Immune Function during Fetal Life

The initial stage of hematopoiesis in the human fetus occurs in extraembryonic mesenchymal tissue and in the mesoderm of the yolk sac. Pluripotent erythroid and granulo-macrophage progenitors are detectable in the latter at around the fourth week of gestation ( Box 6-1 ). These cells appear subsequently in the fetal circulation and by weeks 5 to 6 in the liver, which at that stage of development is the major site of hematopoiesis. Within the liver, the interactions between stromal cells and hematopoietic cells play an important role in regulation. Expression of fibronectin by stromal cells is increased during the second trimester and is believed to result in enhanced proliferation and differentiation of hematopoietic cells. The spleen and thymus are seeded from the liver, and by the eighth week of development CD7 ⁺ precursor cells are found in the thymus, whereas stem cells do not appear in bone marrow until around the 12th week of gestation. T cells recognizable by expression of characteristic TcR/CD3 are found in peripheral lymphoid organs from weeks 13 to 15 of gestation onwards, despite the lack of well-defined thymic cortical and medullary regions and mature epithelial components. These early T cells also express CD2 and CD5. The maturation of nonlymphoid components within peripheral lymphoid tissues progresses even more slowly and takes up to 20 weeks.

It is feasible that the fetal gastrointestinal tract may be an additional site for extrathymic T cell differentiation in the human fetus, as has been reported in the mouse. T cells are detectable in the intestinal mucosa by 12 weeks of gestation, and many of these express the CD8αα phenotype, in particular within Peyer’s patches. In the mouse, CD8αα cells appear to be thymus independent and are believed to develop in the gut. Although there is no direct evidence for this in humans, it is noteworthy that fetal gut lamina propria lymphocytes are initially an actively proliferating population as indicated by constitutive expression of Ki67, and there is little or no overlap between gut-derived and blood-derived TcRβ transcripts.

The gut mucosa may also be a major site for differentiation of TcRγ/δ cells during fetal life. Rearranged TcRδ genes are first detectable in the gut at 6 to 9 weeks of gestation, which is earlier than is observed in the thymus. The liver is another significant extrathymic site for TcRγ/δ differentiation in humans, including a unique subset expressing CD4.

The capacity to respond to polyclonal stimuli such as phytohemagglutinin (PHA) is first seen at 15 to 16 weeks of gestation. The degree to which the fetal immune system can respond to foreign antigens has not been clearly established. On the one hand, the offspring of mothers infected during pregnancy with a range of pathogens including mumps, ascaris, malaria, schistosomes and helminths display evidence of pathogen-specific T cell reactivity at birth, whereas infection with other organisms such as toxoplasma may induce tolerance. However, more recent studies have detected significant populations of T cells in cord blood that express the effector memory phenotype, even in the absence of any evidence of previous maternal infection. Additionally, vaccination of pregnant women with tetanus toxoid results in the appearance of IgM in the fetal circulation that is indicative of fetal T cell sensitization. Similarly, vaccination of pregnant women against influenza results in the presence of influenza-specific IgM in cord blood, and virus-specific CD8 ⁺ T cells detected by the use of MHC tetramers. There is also a variety of evidence based on in vitro lymphoproliferation of cord blood mononuclear cells and recently the presence of low levels of IgE in cord blood which suggests that environmental antigens (including dietary and inhalant allergens) to which pregnant women are exposed may in some circumstances prime T cell responses transplacentally. However these conclusions have been challenged on the basis of a variety of evidence of low specificity of cord blood responses to allergen (further discussion below) and on the kinetics of postnatal development of allergen-specific Th memory, and the issue remains contentious. The recent discovery that the placenta is not sterile, but rather hosts a distinct microbiome in healthy pregnancy, is sure to stimulate much additional research into fetal immune function.

Studies examining lymphocyte subsets in cord blood from babies born at gestational ages between 20 and 42 weeks found that the proportion of cord natural killer (NK) cells increased with gestational age, while the proportion of CD4 ⁺ cells and the ratio of CD4 ⁺ : CD8 ⁺ cells decreased. Despite the lack of significant numbers of CD4 ⁺ and CD8 ⁺ CD45RO ⁺ T cells in cord blood, fetal spleen and cord blood samples from premature infants contain these cells in relatively high frequency. These ‘postactivated’ or ‘memory’ T cells were unresponsive to recombinant IL-2, suggesting they may have been anergized by earlier contact with self or environmental antigens. CD4 ⁺ CD25 ⁺ T regulatory cells are detected in fetal lymphoid tissue, and they have been shown to have a suppressive effect on fetal CD4 ⁺ and CD8 ⁺ T cells expressing the activation antigen CD69. Fetal thymic exposure to high-avidity TcR ligand has been shown to promote development of T regulatory cells in mice, while exposure to low-affinity TcR ligand did not; it appears that T regulatory cells require a higher ligand avidity for positive selection than conventional T cells. Interestingly, expression and function of T regulatory cells has been found to be impaired at birth in the offspring of atopic mothers.

These findings collectively suggest that the fetal immune system develops at least partial functional competence before birth but lacks the full capacity to generate sustained immune responses; although IgM responses develop in the fetus following maternal tetanus vaccination, there is no evidence of class-switching in the offspring until they are actively vaccinated. Given the fact that the fetal immune system can generate at least primary immune responses against external stimuli, the question arises as to how immune responses within or in close contact with the fetal compartment are regulated. The necessity for tight control of these responses becomes obvious in light of findings that a variety of T cell cytokines are exquisitely toxic to the placenta. Part of this control may be at the level of transcription factor expression.

