subset consists of naive recent thymic emigrants, with effector memory T cells (TEM) found only in the lungs and small intestine. Additionally, regulatory T (Treg) 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 Treg cells suppress endogenous T cell activation, and early T cell functionality is confined to the mucosal sites that have the lowest Treg:TEM cell ratios, which suggests control in situ of immune responses in early life. Knowledge of human immune responses during early life remains sparse, owing to the difficulty and impracticality of obtaining blood and tissue samples from infants. Our current view of normal infant immune responses, including T cell differentiation and function, is based mainly on the sampling of umbilical cord blood9–11 or fetal tissue12,13, which reflect immune responses in utero but not responses to the diverse antigens encountered in early life. Fundamental information about the establishment of adaptive immunity during infancy, including how T cells respond, differentiate and populate tissue sites, remains undefined. Through collaboration with organ procurement agencies, we have established protocols for obtaining lymphoid and mucosal tissues from individual organ donors for whom consent has been given for the use of tissues for research 7,8. We showed previously that distinct subsets of T cells, including naive and previously activated effector and memory T cell subsets, are differentially compartmentalized in the lymphoid and mucosal tissues of older children and adults7,8,14,15. To investigate T cell immunity in tissues during early life, we obtained multiple lymphoid and mucosal tissues (and blood) from a cohort of infant and pediatric organ donors in the first 2 years of life (n = 17, Supplementary Table 1). We compared these to tissues from a cohort of adult donors stratified into young adults, aged 15–25 years (n = 23), and adults, aged >25 years (n = 24) (Supplementary Table 2). The tissues analyzed are representative of circulatory, lymphoid and mucosal sites and included blood, spleen, inguinal lymph nodes (ILNs), lung-draining lymph nodes (LLNs), mesenteric lymph nodes (MLNs), lungs, jejunum, ileum and colon. Samples from pediatric donors contained lower T cell:B cell ratios than those from adult donors, in the blood, spleen, MLNs and intestinal sites, which indicated that T cell numbers in these tissues are reduced in early life. The CD4+:CD8+ T cell composition, by contrast, was similar in pediatric and young adult donor tissues, showing a predominance of CD4+ T cells in lymphoid tissues (Supplementary Fig. 1a). In pediatric tissues, however, major functional subsets of T cells that are delineated by their expression of the cell surface CD45 isoform CD45RA and the lymph node homing/chemokine receptor CCR7, including naive (CD45RA+CCR7+) and previously activated T effector memory (CD45RA−CCR7−, TEM) cell subsets, exhibited distinct frequencies and distributions in pediatric as compared to young adult tissues, whereas central memory (CD45RA−CCR7+, TCM) and terminal effector (CD45RA+CCR7−, TEMRA) cell subsets were similar in both age groups (Fig. 1a,b). In pediatric donors (2 months to 2 years of age), naive T cells represented the predominant population (70–95%) of CD4+ and CD8+ T cells in the blood, spleen, lymph nodes and colon, whereas in young adults, naive T cells were present only in blood and lymphoid sites and at frequencies of <50% (Fig. 1a,b). CD4+ T cells in pediatric tissues also exhibited a high proportion of CD31+ cells, denoting recent thymic emigrants (RTEs)16 (>60% in lymphoid tissues; 25–40% in mucosal sites, Fig. 1c), which 1Columbia Center for Translational Immunology, Columbia University Medical Center, New York, New York, USA. 2Department of