An updated management approach of Pompe disease patients with high-sustained anti-rhGAA IgG antibody titers: experience with bortezomib-based immunomodulation.

Introduction: High sustained anti-rhGAA antibody titers (HSAT; ≥12,800) are directly linked to reduced efficacy of enzyme replacement therapy (ERT) and subsequent clinical deterioration in infantile-onset Pompe disease (IOPD). We have previously demonstrated the safety and effectiveness of a bortezomib-based immune-tolerance induction (ITI) regimen (bortezomib, rituximab, methotrexate, and IVIG) in eliminating HSAT. Methods: Here, we describe two IOPD cases (patients 6 and 8) who developed HSAT at 8 and 10 weeks on ERT despite transient low-dose methotrexate ITI administration in the ERT-naïve setting and were treated with a bortezomib-based ITI regimen, and we compare their courses to a series of six historical patients (patients 1-5, and 7) with a similar presentation who exemplify our evolving approach to treatment. Results: In total, patients 6 and 8 received 16 and 8 doses of bortezomib (4 doses=1 cycle) respectively reducing titers from 25,600 to seronegative, but differences in the course of their therapy were instructive regarding the optimal approach to initial treatment of HSAT; specifically, patient 6 was treated initially with only a single course of bortezomib rescue therapy, while patient 8 received two back-to-back courses. Patient 8 received IVIG therapy throughout the immunosuppression whereas patient 6 received IVIG therapy and was switched to subcutaneous IgG replacement. Patient 6 had a transient reduction in anti-rhGAA antibodies, after receiving a single initial cycle of bortezomib, but had a recurrence of high anti-rhGAA antibody titer after 160 weeks that required 3 additional cycles of bortezomib to ultimately achieve tolerance. In contrast, patient 8 achieved tolerance after being given two consecutive cycles of bortezomib during their initial treatment and had B cell recovery by week 54. Since the reduction in anti-rhGAA antibodies, both patients are doing well clinically, and have decreasing ALT, AST, and CK. No major infections leading to interruption of treatment were observed in either patient. The bortezomib-based ITI was safe and well-tolerated, and patients continue to receive ERT at 40 mg/kg/week. Discussion: These case studies and our previous experience suggest that to achieve an effective reduction of anti-rhGAA antibodies in the setting of HSAT, bortezomib should be initiated at the earliest sign of high anti-rhGAA antibodies with a minimum of two consecutive cycles as shown in the case of patient 8. It is important to note that, despite initiation of ERT at age 2.3 weeks, patient 8 quickly developed HSAT. We recommend close monitoring of anti-rhGAA antibodies and early intervention with ITI as soon as significantly elevated anti-rhGAA antibody titers are noted.

Subventricular zone stem cell niche injury is associated with intestinal perforation in preterm infants and predicts future motor impairment.

Brain injury is highly associated with preterm birth. Complications of prematurity, including spontaneous or necrotizing enterocolitis (NEC)-associated intestinal perforations, are linked to lifelong neurologic impairment, yet the mechanisms are poorly understood. Early diagnosis of preterm brain injuries remains a significant challenge. Here, we identified subventricular zone echogenicity (SVE) on cranial ultrasound in preterm infants following intestinal perforations. The development of SVE was significantly associated with motor impairment at 2 years. SVE was replicated in a neonatal mouse model of intestinal perforation. Examination of the murine echogenic subventricular zone (SVZ) revealed NLRP3-inflammasome assembly in multiciliated FoxJ1+ ependymal cells and a loss of the ependymal border in this postnatal stem cell niche. These data suggest a mechanism of preterm brain injury localized to the SVZ that has not been adequately considered. Ultrasound detection of SVE may serve as an early biomarker for neurodevelopmental impairment after inflammatory disease in preterm infants.

Homeostatic cytokines reciprocally modulate the emergence of prenatal effector PLZF+CD4+ T cells in humans.

