It is estimated that 2.5% of young children have milk allergy, 1–2% have egg allergy and 1% have peanut allergy.(2
) and that these allergies are increasing in prevalence and becoming more persistent.(3
) However, little is known about the underlying mechanisms resulting in the development of allergy or tolerance to these foods.(2
) The cohort described herein was established to evaluate and elucidate environmental, genetic and immunologic factors associated with the development or persistence of these common food allergies with the goal of determining better diagnostic and therapeutic modalities. The cohort will be followed longitudinally in this observational study to characterize the outcomes of milk, egg, and peanut allergy, including the conduct of oral food challenges as clinically indicated. The current study focuses upon immune characteristics associated with clinical allergy and sensitization in these infants at enrollment.
We undertook analysis of activated mononuclear cell expression of several key markers of immune regulation and Th1/Th2 bias. We found that allergen-induced IL-4 expression in peripheral mononuclear cells of this high risk cohort was associated with clinical allergy to milk and IgE-sensitization to milk and peanut. This is consistent with other reports of a Th2 bias in food allergy as well as with our current understanding of the role of IL-4 in inducing IgE class switching in activated B cells.(27
) Differentiated CD4 Th2 cells have been shown to express high levels of GATA-3 and low levels of TBET -- master transcriptional regulators for many Th2 and Th1 genes, respectively.(28
) We hypothesized that allergen-specific T cells activated in vitro
would be predominantly Th2 effector memory cells expressing a high GATA-3/TBET ratio and that selection of activated cells on the basis of their upregulation of CD25 would be reflected by an increase in the GATA-3/TBET ratio of transcripts detectable by PCR. Though previous studies have associated Th2 immune responses with food allergy, the immune phenotype of such young children with early evidence of food allergy or sensitization has not been previously studied on this scale. We did not find evidence of an increased GATA-3/TBET ratio with allergen activation. The lack of detectable enrichment for GATA-3 expressing cells was not due to a failure of detecting GATA-3, which was readily detectable and expressed at higher levels on average than IL-4 and several other transcripts (see Table E5
, ONLINE REPOSITORY). Activation of cells, reflected by an increase in CD25 expression over medium alone was also detectable (not shown) though it did not differ significantly between clinical groups (). This finding suggests that strongly polarized allergen-specific T cell differentiation may not have occurred in these subjects, even though they have produced IgE in vivo and allergen stimulation in vitro induced IL-4. In this study we looked specifically at CD25+ cells isolated following stimulation with antigen in order to capture both potential regulatory (constitutive CD25 expression) and activated (induced CD25 expression) T cell populations, however it is known that CD25 is also induced or constitutively expressed on many non-lymphoid cells, including NK T cells and basophils, potent potential sources of IL-4.
Basophils constitutively express CD25,(29
) are enriched with mononuclear cells during density gradient isolation and produce high levels of IL-4 in sensitized subjects.(30
) Recently, basophils have been implicated in allergy model systems for playing an important role in priming and enhancing memory Th2 responses.(31
) Murine basophils have been shown to express IL-4 in the absence of detectable GATA-3 or c-maf, expression, suggesting that IL-4 may be regulated distinctly in these cells.(29
) Thus, new paradigms are emerging that basophils play a key role in directing Th2 responses and our data may provide additional support for this observation. Preliminary studies utilizing flow cytometry revealed that basophils (CD3- CD123+ CD203+ HLA-DR dim and IL4+, data not shown) are present in the CD25 preparation. NKT cells can also produce significant IL-4, although the role of these cells in allergic sensitization or disease is controversial.
We cannot rule out the possibility that the frequency of Th2 effector subsets is so low in the peripheral blood that as a fraction of total CD25+ cells enriched by selection, the overall impact on GATA-3 transcript number on the subsequently isolated mRNA is too small to be detected, while the activation-induced change in IL4 in those same few cells is detectable. There were not sufficient cell numbers to both phenotype the CD25+ fraction and perform RT-PCR in our initial protocol, and therefore we cannot draw clear conclusions regarding the potential role of non-T cells. We are currently investigating the potential role for these populations in the observed in vitro IL-4 response. It will also be interesting to observe in this cohort, whether a T cell signature of Th2 polarization develops over time and if the early IL-4 response, potentially from other cells, proves to be important for priming clinical allergy. If so, this would corroborate findings in animal models and suggest new targets for primary prevention of disease.
