The neonatal immune system has been shown to respond differently when compared with the adult system. Most research on this phenomenon has been performed with cord blood (CB) cells as a representative for the neonatal immune system, showing differences in cytokine production,1Belderbos M.E. Levy O. Stalpers F. Kimpen J.L. Meyaard L. Bont L. Neonatal plasma polarizes TLR4-mediated cytokine responses towards low IL-12p70 and high IL-10 production via distinct factors.PLoS ONE. 2012; 7: e33419Crossref PubMed Scopus (55) Google Scholar receptor expression,2de Roock S. Hoeks S.B.E.A. Meurs L. Steur A. Hoekstra M.O. Prakken B.J. et al.Critical role for programmed death 1 signaling and protein kinase B in augmented regulatory T-cell induction in cord blood.J Allergy Clin Immunol. 2011; 128: 1369Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar and cell differentiation capacity.3Kollmann T.R. Crabtree J. Rein-Weston A. Blimkie D. Thommai F. Wang X.Y. et al.Neonatal innate TLR-mediated responses are distinct from those of adults.J Immunol. 2009; 183: 7150-7160Crossref PubMed Scopus (363) Google Scholar Because of ethical restrictions, it is hard to obtain cells from healthy newborns and infants. Children prenatally diagnosed with an orofacial cleft will have several surgical corrections in the first year of life, providing the opportunity to study immune maturations with longitudinal samples (see Table E1 and additional information on methods in this article's Online Repository at www.jacionline.org). To provide an insight into the changes in the composition of peripheral blood cells after birth, we analyzed the relative fractions of T cells, monocytes, and B cells of all infants at different ages. Although the total number of T cells did not change, the ratio between CD4 and CD8 cells decreased after birth. The number of monocytes gradually decreased over the first year of life but was higher again in adults. B cells showed an inverse pattern (see Fig E1, A-E, in this article's Online Repository at www.jacionline.org). We further characterized subtypes of CD4+ T-helper cells. Most of these cells had a naive (CD45RA+) phenotype in CB (Fig 1). Although we hypothesized that colonization of the mucosal tissues early after birth would result in a rapid increase in cells with a memory (CD45RO+) phenotype, we observed only a slight change over the first year of life (Fig 1, A). No differences were found in the percentages of Forkhead box protein 3 (FOXP3+) regulatory T (Treg) cells, but we did observe differences in the ratios of subpopulations as described by Miyara et al.4Miyara M. Yoshioka Y. Kitoh A. Shima T. Wing K. Niwa A. et al.Functional delineation and differentiation dynamics of human CD4+ T cells expressing the FoxP3 transcription factor.Immunity. 2009; 30: 899Abstract Full Text Full Text PDF PubMed Scopus (1742) Google Scholar Resting Treg cells in population I are described as potent suppressors that are not in cell cycle. These cells were significantly more abundant throughout the first year of life. Activated Treg cells are not only potent suppressors but also express proliferation markers. These cells were present in equal proportions in all age groups. Population III has been described as consisting of less stable Treg cells with regard to the DNA methylation status of the FOXP3 locus and containing IL-17–producing cells while their suppressive capacity is under debate.4Miyara M. Yoshioka Y. Kitoh A. Shima T. Wing K. Niwa A. et al.Functional delineation and differentiation dynamics of human CD4+ T cells expressing the FoxP3 transcription factor.Immunity. 2009; 30: 899Abstract Full Text Full Text PDF PubMed Scopus (1742) Google Scholar, 5Afzali B. Mitchell P.J. Edozie F.C. Povoleri G.A.M. Dowson S.E. Demandt L. et al.CD161 expression characterizes a sub-population of human regulatory T cells that produces IL-17 in a STAT3 dependent manner.Eur J Immunol. 2013; 43: 2043Crossref PubMed Scopus (105) Google Scholar These cells were significantly more abundant in adult samples. Next, we assessed the cytokine production of isolated PBMCs after activation by phorbol 12-myristate 13-acetate and ionomycin for 4 hours. Very low concentrations of T-cell cytokines IL-17, IL-13, and IL-10 were found, without age-dependent differences (not shown). IL-1β, IL-6, and TNF-α were significantly lower in CB samples as shown before6de Roock S. Stoppelenburg A.J. Scholman R. Hoeks S.B.E.A. Meerding J. Prakken B.J. et al.Defective TH17 development in human neonatal T cells involves reduced RORC2 mRNA content.J Allergy Clin Immunol. 