In healthy children, overall IgG4 antibody concentrations drop from birth to a nadir around 6 months of age as maternal IgG4 concentrations in the infant’s system decline. IgG4 concentrations usually increase as infants and young children are exposed to allergen-containing foods, then plateau around age 6–8 years 
, and finally reach adult concentrations by age 13 years 
, revealing the gradual IgG4 rise throughout childhood and the long-lasting effects of infant dietary exposures to food allergens. IgG4 antibodies to ovalbumin follow this pattern 
, while IgG4 antibodies to β-lactoglobulin have been shown to peak in early childhood and drop thereafter 
We hypothesized that increased IgG4 concentrations against multiple dietary antigens, indicating increased gut permeability and subsequent mucosal immune response, would be associated with earlier IA development, and more rapid progression to T1D in children with IA. We approached this research question in three ways. First, we analyzed the IgG4 concentrations throughout childhood (as time-varying covariates). We then explored a combined measure of antigen response by ranking and summing quartiles of IgG4 concentrations of the three IgG4 antibodies. Finally, we examined infancy as a critical time period.
Before analyzing IA and T1D risk, we defined the association between relevant infant dietary exposures and childhood IgG4 concentrations to ß-lactoglobulin, gluten, and ovalbumin. In our cohort of children with increased genetic risk for T1D, circulating concentrations of the three dietary IgG4 antibodies measured (dairy, gluten, and eggs) were strongly correlated with each other. We found that ß-lactoglobulin concentrations were inversely associated with total breastfeeding duration and with age at introduction to cow’s milk. This finding is consistent with other studies, in which early exposure to cow’s milk in infancy was associated with higher IgG antibody concentrations throughout infancy and early childhood 
Previous research has found T1D to be associated with increased IgG antibodies to ß-lactoglobulin 
, but we are the first to examine the association between ß-lactoglobulin IgG4 concentrations and IA development. Our analyses showed that neither development of IA, nor progression to T1D in IA positive subjects, were associated with increased IgG4 antibodies to β-lactoglobulin. Our prospective findings agree with a previous case-control study 
, and disagree with the case-control studies that found an association between ß-lactoglobulin IgG and T1D 
. Our findings also disagree with the prospective findings of Luopajarvi et al
. This discrepancy may be due to our examination of ß-lactoglobulin levels throughout childhood, while Luopajarvi et al
. examined ß-lactoglobulin levels in the first 3 years of life. Also, Luopajarvi and colleagues measured ß-lactoglobulin IgG, while we measured the IgG4 isotype.
Gluten consumption has been shown to initiate an inflammatory response in the gut, which may compromise intestinal barrier function and increase gut permeability 
. However, our findings in regards to an association between gluten IgG4 concentrations and IA or T1D risk were null, in agreement with previous research which showed that eliminating gluten from the diet does not reduce islet autoantibody titers 
. Previous research on the DAISY cohort has also shown that IgG antibodies to Glo-3A, a wheat storage globulin, were not associated with IA development 
, although there was a subset of responders, which may indicate etiologic heterogeneity. In other studies, an immune response to gluten has been observed in children with newly diagnosed T1D, but not in children with a longer duration of T1D 
. However, Mojibian et al.
found an immune response to dietary wheat polypeptides and gliadin in transglutaminase-negative young adults with long duration T1D 
Our study found higher ovalbumin IgG4 antibody concentrations at the first IA positive visit was marginally associated with faster progression to T1D. Our prospective study results confirm the findings of two previous case-control studies, which found higher IgG antibodies to ovalbumin in T1D patients 
. Greater immune response to ovalbumin has also been observed in patients with long-term T1D 
The immune factors influencing autoimmune disease risk are complex. In our study, the increased ovalbumin IgG4 concentrations may indicate an immunological tolerance to foreign proteins, resulting from a greater Th2 response 
in children that subsequently develop T1D. Th2 cells have been shown to inhibit regulatory T cell development 
, potentially increasing risk of autoimmune disease. Alternatively, regulatory T cells have also been shown to induce IgG4 antibodies 
. Therefore, low IgG4 concentrations could indicate low or dysfunctional regulatory T cells, which would in turn increase risk of autoimmune disease 
. However, our analysis did not reveal inverse associations between IgG4 concentrations and development of either IA or T1D.
Our finding of a significant association between T1D risk and ovalbumin IgG4 concentrations, but not β-lactoglobulin or gluten IgG4 concentrations, does not agree with our hypothesis of a greater generalized mucosal immune response, indicating increased intestinal permeability, in children that develop T1D. Our discrepant finding may be due to the greater potency of the ovalbumin antigen. IgG4 antibodies to ovalbumin can be found in infants prior to egg exposure 
, possibly due to exposure to the antigen via breastmilk 
, whereas β-lactoglobulin IgG4 response is higher in infants directly exposed to the antigen compared to breastfeeding infants 
. The greater potency of the ovalbumin antigen, paired with our relatively small sample size, may explain why we only found an association between T1D risk and ovalbumin, but not β-lactoglobulin or gluten, IgG4 concentrations. It is possible that a larger sample size would reveal associations of smaller magnitude between T1D risk and β-lactoglobulin or gluten IgG4 concentrations.
Our mostly negative findings may also be due to the very gradual rise in IgG4 antibody concentrations throughout childhood: IgG4 concentrations do not reach adult levels until age 13 years 
. IgG4 concentrations in our analysis may be generally low due to the young age of the population (mean age at last follow-up: 9.2 years). The association we were looking for may be more evident in a cohort of older children or young adults with increased genetic risk for T1D.
Strengths of this study include the prospective, longitudinal nature of the data, with frequent follow-up intervals and collection of exposure variables prior to IA or T1D onset. No other studies have prospectively examined the association between IgG4 antibodies to multiple dietary factors and earlier IA or T1D development. The case-cohort analysis structure allowed us to efficiently conduct a resource-intensive analysis on a sub-sample of the full cohort, without compromising the strength of the prospective cohort design. Limitations of this analysis include lack of generalizability to the US pediatric population, since the DAISY cohort was selected for having increased genetic risk of developing T1D. We did not measure intestinal permeability directly; instead we measured circulating IgG4 antibodies to dietary components in hope of observing an indirect measure of intestinal permeability. The DAISY study was not designed to specifically answer this research question, and the analysis may have been underpowered. However, the effect sizes of our negative findings were small. It is likely that, if a similar analysis conducted on a larger cohort found significant associations, they would be of modest effect size and unlikely to be clinically significant. Finally, we were unable to explore the presence of food allergies in the DAISY population for two reasons. First, DAISY collects parent-reported food allergies, but these reported allergies are not confirmed with clinical allergy testing. Second, we did not measure IgE antibody concentrations, as the measurement of food allergies was not the aim of this investigation.
In conclusion, our results suggest that dietary IgG4 antibody concentrations in childhood may be related to timing of infant diet exposures. This study also demonstrates that IgG4 antibodies against common dietary antigens are relatively poor biomarkers of IA risk. We did find evidence that ovalbumin IgG4 antibody concentrations may be increased at IA development in children who subsequently develop T1D. This novel observation should be explored in other populations. Our examination of the individual dietary IgG4 antibody concentrations, as well as our composite measure of ß-lactoglobulin, gluten, and ovalbumin IgG4 antibodies in childhood, provided no evidence of a greater generalized immune response in children developing IA or progressing to T1D.