Microbiology and Immunology, Columbia University Medical Center, New York, New York, USA. 3Department of Surgery, Columbia University Medical Center, New York, New York, USA. 4Department of Medicine, Columbia University Medical Center, New York, New York, USA. 5LiveOnNY, New York, New York, USA. Correspondence should be addressed to D.L.F. ([email protected]). Received 30 June; accepted 12 November; published online 14 December 2015; doi:10.1038/nm.4008 nature medicine advance online publication letters exceeded the proportion of CD4+ RTEs in young adult lymphoid tissue (<30%) and at mucosal sites (<10%) (Fig. 1c). Pediatric and young adult thymii exhibited similar percentages of CD4+CD8+ (double positive, DP) thymocytes (Fig. 1d), and naive T cells were largely RTEs in both groups (Fig. 1c). Therefore, the reduction in tissue RTEs in young adults does not correlate with differences in thymopoiesis; CD4+ T cells Blood Jejunum 87 3 77 9 10 Spleen 10 41 Lung 78 9 27 12 19 MLN 6 9 11 1 90 5 7 3 Jejunum 93 15 2 Spleen 57 24 19 5 70 29 ILN Blood 62 11 IIeum 59 11 16 Colon 89 2 95 1 95 3 7 1 2 1 95 ** ** * * 40 * 20 60 40 * * * 20 11 Jejunum 75 12 16 Spleen 27 32 25 26 Colon 70 58 12 10 9 21 20 22 22 1 86 Lung IIeum 36 50 6 10 43 ILN 83 * * 40 20 Bl o Sp od le en IL N LL N Lu ng Je ML ju N nu m IIe u C m ol on 0 40 * ** 60 ** * ** ** 20 100 100 * 80 * ** 80 60 40 20 0 ** 80 60 * 40 20 * * * * 0 c Bl o Sp od le en IL N LL N Lu ng M Je LN ju nu m IIe u C m ol on * * 0 CD8+ TEM cells (%) 60 80 Bl o Sp od le en IL N LL N Lu ng M Je LN ju nu m IIe u C m ol on 80 20 47 MLN 100 0 0 * 18 LLN 68 7 12 73 MLN 63 5 Colon 59 8 28 57 29 100 Pediatric 80 Young adult 60 40 20 * 0 * 100 Pediatric Young adult 80 60 40 20 * 0 Bl o Sp od le en IL N LL N Lu ng Je ML ju N nu m IIe u C m ol on 100 ** ** 60 100 18 CD4+ TEMRA cells (%) ** CD4+ TEM cells (%) ** CD4+ TCM cells (%) 80 6 12 IIeum 16 22 63 Blood CCR7 CD8+ TCM cells (%) CD4+ naive T cells (%) 100 13 69 Lung ILN CCR7 b 23 8 62 8 MLN Jejunum 79 Spleen 76 CD8+ T cells LLN 65 22 Lung 11 Colon 88 11 4 46 CD4+ T cells LLN CD8+ TEMRA cells (%) CD45RA 83 Blood IIeum 57 ILN CD8+ naive T cells (%) d DP thymocytes (%) Total CD4+CD31+ T cells (%) Figure 1 Naive T cells predominate in 80 ** ** ** ** ** ** infant tissues, with TEM cells confined 60 72 5 to mucosal sites. (a) Flow cytometry plots ** ** * 40 of CD45RA and CCR7 expression by CD4+ 80 4 months old and CD8+ T cells in pediatric blood and 20 60 tissues from a representative infant (left, 0 20 donor 118, 2 months) and from an older baby 40 80 (right, donor 213, 2 years). (b) Mean 84 7 20 frequency ± s.e.m. of each T cell subset in 60 the order (left to right) naive; central 22 years 0 40 old memory (TCM); effector memory (TEM); Pediatric Young Pediatric terminal effector (TEMRA) expressed as a adult 20 Young adult 8 + + percent of CD4 (top) or CD8 (bottom) T cells CD4 0 in circulation, lymphoid and mucosal tissues (ordered from circulation, red; to mucosal sites, green) and separated by age group (pediatric (0–2 years) 15 donors, red; young adult (15–25 years), 9 donors, black). (c) CD31 expression (mean frequency ± s.e.m.) by total CD4 T cells (top) and naive CD4+ T cells (bottom) from eight pediatric and nine young adult donors as in b. (d) CD4 and CD8 expression by thymocyte populations in the thymic tissue of an infant (top, donor 107, 4 months) and a young adult (bottom, donor 102, 22 years) and compiled frequencies of CD4 +CD8+ (double positive, DP) cells in the thymii of six pediatric and five young adult donors (right). Statistical significance represents comparisons between the indicated frequencies in pediatric and young adult donors at the same tissue sites, measured by multiple t-tests adjusted for multiple comparisons. *P < 0.05; **P < 0.001. Individual donors used, descriptive statistics and individual P values are shown in Supplementary Tables 2–4. ILN, inguinal lymph nodes; LLN, lung-draining lymph nodes; MLN, mesenteric lymph nodes. N n LL N Lu ng M L Je N ju nu m IIe um C ol on IL ee Sp l Bl oo d CD8 Naive CD4+CD31+ T cells (%) © 2015 Nature America, Inc. All rights reserved. 