It is also pertinent to question how potential immunostimulatory interactions between cells derived from fetal and maternal bone marrow are regulated at the fetomaternal interface. It has been clearly demonstrated that fetal cells readily traffic into the maternal circulation, potentially sensitizing the maternal immune system against paternal HLA antigens present on the fetal cells. However, it is clear that the maternal immune system in the vast majority of circumstances successfully eradicates fetal cells from the peripheral circulation while remaining functionally tolerant of the fetus. This suggests that tolerance of the fetal allograft is a regionally controlled process that is localized to the fetomaternal interface.

The mechanisms that regulate the induction and expression of immune responses in this milieu are complex and multilayered. The first line of defense appears to involve tolerogenic HLA-G-expressing, IL-10-producing dendritic cells detectable in the maternal circulation and in the decidua, and the activity of these is complemented by decidual macrophage populations. The tolerogenic activity of these cell populations is supported via an immunosuppressive ‘blanket’ maintained through the local production within the placenta by trophoblasts and macrophages of metabolites of tryptophan generated via indolamine 2,3-dioxygenase, which are markedly inhibitory against T cell activation and proliferation. Constitutive production of high levels of IL-10 by placental trophoblasts provides a second broad-spectrum immunosuppressive signal to dampen local T cell responses, as well as the homeostatic function of alternatively activated macrophages.

An additional line of defense involves mechanisms that operate to protect against T cell activation events, which evade suppression via these pathways. These include the expression of FasL on cells within the placenta providing a potential avenue for apoptosis-mediated elimination of locally activated T cells, NK cells that selectively antagonize Th-17 cell activation, and the presence of maternally derived CD4 ⁺ CD25 ⁺ T regulatory cells, which are recruited to the maternofetal interface where they act to dampen fetus-specific responses. These mechanisms are complemented by a series of pathways that operate to selectively dampen production at the fetomaternal interface of Th1 cytokines, in particular interferon-gamma (IFN-γ). This cytokine plays an important role in implantation; however, if it is produced in suprathreshold levels at later stages of pregnancy, triggered for example by local immune responses against microbial or allo-antigens, IFN-γ (and other Th1 cytokines) can potentially cause placental detachment and fetal resorption. These Th2-trophic mechanisms involve local production of a range of immunomodulators including IL-10, which programs antigen-presenting cells (APCs) for Th2 switching; progesterone, which directly inhibits IFN-γ gene transcription; progesterone and estradiol, which both inhibit the NF-κB pathway in monocytes; and PGE ₂ , which promotes Th2 switching via effects upon APCs, in particular dendritic cells. Circulating myeloid and plasmacytoid dendritic cells (mDC and pDC) in late pregnancy appear to be constrained with respect to the level of activation they can achieve, and it has been suggested that this mechanism may be mediated via the glycoprotein hormone activin-A.

Resistance to Infection during Infancy

Infancy represents a period of high susceptibility to infection with a range of pathogens including bacteria and fungi and, in particular, viruses. The expression of cell-mediated immunity during active viral infection is attenuated in infants compared to older age groups, and the subsequent generation of virus-specific immunologic memory is also inefficient. These findings suggest that a range of developmentally related deficiencies in innate and adaptive immunologic mechanisms are operative in neonates.

Surface Phenotype of T Cells in Early Life

Total lymphocyte counts in peripheral blood are higher in infancy than in adulthood, and at birth T cell levels are twice those of adults. Longitudinal studies on individual infants indicate a further rapid doubling in T cell numbers in the circulation during the first 6 weeks of life, which is maintained throughout infancy. Surface marker expression on infant T cells differs markedly from that observed in adults. The most noteworthy characteristics are frequent expression of CD1, PNA, CD31 and CD38. These four antigens are considered to mark mature thymocytes as opposed to circulating ‘mature’ naïve T cells.

Analyses performed on CD38 ⁺ cord blood cells have reinforced this view. In particular, animal model studies on thymic output have led to the development of an accurate technique for phenotypic identification of recent thymic emigrants (RTE), which are newly produced peripheral naïve T cells that retain a distinct phenotypic signature of recent thymic maturation that distinguishes them from long-lived naïve T cells produced at remote sites. This approach involves the measurement of T cell receptor excision circles (TRECs), which are stable extrachromosomal products generated during the process of variable/diverse/joining (VDJ) TcR gene rearrangement. TRECs are not replicated during mitosis, becoming diluted with each round of cell division. Hassan and Reen have demonstrated that the majority of circulating CD4 ⁺ CD45RA ⁺ human T cells at birth are RTE, as reflected by their high level of TRECs. These researchers also demonstrated that, analogous to thymocytes, the RTE were highly susceptible to apoptosis, and unlike mature adult-derived CD4 ⁺ CD45RA ⁺ naïve T cells, they were uniquely responsive to common γ-chain cytokines, particularly IL-7. Whereas IL-7 promotes their proliferation and survival, IL-7-exposed RTE could not reexpress recombination-activating gene-2 gene expression in vitro. These findings suggest that postthymic naïve peripheral T cells in early infancy are at a unique stage in ontogeny as RTE, during which they can undergo homeostatic regulation including survival and antigen-independent expansion, while maintaining their preselected TcR repertoire.