The development of human prenatal adaptive immunity progresses faster than previously appreciated, with the emergence of memory CD4+ T cells alongside regulatory T cells by midgestation. We previously identified a prenatal specific population of promyelocytic leukemia zinc finger-positive (PLZF+) CD4+ T cells with heightened effector potential that were enriched in the developing intestine and accumulated in the cord blood of infants exposed to prenatal inflammation. However, the signals that drive their tissue distribution and effector maturation are unknown. Here, we define the transcriptional and functional heterogeneity of human prenatal PLZF+CD4+ T cells and identify the compartmentalization of T helper-like (Th-like) effector function across the small intestine (SI) and mesenteric lymph nodes (MLNs). IL-7 was more abundant in the SI relative to the MLNs and drove the preferential expansion of naive PLZF+CD4+ T cells via enhanced STAT5 and MEK/ERK signaling. Exposure to IL-7 was sufficient to induce the acquisition of CD45RO expression and rapid effector function in a subset of PLZF+CD4+ T cells, identifying a human analog of memory phenotype CD4+ T cells. Further, IL-7 modulated the differentiation of Th1- and Th17-like PLZF+CD4+ T cells and thus likely contributes to the anatomic compartmentalization of human prenatal CD4+ T cell effector function.

In utero human cytomegalovirus infection expands NK cell-like FcγRIII-expressing CD8+ T cells that mediate antibody-dependent functions.

Human cytomegalovirus (HCMV) profoundly modulates host T and natural killer (NK) cells across the lifespan, expanding unique effector cells bridging innate and adaptive immunity. Though HCMV is the most common congenital infection worldwide, how this ubiquitous herpesvirus impacts developing fetal T and NK cells remains unclear. Using computational flow cytometry and transcriptome profiling of cord blood from neonates with and without congenital HCMV (cCMV) infection, we identify major shifts in fetal cellular immunity marked by an expansion of Fcγ receptor III (FcγRIII)-expressing CD8+ T cells (FcRT) following HCMV exposure in utero. FcRT cells from cCMV-infected neonates express a cytotoxic NK cell-like transcriptome and mediate antigen-specific antibody-dependent functions including degranulation and IFNγ production, the hallmarks of NK cell antibody-dependent cellular cytotoxicity (ADCC). FcRT cells may represent a previously unappreciated effector population with innate-like functions that could be harnessed for maternal-infant vaccination strategies and antibody-based therapeutics in early life.

Human fetal T cells: Insights into developmental specialization and mechanisms of lineage transition.

The switch from primitive to definitive hematopoiesis occurs early in development through the emergence of a wave of definitive hematopoietic stem cells from intraembryonic sites, supplanting the original primitive population of extraembryonically derived stem cells. When it became clear that unique features of the fetal immune system could not be reproduced by adult stem cells, it was hypothesized that a lineage of definitive fetal hematopoietic stem cells predominates antenatally, ultimately giving way to an emerging wave of adult stem cells and resulting in a "layered" fetal immune system consisting of overlapping lineages. It is now clear, however, that the transition from human fetal-to-adult T cell identity and function does not occur due to a binary switch between distinct fetal and adult lineages. Rather, recent evidence from single cell analysis suggests that during the latter half of fetal development a gradual, progressive transition occurs at the level of hematopoietic stem-progenitor cells (HSPCs) which is reflected in their T cell progeny. At a transcriptional level, clusters of genes are up- and down-regulated with sequenced timing, suggesting that the transition is under the control of master regulatory factors, including epigenetic modifiers. The net effect is still one of "molecular layering," that is, the continuous layering of iterative generations of HSPCs and T cells that arise through progressive changes in gene expression. This review will focus on recent discoveries that elucidate mechanisms of fetal T cell function and the transition from fetal to adult identity. The epigenetic landscape of fetal T cells facilitates their ability to fulfill the dominant fetal mandate of generating tolerance against self, maternal, and environmental antigens by supporting their predisposition to differentiate into CD25+ FoxP3+ regulatory T cells (TRegs ). We will explore how the coordinated development of two complementary populations of fetal T cells-conventional T cells dominated by TRegs and tissue-associated memory effector cells with innate-like inflammatory potential-is crucial not only for maintaining intrauterine immune quiescence but also for facilitating an immune response that is appropriately tuned for the bombardment of antigen stimulation that happens at birth.

Immunophenotype associated with high sustained antibody titers against enzyme replacement therapy in infantile-onset Pompe disease.