11 CD8+ T cells LLN CD45RA a it could derive from differential seeding, retention or activation at these sites. In pediatric tissues, memory T cells (mainly TEM cells) were found in frequencies >20% only in the lungs, jejunum and ileum, and they were significantly less abundant (P < 0.05) in the blood, spleen, lymph nodes and colon (Fig. 1a,b, Supplementary Fig. 1b and advance online publication nature medicine Supplementary Table 3). By contrast, in young adults, TEM cells predominated in all mucosal sites (>90%), and they comprised 30–40% of lymphoid T cells (Fig. 1b). The compartmentalization of early T cell memory to the lungs and small intestines, but not to the draining lymph nodes, suggests local in situ priming to inhaled and ingested antigens, respectively. We examined whether infant tissue TEM cells express markers of tissue resident memory T (TRM) cells1,3,14, including the activation marker CD69 and the integrin CD103, which are both involved in tissue retention17,18. CD69 was expressed by the majority of TEM cells in all pediatric tissues, similarly to TEM cells in adult tissues, whereas blood TEM cells were CD69− (Fig. 2a,b)8. However, in pediatric mucosal sites, a lower frequency of CD8 + TEM cells coexpressed CD69 and CD103 compared to higher frequencies in adult mucosal sites (Fig. 2c,d and Supplementary Table 3)8, suggesting that mucosal memory T cells in early life have not yet fully differentiated into TRM cells. To further establish the differentiation status of T cells in pediatric tissues, we analyzed their capacity for effector cytokine production. Lymph node T cells from adult tissues produced substantial levels of interleukin 2 (IL-2), interferon-γ (IFN-γ) and IL-4 after stimulation mediated by the T cell receptor (TCR)-CD3 complex, whereas a Blood Spleen 3 LLN 59 Lung 59 pediatric T cells produced lower levels of both IL-2 and IL-4 and negligible levels of IFN-γ (Fig. 2e). Low-level IFN-γ production by pediatric lymphoid T cells, compared to those of adults, was also observed after TCR-independent stimulation of the cells with phorbol 12-myristate 13-acetate (PMA) and ionomycin (Fig. 2f), which suggests that there are cell-intrinsic differences. Nor did pediatric lymphoid T cells produce high levels of IL-8 (Supplementary Fig. 2), as observed in T cells from newborn cord blood9. However, pediatric memory T cells in the small intestines produced IFN-γ rapidly in response to PMA stimulation, exceeding the low proportion of IFN-γ producers in the LN and in the colon (Fig. 2g). Thus, memory T cells that are generated during early life in mucosal sites acquire the capacity to secrete proinflammatory cytokines. We considered whether Treg cells have differential distribution or function in pediatric, as opposed to adult, tissues. Treg cells are a subset of CD4+ T cells that express the transcription factor forkhead box P3 (FOXP3) and the IL-2Rα chain (CD25), and they are essential for the establishment of self-tolerance and immune homeostasis (for reviews, see refs. 19–21). In humans, there are few studies of Treg cells in tissues22,23, and the role of Treg cells in early immune responses is unknown. In infant tissues, a high proportion (20–40%) of CD4+ c Jejunum 78 Lung Jejunum 20 98 lleum 45 Colon 33 29 2 years old + CD4 T cells Naive TEM 13 65 68 86 % maximum 93 60 49 60 61 40 90 87 57 20 years old CD103 + CD8 T cells 31 8 11 35 CD69 d 100 80 CD8+CD69+ CD103+ TEM (%) CD4+CD69+ T cells (%) b * 100 Pediatric Young adult 100 ** 80 CD8+CD69+ CD103– TEM (%) CD69 80 on um ol C lle um ng Lu Je ju n Bl oo d Sp le en M LN sp Ad le ult en IFN-γ, IL-2 (pg/ml) IL N LL N Lu ng M Je LN ju nu m lle um C ol on f IL-4 (pg/ml) Bl oo d Sp le en e CD3+ lFN-γ+ T cells (%) on ol C um lle um Je j un Lu ng Figure 2 Distinct properties 60 ** ** * 60 ** 60 of early tissue memory T cells * 40 Pediatric ** 40 in lymphoid and mucosal sites. 