Analysis of Allergen-Specific T Cell and IgE Reactivity to Different Preparations of Cow's Milk-Containing Food Extracts

Abstract

Background: cow's milk allergy (CM) is among the most common food allergies in young children and is often outgrown by adulthood. Prior to developing a tolerance to CM, a majority of CM-allergic children may tolerate extensively-heated CM. This study aims to characterize the IgE- and T cell-reactivity to unheated CM and the progressively more heated CM-containing foods.

Methods: CM-containing food extracts from muffin, baked cheese, custard and raw, pasteurized CM commercial extract were tested for skin prick test reactivity, IgE binding and T cell reactivity as assessed by IL-5 and IFNγ production.

Results: the skin prick test (SPT) reactivity was significantly decreased to muffin extract compared to raw, pasteurized CM. Both IgE- and T-cell reactivity were readily detectable against food extracts from all forms of CM. Western blot analysis of IgE reactivity revealed variability between extracts that was protein-specific. T cell-reactivity was detected against all four extracts with no significant difference in IL-5 or IFNγ production between them.

Conclusion: our data indicate that despite reduced clinical reactivity, extracts from heated CM-containing foods retain immunogenicity when tested in vitro, particularly at the T cell level.

Keywords: IgE; T cells; allergen extract; baked milk; cow’s milk allergy; extensively-heated milk.

Figure 1

Skin prick test wheal diameters of CM-allergic subjects in response to CM (n = 15), custard (n = 11), baked cheese (n = 11) and muffin extracts (n = 11). The left panel shows a bar graph, with bars indicating median with interquartile range. The right panel shows a line graph with each individual represented by a connected line. Note, four donors tested for raw, pasteurized CM were not tested for the other three extracts. Statistical comparison was performed with the Friedman test (two-tailed), only considering the 11 subjects for whom full data sets were available. p < 0.05 is considered significant.

Figure 2

Immunoblot analysis of raw, pasteurized CM and CM-containing food extracts. (A) 1-D immunoblot analysis of raw, pasteurized CM, custard, baked cheese and muffin extracts showing IgE-reactive protein bands (red) and (B) a 2-D immunoblot analysis of raw, pasteurized CM extract showing protein spots (green) reactive with IgE (blue) and/or IgG (red) from a serum pool from 10 CM-allergic subjects.

Figure 3

Quantification of IgE reactivity of allergen-corresponding protein bands in raw, pasteurized CM, custard, baked cheese and muffin extracts. (A) Representative western blot using an individual plasma sample, showing IgE-reactive protein bands. Bands that were quantified are shown in the raw, pasteurized CM extract lane, indicated by yellow boxes. Molecular weight markers (KD) are shown in the left side. (B) Bar graphs showing quantification of the eight protein bands (relative band intensities expressed as fold difference to mean) in the four extracts from western blots of individual subjects. Bars represent median, with error bars showing interquartile range, (n = 12).

Figure 4

T cell cytokine production in response to raw, pasteurized CM and heated CM-containing food extracts. (A) A bar graph showing IL-5 and IFNγ production in response to re-stimulation with raw, pasteurized CM extract after a two-week expansion culture (allergic subjects n = 20, non-allergic subjects n = 14). (B) Bar graphs showing IL-5 (left panel) and IFNγ (right panel) production in response to re-stimulation with raw, pasteurized CM (black), custard (blue), baked cheese (red) and muffin (green) extracts after two-week expansion culture (n = 10). Bars represent medians, with error bars showing interquartile means. Statistical analysis was performed by Mann-Whitney test (two-tailed), p < 0.05 considered significant.