Studies examining the patterns of postnatal change in T cell surface marker expression have identified relatively high numbers of T cells coexpressing both CD4 and CD8 during infancy, which is also a hallmark of immaturity. In contrast, the expression of CD57 on T cells, which marks non-MHC-restricted cytotoxic cells, is infrequent, as are T cells coexpressing IL-2 and HLA-DR, which is indicative of recent activation. The expression of other activation markers such as CD25, CD69 and CD154 is also low.

Of particular interest in relation to the understanding of overall immune competence during postnatal life are changing patterns of surface CD45RA and CD45RO on T cells. T cells exported from the thymus express the CD45RA isoform of the leukocyte common antigen CD45, and after activation switch to CD45RO expression. Most postactivated neonatal CD4 ⁺ CD45RO ⁺ T cells are short-lived and die within a matter of days, but a subset of these is believed to be programmed to enter the long-lived recirculating T cell compartment as T memory cells. The proportion of CD45RO ⁺ cells within the CD4 ⁺ T cell compartment progressively increases from a baseline of less than 10% at birth, up to 65% in adulthood, reflecting age-dependent accumulation of antigenic exposure. The rate of increase within the TcRα/β and TcRγ/δ populations is approximately equivalent and is slightly more rapid for CD4 ⁺ T cells relative to CD8 ⁺ T cells. The relative proportion of CD45RO ⁺ putative memory T cells attains adult-equivalent levels within the teen years, although the population spread during the years of childhood is very wide. This suggests substantial heterogeneity within the pediatric population in the efficiency of mechanisms regulating the generation of T helper memory.

Functional Phenotype of T Cells during Infancy and Early Childhood

T cell function during infancy exhibits a variety of qualitative and quantitative differences relative to that observed in adults. It has been demonstrated when employing a limiting dilution analytic system that at least 90% of peripheral blood CD4 ⁺ T cells from adults can give rise to stable T cell clones, whereas the corresponding (mean) figure for immunocompetent T cell precursors in infants was less than 35%. Moreover, the cytokine production profile of T cell clones from infants displayed a prominent Th2 bias, which may be related to the recently described predilection of T cells from this age group for preferential expression of the master Th2 regulator GATA3. Cloning frequencies within the infant population were bimodally distributed, with a significant subset of ostensibly normal healthy subjects displaying particularly low cloning frequencies of no more than 20%.

In apparent contrast to these findings, the magnitude of initial T cell proliferation induced by polyclonal T cell mitogens such as PHA in short-term cultures is higher at birth than subsequently during infancy and adulthood. However, proliferation is not sustained, which may reflect the greater susceptibility of neonatal T cells to apoptosis post activation and/or decreased production of IL-2. In contrast, activation induced by TcR stimulation and cross-linking CD2 or CD28 is reduced.

In addition to these deficiencies, neonatal T cells are hyperresponsive to IL-4 and hyporesponsive to IL-12 compared to adults, the latter being associated with reduced IL-12 receptor expression. Neonates also have reduced capacity to produce IL-12, which can last into childhood; work from our laboratory has suggested that slow maturation of IL-12 synthetic capacity can be attributed to deficiencies in the number and/or function of dendritic cells.

Neonatal T cells exhibit heightened susceptibility to anergy induction post stimulation with bacterial superantigen, employing protocols that do not tolerize adult T cells. This has been ascribed to deficient IL-2 production but may alternatively be related to developmentally related deficiencies in the Ras signaling pathway, which have been associated with secondary unresponsiveness to alloantigen stimulation by T cells from neonates. Additional aberrations in intracellular signaling pathways reported in neonatal T cells include phospholipase C and associated Lck expression, protein kinase C and CD28, which is associated with dysfunction in FasL-mediated cytotoxicity and reduced NFκB production.

Profiles of chemokine receptor expression and responsiveness in neonatal T cells have been observed to differ distinctly from those in adults. In particular, adult peripheral blood T cells expressed CCR-1, -2, -5, -6 and CXCR-3 and CXCR-4, whereas those from cord blood expressed only CXCR-4, reflecting markedly attenuated capacity to respond to signals from inflammatory foci. Additional differences have been observed between T cells from normal and preterm infants, particularly with respect to CCR4 and α4β7 expression.

Evidence from a range of studies indicates that both cytotoxic effector functions and capacity to provide help for B cell immunoglobulin production are attenuated during infancy. These functional deficiencies are likely to be the result of a combination of factors that include decreased expression of CD40L, reduced expression of cytokine receptors and decreased production of a wide range of cytokines following stimulation. The mechanism(s) underlying these reduced cytokine responses are unclear, but factors intrinsic to the T cells themselves, as well as those involving accessory cell functions, appear to be involved.

The IFN-γ gene is under tight regulation during fetal development, presumably to prevent rejection of the fetus by the mother’s immune system that may result from excessive IFN-γ in the uterine environment. Expression of IFN-γ is modulated in part at the epigenetic level via gene methylation, with transcriptional activity inhibited by hypermethylation of DNA. This laboratory has demonstrated hypermethylation at multiple CpG sites in the proximal promoter region of the IFN-γ gene in CD4 ⁺ CD45RA ⁺ T cells in cord blood relative to their adult counterparts. We subsequently demonstrated that in vitro differentiation of CD4( ⁺ ) T cells down the Th1, but not Th2, pathway is accompanied by progressive demethylation of CpG sites in the IFN-γ promoter, which is most marked in neonatal cells. While atopy development by age 2 was not associated with variations in methylation patterns in cord blood T cells, IFN-γ promoter methylation was reduced in CD8( ⁺ ) T cells from atopic children in the age range in which hyperproduction of IFN-γ has recently been identified as a common feature of the atopic phenotype.