40 Young adult 20 (a) Representative histograms 20 20 0 of CD69 expression by naive 0 0 + 100 (red) and TEM (blue) CD4 (top) and CD8+ (bottom) T cells from 80 an infant donor (donor 127, 16 60 months). (b) Mean frequencies ± 40 50 + + s.e.m. of CD4 CD69 (top) or 20,000 Pediatric 80 20 of CD8+CD69+ (bottom) TEM cells Young adult 40 0 15,000 60 from 15 pediatric donors (0–2 years, 30 red) or nine young adults (15–25 years, 10,000 40 black). (c) Representative flow cytometry 20 * plots of CD69 and CD103 expression on 5,000 20 10 CD8+ TEM cells from a pediatric donor (top, donor 63, 26 months) * 0 0 and a young adult donor (bottom, donor 105, 20 years). (d) Mean 0 IL-2 IL-4 IFN-γ frequencies ± s.e.m. of CD8+CD69+CD103+ (left) and CD8+CD69+CD103− (right) TEM in nine pediatric (white) donors and eight young adult (black) donors. Statistical significance for each site is indicated as Pediatric *P < 0.05; **P < 0.001. (e) Cytokine content in culture supernatants ILN MLN Jejunum lleum Colon (pg per ml, mean ± s.e.m.) of infant (n = 4, white) and adult 5 22 5 3 58 3 9 2 3 5 (n = 3, black) mesenteric lymph node (MLN) T cells after stimulation with anti-CD2/CD3/CD28–coupled beads for 4 days. Unstimulated cells produced <1 pg per ml of cytokines (data not shown). Statistical significance is indicated as *P < 0.05. (f) Average interferon-γ (IFN-γ) 32 59 29 3 52 46 59 35 22 47 production from indicated tissues of five pediatric donors and three CD45RA adult donors stimulated with phorbol 12-myristate 13-acetate (PMA) and ionomycin for 4 h. (g) IFN-γ production versus CD45RA expression from CD3+ T cells of donor 213 (2 years) stimulated with PMA and ionomycin for 4 h. Individual donors used, descriptive statistics and individual P values are shown in Supplementary Tables 2 and 3. CD8+CD69+ T cells (%) g IFN-γ © 2015 Nature America, Inc. All rights reserved. letters nature medicine advance online publication letters 7 Jejunum 2 – CD25 + CD127 + CD4 T cells 3 34 lleum 27 Spleen 3 Lung 5 lleum 2 ILN 14 Colon 15 ILN 4 MLN 5 Colon 4 6 months old CD25 5 0 0 Spleen MLN 0.2 <0.1 0.5 2 30 Lung 20 41 75 2 10 0 10 8 0.4 5 0.5 43 75 5 75 CD69 8 + 6 4 2 0 Bl Sp ood le en IL LLN Lu N n Je M g ju LN nu lle m C um ol on Colon lleum 100 80 60 40 20 0 CD69 CD103 – CD69+CD103+ Bl o Sp od le en IL N LL N Lu ng Je ML ju N nu m lle u C m ol on * * * 20 <0.1 0.9 CD103 ** ** ** ** ** * * 40 Pediatric Adult 40 + ** 60 Blood 1 Bl Sp ood le en IL LLN Lu N n Je M g ju LN nu lle m C um ol on 10 * f CD3 Bl Sp ood le en IL LLN Lu N n Je M g ju LN nu lle m C um ol on 20 15 ** Bl o Sp od le en IL N LL Lu N ng Je ML ju N nu lle m u C m ol on Figure 3 Elevated frequency and broad tissue distribution of Treg cells in pediatric, as compared to adult, tissues. Treg (a) Expression of CD25 and FOXP3 by CD4+ T cells in tissues from an infant (donor 118, 2 months, left) and from 30 Non-Treg an adult (donor 105, 20 years, right) with frequency of CD25 +FOXP3+ cells indicated. (b) Gating strategy for * Treg cells from pediatric (top, donor 68, 6 months) and from adult (bottom, donor 110, 40 years) lung-draining 20 ** lymph node (LLN) tissue, showing an initial CD25+CD127− gate (left) subsequently gated on FOXP3 expression, ** ** ** ** ** ** ** versus non-Treg (CD25−CD127+) cells as a