T cell-mediated immune responses in human newborns: ready to learn?

Abstract

Infections with intracellular pathogens are often more severe or more prolonged in young infants suggesting that T cell-mediated immune responses are different in early life. Whereas neonatal immune responses have been quite extensively studied in murine models, studies of T cell-mediated immunity in human newborns and infants are scarce. Qualitative and quantitative differences when compared with adult immune responses have been observed but on the other hand mature responses to certain vaccines and infectious pathogens were demonstrated during the postnatal period and even during foetal life. Herein, we review the evidence suggesting that under appropriate conditions of stimulation, protective T cell-mediated immune responses could be induced by vaccines in early life.

Introduction

Young infants are exposed to a large number of infectious microorganisms, including viruses, bacteria and parasites. During the first months of life, maternal antibodies transferred to the foetus and infant attenuate the severity of infectious episodes caused by microorganisms against which the mother has developed immunological memory [1]. In contrast, maternal lymphocytes normally do not cross the placental barrier so that T cell-mediated immunity is not transferred to the offspring. Maternal T lymphocytes are present at relatively low concentrations in breastmilk but their role in the recipient infant is currently unknown [2]. Therefore, young infants are dependent on their own immune system to fight infections caused by intracellular pathogens for which immune defences merely depend on T lymphocytes. Viruses, including respiratory syncytial virus (RSV), herpes simplex virus (HSV), human immunodeficiency virus (HIV) and human cytomegalovirus (HCMV), Mycobacterium tuberculosis and Plasmodium falciparum are common causes of severe infections in early life, particularly in developing countries [3–7]. Prolonged replication of hepatitis B virus (HBV) and HCMV is also observed when infection occurs in early life [6,8]. These clinical observations suggest that T cell-mediated immune responses are different in young infants [3,4,9]. This would limit the efficacy of vaccines against intracellular pathogens. Whereas neonatal immune responses have been quite extensively studied in murine models (reviewed in 4,9), studies comparing T cell-mediated immunity in human infants and adults are scarce. Both qualitative and quantitative differences have been observed but on the other hand adult-like immune responses to certain infectious pathogens and vaccines were demonstrated during the postnatal period and even during foetal life. These observations suggest that under appropriate conditions of stimulation neonatal T lymphocytes can be instructed to fight intracellular pathogens.

In vitro studies of human neonatal T cells

Circulating neonatal T lymphocytes are fundamentally different from naïve adult T cells and have characteristics of recent thymic emigrants (Table 1). They contain high concentrations of T-cell-receptor excision circles (TRECs), episomal DNA byproducts of TCR α-chain rearrangement that are not replicated but diluted during cell division [10,11]. Like adult naïve cells, most neonatal T lymphocytes express the CD45 RA⁺ isoform and the costimulatory molecules CD27 and CD28. In contrast to adult naïve lymphocytes, neonatal lymphocytes express the CD38 molecule. In addition, a high proportion of circulating neonatal T cells are in cycle and display an increased susceptibility to apoptosis indicating high cell turn-over [10–14]. Proliferation of naïve T lymphocytes can also be detected during fetal life (our unpublished observation) and could last up to five years of age [15]. The high cell turn-over observed in early life probably plays a central role in the establishment of the T cell repertoire. Despite their high turn-over, T cells preserve long telomeric sequences through a high constitutive telomerase activity [10]. In vitro apoptosis of neonatal T lymphocytes can be prevented by cytokines signalling through the γ-chain of the IL-2 receptor, namely IL-2, IL-4, IL-7 and IL-15 [11,14,16]. Among these cytokines, IL-7 and IL-15 also induce the proliferation of neonatal T lymphocytes in the absence of other stimuli [10,11,13,14,16,17]. IL-7 is involved in thymocyte development at a stage preceding the T cell receptor rearrangement [18]. Circulating neonatal T cells express higher levels of the IL-7 receptor α-chain (CD127) than adult naïve T cells [10,16]. Interestingly, IL-7-induced proliferation of newborn T lymphocytes is dependent on caspases, molecules that are classically involved in activation-induced cell death [19]. Proliferation induced by IL-7 is not associated with T cell differentiation [10,13]. IL-15 preferentially stimulates the proliferation of CD8 rather than CD4 T cells [10]. In contrast to IL-7, IL-15 induces the differentiation of CD8 T lymphocytes in vitro[10,20]. Although IL-7 is likely to play a role in the high turn-over of neonatal T lymphocytes, other factors are also probably involved. In neonatal mice, homeostatic proliferation of CD4 T lymphocytes is critically dependent on class II MHC—TCR interaction and only partly involves IL-7 [21].