It has been proposed that many naïve neonatal T cells may have low-affinity TcRs and reduced affinity for T cell activation, and that expansion may take place without production of conventional memory T cells. If this is the case, cytokine responses to antigens in cord blood might have little relevance to immune responses to the same antigens later in childhood. It is possible that the relevance of cord blood responses to those in later life varies according to antigen. Findings from our laboratory have suggested that the allergen reactivity of neonatal T cells consists predominantly of a default response by recent thymic emigrants, which provide an initial burst of short-lived cellular immunity in the absence of conventional T cell memory. This response appears to be limited by parallel activation of regulatory T cells, which arise as a result of these initial allergen encounters. There is an inverse relationship between the numbers of circulating regulatory and memory CD4 ⁺ T cells both during pregnancy and postnatally. The frequency of regulatory T cells at birth is inversely associated with gestational age and this difference may persist for a significant period into infancy.

Our studies in a longitudinal birth cohort comprising children at high risk (i.e. one or both parents allergic) examined how immune function in early childhood relates to infection and development of allergy. We found that priming of Th2 responses associated with persistent house dust mite (HDM)-IgE production in a high-risk cohort occurred entirely postnatally, as HDM reactivity in cord blood appeared to be nonspecific and was unrelated to subsequent development of allergen-specific Th2 memory or IgE. However, a different picture emerged when polyclonal responses to mitogen were assessed by measuring PHA-induced cytokines from cultured cord blood mononuclear cells (CBMC) from cohort subjects, which correlated with frequency/intensity of respiratory infections up to age 5. The ratio of PHA-induced IL-10 : IL-5 was highly predictive of subsequent severe infection, with high IL-5 responses associated with increased infection risk and the converse for high IL-10 responses. We suggest that the relevant underlying mechanisms may involve IL-10-mediated feedback inhibition of IL-5-dependent eosinophil-induced inflammation, which is a common feature of antiviral responses in early childhood. Additionally, the same immunophenotype appears to be associated with reduced capacity to produce IL-21, and it is significant that a series of studies point to a crucial role for this cytokine in resistance to persistent viral infection. The relevance of cord blood responses to immune function in later life may depend upon environmental factors and associated exposures to infection during pregnancy. A study performed in a malaria-endemic region of Kenya examining mononuclear cell responses to malaria antigen found that the fine specificity of lymphocyte proliferation and cytokine secretion was similar in cord and adult blood mononuclear cells. Stimulation with overlapping peptides to identify dominant malaria T cell epi­topes also showed that cord blood cells from neonates whose mothers who had been malaria-infected during pregnancy were 4-fold more likely to acquire a peptide-specific immune response. It was therefore proposed that the fetal malaria response functions in a competent adaptive manner, which may help to protect neonates from severe malaria during infancy.

Recent research has identified a new subset of T helper cells that produce IL-17. These ‘Th17’ cells appear to mediate tissue inflammation by supporting neutrophil recruitment and survival, proinflammatory cytokine production by structural cells and matrix degradation (reviewed in reference ). Studies have shown that all IL-17-producing cells originate exclusively from CD161 ⁺ naïve CD4 ⁺ T cells of umbilical cord blood and the postnatal thymus in response to a combination of IL-1β and IL-23. It has also been shown that human naïve CD4 ⁺ + T cells can give rise to either Th1 or Th17 cells in the presence of IL-1β and IL-23, with IL-12 presence determining Th1 development. Additionally, a subset of IL-17-producing cells possessed the ability to produce IFN-γ even after their development from CD4 ⁺ T cells, perhaps representing an intermediate Th1/Th17 phenotype. A recent study comparing T cells from preterm and term infants with those from adults also suggests that Th17 cell capacity may be inversely related to developmental age, leading to a relative Th17 bias in early life, and this may reflect parallel developmental kinetics for the Th17-trophic cytokines IL-6 and IL-23. Expression of full Th17 activity in infants may require specific stimuli such as viral infection, exemplified by respiratory syncytial virus (RSV).

Innate Immunity in Neonates

There is a high level of interconnectivity between the innate and adaptive arms of the immune system. Competent adaptive immune function is important for switching off innate immune responses to prevent them from overshooting and causing bystander damage to host tissues, while defects in innate immunity appear to play a role in the development of a number of inflammatory diseases including allergy. Toll-like receptors (TLRs) are central to the function of the innate immune system, and there are at least 10 known human TLRs that recognize pattern motifs present in bacteria, viruses or other prokaryotes. Many aspects of TLR-associated functions are inefficient in early life, though patterns of functional maturation across the population are complex and heterogeneous.

The capacity of the innate immune system to recognize and rapidly respond to pathogens via TLRs is a major determinant of risk for infection during this crucial period, exemplified by recent findings linking respiratory-related hospitalization in infants and reduced capacity of their monocytes for viral-induced IFN-γ production. Infants, especially those born prematurely, are particularly susceptible to severe bacterial infections. A study investigating mechanisms behind this phenomenon demonstrated that TLR4 expression is dependent on gestational aging: preterm infants show decreased expression of TLR4 on monocytes compared to full-term newborns; both showed lower expression than adults. Similarly, cytokine production following lipopolysaccharide (LPS) stimulation was significantly lower in whole blood cultures from preterm compared to full term infants; both had lower production than adults. Subsequent studies examining TLR2 expression found that although TLR2 levels did not differ between preterm and full-term neonates, levels of the proximal downstream adapter molecule myeloid differentiation factor MyD88 were significantly reduced in preterm newborns, along with cytokine responses to TLR2 ligand.