control. (c) Treg cell frequency (mean ± s.e.m.) in tissues from 10 13 pediatric (red) and 24 adult (black) donors, expressed as the percent of CD25 +CD127−FOXP3+ cells from 0 total CD4+ T cells. *P < 0.05; **P < 0.001. (d) Treg cell tissue distribution in pediatric and adult donors. Graphs show Treg cell frequency tissues from individual pediatric (top row, 2 months, 4 months, 1 year) and adult (lower, 20 years, 38 years, 51 years) donors. (e) CD45RA expression by Treg cells from 13 pediatric (red) and 24 adult (black) donors. *P < 0.05. (f) CD69 and CD103 expression on Treg cells, shown as representative flow cytometry plots (top) and compiled percentages (mean ± s.e.m.) of CD69+CD103− (white) and CD69+CD103+ (gray) populations from 16 donors (bottom). (g) Percent Ki67 expression (mean ± s.e.m.) by Treg (purple) and by non-Treg (CD4+CD25−CD127+) cells (black) in blood and tissues from 12 pediatric and adult donors. Statistical significance indicated as *P < 0.05; **P < 0.001. Tissues displayed are ordered from blood, red, to mucosal sites, green. Individual donors used, descriptive statistics and P values are shown in Supplementary Tables 2, 4 and 5. + g Bl Sp ood le en IL LLN Lu N n Je M g ju LN nu lle m C um ol on + % Ki67 CD4 Treg (%) © 2015 Nature America, Inc. All rights reserved. + CD4 Treg (%) 25 Adult + CD4 Treg (%) Pediatric Adult Pediatric + CD4 Treg (%) d 40 years old FOXP3 CD25 c 39 16 CD127 FOXP3 2 18 MLN 80 77 44 Spleen 29 Lung e + CD25 – CD127 Bl o Sp od le en IL N LL Lu N ng Je M ju LN nu lle m u C m ol on 5 LLN + Blood CD4 Treg (%) 31 Jejunum 32 + b 31 LLN Blood CD45RA CD4 Treg (%) a T cells were CD25+FOXP3+, whereas the frequency of these cells was lower in young adult tissues (Fig. 3a). Quantitation of Treg cells on the basis of gating of CD25+CD127−FOXP3+ CD4+ T cells24,25 (Fig. 3b and Supplementary Fig. 3) revealed an elevated frequency of Treg cells (10–30%) in all infant and pediatric tissues, which contrasted with the 6- to 10-fold lower frequency of Treg cells (2–5% CD4+ T cells) seen in adult tissues (Fig. 3c, Supplementary Fig. 4a and Supplementary Table 4). Treg cell distribution in tissues also differed between pediatric and adult donors, with high frequencies of Treg cells in pediatric mucosal and lymphoid tissues, whereas in adults, Treg cell frequencies were highest in the lymphoid tissues (often in the LLN) as compared to mucosal sites (Fig. 3c,d and Supplementary Fig. 4b). A heat map of tissue Treg cell frequencies in individual donors ranging from infants to adults 63 years of age shows the sharp reduction in Treg cell frequencies that occurs after childhood (17–20 years) (Supplementary Fig. 4a). We examined potential differences in Treg cell phenotypes and turnover between tissues and the circulation. In pediatric and adult tissues, Treg cells in mucosal tissues were predominantly CD45RA−, whereas 40–60% Treg cells in lymphoid tissues and blood were CD45RA+ (Fig. 3e and Supplementary Fig. 5a), suggesting that there are tissue-intrinsic differences in the activation state26,27. Pediatric and adult tissue Treg cells also expressed CD69 (similarly to TRM cells) but not CD103, whereas blood Treg cells were CD69−CD103− (Fig. 3f). All tissue and circulating Treg cells showed extensive proliferation compared to their non-Treg cell counterparts (Fig. 3g, Supplementary Fig. 5b and Supplementary Table 5). Thus, Treg cells in human tissues exhibit distinct properties that are independent of age and are instead influenced by CD45RA− phenotypes28 and/or tissue-specific signals. Pediatric Treg cells expressed higher amounts of FOXP3 (which is associated with increased suppressive capacity29) than did adult Treg cells in lymphoid tissues, whereas levels of FOXP3 expression in adult and pediatric blood