2013; 132: 754Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar and increased with age (Fig 1, C-E). IFN-γ was the only T-cell cytokine observed in considerable amounts in adult samples, but not in CB and 3-month samples (see Fig E2 in this article's Online Repository at www.jacionline.org). So, the ex vivo phenotype of all infant samples showed a more naive and regulatory phenotype and an increasing capacity of inflammatory cytokine production with age. To investigate the capacity of T cells to become activated and differentiate into different T-cell subsets, we cultured PBMCs for 6 days with anti-CD3. We focused on Treg cells and TH17 cells because it has been shown that CB cells differ from adult cells in their capacity to differentiate into these cells.2de Roock S. Hoeks S.B.E.A. Meurs L. Steur A. Hoekstra M.O. Prakken B.J. et al.Critical role for programmed death 1 signaling and protein kinase B in augmented regulatory T-cell induction in cord blood.J Allergy Clin Immunol. 2011; 128: 1369Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar, 6de Roock S. Stoppelenburg A.J. Scholman R. Hoeks S.B.E.A. Meerding J. Prakken B.J. et al.Defective TH17 development in human neonatal T cells involves reduced RORC2 mRNA content.J Allergy Clin Immunol. 2013; 132: 754Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar In addition, the gut microbiota has an important role in the development of these cells and colonization occurs soon after birth.7Ivanov I.I. Frutos Rde L. Manel N. Yoshinaga K. Rifkin D.B. Sartor R.B. et al.Specific microbiota direct the differentiation of IL-17-producing T-helper cells in the mucosa of the small intestine.Cell Host Microbe. 2008; 4: 337-349Abstract Full Text Full Text PDF PubMed Scopus (1356) Google Scholar, 8Wu H.J. Ivanov I.I. Darce J. Hattori K. Shima T. Umesaki Y. et al.Gut-residing segmented filamentous bacteria drive autoimmune arthritis via T helper 17 cells.Immunity. 2010; 32: 815-827Abstract Full Text Full Text PDF PubMed Scopus (1248) Google Scholar, 9Round J.L. Mazmanian S.K. Inducible Foxp3+ regulatory T-cell development by a commensal bacterium of the intestinal microbiota.Proc Natl Acad Sci U S A. 2010; 107: 12204-12209Crossref PubMed Scopus (1677) Google Scholar We therefore hypothesized that the infant samples would reflect an adult phenotype with regard to TH17 and Treg-cell differentiation. First, we assessed the proliferation of PBMCs on anti-CD3 activation by 3H-labeled thymidine incorporation. Cells derived from adults showed the highest proliferation, although only significantly different from the samples obtained at 3 months (see Fig E3, A, in the Online Repository at www.jacionline.org). The inability of CB cells to produce IL-17 was confirmed in these assays (Fig 2, A and B). The number of T cells producing IL-17 was already increased at age 3 months, and the amount of IL-17 in the supernatant of these cell cultures gradually increased from CB to adulthood. On activation, CB cells easily upregulated FOXP3 when compared with adult cells (Fig 2, C). In contrast to the differences in TH17 development, this phenomenon was still observed in samples derived from children aged 3 to 12 months. So, the propensity of CB T cells to become Treg cells remained until at least age 1 year, while the inability to become TH17 is overcome within the first months of life. Furthermore, we investigated the production of other T-cell–derived cytokines in these cultures. No difference in IL-13 production was found. The assumed TH2 bias in infants was mainly present in the form of a significantly reduced IFN-γ production, corresponding with a lower IFN-γ/IL-13 ratio compared with that in adults (see Fig E3, B-D). Antigen-presenting cells (APCs) in CB have an important role in the induction of Treg cells from naive T cells. Alloreactions between naive T cells and APCs show higher numbers of Treg cells when CB-derived APCs are used.2de Roock S. Hoeks S.B.E.A. Meurs L. Steur A. Hoekstra M.O. Prakken B.J. et al.Critical role for programmed death 1 signaling and protein kinase B in augmented regulatory T-cell induction in cord blood.J Allergy Clin Immunol. 