Table 1.

In vitro studies of human neonatal T cells

	References
Contain high concentrations of TRECs	[10,11]
High cell turn-over	[10–14]
Have high constitutive telomerase activity and long telomeric sequences	[10]
Have increased susceptibility to apoptosis, prevented by cytokines signalling through the IL-2 receptor γ-chain	[11,14,16]
Proliferation induced by IL-7 and IL-15	[10,11,13,14,16,17,19,20]
Low production of IFN-γ by CD4 T lymphocytes associated with hypermethylation of IFN-γ promoter	[25,26,28]
Expression of CD40 ligand by CD4 T lymphocytes is controversial	[29,30,31,32,33,34,35]
Expression of NFAT by CD4 T lymphocytes is controversial	[36–38]
CD25+ regulatory T lymphocytes occur spontaneously during foetal life	[41–44]

Several mechanisms limit T helper 1 (Th1) type responses in early life. In utero, Th1 responses are toxic to the placenta and are inhibited by trophoblast-derived IL-10 and progesterone [22–24]. At birth, Th1 responses are still of lower magnitude than later in life. In vitro, newborn CD4 T cells produce lower levels of IFN-γ than adult naïve T cells and are hypermethylated at CpG and non-CpG sites within the IFN-γ promoter [25]. In the presence of suboptimal CD28 costimulation, IL-12 stimulates the production of both IL-4 and IFN-γ by neonatal CD4 T lymphocytes whereas adult cells do not produce IL-4 under similar conditions [26]. In response to polyclonal or superantigen activation, postnatal thymocytes develop into Th2 cytokine producing CD4 cells whereas IL-12 is required to stimulate the production of IFN-γ[27]. In contrast, neonatal CD8 T cells produce similar levels of IFN-γ and have a pattern of IFN-γ promoter methylation comparable to that of naïve adult cells [25]. In addition, neonatal CD8 T lymphocytes are strictly dependent on the presence of IL-4 at the time of priming to differentiate into IL-4-producing cells [28].

The capacity of neonatal CD4 T lymphocytes to express CD40 ligand (CD154), a molecule playing a critical role in the help to B lymphocytes and CD8 T cells, remains controversial. Several authors reported a lower expression of CD154 by cord blood CD4 T cells activated by polyclonal stimuli as compared to adult cells [29–33]. In contrast, Splawski et al. [34] reported high levels of CD154 expression by newborn CD4 T cells stimulated with anti-CD3 Ab but a low expression when cells were stimulated with PMA and ionomycin. More recently, Suarez et al. [35] demonstrated that cAMP-inducing agents combined to ionomycin induce the expression of high levels of CD154 by neonatal CD4 T cells.