Introduction: The efficacy of enzyme replacement therapy (ERT) with alglucosidase alfa for infantile-onset Pompe disease (IOPD) is limited in some patients due to the development of high and sustained antibody titers (HSAT; ≥12,800). Methods: We carried out detailed immunophenotyping of IOPD patients (n=40), including analysis of circulating cell populations by flow cytometry and plasma cytokines by multiplex array, to determine whether patients with HSAT have unique immunological characteristics compared to those with low titers (LT; <12,800). Results: Compared to patients with LT, patients who develop HSAT were skewed toward a type 2 immune profile, with an increased frequency of Th2 cells that was positively correlated with levels of Th2 (IL-4, IL-5, IL-13) and pro-inflammatory (IL-6, TNF-α, MIP-1α, MIP-1ß) cytokines. B cells were increased in HSAT patients with a decreased fraction of unswitched memory B cells. Plasma GM-CSF concentrations were lower on average in HSAT patients, while CXCL11 was elevated. Finally, using principal components analysis, we derived an HSAT Signature Score that successfully stratified patients according to their antibody titers. Discussion: The immune profiles revealed in this study not only identify potential biomarkers of patients that developed HSAT but also provide insights into the pathophysiology of HSAT that will ultimately lead to improved immunotherapy strategies.

Heritable vaginal bacteria influence immune tolerance and relate to early-life markers of allergic sensitization in infancy.

Maternal asthma status, prenatal exposures, and infant gut microbiota perturbation are associated with heightened risk of atopy and asthma risk in childhood, observations hypothetically linked by intergenerational microbial transmission. Using maternal vaginal (n = 184) and paired infant stool (n = 172) samples, we identify four compositionally and functionally distinct Lactobacillus-dominated vaginal microbiota clusters (VCs) that relate to prenatal maternal health and exposures and infant serum immunoglobulin E (IgE) status at 1 year. Variance in bacteria shared between mother and infant pairs relate to VCs, maternal allergy/asthma status, and infant IgE levels. Heritable bacterial gene pathways associated with infant IgE include fatty acid synthesis and histamine and tryptophan degradation. In vitro, vertically transmitted Lactobacillus jensenii strains induce immunosuppressive phenotypes on human antigen-presenting cells. Murine supplementation with L. jensenii reduces lung eosinophils, neutrophilic expansion, and the proportion of interleukin-4 (IL-4)+ CD4+ T cells. Thus, bacterial and atopy heritability are intimately linked, suggesting a microbial component of intergenerational disease transmission.

Single-cell transcriptional profiling of human thymic stroma uncovers novel cellular heterogeneity in the thymic medulla.

The thymus' key function in the immune system is to provide the necessary environment for the development of diverse and self-tolerant T lymphocytes. While recent evidence suggests that the thymic stroma is comprised of more functionally distinct subpopulations than previously appreciated, the extent of this cellular heterogeneity in the human thymus is not well understood. Here we use single-cell RNA sequencing to comprehensively profile the human thymic stroma across multiple stages of life. Mesenchyme, pericytes and endothelial cells are identified as potential key regulators of thymic epithelial cell differentiation and thymocyte migration. In-depth analyses of epithelial cells reveal the presence of ionocytes as a medullary population, while the expression of tissue-specific antigens is mapped to different subsets of epithelial cells. This work thus provides important insight on how the diversity of thymic cells is established, and how this heterogeneity contributes to the induction of immune tolerance in humans.

Single-Cell Mapping of Progressive Fetal-to-Adult Transition in Human Naive T Cells.

Whereas the human fetal immune system is poised to generate immune tolerance and suppress inflammation in utero, an adult-like immune system emerges to orchestrate anti-pathogen immune responses in post-natal life. It has been posited that cells of the adult immune system arise as a discrete ontological "layer" of hematopoietic stem-progenitor cells (HSPCs) and their progeny; evidence supporting this model in humans has, however, been inconclusive. Here, we combine bulk and single-cell transcriptional profiling of lymphoid cells, myeloid cells, and HSPCs from fetal, perinatal, and adult developmental stages to demonstrate that the fetal-to-adult transition occurs progressively along a continuum of maturity-with a substantial degree of inter-individual variation at the time of birth-rather than via a transition between discrete waves. These findings have important implications for the design of strategies for prophylaxis against infection in the newborn and for the use of umbilical cord blood (UCB) in the setting of transplantation.

dittoSeq: universal user-friendly single-cell and bulk RNA sequencing visualization toolkit.

SUMMARY: A visualization suite for major forms of bulk and single-cell RNAseq data in R. dittoSeq is color blindness-friendly by default, robustly documented to power ease-of-use and allows highly customizable generation of both daily-use and publication-quality figures. AVAILABILITY AND IMPLEMENTATION: dittoSeq is an R package available through Bioconductor via an open source MIT license. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Understanding Early-Life Adaptive Immunity to Guide Interventions for Pediatric Health.