Treg cells were similar (Fig. 4a,b). Functionally, the depletion of CD25+CD4+ Treg cells from pediatric lymph node T cell cultures resulted in increased T cell proliferation (Fig. 4c) and greater cytokine production (Fig. 4d and Supplementary Table 6); adult LN T cells, by contrast, were activated similarly whether or not CD25 was depleted (Fig. 4c,d). A comparable increase in T cell proliferation was observed when Treg cells were depleted from cultures derived from pediatric lungs and colon (Supplementary Fig. 6). Together, these results suggest that early in life, Treg cells may be suppressing tissue immune responses to a greater extent than they do later in life. The findings are consistent with recent results in advance online publication nature medicine letters Non-Treg 22 years old c 2 months ∆MFl 2,064 ∆MFl 3,819 4 months 2 years 16 months 22 years CD4+ T cells ** 3,000 * n.s. n.s. CD8+ T cells CFSE CD25-depleted N LL M LN Sp le e n d 0 Total T cells 60 40 20 d 3 80 60 40 20 0 Pediatric Adult * Fold change in cytokine production Unstimulated 1,000 Bl oo Undivided CD8+ T cells (%) Pediatric Adult % max FOXP3 MFI b 4,000 e ** 2 4 16 Age (months) 2 22 Age (years) 4 3 Note: Any Supplementary Information and Source Data files are available in the online version of the paper. Acknowledgments This work was supported by the US National Institutes of Health (NIH) (grant no. AI100119; D.L.F., AI106697: D.L.F., F31AG047003; J.J.C.T., AI083022; K.L.B.) and a BD Bioscience Research Grant (J.J.C.T.). These studies were performed in the Columbia Center for Translational Immunology (CCTI) Flow Cytometry Core, funded in part through an S10 Shared Instrumentation Grant from the NIH (grant no. S10RR027050), with the excellent technical assistance of S.-H. Ho. nature medicine advance online publication on ol C m um lle LN nu M ju Je N ng Lu N IL LL en oo Sp Bl -2 d Methods Methods and any associated references are available in the online version of the paper. IL -γ N mice that show distinct roles for perinatal Treg cells in establishing self-tolerance30. Our results provide a new view of infant T cell distribution and function throughout the body, with important implications for promoting immune health and protection in early life. We show that naive and Treg cells populate the lymphoid and mucosal tissues during infancy, and that differentiation of functional memory T cells occurs in certain mucosal sites. We propose that the higher Treg:TEM cell content seen in infant as compared to adult tissues (Fig. 4e) leads to in situ regulation of T cell differentiation; this confines early T cell differentiation activation and memory formation to the lungs and small intestines, both sites of continuous, high-level antigen exposure. Our results suggest that immunization may be targeted during infancy to sites with a low ratio of Treg:TEM cells for the promotion of immune protection during the early years and beyond. le Treg / TEM Figure 4 Pediatric tissue Treg cells suppress endogenous T cell proliferation 2 and function. (a) FOXP3 expression by pediatric and adult Treg cells. 2 Representative FOXP3 expression by mesenteric lymph node (MLN) 1 1 CD4+CD25+CD127− cells (red) compared to CD25−CD127+FOXP3− cells (black dashed) from a pediatric (16 months, left) donor and an adult (22 years, 0 0 right) donor. ∆MFI (change in mean fluorescence intensity) = MFI(Treg cells)–MFI(control non-Treg cells). (b) Normalized FOXP3 MFI (± s.e.m.) as in a of Treg cells from the blood and lymphoid tissues of ten 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 MLN tissue from pediatric and adult donors of the indicated ages after anti-CD3/CD28 stimulation. Left, CFSE dilution of activated compared to unstimulated (gray shaded) CD4+ (top) and CD8+ (bottom) T cells. Right, percent of CFSE+ (undivided) CD4+ (top) or CD8+ (bottom) T cells stimulated either with or without CD25-depletion for each individual infant and adult donor. (d) Cytokine production by spleen and MLN T cells in the presence or absence of Treg cells. T cells from pediatric and adult spleen and MLN were activated as in c with or without CD25 depletion, and interferon-γ (IFN-γ) and interleukin-2 (IL-2) in supernatants were measured after 48 h. Graph shows mean fold change (± s.e.m.) calculated by dividing the cytokine level produced by the CD25-depleted by the level obtained from unfractionated pediatric (white, n = 4) and adult (black, n = 4) T cells. *P < 0.05; **P < 0.001. (e) Average Treg:TEM cell ratios in tissues from pediatric donors (0–2 yrs, n = 17). Red dashed line indicates the maximum Treg:TEM cell ratio in adult tissues. Tissues displayed are ordered from blood, red, to mucosal sites, green. lF © 2015 Nature America, Inc. All rights reserved. CD25-depleted 80 0 100 FOXP3 2,000 Unfractionated 100 Undivided CD4+ T cells (%) Treg 16 months old % max a We gratefully acknowledge the generosity of the organ donor families and the efforts of the LiveOnNY transplant coordinators and staff for making this study possible. We also wish to thank S. Mickel and B. Kumar for their assistance with tissue processing, B. Levin and N. Yudanin for their assistance with statistical analyses and K. Zens, N. Yudanin and E. 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Science 348, 589–594 (2015). advance online publication nature medicine ONLINE METHODS © 2015 Nature America, Inc. All rights reserved. Acquisition of human tissue. Human tissues were obtained from deceased (brain dead) organ donors at the time of organ acquisition for life-saving clinical transplantation. Infant donor tissue was obtained in collaboration with the Columbia University/New York Presbyterian Hospital pediatric liver transplant program. Adult donor tissues were obtained through an approved protocol and material transfer agreement with LiveOnNY (formerly the New York Organ Donor Network, NYODN). Organ donors were free of chronic disease and cancer, negative for hepatitis B, hepatitis C and HIV and 79% male. The study does not qualify as human subjects research, as confirmed by the Columbia University IRB, because tissue samples were obtained from deceased individuals. Cord blood was obtained as discarded samples from K. Liu, Columbia University. Adult blood and pediatric thymus tissues were acquired through the Columbia Center for Translational Immunology (CCTI) human studies core, which operates in accordance with the Columbia University human research protection office institutional review board. Lymphocyte isolation from human lymphoid and non-lymphoid tissues. Tissue samples were maintained in cold saline and brought to the laboratory within 2–4 h of organ procurement. Samples were rapidly processed using enzymatic and mechanical digestion to obtain lymphocyte populations with high viability, as described in detail7,8. Tissues were minced and incubated at 37 °C in enzymatic digestion media: RPMI (Thermo Fisher, Waltham, MA) containing 10% FBS (Thermo Fisher), l-glutamate (Thermo Fisher), sodium pyruvate (Thermo Fisher), nonessential amino acids (Thermo Fisher), penicillin-streptomycin (Thermo Fisher), collagenase D (1 mg/ml, Roche, Indianapolis, IN), trypsin inhibitor (1 mg/ml, Thermo Fisher) and DNase I (0.1 mg/ml, Roche). Digested tissue was further disrupted using the gentleMACS tissue dissociator (Miltenyi Biotech, San Diego, CA); the resulting suspension was passed through a tissue sieve (10–150 mesh size) and then pelleted through centrifugation. Residual red blood cells (RBC) were lysed using AKC lysis buffer (Corning Cellgro, Manassas, VA), and dead cells and debris were removed via centrifugation through 30% Percoll (GE Healthcare Life Sciences, Pittsburgh, PA). Lymphocytes were isolated from blood using lymphocyte separation media (Cellgro) and AKC lysis buffer as described7. Flow cytometry analysis. The following fluorochrome-conjugated antibodies were used for surface staining: anti-human CD3 (Brilliant Violet 650, 1:100, OKT3, BioLegend, San Diego, CA), CD4 (PeCy7, 1:100, SK3, BioLegend), CD8 (APC-Cy7, 1:100, SK1, BD Biosciences), CD19 (PE Texas Red, 1:100, SJ25-C1, Invitrogen or APC, HIB19, BioLegend), CD25 (FITC, 1:50, 2A3, BD Biosciences, San Jose, CA), CD31 (APC, 1:100, WM59, eBioscience, San Diego, CA), CD45RA (Brilliant Violet 605, 1:100, HI100, BioLegend), CD45RO (PerCpFl710, 1:100, UCHL1, eBioscience), CD69 (Brilliant Violet 421, 1:100, doi:10.1038/nm.4008 FN50, BioLegend or BUV395, 1:100, FN50, BD Biosciences), CD103, (Alexa Fluor 647, 1:100, Ber-ACT8, BioLegend), CD127 (BV711, 1:100, A019D5, BioLegend), CCR7 (Alexa Fluor 488, 1:100, TG8, BioLegend). For intracellular staining, surface stained cells were resuspended and incubated in fixation buffer (eBioscience), washed, resuspended in 0.1 ml permeabilization buffer (eBioscience) and stained with anti-FOXP3 antibodies (PE, 1:20, 236A/E7, eBioscience) and Ki67 (α700, 1:100, Ki-67, BioLegend) for 30 min at room temperature and washed twice with permeabilization buffer. Stained cells were acquired on a 6-laser LSRII analytical flow cytometer (BD Biosciences) in the CCTI flow cytometry core and analyzed using FlowJo software (Treestar, Ashland, OR). T cell proliferation and Treg cell depletion. T cells isolated from tissue suspensions as above were purified using a human T cell enrichment kit (STEMCELL Technologies, Vancouver, BC). A portion of these cells was subjected to CD25 depletion using BD IMag CD25 Magnetic Particles (BD Biosciences), with the CD25-depleted population being collected in the column flow-through. Purified T cells (unfractionated and CD25-depleted) were labeled with 2.5 µM CFSE for 15 min at 37 °C, washed with media + FBS to quench excess CFSE and plated in 96-well round bottom plates (100,000 cells per well) +/− antiCD2/CD3/CD28-coupled beads (one bead/cell; T cell activation/expansion kit, Miltenyi Biotech) for 4 days at 37 °C incubator with 5% CO2, after which cells were harvested and analyzed by flow cytometry. Cytokine analysis. Supernatants from T cell cultures treated as above were analyzed for cytokine content by Luminex using a cytokine human 25-plex panel (Life Technologies, Grand Island, NY), as performed by the Human Immunology Core (University of Pennsylvania, Philadelphia, PA). For intracellular cytokine staining, isolated T cells were cultured for 4 h with PMA/ ionomycin in the presence of Brefeldin A as described7. Cells were then stained for surface markers, fixed, permeabilized as above and stained with antibodies for IL-8 (PerCP, 1:20, BH0814, BioLegend) and IFN-γ (Alexa Fluor 700, B27, BD Biosciences). Cytokine concentrations were also measured in supernatants using cytometric bead array with the BD Biosciences Human Th1/Th2 cytokine kit II following stimulation with anti-CD2/CD3/CD28–coupled beads, as indicated above, for 2 days. Statistical analysis and data visualization. Descriptive statistics were calculated using Microsoft Excel. Statistical significance was calculated for each subset using Student’s t tests or by two-way ANOVA assuming unequal variance and adjusted for multiple comparisons by Holm-Sidak using GraphPad PRISM (GraphPad Software, Inc., La Jolla, CA). We did not use statistical methods to predetermine sample size, nor were the investigators blinded to sample identity or results. nature medicine