Together, the available data indicate that naïve T lymphocytes are differently programmed in neonates and in adults. Recent studies have attempted to determine whether this different program involves specific signal transduction pathways. The group of Laughlin reported that neonatal CD4 T cells express lower levels of the nuclear factors of activated T cell (NFAT) than adult cells, including NFAT-1 a transcription factor involved in the activation of CD154 and IFN-γ genes [36,37]. In contrast, O'Neill et al. [38] reported similar levels of NFAT-1 in resting or activated neonatal and adult CD4 T cells. The basis of these conflicting results remains unclear. Importantly, most of these experiments involved the comparison of cord blood cells, including mostly naïve lymphocytes, with adults cells that include both naïve and memory lymphocytes. Direct comparison of cord cells with naïve adult cells is needed. In order to define the profiles of genes expressed during human T cell differentiation, Lee et al. [39] performed microarray gene analysis on resting thymocytes, cord blood CD4 T lymphocytes and adult naïve CD4 T lymphocytes. Interestingly, they observed that the greatest degree of expression change occurs at the transition between double positive and single positive CD4 thymocytes while the least amount of changes exists between cord blood and naïve adult CD4 lymphocytes. Yet, some markers of cord blood cells were identified, including CCR9, a chemokine receptor binding the thymus-expressed chemokine (TECK) and previously been proposed as a marker of recent thymic emigrants [40].

Essential regulatory mechanisms controlling T cell responses are in place in early life. CD25⁺ CD4 T lymphocytes naturally develop in the human foetus and newborn [41–44]. This subset inhibits the proliferation of CD4 and CD8 T lymphocytes and thereby controls autoimmune reactions as well as allogeneic and antimicrobial immune responses. In contrast to adult cells, cord blood CD25⁺ CD4 lymphocytes have a naïve phenotype. Interestingly, CD25⁺ regulatory cells are detected at higher frequencies in preterm as compared to term newborns, suggesting that this subset could play an important immunoregulatory role during intrauterine life (43 and our unpublished observations). Regulatory properties can also be acquired in vitro by CD4 lymphocytes cultured in the presence of IL-10 and IFN-α[45].

Vaccines as models of controlled antigenic challenges in young infants

In order to determine whether differences exist between neonatal and adult T cell-mediated immune responses in vivo, vaccines currently administered to young infants have been used as models of controlled antigenic exposures (Table 2). Following World Health Organization recommendations, infants living in tropical countries are immunized at birth with the antituberculosis vaccine BCG, the oral poliovirus vaccine (OPV) and the anti-hepatitis B vaccine (HBVac) [3,4]. In countries were these diseases are not endemic, infants receive their first vaccines around 2 months of age.

Table 2.

Immune responses to vaccines and infectious pathogens in early life

Vaccine	Immune responses	References
Hepatitis B vaccine	Defective early Th1 and increased memory Th2 responses in newborns as compared to naïve adults; higher antibody response than adults	[46]
Oral poliomyelitis vaccine	Defective Th1 response and similar antibody response in newborns as compared to immune adults	[47]
M. bovis BCG vaccine	Adult-like Th1 response in newborns; promotes antibody, Th1 and Th2 responses to unrelated vaccines	[48–50,58]
Whole cell pertussis vaccine	Th1 response in 2 months-old infants	[52]
Measles vaccine	Lower Th1 response in 6–12 months-old infants as compared to immune adults	[53]
Human cytomegalovirus	Mature CD8 T cell response and defective CD4 T cell response in fetuses and infants as compared to adults	[70,71,75,98,99]
Human immunodeficiency virus	Defective CD4 and CD8 T cell responses in newborns and infants as compared to adults	[76,77,78,79,80,81,82,100,101]
Trypanosoma cruzi	Adult-like CD8 T cell response	[88]
Herpes simplex virus	Delayed IFN-γ response in infants as compared to adults	[102]
Bordetella pertussis	Th1 response in infants	[52]

Infants immunized at birth with HBVac or OPV develop lower Th1 type responses than adults [46,47]. These lower Th1 responses are associated with higher antibody responses than in adults. During the early phase of the response, Th2 type cytokine production is similar in infants and adults but memory responses to HBVac is characterized by higher Th2 cytokine production in infants [46]. The low Th1 responses to HBVac and OPV contrast with the mature Th1 response induced by BCG in newborns [48–50]. The response induced by BCG at birth is quantitatively and qualitatively similar to that observed in immune adults. In tuberculosis endemic countries, adults develop relatively weak immune responses to BCG, probably because of their chronic exposure to environmental mycobacteria [51]. In these countries, newborns who have not yet been chronically exposed to mycobacteria actually develop stronger Th1 responses to BCG than adults. Th1 responses were detected in 2 months-old infants immunized with the whole cell pertussis vaccine [52] but comparison with naïve adults could not be performed. Lower IFN-γ responses to measles vaccine were detected in 6–12 months-old infants as compared to immune adults [53].