2011; 128: 1369Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar We wondered whether this capacity of CB APCs is also observed in infant-derived APCs. We first tested the number of induced Treg cells when fluorescence activated cell sorting–sorted CD4+CD45RA+CD45RO− naive T cells were activated with autologous APCs (Fig 2, D). In contrast to CB samples, all other samples had Treg-cell numbers comparable to adult samples. Next, we activated naive T cells from samples from donors aged 3 to 12 months with autologous APCs from CB and vice versa. Because of low cell numbers, we had to pool results from all infant samples. CB APCs induced more FOXP3+ T cells than did APCs derived from older donors (Fig 2, E). Nevertheless, CB T cells always showed a higher percentage of FOXP3+ cells than did T cells from 3- to 12-month-old donors. So, the propensity of CB naive T cells to become Treg cells was dependent on the interaction between T cells and APCs. We conclude that neonatal T cells develop the capacity to differentiate into TH17 cells before the age of 3 months and retain a propensity to become Treg cells until the age of at least 12 months. So, immunity against pathogens is being raised while at the same time the immune system remains to have a regulatory profile. This latter fact might be important in the maintenance of tolerance toward allergens and food antigens encountered for the first time. We thank all of the infants and their parents who took part in this study, Dr L. Pistorius and Dr G. T. R. Manten for their help with the inclusion of the participants, and Mariska van Dijk for performing the Luminex assay. Between June 2011 and January 2013, 7 children were enrolled in the study. Infant characteristics are presented in Table 1. All mothers were healthy and had uncomplicated pregnancies. All children were prenatally diagnosed with a different severity of orofacial cleft that required 1 or multiple chirurgical corrections. Blood samples were drawn from the umbilical cord directly after birth and from peripheral blood during surgical interventions that took place at regular intervals after birth. Additional control samples were taken by venapuncture from 6 unrelated healthy volunteers. This study was approved by the local medical committee of the University Medical Center Utrecht (08-331). Written informed consent was obtained from all parents before inclusion in the study. All procedures adhered to the principles of the Declaration of Helsinki. After collection of the blood samples, CBMCs or PBMCs were isolated within 24 hours by using Ficoll-Isopaque density gradient centrifugation (Ficoll-Isopaque, Pharmacia, Upsalla, Sweden). Cells were washed and frozen in 90% FCS and 10% dimethyl sulfoxide at −80°C. Cells stored for more than 1 month were transferred to −150°C. All samples from one donor were thawed simultaneously together with a sample from an adult donor. Cells were subsequently aliquoted for different experiments. For ex vivo cytokine production measurements, 1 × 105 PBMCs were cultured in culture medium (RPMI 1640 supplemented with 1% l-glutamine and 100 mg/mL streptomycin and 100 U/mL penicillin; Gibco, Breda, The Netherlands) with 20 ng/mL phorbol myristate acetate and 1 μg/mL ionomycin at 37°C. Then, either after 4 hours, supernatants were harvested for cytokine analysis, or after 30 minutes, monensin (Golgistop, BD Bioscience, San Jose, Calif) was added and cells were cultured for another 3.5 hours and stained for IL-17 production. For measurement of proliferation, differentiation, or cytokine production on immune activation, 1 × 105 PBMCs were activated by plate-bound anti-CD3 (1 μg/mL, clone OKT3; eBioscience, San Diego, Calif) at 37°C. After 4 days, 3H-labeled thymidine was added for another 16 hours for the measurement of proliferation. After 6 days of culture with plate-bound anti-CD3, supernatant was harvested for cytokine measurement and cells were used for flow cytometric analysis after restimulation with 20 ng/mL phorbol 12-myristate 13-acetate and 1 μg/mL ionomycin for 4 hours in the presence of monensin. Naive T cells and APCs were fluorescence activated cell sorting–sorted on an ARIA II (BD Bioscience) as CD4+CD45RA+CD45RO− and CD3− fractions, respectively. A total of 5 × 104 naive T cells and 5 × 104 APCs were activated by 1 μg/mL plate-bound anti-CD3 for 6 days and subsequently analyzed for FOXP3 expression by using flow