Invited Symposium: Novel Cellular and Molecular Mechanisms in Allergic Inflammation
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Allergen-specific T-cell priming
T-CELL PRIMING AGAINST ENVIRONMENTAL ALLERGENS: LYMPHOPROLIFERATIVE STUDIES IN CORD BLOOD (CB)
Until comparatively recently, atopy was considered to be synonymous with hyperresponsiveness to a range of non-pathogenic environmental antigens (allergens), which were ostensibly “ignored” by the healthy/non-atopic immune system. However, it is now clear that qualitative as opposed to quantitative aspects of T-cell immunity to these allergens are the principal determinants of responder phenotype. The murine Th1/Th2 paradigm (1) as reinterpreted for human (2-4), remains the most useful model for describing the difference in allergen-specific T-cell responsiveness between atopics and non-atopics, despite justifiable reservations concerning the rigidity of the model (5). For example, many authors (e.g. (6, 7)) point to the fact that a common response pattern amongst atopics is that of a mixture of Th1 and Th2 cytokines, resembling the murine Th0 cytokine phenotype. The defining feature of the atopic response appears to be the contribution from key Th2 cytokines (in particular IL-4 and IL-5), which may or may not be accompanied by IFNg, in contrast to the more restricted Th1-like pattern in non-atopics.
In relation to the aetiology of atopy, the central question is accordingly: how are these divergent cytokine response patterns programmed into long term immunological memory, in atopics and non-atopics?
In addressing this issue, one research stream that is growing rapidly in the current literature relates to the initial confrontations between the naive immune system and environmental allergens, which occur in very early life. As discussed below, the outcome of these early responses appear likely to play a major role in determining patterns of allergen reactivity in later life.
The initial findings suggestive of transplacental priming against environmental allergens date back to the 1970s from studies with ragweed pollen (8, 9). These findings were not followed up until comparatively recently, but have now been confirmed by a number of groups, employing a range of dietary and inhalant allergens (e.g. (10-15)). The interpretation of these results has been widely disputed, given the possible contamination of CB with maternal T-cells. However, a recent study from our group employing DNA genotyping of CB-derived allergen-specific T-cell clones has formally demonstrated their fetal origin (16).
The issue of whether there are quantitative or qualitative differences in this initial T-cell priming process between atopic family history positive (FH positive) and FH negative subjects remains controversial. There are some published data on lymphoproliferation suggesting that fetal priming is associated with atopic risk (17), whereas our recent studies, in particular those on CB cytokine responses (detailed below), suggest a more complex picture.
Postnatal maturation of T-cell immunity
POSTNATAL MATURATION OF ALLERGEN-SPECIFIC T-CELL IMUNITY: DIVERGENCE OF RESPONSE PATTERNS FOR DIETARY VERSUS INHALANT ALLERGENS
Cross-sectional studies from our group have demonstrated that responses to these two classes of allergens follow distinct pathways after birth. Responses to inhalant allergens, exemplified by house dust mite (HDM), increase in frequency and intensity postnatally, and by adulthood are present in close to 100% of subjects (15, 18). As noted above, T-cell cloning studies from several groups indicate that these responses partition into Th1-like (normals) or Th2-like (atopics). This pattern contrasts markedly with that observed in studies on allergens presented via the gastrointestinal tract (GIT). Lymphoproliferative responses to the archetypal dietary allergen ovalbumin (OVA) peak in frequency and intensity during infancy, but decline markedly by age 2-5 yrs and remain at low frequency in adulthood (15, 19). Epitope mapping of these T-cell responses indicates that several regions of the OVA molecule are recognised by fetal T-cells, and the range of epitopes recognized contracts rapidly postnatally (19).
It is of interest to note in this context that low-level IgE production against allergens such as OVA is common in FH positive and FH negative infants (peak levels generally higher in the FH positive group), typically peaking before one year of age and declining to below detectable limits by age 2 years, except in a small subset of children who develop persistent food allergy (20, 21). This contrasts with findings relating to inhalants, where the typical response pattern involves initiation of IgE responses beyond age one, and with a much higher frequency of these responses persisting into later childhood (20, 21).
We have suggested (15, 22) that these disparate patterns of postnatal immune responsiveness may reflect the operation of two distinct classes of T-cell regulatory mechanisms, high zone tolerance (T-cell anergy and/or deletion) driven by high level exposure to dietary allergens, versus low zone tolerance (immune deviation) in the case of low level exposure to inhalants.
Cross-sectional studies
CROSS-SECTIONAL STUDIES ON ALLERGEN-SPECIFIC CYTOKINE RESPONSES DURING CHILDHOOD
We have recently published a series of findings demonstrating the overall Th2 polarity of allergen-specific CBMC responses (16, 23). These studies deliberately focused upon cytokine protein or mRNA production at the 24 hr time point post stimulation, in order to minimise distortion of in vivo cytokine gene expression patterns as a result of in vitro T-cell differentiation in the cultures. The key findings were that IL-6, IL-10 and IL-13 were readily detectable at the protein level, and IL-4, IL-5 and IL-9 at the mRNA level. IFNg was also detectable in a subset of samples, but only employing extremely high PCR cycle numbers (47 cycles, as opposed to 35 cycles with adults).