We observed several differences in gene expression in response to egg stimulation compared to stimulation with milk or peanut. CD25 expression was significantly but only slightly increased for those with egg allergy but IL4 expression was not increased. The observed increase in CD25 expression following egg stimulation in vitro was marginal and did not reach the magnitude of significance observed for IL4 expression following in vitro stimulation with milk or peanut. The explanation for the difference in response to egg compared to milk and peanut (e.g., lack of IL4 increase) remains uncertain, but may be related to particular proteins used in the stimulation experiments. For example, the antigens used for milk stimulation were comprised of caseins, the major milk allergens, whereas the egg stimulation was performed with whole egg protein extracts. In future studies we will explore whether enhancement with specific egg proteins (e.g., Gal d 1) results in different patterns of response.
In addition to the observed immunologic characteristics, several key clinically-relevant observations were made. Although children with known peanut allergy or previously known elevated (> 5 kUA
/L ) peanut-specific IgE were not eligible for enrollment, there was a strikingly high rate of peanut sensitization and likely peanut allergy: 69% of the cohort were sensitized and 28% of 503 with IgE quantization had IgE concentrations to peanut over 5 kUA
/L, which we estimate is likely associated with clinical peanut allergy (as described in the ONLINE REPOSITORY). Although there are no prior studies with precisely our enrollment criteria, a large international study of infants with active AD where 68% had moderate-severe eczema, 24% were sensitized to peanut.(15
) Our observation indicates that infants presenting with likely milk/egg allergy without already known peanut allergy are at high risk of also having peanut sensitization and, therefore, possible peanut allergy. A recent report concerning the young age of presentation of children with peanut allergy supports the concern that young egg or milk allergic children, or eczematous children, are at high risk for exhibiting peanut allergy on their first known ingestion.(34
We evaluated the relative sensitivity of PST and serum IgE in detecting sensitization (not clinical allergy) in this cohort because there are currently very limited data about these relationships in young potentially food-allergic infants/children. We found a strong relationship between PST wheal size and serum IgE concentration for the 3 foods. As indicated in and Figures E1a–c
, when tests were discordant for peanut, the serum IgE test was more sensitive than the skin tests in detecting peanut sensitization (i.e., 15% of those tested had detectable peanut-specific serum IgE but negative peanut PST compared to 8% who had undetectable peanut IgE and a positive peanut PST, p < 0.001), which is in contrast to general conceptions that PST are more sensitive than serum tests. The reason for this observation could include our use of a more sensitive serum assay than previous generation tests,(35
) reduced skin test reactivity in infants,(36
) or other factors such as differences in the proteins being tested in the 2 methods. These findings are important when selecting tests to evaluate infants for possible food hypersensitivity, and substantially dispel the long held notion that skin testing is uniformly more sensitive (more likely to be positive) than serologic testing, especially in younger children.(37
) Unlike the test results for peanut, those for milk and egg must be interpreted with caution and may be biased because a positive PST to milk or egg was required for enrollment into the study. Conversely, we did not enroll infants with serum testing strongly positive (>5 kUA
/L) to peanut prior to their screening, so the bias would have been against our finding the serum test to be a more sensitive indicator of sensitization. We emphasize that sensitization to peanut is not indicative of peanut allergy.(38
) We are therefore not discussing clinical allergy outcomes but detection of IgE by these methods. As this cohort ages, we will determine whether elevated IgE levels in the infants are associated with clinical reactions.
In summary, we have established a cohort of infants with likely egg and/or milk allergy who will be studied longitudinally for the resolution of egg and milk allergies or the development peanut allergy. We have focused upon relationships of concurrent immune determinants with clinical allergy to milk/egg and sensitization to the 3 most common early childhood food allergens, egg, milk and peanut. Data about parallel PST and serum IgE tests reveal that these tests are highly correlated, but there are also potentially significant discrepancies between them. Mononuclear cell stimulation results indicate that IL-4 expression is associated with allergy to milk and sensitization to milk/peanut in the absence of detectable induction of GATA-3 in these cells. Lastly, we documented an alarmingly high rate of peanut sensitization (68.8%), including 26.6% with a significantly elevated peanut-specific IgE level > 5 kUA/L. This observation may have clinical implications for pediatricians and allergists evaluating young children with evidence of milk and/or egg allergy or more than mild AD and evidence of sensitization to milk or egg. The results underscore the need for caution in managing these infants’ early diet. Specifically, children with these clinical features may represent those for whom testing for food allergy is appropriate prior to advancing the diet.