Studies examining the effect of breastfeeding on neonatal innate immune response have found that breast milk from days 1 to 5 postpartum negatively modulated TLR2 and TLR3 ligand responses, while enhancing those of TLR4 and 5. Breast milk has been found to contain sCD14 and sTLR2 in addition to unidentified TLR-modulatory factors. It has been suggested that the differential modulation of TLR function by breast milk may serve to promote efficient response to potentially harmful LPS-producing Gram-negative bacteria via TLR4 while allowing the establishment of Gram-positive bifidobacteria as the predominant intestinal microflora.

Neonatal immune responses to microbial stimuli appear to be affected by maternal allergy. Children with atopic mothers have been observed to have significantly lower expression in cord blood monocytes of TLR2 and TLR4 than their mothers both before and after microbial stimulation, a disparity that was not seen between nonatopic mothers and their children. In addition, CMBC from children with atopic mothers produced less IL-6 in response to peptidoglycan stimulation than those from children with nonatopic mothers. In another study, CBMC stimulation with the TLR2 ligand peptidoglycan led to secretion of IL-10 and induction of FOXP3 that varied according to maternal atopy; CBMC from newborns with maternal atopy showed reduced induction of these cytokines compared to those without maternal atopy.

A study from our laboratory focussed on the ontogeny of the innate immune system and examined the cytokine secretory capacity of mononuclear cells from subjects at various ages between birth and adulthood. Cells were primed with IFN-γ then stimulated with LPS; production of IL-6, IL-10, IL-12, IL-18, IL-23, TNF-α and myxovirus resistance protein A (MXA: a cytokine induced by type I interferon in response to virus infection) was measured and compared. The developmental pattern between 1 year and 13 years showed that levels of all cytokines increased with age, with levels of some cytokines further increasing in adulthood. However, a subset of cytokines showed hyperexpression in CBMC. There appeared to be major differences in developmental regulation between the MyD88-dependent (TNF-α, IFN-γ, IL-6 and IL-10) cytokines, which were hyperexpressed by CBMC relative to infant peripheral blood mononuclear cells (PBMC), compared to the MyD88-independent cytokines (IL-12, IL-18, IL-23 and MXA) which were expressed at lower levels in both CBMC and PBMC from infants than in PBMC from older age groups, and similar dichotomous patterns of production capacity in neonates between different classes of cytokines have been reported in several more recent studies. A factor present in neonatal plasma has recently been implicated in the polarization of neonatal cells toward the low IL-12/high IL-10 producer phenotype, but this finding has yet to be confirmed.

There appears to be a gradual maturation of phagocytic capacity by innate immune cells over time. The phagocytic activity of fetal neutrophils and monocytes has been observed to be significantly lower than that of healthy neonates and adults, and a direct relationship between gestational age and number of phagocytosing granulocytes has been demonstrated. Similarly, the activity of NK cells in infants is correlated with gestational age and is significantly impaired at baseline compared to children and adults. However, following stimulation with priming agents exemplified by IL-18 and IL-12 or single-stranded RNA, neonatal cells rapidly develop higher levels of IFN-γ and cytolytic activity than are seen in adults, suggesting that this arm of innate immunity may play a significant role in host defense during this life period.

A series of recent studies have added a further layer of complexity to this picture, with the demonstration that developmental heterogeneity across the spectrum of innate immune functions also varies in relation to ethnic background and/or geographic location of study groups; similar observations apply to innate regulatory T cell activity in early life.

B Cell Function in Early Life

Certain aspects of B cell function in neonates appear unique in relation to adults. In particular, large numbers of neonatal B cells express CD5, together with activation markers such as IL-2R and CD23. It has been postulated that these CD5 ⁺ B cells act as a ‘first line of defense’ in primary antibody responses in neonates utilizing a preimmune repertoire, in contrast to CD5 ⁻ B cells in which response patterns are acquired following antigen contact. Unlike adult B cells, these neonatal B cells proliferate readily in the presence of IL-2 or IL-4 without requirement of further signals. An additional (albeit less frequent) neonatal B cell subset expresses IgD, IgM, CD23 and CD11b, is CD5 variable, and spontaneously secretes IgM antibodies against a range of autoantigens.

Conventional B cell function, that is, antibody production following infection or vaccination, is reduced in infants relative to adults, and some in vitro studies suggest that this may be related to a defect in isotype switching. The relative contributions of the T cell and B cell compartments to this deficiency in immunoglobulin production are widely debated, but the consensus is that both cell types play a role.

As noted previously, T cells in infants do not readily express high levels of CD40L unless provided with particularly potent activating stimuli. CD40L represents a critical signal for T helper cell-induced class switching and the generally low expression on neonatal T cells may thus be a limiting factor in the process. Reduced T cell cytokine production may further exacerbate the problem. However, although immunoglobulin production by neonatal B cells is low in the presence of neonatal T helper cells, production levels can be markedly improved if mature T helper cells or adequate soluble signals are provided. However, the neonatal B cells still fail to reach adult-equivalent levels of production, suggesting that an intrinsic defect also exists. In this regard, it is pertinent to note that functional immaturity within the B cell compartment has recently been identified as a predictor of high risk for later development of allergic disease.