Infants are capable of mounting adaptive immune responses, but their ability to develop long-lasting immunity is limited. Understanding the particularities of the neonatal adaptive immune system is therefore critical to guide the design of immune-based interventions, including vaccines, in early life. In this review, we present a thorough summary of T cell, B cell, and humoral immunity in early life and discuss infant adaptive immune responses to pathogens and vaccines. We focus on the differences between T and B cell responses in early life and adulthood, which hinder the generation of long-lasting adaptive immune responses in infancy. We discuss how knowledge of early life adaptive immunity can be applied when developing vaccine strategies for this unique period of immune development. In particular, we emphasize the use of novel vaccine adjuvants and optimization of infant vaccine schedules. We also propose integrating maternal and infant immunization strategies to ensure optimal neonatal protection through passive maternal antibody transfer while avoiding hindering infant vaccine responses. Our review highlights that the infant adaptive immune system is functionally distinct and uniquely regulated compared to later life and that these particularities should be considered when designing interventions to promote pediatric health.

Helios enhances the preferential differentiation of human fetal CD4+ naïve T cells into regulatory T cells.

T cell receptor (TCR) stimulation and cytokine cues drive the differentiation of CD4+ naïve T cells into effector T cell populations with distinct proinflammatory or regulatory functions. Unlike adult naïve T cells, human fetal naïve CD4+ T cells preferentially differentiate into FOXP3+ regulatory T (Treg) cells upon TCR activation independent of exogenous cytokine signaling. This cell-intrinsic predisposition for Treg differentiation is implicated in the generation of tolerance in utero; however, the underlying mechanisms remain largely unknown. Here, we identify epigenetic and transcriptional programs shared between fetal naïve T and committed Treg cells that are inactive in adult naïve T cells and show that fetal-derived induced Treg (iTreg) cells retain this transcriptional program. We show that a subset of Treg-specific enhancers is accessible in fetal naïve T cells, including two active superenhancers at Helios Helios is expressed in fetal naïve T cells but not in adult naïve T cells, and fetal iTreg cells maintain Helios expression. CRISPR-Cas9 ablation of Helios in fetal naïve T cells impaired their differentiation into iTreg cells upon TCR stimulation, reduced expression of immunosuppressive genes in fetal iTreg cells such as IL10, and increased expression of proinflammatory genes including IFNG Consequently, Helios knockout fetal iTreg cells had reduced IL-10 and increased IFN-γ cytokine production. Together, our results reveal important roles for Helios in enhancing preferential fetal Treg differentiation and fine-tuning eventual Treg function. The Treg-biased programs identified within fetal naïve T cells could potentially be used to engineer enhanced iTreg populations for adoptive cellular therapies.

Landscape of stimulation-responsive chromatin across diverse human immune cells.

A hallmark of the immune system is the interplay among specialized cell types transitioning between resting and stimulated states. The gene regulatory landscape of this dynamic system has not been fully characterized in human cells. Here we collected assay for transposase-accessible chromatin using sequencing (ATAC-seq) and RNA sequencing data under resting and stimulated conditions for up to 32 immune cell populations. Stimulation caused widespread chromatin remodeling, including response elements shared between stimulated B and T cells. Furthermore, several autoimmune traits showed significant heritability in stimulation-responsive elements from distinct cell types, highlighting the importance of these cell states in autoimmunity. Allele-specific read mapping identified variants that alter chromatin accessibility in particular conditions, allowing us to observe evidence of function for a candidate causal variant that is undetected by existing large-scale studies in resting cells. Our results provide a resource of chromatin dynamics and highlight the need to characterize the effects of genetic variation in stimulated cells.

CD161 contributes to prenatal immune suppression of IFNγ-producing PLZF+ T cells.