The variable capacity of different vaccines to induce Th1 responses in young infants suggests that the quality of the signal delivered by antigen-presenting cells to T lymphocytes plays an important role. Mycobacteria as well as Bordetella pertussis toxin are potent activators of dendritic cells (DC), the antigen-presenting cells required for priming of naïve T lymphocytes [54–57]. The Th1 response induced by BCG and whole cell pertussis vaccines in early life could therefore be related to their potent DC activating properties. As it will be discussed below, neonatal DCs have a lower capacity to produce cytokines promoting Th1 responses. The induction of Th1 responses in early life would therefore require vaccines that are particularly efficient at promoting this type of response. In order to determine whether the potent Th1 response induced by BCG could influence the response to the other vaccines, Ota et al. [58] conducted a randomized controlled trial measuring the response to HBVac and OPV in infants who received BCG at the time of priming or after the last dose of HBVac and OPV. This study showed that BCG increases the cytokine and antibody responses to unrelated vaccine antigens. Surprisingly, the production of both Th1 and Th2 cytokines was promoted suggesting that the magnitude but not the quality of neonatal responses to vaccines was influenced by BCG. Whether this phenomenon is related to the age of the study population and whether Th1 polarization of vaccine responses would be induced by BCG in adults has not yet been determined. Several epidemiological studies have suggested that BCG immunization in early life could influence the immune response to allergens and infectious pathogens. BCG vaccination of young infants was associated with a reduced risk of atopy in some populations but not in others [59–64]. In Guinea Bissau, BCG immunization was associated with a reduced risk of acute respiratory tract infections and of all causes mortality [65,66]. The possibility that vaccines such as BCG may have nonspecific clinical effects remains a subject of intense controversy [67–69]. Controlled epidemiological and immunological studies conducted in different populations are needed to answer this important question. The observation that BCG increases both Th1 and Th2 cytokine responses to unrelated vaccines in infants indicates that its potential nonspecific effects are unlikely to be related to a shift from Th2 to Th1 type immune responses [58].

T cell-mediated immune responses to infectious pathogens in early life

Only a restricted number of studies have described T cell responses to infectious pathogens in early life (Table 2). As observed with infant vaccines, the magnitude and the quality of cellular immune responses vary from one pathogen to another. HCMV is the most common cause of congenital infection. Acute HCMV infection is most often asymptomatic in immunocompetent adults. In contrast, about 10% of infected newborns develop severe symptoms [6]. If the pathogenesis of symptomatic infections remains poorly understood, the cellular immune response to HCMV has been recently studied in asymptomatic newborns and infants. Following congenital infection, newborns develop a mature CD8 T cell response to HCMV [70,71]. This response is similar to that detected in adults and is characterized by large oligoclonal expansions directed against a restricted number of immunodominant antigens. HCMV-specific CD8 T cells express a late differentiation phenotype and have antiviral effector functions, producing IFN-γ and having perforin-dependent cytolytic activity [72–75]. CD8 T cell responses to HCMV have been measured in infected foetuses as early as after 28 weeks of gestation [71]. This mature CD8 T cell response to HCMV contrasts with the low CD8 T cell response to HIV. In HIV-infected infants, CD8 T cell responses are of lower magnitude and are less commonly detected than in infected adults [76–81]. In coinfected infants, high frequencies of HCMV-specific CD8 T cells are detectable but anti-HIV-specific CD8 T cells are infrequently detected [82]. Similar results were obtained using IFN-γ Elispot and HLA class I-peptide multimers. The low CD8 T lymphocyte responses to HIV could play an important role in the rapid disease progression observed in young children. An estimated 600 000 new paediatric infections occur each year. Whereas children account for 4% cases of HIV infection, they represent 20% of AIDS deaths [5].