cytometry. After thawing, an aliquot of cells was immediately stained with antibodies directed against CD3, CD4, CD8, CD14, and CD19 to quantify relative numbers of CD4 and CD8 T cells, monocytes, and B cells, respectively. Naive and memory cells were quantified by staining with CD45RA and CD45RO. For Treg-cell quantification, cells were permeabilized by using the FOXP3 staining kit from eBioscience and subsequently stained with an antibody against FOXP3 (clone PCH101, eBioscience). IL-17 (clone eBio64DEC17, eBioscience) was stained after restimulation with phorbol 12-myristate 13-acetate and ionomycin in the presence of monensin and permeabilization with the FOXP3 staining kit. After culture, cells were stained with a fixable viability dye (eBioscience) to discriminate between dead and viable cells. Supernatants of cell cultures were harvested and frozen at −80°C until analysis. Cytokine concentrations were measured by using Multiplex analysis (Luminex, Austin, Tex) as described elsewhere.E1de Jager W. Prakken B.J. Bijlsma J.W.J. Kuis W. Rijkers G.T. Improved multiplex immunoassay performance in human plasma and synovial fluid following removal of interfering heterophilic antibodies.J Immunol Methods. 2005; 300: 124Crossref PubMed Scopus (209) Google Scholar Significance of the differences observed was tested by using the Kruskal Wallis test and Dunn's Multiple comparisons test using GraphPad Prism 5.03 (GraphPad Software, San Diego, Calif) for Windows. CB and infant samples were compared with samples derived from adult controls.Fig E2T-cell subsets ex vivo. A, CD4+FOXP3+ cell percentages ex vivo. B, PBMCs were stimulated with phorbol 12-myristate 13-acetate and ionomycin. Supernatants were collected and analyzed for IFN-γ. *P < .05.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig E3Proliferation and cytokine production after activation with anti-CD3. PBMCs were activated by plate-bound anti-CD3. A, Proliferation was measured after 4 days of culture by 3H-labeled thymidine incorporation for 16 hours. B and C, After 6 days, culture supernatants were harvested and analyzed for IFN-γ and IL-13 production, respectively. D, Ratio between IFN-γ and IL-13 in the culture supernatant. *P < .05.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Table E1Infant and sample characteristicsInfant characteristicsMode of delivery Vaginal (%)71 Cesarean section (%)29Sex Female (%)29 Male (%)71Prematurity (%, GA below 37 wk)14 (GA 34 + 4 wk)Birth weight (g), mean ± SD3307 ± 819Infant samplesCB (%)863 mo (%)576 mo (%)4312 mo (%)43GA, Gestational age. Open table in a new tab GA, Gestational age.

Cytokines are key components of the innate and adaptive immune system. As pivotal players in the progression or regression of a pathological process, these molecules provide a window through which diseases can be monitored and can thus act as biomarkers. In order to measure cytokine levels, a plethora of protocols can be applied. These methods include bioassays, protein microarrays, high-performance liquid chromatography (HPLC), sandwich enzyme-linked immunosorbent assay (ELISA), Meso Scale Discovery (MSD) electrochemiluminescence and bead based multiplex immunoassays (MIA). Due to the interaction and activity of cytokines, multiplex immunoassays are at the forefront of cytokine analysis by allowing multiple cytokines to be measured in parallel. However, even with optimized protocols, sample standardization needs to occur before these proteins can optimally act as biomarkers. This review describes various factors influencing the levels of cytokines measured in plasma, serum, dried blood spots and tissue biopsies, focusing on sample collection and handling, long term storage and the repetitive use of samples. By analyzing how each of these factors influences protein levels, it is concluded that samples should be stored at low temperatures in order to maintain cytokine stability. In addition, within a study, sample manipulations should be kept the same, with measurement protocols being chosen for their compatibility with the research in question. By having a clear understanding of what factors influence cytokine levels and how to overcome these technical issues, minimally confounded data can be obtained and cytokines can achieve optimal biomarker activity.