Growing interest in the human microbiome in health and disease has reawakened interest in the role of gut flora in the development of the overall B cell repertoire, in particular the stimulatory effect of early gut colonization on B memory cell expansion. An additional area of B cell immunobiology that is set to have a major impact in the area of immune development and allergy pertains to the activity of regulatory B cell populations, which are hypothesized to play a direct role in control of allergic inflammation; moreover, a related population of B cells has been identified in neonatal thymic tissue, which appears to stimulate the generation of regulatory T cells.

Antigen-Presenting Cell Populations

The key ‘professional’ antigen-presenting cell (APC) populations in this context are the mononuclear phagocytes (MPCs), dendritic cells (DCs) and B cells. The precise role of each cell type in different types of immune response is not completely clear, although it is evident that DCs represent the most potent APC for priming the naïve T cell system against antigens encountered at low concentrations (e.g. virus and environmental allergens).

Ontogenic studies on human MPCs have been essentially limited to blood monocytes. Although neonatal populations appear comparable to those of adults in number and phagocytic activity, they display reduced chemotactic responses and reduced capacity for secretion of inflammatory cytokines such as TNF-α. Their capacity to present alloantigen to T cells is reportedly normal, but they display reduced levels of MHC class II expression. Several studies have implicated poor accessory cell function of infant blood monocytes as a co-factor in the reduced IFN-γ responses of infant T cells to polyclonal mitogens such as PHA, possibly as a result of diminished elaboration of co-stimulator signals. Macrophage populations at mucosal sites such as the lung and airways have important immunoregulatory roles in adults, but it is not clear whether these mechanisms are operative in early life. A murine study from our group indicates lower levels of expression of immunomodulatory molecules, including IL-10 and nitric oxide, by lung macrophages during the neonatal period.

B cells are also recognized as important APCs, in particular for secondary immune responses. In murine systems, it has been demonstrated that neonatal B cells function poorly as APCs relative to their adult counterparts and do not reach adult-equivalent levels of activity until after weaning.

As noted previously, DCs are the most potent APC population in adult experimental animals for initiation of primary immunity and in this regard have been designated as the ‘gatekeepers’ of the immune response. The distribution and phenotypes of these cells appear comparable in murine and human tissues, and it is accordingly reasonable to speculate that the proposed role of murine DCs as the link between the innate and adaptive arms of the immune system is also applicable in man. Importantly, in the context of allergic disease, comparative studies on DCs from mucosal sites in humans and experimental animals suggest very similar functional characteristics.

DCs commence seeding into peripheral tissues relatively early in gestation, and at birth recognizable networks of these cells can be detected in a variety of tissues including epidermis, intestinal mucosa and the upper and lower respiratory tract. The cells within these DC networks in perinatal tissues are typically present at lower densities and express lower levels of surface MHC class II relative to adults, hinting at developmentally related variations in function phenotype. Recent murine studies have emphasized these differences. Notably, the phenomenon of neonatal tolerance in mice has recently been ascribed to the relative inability of neonatal DCs from central lymphoid organs to present Th1-inducing signals to T cells, leading to the preferential generation of Th2-biased immune responses. Of particular relevance to studies on susceptibility to infectious and allergic diseases in infancy, our group has demonstrated that in the rat the airway mucosal DC compartment develops very slowly postnatally, not attaining adult-equivalent levels of tissue density, MHC class II expression or capacity to respond to local inflammatory stimuli until after biologic weaning.

Data based initially on immunohistochemical studies of autopsy tissues and subsequently verified by airway biopsy studies suggest that the kinetics of postnatal maturation of airway DC networks in humans may be comparably slow.

Reports suggest that the numbers of circulating HLA-DR ⁺ plasmacytoid and myeloid DCs are reduced at birth relative to adults, with mDCs showing diminished APC activity. Additionally, analysis of cord blood monocyte-derived DC functions indicates diminished expression of HLA-DR, CD80 and CD40 and attenuated production of IL-12p35 in response to stimuli such as LPS, poly(I : C) and CD40 ligation. However, studies using human CD8 ⁺ T cell clones to compare the ability of neonatal and adult DCs to present and process antigen using the MHC class I pathway found that neonatal DCs were not defective in their ability to perform these functions. Studies have shown that synergistic stimulation of neonatal DCs by ligands for multiple TLRs is required for efficient differentiation, signaling and T cell priming; membrane-associated TLR4 and intracellular TLR3 were found to act in synergy with endosomal TLR4 to induce functional maturation of neonatal DCs. Interestingly, cord blood monocyte-derived DCs have also been shown to express higher levels of IL-27 following TLR stimulation, which may compensate for the diminished ability of neonatal DCs to produce IL-12.

Granulocyte Populations

Eosinophils, mast cells and basophils play key roles in the pathogenesis of allergic disease, performing important functions in relation to host resistance to certain pathogens, and are thus relevant to this discussion. In particular, hyperreactivity within this granulocyte compartment, exemplified by exaggerated airway eosinophil responses to viral infections as highlighted in recent studies on RSV, is widely considered a harbinger of the early stages of asthma pathogenesis in children.

Eosinophilia in the first year of life has been linked to enhanced risk for later development of atopic diseases in a range of studies but few direct mechanistic data are available. Several earlier observations are suggestive of developmentally related problems in eosinophil trafficking in early life; inflammatory exudates in neonates frequently contain elevated numbers of eosinophils, and eosinophilia is common in premature infants. The mechanisms underlying these developmental variations in eosinophil function are unclear, but some evidence suggests a role for integrin expression involving Mac-1 and L-selectin. Developmental defects in this compartment may additionally be more frequent in the offspring of atopic mothers and may thus be part of the suite of mechanisms that mediate genetically determined high risk for allergic disease.