The mechanisms underlying the difference in the capacity of young infants to develop CD8 T cell responses to HIV and HCMV is unclear. They could be related to differences in the quality of T cell priming. Indeed, HCMV-specific CD8 T cells display a more advanced differentiation phenotype and express higher levels of perforin than HIV-specific cells [72,83,84]. Recent data indicate that the intensity of T cell activation plays a critical role in CD8 T cell differentiation [85]. The poor CD8 T cell response to HIV in infants could also be related to a low CD4 T cell help; interestingly, the potent CD8 T cell response to HCMV may not require CD4 help. Alternatively, the transmission of HIV mutants that have escaped maternal CD8 T cells could prevent the development of infant CD8 T cell responses to dominant epitopes [86]. Epidemiological studies have shown that HLA concordance between mother and child increases the risk of HIV transmission [87]. Finally, specific immune evasion mechanisms may be expressed by HIV that are particularly efficient at inhibiting the response of neonatal T cells. HCMV is not the only infectious pathogen stimulating potent CD8 T cell responses in early life. Newborns with congenital Trypanosoma cruzi infection also display large clonal expansions of CD8 T cells recognizing parasite antigens and producing IFN-γ[88]. CD8 T cell responses to RSV and measles can be detected in infants but whether these responses are of similar quality and magnitude than those of adults has not yet been established [89–91].

Our knowledge of CD4 T lymphocyte responses to infectious pathogens in early life is even more restricted than that of CD8 T cells. Although foetuses and infants develop mature CD8 T cell responses to HCMV, they have lower CD4 T cell responses to this virus than adults. Adults develop strong CD4 T cell responses during the acute and persistent phases of HCMV infection [92–96]. In contrast, children who acquired HCMV in utero or during infancy have very low proliferative and IFN-γ responses to HCMV antigens [97,98]. Intriguingly, these low responses can persist for several years post infection. In parallel, children infected in early life excrete the virus for prolonged periods of time. In the mouse model of CMV infection CD4 T lymphocytes are required to control viral replication in salivary glands [99]. The low CD4 T cell response to HCMV infection observed in infants could be involved in their persistent viral excretion and thereby allow virus spread in the population [6]. Low CD4 T cell responses are also detected in HIV-infected infants whereas CD4 T cell responses to this virus can be detected in older children and in adults [100,101]. The mechanisms involved in the low CD4 T cell responses to HCMV and HIV are not yet understood. They probably involve both intrinsic T cell characteristics previously mentioned as well as immune evasion mechanisms. Indeed, as discussed above, mature CD4 T cell responses can be triggered by vaccines in early life and Th1 type CD4 T cell responses develop following whooping cough in young infants [52]. Along the same line, IFN-γ-producing CD4 T cells were detected in HSV-infected infants, although their responses were established with a slower rate than in adults [102]. Also, following in utero exposure to helminth antigens newborn T lymphocytes produce similar cytokines as those produced by T lymphocytes obtained from infected adults [103,104].