In experiments focusing on these responses in older age groups, we have demonstrated that at age 18 months, allergen responsiveness within the population segregates on the basis of FH, with FH positive subjects typically manifesting a mixed (Th0-like) cytokine pattern including IL-4, IL-5 and IFNg, in contrast to much weaker cytokine responses amongst FH negative subjects which most frequently do not attain statistical significance (19). By age 5-6 years, <2% of 5-6 yr olds are SPT positive to the dietary allergen OVA, and in vitro PBMC cytokine responses are restricted to IL-6 (of non T-cell origin) and low level T-cell derived IL-10 (unpublished observations). In contrast, approximately 30% of the 5-6 yr old population are SPT positive to inhalants such as HDM, and their responses are dominated by the cytokines IL-4, IL-5, IL-6, IL-9, IL-10 and IL-13, often with low level IFNg, whereas the responses in the SPT negative non-atopic population are principally IFNg in combination with IL-10 (19, 24). IL-10 production here is principally of T-cell origin (24), possibly from the same Th1 cells which are producing the IFNg.
These findings collectively are consistent with the generalised model (illustrated in (22)) referred to above, in which initial T-cell priming against allergens occurs within the Th2-polarised fetal microenvironment, with these responses subsequently being reshaped via high zone/low zone tolerance mechanisms which are driven by postnatal allergen exposure. “Normality” in this context equates to functional silencing of the bulk of food allergen reactivity during early infancy, versus immune deviation towards the Th1 (or Th0) cytokine phenotype for responses to inhalants; atopy in the context of Th2-mediated allergy to inhalants can thus be viewed as a failure of this latter immune deviation process.
Genetic basis for atopic risk
MECHANISM(S) UNDERLYING GENETIC PREDISPOSITION TO ATOPY: CLUES FROM IN VITRO CYTOKINE STUDIES IN NEONATES AND INFANTS
We have previously demonstrated that T-cells from FH positive infants are deficient in capacity to generate T-cell clones, and in capacity to produce Th1 and Th2 cytokines, relative to their FH negative counterparts (25). The cytokine defect was most pronounced for IFNg (25), and may represent postnatal persistence of the normal Th1-damping mechanisms which are operative in the fetal microenvironment (26). Confirmatory findings relating to this atopy-associated maturational defect in IFNg production have been reported employing CBMC (12, 27-31). It has also been observed that responsiveness to BCG vaccination during infancy and early childhood, an indirect measure of Th1 function, is compromised in atopic children (32), and we have observed similar differences in regard to responses to the DPT vaccine (33).
We have hypothesized that the delayed postnatal maturation of Th1-associated function(s) in children with the atopic genotype may compromise their capacity to immune deviate fetally-primed allergen-specific Th2 responses towards the Th1 cytokine phenotype, resulting in the consolidation of Th2-polarised immunological memory during early postnatal life (22, 31). This hypothesis is currently being tested in a series of prospective studies, the first of which is described briefly below.
Prospective studies
PROSPECTIVE STUDIES ON ALLERGEN-SPECIFIC T-CELL IMMUNITY DURING EARLY CHILDHOOD
The published information available on prospective analysis of postnatal T-cell responses to allergens in individual children is limited to studies involving small subject panels, and a restricted range of cytokines (12, 34). These provide suggestive evidence that children who express atopy symptoms during early childhood tend to express Th2-like immunity and/or inefficient Th1 immunity.
A recent study from our group (31) provides significant new information relevant to the mechanisms underlying this process. In this study, a cohort of subjects were followed to age 2 years, and two groups selected who expressed definite atopic disease at the outcome age (n=18) or who were symptom free (n=13). Allergen-specific T-cell responses in these subjects were then analysed employing cryopreserved PBMC samples which had previously been collected at 6 month intervals commencing with CBMC.
Several findings emerged from the study. Firstly, it was clear that CBMC from children who did not develop atopy expressed higher allergen-specific cytokine responses measured either as protein (IL-6, IL-10, IL-13) or mRNA (IL-4, IL-5 and IL-9), relative to their atopic counterparts. However, the postnatal course followed by these responses varied markedly between the two groups, the atopics demonstrating age-dependent increases in IL-4 and IL-13, in marked contrast to the inverse pattern (age-dependent decrease) in the group who did not develop atopy. Moreover, the non-atopic group exhibited rapid postnatal upregulation of allergen specific IFNg mRNA responses, compared to a very slow rise in the atopics.
Additionally, APC-independent stimulation of purified CD4+ T-cells from CB samples from both groups revealed markedly reduced IFNg production in the group who subsequently developed atopy, confirming earlier observations made on unfractionated CBMC (28).
These findings are consistent with the general model proposed above i.e. that the postnatal development of atopy is associated with a failure to “silence” (or immune deviate) fetally primed allergen-specific Th2 responses, which is in turn associated with deficient expression of Th1 function during the neonatal period.
Further prospective studies are in progress to analyse this model in more detail.
ADDENDUM
An issue of growing importance in this field is the interaction between infections during early life, and atopy development. We have argued previously that infection(s), or more precisely microbial contact per se, is likely to indirectly influence the development of allergen-specific T-cell memory during early childhood via provision of essential stimuli required for postnatal maturation of Th1 function (20, 22). We have furthermore argued that in addition to possible effects of respiratory infections (35), it is likely that the major contributions to this natural process may come from GIT bacterial flora (20, 22, 36), particularly via stimulation by LPS. Of interest to note in this context is a recent study demonstrating a polymorphism in the human CD-14 gene encoding the high affinity receptor for LPS, which is associated with variations in serum IgE levels and SPT reactivity.
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