BACKGROUND: While the human fetal immune system defaults to a program of tolerance, there is concurrent need for protective immunity to meet the antigenic challenges encountered after birth. Activation of T cells in utero is associated with the fetal inflammatory response with broad implications for the health of the fetus and of the pregnancy. However, the characteristics of the fetal effector T cells that contribute to this process are largely unknown. METHODS: We analyzed primary human fetal lymphoid and mucosal tissues and performed phenotypic, functional, and transcriptional analysis to identify T cells with pro-inflammatory potential. The frequency and function of fetal-specific effector T cells was assessed in the cord blood of infants with localized and systemic inflammatory pathologies and compared to healthy term controls. RESULTS: We identified a transcriptionally distinct population of CD4+ T cells characterized by expression of the transcription factor Promyelocytic Leukemia Zinc Finger (PLZF). PLZF+ CD4+ T cells were specifically enriched in the fetal intestine, possessed an effector memory phenotype, and rapidly produced pro-inflammatory cytokines. Engagement of the C-type lectin CD161 on these cells inhibited TCR-dependent production of IFNγ in a fetal-specific manner. IFNγ-producing PLZF+ CD4+ T cells were enriched in the cord blood of infants with gastroschisis, a natural model of chronic inflammation originating from the intestine, as well as in preterm birth, suggesting these cells contribute to fetal systemic immune activation. CONCLUSION: Our work reveals a fetal-specific program of protective immunity whose dysregulation is associated with fetal and neonatal inflammatory pathologies.

In utero priming of highly functional effector T cell responses to human malaria.

Malaria remains a significant cause of morbidity and mortality worldwide, particularly in infants and children. Some studies have reported that exposure to malaria antigens in utero results in the development of tolerance, which could contribute to poor immunity to malaria in early life. However, the effector T cell response to pathogen-derived antigens encountered in utero, including malaria, has not been well characterized. Here, we assessed the frequency, phenotype, and function of cord blood T cells from Ugandan infants born to mothers with and without placental malaria. We found that infants born to mothers with active placental malaria had elevated frequencies of proliferating effector memory fetal CD4+ T cells and higher frequencies of CD4+ and CD8+ T cells that produced inflammatory cytokines. Fetal CD4+ and CD8+ T cells from placental malaria-exposed infants exhibited greater in vitro proliferation to malaria antigens. Malaria-specific CD4+ T cell proliferation correlated with prospective protection from malaria during childhood. These data demonstrate that placental malaria is associated with the generation of proinflammatory malaria-responsive fetal T cells. These findings add to our current understanding of fetal immunity and indicate that a functional and protective pathogen-specific T cell response can be generated in utero.

Lin28b Regulates Fetal Regulatory T Cell Differentiation through Modulation of TGF-ß Signaling.

Immune tolerance between the fetus and mother represents an active process by which the developing fetus must not mount immune responses to noninherited Ags on chimeric maternal cells that reside in fetal tissue. This is, in part, mediated by the suppressive influence of CD4+FOXP3+CD25+ regulatory T cells (Tregs). Fetal secondary lymphoid organs have an increased frequency of Tregs and, as compared with adult T cells, fetal naive CD4+ T cells exhibit a strong predisposition to differentiate into Tregs when stimulated. This effect is mediated by the TCR and TGF-ß pathways, and fetal T cells show significantly increased Treg differentiation in response to anti-CD3 and TGF-ß stimulation. Naive fetal T cells also exhibit increased signaling through the TGF-ß pathway, with these cells demonstrating increased expression of the signaling mediators TGF-ßRI, TGF-ßRIII, and SMAD2, and higher levels of SMAD2/SMAD3 phosphorylation. Increased fetal Treg differentiation is mediated by the RNA-binding protein Lin28b, which is overexpressed in fetal T cells as compared with adult cells. When Lin28b expression is decreased in naive fetal T cells, they exhibit decreased Treg differentiation that is associated with decreased TGF-ß signaling and lowered expression of TGF-ßRI, TGF-ßRIII, and SMAD2. Lin28b regulates the maturation of let-7 microRNAs, and these TGF-ß signaling mediators are let-7 targets. We hypothesize that loss of Lin28b expression in fetal T cells leads to increased mature let-7, which causes decreased expression of TGF-ßRI, TGF-ßRIII, and SMAD2 proteins. A reduction in TGF-ß signaling leads to reduced Treg numbers.

Levels of circulating myeloid subpopulations and of heme oxygenase-1 do not predict CD4(+) T cell recovery after the initiation of antiretroviral therapy for HIV disease.