Properties of neonatal dendritic cells

The variable T cell responses induced by vaccines and infectious pathogens in early life suggest that the function of antigen-presenting cells, in particular DCs, could be different in newborns and adults. Recent studies have compared the responses of neonatal and adult myeloid and plasmacytoid DCs to Toll-like receptor (TLR) ligands. TLRs are relatively conserved molecules recognizing microbial products that stimulate DCs and influence their ability to activate T lymphocytes [105]. A number of studies indicate that neonatal DCs have a lower capacity to produce cytokines promoting Th1 responses than adult DCs. Neonatal monocytes-derived DC (mDC) produce low concentrations of IL-12(p70) in response to lipopolysaccharide (LPS), poly I:C, Bordetella pertussis toxin or CD40 ligation [56,106]. This phenomenon is related to a reduced expression of the gene coding for the p35 subunit of IL-12 whereas IL-12 (p40) gene expression is comparable to that induced in adult cells. The low IL-12 (p35) synthesis depends on a reduced nucleosomal remodelling process that is required for IL-12(p35) gene transcription [107,108]. Importantly, addition of IFN-γ to LPS-activated DCs increases nucleosomal remodelling and IL-12 (p35) synthesis by neonatal mDC to adult-like levels [108]. Low IL-12 production by neonatal mDCs was also observed by Langrish et al. [109]. On the other hand, Upham et al. [110] observed that the production of IL-12 by neonatal monocytes is lower than in adults but that differentiation of monocytes into mDCs increases IL-12 production to the levels detected in adults. These discrepant results are probably related to differences in the conditions under which DCs were cultured and are compatible with the notion that neonatal mDCs are able to produce adult-like levels of IL-12 under some experimental conditions, including the addition of IFN-γ.

The function of neonatal plasmacytoid (p)DCs remains largely unexplored. In order to assess the ability of these cells to produce IFN-α, we used as stimulus CpG oligodeoxynucleotides which are considered as potential adjuvants to prime Th1 responses. In response to both stimuli, neonatal pDC were virtually incapable to synthesize IFNα although they expressed adult-like levels of TLR9 mRNA [111]. Together these results indicate that neonatal DCs have different characteristics than adult DCs. The mechanisms involved in their maturation have not been defined. Intriguingly, recent data indicate that adult-like levels of IL-12 are produced only after several years of life (110 and our own unpublished observation). This slow maturation of DC functions may be involved in the persistent defects in the CD4 T cell responses to HCMV and HIV [97,98,100,101]. Indeed, these viruses impair the function of DCs and reduce their capacity to produce IL-12 and activate T cells. In adults, the function of DCs may be sufficient to allow T cell priming even when infected with suppressive pathogens. In young infants, HCMV or HIV infection of immature DCs may suppress their costimulation capacity below the threshold required to activate CD4 T cells. The intensity and/or the nature of the costimulatory signals required to prime neonatal CD8 T lymphocytes may be lower or different than for CD4 T lymphocytes. Indeed, newborns with congenital HCMV infection have mature CD8 but lower CD4 T cell responses [70,71,97,98]. Also, in vitro neonatal mDCs are less efficient than adult mDCs at stimulating allogeneic CD4 T cells but are able to prime antigen-specific CD8 T lymphocytes at similar levels than adult mDCs [112].

Conclusion

In vitro investigations established that the capacity of neonatal CD4 T cells to produce IFN-γ and of neonatal DCs to promote Th1 responses is lower in infants as compared to adults. In vivo, Th1 responses to a number of vaccines and infectious pathogens are poor during early life. However, mature Th1 responses can develop in certain conditions such as neonatal BCG vaccination and Bordetella pertussis infection, probably in relation with a more efficient activation of DCs. In vitro studies as well as clinical investigations also suggest that the priming of neonatal CD8 T cell responses may require a lower threshold of DC activation than that required for Th1 responses. Other features of neonatal T lymphocytes include homeostatic proliferation and increased susceptibility to apoptosis, but the impact of this high cell turn-over on the establishment of memory responses remains to be clarified.

The classical paradigm that newborns have incompetent T lymphocytes developing only weak or even tolerogenic responses should clearly be reconsidered. The observation that mature cellular immune responses can be developed in early life suggests that under appropriate conditions of stimulation neonatal T lymphocytes can be instructed to fight intracellular pathogens (Fig. 1). We can therefore hope that the identification of molecular pathways leading to DC and T cell activation in human neonates will lead to the development of new vaccines eliciting efficient and safe protective responses against these agents early after birth.

Fig. 1.

Youth denotes aptness to apprehend and learn. From the Iconologia or Moral Emblems by Cæsar Ripa, London, 1709; reproduced with the permission of the Special Collections Library, the Pennsylvania State University Libraries.