To the Editor: In newborns the induction of productive immune responses is generally blunted in comparison with adults, resulting in tolerogenic immune reactivity.1Billingham R.E. Brent L. Medawar P.B. Actively acquired tolerance of foreign cells.Nature. 1953; 172: 603-606Crossref PubMed Scopus (2257) Google Scholar This immune status results in an increased potency to engraft neonatal animals, ineffective vaccination responses in newborns, and reduced occurrence of graft-versus-host disease when cord blood (CB)–derived allografts are used.2Wang J. Zhan P. Ouyang J. Chen B. Zhou R. Yang Y. Unrelated donor umbilical cord blood transplantation versus unrelated donor bone marrow transplantation in adult and pediatric patients: a meta-analysis.Leuk Res. 2010; 34: 1018Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar, 3Levy O. Innate immunity of the newborn: basic mechanisms and clinical correlates.Nat Rev Immunol. 2007; 7: 379Crossref PubMed Scopus (915) Google Scholar We hypothesized that forkhead box protein 3 (FOXP3)–positive regulatory T (Treg) cells are pivotal in this phenomenon because these cells are key players in immune homeostasis.4Bennett C.L. Christie J. Ramsdell F. Brunkow M.E. Ferguson P.J. Whitesell L. et al.The immune dysregulation, polyendocrinopathy, enteropathy, X-linked syndrome (IPEX) is caused by mutations of FOXP3.Nat Genet. 2001; 27: 20-21Crossref PubMed Scopus (2676) Google Scholar When comparing the number of FOXP3+ cells in human CB and adult peripheral blood (APB), we found less FOXP3+ cells in CB than APB (Fig 1, A, ex vivo, uncultured cells). However, when naive (CD25−CD45RO−) T cells were activated by plate-bound anti-CD3, significantly more FOXP3+ T cells were induced from CB precursors. Remarkably, we only observed this difference between CB and APB when viable antigen-presenting cells (APCs; T cell–depleted CB or APB mononuclear cell fraction) were included in the culture; replacement with irradiated APCs abrogated the effect, whereas costimulation by soluble anti-CD28 showed a less pronounced difference (Fig 1, A). Induced CB FOXP3+ T cells (sorted as CD4+CD25+CD127low cells) were able to suppress dose dependently the proliferation of both CD4 and CD8 effector T cells when cultured together in different ratios (not shown), confirming the Treg nature of these cells. Thus on the first activation, CB T cells have a tendency to become functional FOXP3+ Treg cells. To substantiate the role of CB APCs in the induction of high percentages of Treg cells, we activated naive T cells in an alloreaction with APCs. Again we observed a higher number of Treg cells when CB T cells were cultured. CB APCs were able to induce higher numbers of Treg cells than APB APCs from both CB and APB precursor T cells (Fig 1, B). Nevertheless, CB T cells always contained higher percentages of Treg cells, indicating a T-cell intrinsic mechanism as well. We investigated the percentages of different APC populations because the APCs used in our previous experiments consisted of a mixture of different cell types. In both APB and CB, we found comparable numbers of conventional dendritic cells (HLA-DR+CD11c+) and plasmacytoid dendritic cells (HLA-DR+CD123+), with slightly more CD14+ monocytes in APB (not shown). When we sorted different subsets as APCs and cultured them with anti-CD3–activated T cells, we observed that all APC subsets induced more Treg cells from CB than from APB precursor cells (Fig 1, C). The augmented Treg cell induction was not correlated with a reduced proliferation of CB T cells, which we showed to be identical in CB and APB by using carboxyfluorescein succinimidyl ester dilution assays (data not shown). Moreover, we found no difference in the kinetics of FOXP3 upregulation when we measured the percentage of FOXP3+ cells daily; both APB and CB showed a peak of FOXP3+ cells around 4 days of culture, after which the percentage of FOXP3+ cells decreased to stable expression at day 6 (Fig 1, D). Both APB and CB cells had a similar stable FOXP3-expressing population compared with the initial peak around day 4; 65% of the FOXP3+ T cells at day 4 remain stable FOXP3-expressing cells during the 8-day culture. The induction of Treg cells in the periphery on activation can be mediated by several factors. Cytokines like TGF-β and IL-2 are known to induce FOXP3,5Lu L. Zhou X. Wang J. Zheng S.G. Horwitz D.A. Characterization of protective human CD4+ CD25+ FOXP3+ regulatory T cells generated with