The level (or frequency) of circulating monocyte subpopulations such as classical (CD14(hi)CD16(-)) and non-classical (CD14(dim)CD16(+)) monocytes varies during the course of HIV disease progression and antiretroviral therapy (ART). We hypothesized that such variation and/or differences in the degree to which these cells expressed the immunoregulatory enzyme, heme oxygenase-1 (HO-1), would be associated with CD4(+) T cell recovery after the initiation of ART. This hypothesis was tested in a cross-sectional study of four groups of HIV-infected subjects, including those who were seronegative, untreated virologic controllers [detectable viral load (VL) of <1000 copies/mL], untreated virologic non-controllers [VL > 10,000 copies/mL], and ART-mediated virologic controllers [VL < 75 copies/mL]. A longitudinal analysis of ART-treated subjects was also performed along with regression analysis to determine which biomarkers were associated with and/or predictive of CD4(+) T cell recovery. Suppressive ART was associated with increased levels of classical monocyte subpopulations (CD14(hi)CD16(-)) and decreased levels of non-classical monocyte populations (CD14(dim)CD16(+)). Among peripheral blood mononuclear cells (PBMCs), HO-1 was found to be most highly up-regulated in CD14(+) monocytes after ex vivo stimulation. Neither the levels of monocyte subpopulations nor of HO-1 expression in CD14(+) monocytes were significantly associated with the degree of CD4(+) T cell recovery. Monocyte subpopulations and HO-1 gene expression were, however, restored to normal levels by suppressive ART. These results suggest that the level of circulating monocyte subpopulations and their expression of HO-1 have no evident relationship to CD4(+) T cell recovery after the initiation of ART.

Distinct functional programming of human fetal and adult monocytes.

Preterm birth affects 1 out of 9 infants in the United States and is the leading cause of long-term neurologic handicap and infant mortality, accounting for 35% of all infant deaths in 2008. Although cytokines including interferon-γ (IFN-γ), interleukin-10 (IL-10), IL-6, and IL-1 are produced in response to in utero infection and are strongly associated with preterm labor, little is known about how human fetal immune cells respond to these cytokines. We demonstrate that fetal and adult CD14(+)CD16(-) classical monocytes are distinct in terms of basal transcriptional profiles and in phosphorylation of signal transducers and activators of transcription (STATs) in response to cytokines. Fetal monocytes phosphorylate canonical and noncanonical STATs and respond more strongly to IFN-γ, IL-6, and IL-4 than adult monocytes. We demonstrate a higher ratio of SOCS3 to IL-6 receptor in adult monocytes than in fetal monocytes, potentially explaining differences in STAT phosphorylation. Additionally, IFN-γ signaling results in upregulation of antigen presentation and costimulatory machinery in adult, but not fetal, monocytes. These findings represent the first evidence that primary human fetal and adult monocytes are functionally distinct, potentially explaining how these cells respond differentially to cytokines implicated in development, in utero infections, and the pathogenesis of preterm labor.

Fetal regulatory T cells and peripheral immune tolerance in utero: implications for development and disease.

The developing fetus must actively learn to tolerate benign antigens or suffer the consequences of broken tolerance. Tolerance of self-antigens prevents development of autoimmune diseases and is achieved by both deletion of autoreactive T cell clones in the thymus (central tolerance) and by the suppressive influence of CD4(+)  CD25(+)  FoxP3(+) regulatory T cells (Tregs) in the periphery. Fetal CD4(+) T cells have a strong predisposition to differentiate into tolerogenic Tregs that actively promote self-tolerance, as well as tolerance to non-inherited antigens on chimeric maternal cells that reside in fetal tissues. As the fetus nears birth, a crucial transition must occur between the tolerogenic fetal immune system and a more defensive adult-type immune system that is able to combat pathogens. This paper will review the unique tolerogenic nature of fetal T cells and will examine evidence for a novel model of fetal immune development: the layered immune system hypothesis.

Fetal and adult hematopoietic stem cells give rise to distinct T cell lineages in humans.

Although the mammalian immune system is generally thought to develop in a linear fashion, findings in avian and murine species argue instead for the developmentally ordered appearance (or "layering") of distinct hematopoietic stem cells (HSCs) that give rise to distinct lymphocyte lineages at different stages of development. Here we provide evidence of an analogous layered immune system in humans. Our results suggest that fetal and adult T cells are distinct populations that arise from different populations of HSCs that are present at different stages of development. We also provide evidence that the fetal T cell lineage is biased toward immune tolerance. These observations offer a mechanistic explanation for the tolerogenic properties of the developing fetus and for variable degrees of immune responsiveness at birth.