Adult mucosal tissues contain discrete populations of mucosal mast cells (MMCs) and connective tissue mast cells (CTMCs), respectively, within epithelia and underlying lamina propria. No direct information is available on the ontogeny of these mast cells (MCs) in human tissues, but indirect evidence suggests that they seed into gut tissues during infancy in response to local inflammatory stimulation. Our group has examined the kinetics of postnatal development of MCs in the rat respiratory tract, and has reported that both MMC and CTMC populations develop slowly between birth and weaning. MC-derived proteases appear transiently in serum around the time of weaning in the rat, suggesting that the immature MC populations may be unstable or are undergoing local stimulation at this time, and a similar transient peak of MC tryptase is observed in human serum during infancy.

Direct functional studies on MCs from immature subjects are lacking. However, a 2001 report employed oligonucleotide microarray technology to examine IL-4-induced gene expression in cultured MCs derived from cord blood versus adult peripheral blood; the results indicate that expression of FcεR1α is 10-fold higher in adult-derived MCs. This suggests that during infancy the capacity to express IgE-mediated immunity may be restricted, but confirmation of this possibility must await further detailed studies.

Postnatal Maturation of Immune Functions and Allergic Sensitization

Studies from a number of groups have highlighted the importance of the early postnatal period in relation to the development of long-lasting response patterns to environmental allergens. In particular, it is becoming clear that initial priming of the naïve immune system typically occurs before weaning and may consolidate into stable immunologic memory before the end of the preschool years. Given that the underlying immunologic processes involve coordinated operation of the full gamut of innate and adaptive immune mechanisms, issues relating to developmentally determined functional competence during this life phase may be predicted to be of major importance.

In relation to initial priming of the T cell system against allergens, reports from numerous groups indicate the presence of T cells responsive to food and inhalant allergens in cord blood. Cloning of these cells and subsequent DNA genotyping indicated fetal as opposed to maternal origin, and the array of cytokines produced in vitro in their responses is dominated by Th2 cytokines, although IFN-γ is also observed, suggestive of a Th0-like pattern. The issue of how initial priming of these cells occurs remains to be resolved. It is possible that transplacental transport of allergen, perhaps conjugated with maternal IgG, may be responsible, and some indirect evidence based on in vitro perfusion studies has been published to support this notion. Moreover, the use of sensitive microassays has detected the presence of low levels of allergen-specific (particularly food allergen) IgE antibodies in cord blood which are unrelated to maternal antibody profiles, arguing against cross-contamination, though this has been disputed. Furthermore, prospective tracking of the postnatal appearance of aeroallergen-specific IgE antibody, and corresponding allergen-specific Th memory, shows strong concordance between these phenomena, commencing some time (depending on the specificity) between birth and age 6 months. Alternatively, initial priming of T helper cells that drive production of these neonatal antibody responses may be against cross-reacting antigens as opposed to native allergen, and the uncertain relationship between maternal allergen exposure and newborn T cell reactivity is consistent with this view. The T cell epitope map of the typical cord blood T cell response to ovalbumin (OVA), involving multiple regions of the OVA molecule, suggests major qualitative differences relative to conventional adult T cell responses.

Regardless of how initial T cell responses are primed, it is clear that direct exposure to environmental allergens during infancy drives the early responses down one of two alternate pathways. In the majority of (nonatopic) subjects, the Th2 cytokine component of these early responses progressively diminishes, and by age 5 years in vitro T cell responses to allergens comprise a combination of low-level IFN-γ and IL-10 production. In contrast, a subset of children develop positive skin prick test (SPT) reactivity to one or more allergens, and in vitro stimulation of PBMC with the latter elicits a mixed or Th0-like response pattern comprising IL-4, IL-5, IL-9, IL-10, IL-13 and IFN-γ. This latter pattern closely resembles that seen in the majority of adult atopic patients, and much more commonly develops in atopic family history positive (AFH ⁺ ) children than in their AFH ⁻ counterparts.

It is increasingly debated whether it remains useful to describe these differing responses in human atopic and nonatopic patients within the framework of the murine Th1/Th2 paradigm, which was based upon the concept of reciprocal and/or antagonistic patterns of Th memory expression. In this context, studies from our group indicate that reciprocal patterns of expression of the transcription factor GATA3, analogous to those that distinguish Th1 from Th2 polarized cell lines (with regard to down-regulation vs up-regulation, respectively, post stimulation), are reiterated during the allergen-specific recall responses of CD4 ⁺ T cells from nonatopic versus atopic subjects. Moreover as noted above, hyperexpression of this Th2 master regulator is a feature of T cells in the infant period during which allergen-specific Th memory priming is most commonly initiated. This suggests that the Th1/Th2 model still provides a potentially useful framework for the study of allergic responses, despite the strong likelihood of significant interspecies differences.

The central issue in relation to understanding the initial phase of allergic sensitization in childhood concerns the molecular basis for genetic susceptibility to development of Th2-polarized memory against inhalant allergens, and the key to the resolution of this puzzle may lie in a more comprehensive understanding of the mechanisms that drive postnatal maturation of adaptive immune function. In this regard, we have reported earlier that genetic risk for atopy was associated with delayed postnatal maturation of Th cell function, in particular Th1 function, and that this may increase risk for consolidating Th2-polarized memory against allergens during childhood. The evidence originally presented was based on decreased peripheral blood T cell cloning frequency and diminished IFN-γ production by T cell clones in AFH ⁺ infants relative to their AFH ⁻ counterparts, and these findings have been substantiated in several independent laboratories employing bulk culture studies with neonatal PBMC.