IL-2, TGF-β and retinoic acid.PLoS ONE. 2010; 5: e15150Crossref PubMed Scopus (111) Google Scholar whereas inflammatory cytokines prevent this. To test whether possible differences in secreted mediators, such as cytokines and chemokines, can explain the difference in Treg cell induction, we exchanged culture supernatants between CB and APB every day during the 6-day culture period of naive T cells with APCs and anti-CD3. This approach did not result in altered differences between CB and APB Treg cell induction, making a role for secreted mediators unlikely (not shown). Another factor influencing FOXP3 induction is the strength of the T-cell receptor (TCR) signal and costimulation.6Gottschalk R.A. Corse E. Allison J.P. TCR ligand density and affinity determine peripheral induction of Foxp3 in vivo.J Exp Med. 2010; 207: 1701-1711Crossref PubMed Scopus (211) Google Scholar, 7Semple K. Nguyen A. Yu Y. Wang H. Anasetti C. Yu X.Z. Strong CD28 costimulation suppresses induction of regulatory T cells from naive precursors through Lck signaling.Blood. 2011; 117: 3096-3103Crossref PubMed Scopus (69) Google Scholar In our settings the TCR signal was standardized by plate-bound anti-CD3, but costimulation is provided by the APCs. High costimulation through CD28 signaling prevents the upregulation of FOXP3. We therefore investigated the role of the CD28 ligands CD80 and CD86 on APCs. We observed no difference in the expression of these molecules. When blocking antibodies toward CD80, CD86, or both were added to the cultures, this did not result in a difference in FOXP3 expression (Fig 2, A). This finding was confirmed by adding increasing concentrations of cytotoxic T lymphocyte–associated antigen 4-immunoglobulin (CTLA4-Ig) to the cultures, blocking both CD80 and CD86, which had no obvious effect (data not shown). Inhibition of downstream signaling pathways of TCR and CD28 on T-cell activation is able to promote FOXP3 induction as well. The phosphoinositide 3-kinase/protein kinase B (PKB) pathway is central in T-cell differentiation.8Haxhinasto S. Mathis D. Benoist C. The AKT-mTOR axis regulates de novo differentiation of CD4+Foxp3+ cells.J Exp Med. 2008; 205: 565-574Crossref PubMed Scopus (609) Google Scholar Prevention of activation of this pathway by limited costimulation or signaling through inhibitory molecules, such as programmed death 1 (PD-1), promotes FOXP3 upregulation.7Semple K. Nguyen A. Yu Y. Wang H. Anasetti C. Yu X.Z. Strong CD28 costimulation suppresses induction of regulatory T cells from naive precursors through Lck signaling.Blood. 2011; 117: 3096-3103Crossref PubMed Scopus (69) Google Scholar, 9Francisco L.M. Salinas V.H. Brown K.E. Vanguri V.K. Freeman G.J. Kuchroo V.K. et al.PD-L1 regulates the development, maintenance, and function of induced regulatory T cells.J Exp Med. 2009; 206: 3015-3029Crossref PubMed Scopus (1511) Google Scholar We hypothesized that differential downstream signaling on activation might be pivotal to the high levels of FOXP3 found on day 6 of activation and onward (Fig 1, D). We therefore investigated the role of PD-1 and PKB in the induction of FOXP3 in CB-naive T cells. On activation, CB CD4+ T cells have an increased expression of the PD-1 molecule compared with APB (Fig 2, B). When we blocked the interaction between PD-1 and its ligand PD-L1 by including a blocking mAb to PD-L1 in the culture, a significant reduction in the percentage of FOXP3+ T cells was observed (Fig 2, C), isotype mAb had no effect (not shown). A PD-1–blocking mAb showed comparable results. As a consequence, the phosphorylation status of PKB on activation by different concentrations of anti-CD3 was lower for CB than APB cells (Fig 2, D). Additionally, CB T cells required approximately 10-fold more TCR triggering than APB T cells before an increase in PKB phosphorylation was observed. Shortly after birth, the immune system of the newborn encounters all kinds of neoantigens. The low percentages of Treg cells found ex vivo in CB are not likely to play a major role in maintaining tolerance to these neoantigens. However, we here show that PD-1 signaling in CB T cells facilitates their differentiation into induced functional FOXP3+ Treg cells through a mechanism involving reduced PKB signaling. This phenomenon might well represent a mechanism that is developed to ensure active tolerance in the neonatal immune system.