We have proposed that this phenomenon may derive from inappropriate postnatal persistence of one or more of the mechanisms responsible for selective damping of T cell function, in particular that relating to Th1 immunity, during fetal life. A possible contributor may be reduced expression of protein kinase C (PKC) ζ in immature T cells, which is associated with prolongation of Th2 polarization, particularly in infants of allergic mothers. Alternatively, given that the postnatal maturation of adaptive immunity is essentially driven by microbial signals from the outside environment, one or more deficiencies in relevant receptors or downstream signaling pathways in microbial sensing cells within the adaptive immune system may retard this process. Genetic variations described in CD14 may be an example, and similar variants in one or more of the TLR genes constitute additional likely candidates. These possibilities are of particular interest in light of reports that environmental exposure to airborne bacterial lipopolysaccharide in childhood may be protective against Th2-mediated sensitization to inhalant allergens. European ‘farmer-mother’ studies have demonstrated that the combination of prenatal and postnatal exposure to inhaled and ingested microbial breakdown products is associated with strong protective effects against development of allergic diseases during early childhood. A range of immune-associated mechanisms may contribute to these effects including modulation of TLR function initially in decidual tissue at the fetomaternal interface and subsequently within the developing innate immune system of the offspring. These exposures appear to stimulate the postnatal development of regulatory T cell function, which has previously been identified as deficient in children at high risk of development of allergic diseases. It is pertinent to note that these exposures have also been observed to enhance postnatal maturation of IFN-γ-producing capacity, a lack of which has been identified as a risk factor for allergy by our group and others. Longitudinal studies have suggested that Th1 deficiency may be transient and reversible, such that by 18 months of age Th1 function in children with atopic family history is equivalent to or greater than that in children without atopic family history. We found in studies focussing on CBMC from AFH ⁺ children that early development of sensitization within this low-IFN-γ-producing group is maximal among those who later show the highest IFN-γ responses, suggesting a potentially dualistic role for IFN-γ in atopy pathogenesis. This conclusion is reinforced by the results of other studies in older (school age) children that suggest a positive role for IFN-γ in airway symptomatology in atopics. It is also feasible that IL-17 and TNF-α responses may play a similar dualistic role in disease pathogenesis.

Further research is required to elucidate the complex regulatory mechanisms that govern generation of different patterns of allergen-specific Th memory during childhood. However, it is also becoming clear that an additional, and related, set of complexities needs to be considered. It is now evident that only a subset of atopic patients progress to development of severe persistent allergic diseases, in particular atopic asthma, and it is likely that these subjects suffer additional and/or particularly intense inflammatory insults to target tissues. In this context, epidemiologic evidence suggests that risk for development of persistent asthma is most marked in children who display early allergic sensitization to inhalants and who develop severe wheezing and lower respiratory tract infections during infancy. This has given rise to the suggestion that development of the airways remodeling characteristic of chronic asthma may, in many circumstances, be the long-term result of inflammation-induced changes in lung and airway differentiation during critical stages of early growth during childhood. Resistance to respiratory infections is also mediated by the same Th1 mechanisms identified as attenuated in children at risk of atopy, suggesting that the same set of genetic mechanisms may be responsible for airways inflammation induced via the viral infection and atopic pathways in children at high risk of asthma ( Box 6-2 ).

In this regard, a rapidly emerging area of interest relates to the role of vitamin D and resistance to infections and allergic diseases during childhood. Vitamin D is a potent immunomodulator; the active form (1,25(OH)2-vitamin D) complexes with its receptor in most known cell types, including immune cells, to initiate transcription of many genes. Vitamin D induces epithelial and immune cells to produce antimicrobial peptides such as cathelicidin and defensin, which mediate killing of viruses and bacteria. This mechanism is active at birth, and low vitamin D at birth has been associated with increased respiratory infections in early life. Vitamin D also promotes immune tolerance to allergens by up-regulating regulatory T cells and suppressing IgE production. A positive correlation has been observed between vitamin D and IL-10 levels in cord blood, though another study found no associations between vitamin D and immune cell populations in cord blood. Supplementation of pregnant mothers with vitamin D has been associated with increased production of tolerogenic antigen-presenting cells in cord blood, identified by expression of ILT3 and ILT4 transcripts. ³⁰²

The question of whether insufficient vitamin D during immune development can predispose children to allergy and asthma is a hot topic, and one that is currently unresolved as cohort studies have yielded conflicting results. While this may in part be explained by inconsistencies in vitamin D measurement, heterogeneity between cohort populations is likely to be an important factor given that some associations between vitamin D and clinical outcomes are modified by specific genotypes.

An additional variable that merits more detailed research in this context is the role of airway DC populations. In the adult, these cells regulate the Th1/Th2 balance in immune responses to airborne antigens and also mediate primary and secondary immunity to viral pathogens. However, airway DC networks develop very slowly postnatally, apparently ‘driven’ by exposure to inhaled airborne irritants including bacterial lipopolysaccharides, and also by viral infections. Hence the rate at which this key cell population gains competence to respond to maturation-inducing stimuli and then to orchestrate appropriately balanced T cell responses against viral pathogens, allergens and also bacterial pathogens within the nasopharyngeal microbiome may be a key determinant of overall susceptibility to allergic disease. Variations in the genes that govern the functions of these cells in early life are thus likely to be of major importance in the etiology of a variety of disease processes, in particular atopic asthma and related syndromes.