Of the 17 EG subjects enrolled, 13 were designated allergic EG (AEG) and 4 non-allergic EG (NA-EG), based on the presence of multiple food hypersensitivities in the former (). PA subjects had significantly higher peanut specific IgE than did AEG subjects (16 vs. 1.2 kIU/L, p= 0.028).
After serial gating to identify viable CD4+ T cells (), we used the rapid upregulation of CD154 and cytokines to identify food Ag specific T cells18, 19
cells were readily apparent in the peanut Ag activated samples () from both PA and AEG subjects, but not in the unstimulated (media control) cultures (). CD8 T cytokine responses to food allergens were not detected (data not shown).
Detection of food allergen specific T cell responses.
Although similar intracellular cytokine staining methods have been validated for pathogen associated Th1 immune responses17, 20
, they have not been previously used to analyze Th2 dominant allergen specific responses. To demonstrate that these food allergen specific responses are due to T cell recognition of MHC bound Ag, we used an anti-MHC class II mAb to block Ag presentation. Addition of anti-MHC class II decreased peanut Ag induced cytokine responses (; 80%, 91%, 94%, and 86% inhibition of IL-4, IL-5, IFN-γ, TNF, respectively). For all subsequent figures, the CD4+
gate was used to enumerate allergen specific cells.
For all cytokines and in all subject groups, the frequency of cytokine producing cells in the media control was exceeding low and was not significantly different among groups (Fig E1
and data not shown). Upon activation with peanut Ag, cytokine expression was highly induced in both the AEG and PA subjects (Fig E1
Both EGIDs and PA are associated with Th2 responses and food allergen specific IgE, yet the two diseases have very different clinical presentations. To explore whether these differences are reflected in the T cell response, we measured the frequency of peanut specific CD4 T cells producing IL-4, IL-5, TNF or IFN-γ. Although the PA group had >10-fold higher peanut specific IgE than the AEG subjects, the frequency of peanut Ag specific IL-4 producing CD4 T cells was not significantly different (, p=0.32). In contrast, AEG subjects had >10 times more IL-5 producing peanut specific T cells relative to the PA group (, p=0.038). IFN-γ and TNF expression was not significantly different between the two groups ().
AEG is associated with a greater frequency of peanut specific IL-5 producing T cells.
None of the NA subjects had detectable peanut specific IL-5 responses and most had undetectable IL-4 expression (). In contrast, small but measurable IFN-γ and TNF responses were detected in about half of NA subjects.
Because previous studies have used ratios of Th1/Th2 frequencies to measure relative Th2 skewing21
, we next analyzed the ratio of peanut Ag specific cytokine producing cells. Both the AEG and PA responses were highly Th2 skewed, with IL-4:IFN ratios of 8.4 and 9.4, respectively (Fig E2A
). The corresponding IL-5:IFN ratios were 4.2 and 0.67, respectively (Fig E2B
). In summary, these results demonstrate that both PA and AEG are associated with increased peanut Ag specific IL-4 responses, whereas IL-5 responses are largely limited to AEG.
We next employed polychromatic flow cytometry to simultaneously analyze IL-4, IL-5, IFN, and TNF at the single cell level. Two major Th2 subpopulations were discernable: IL-5+ Th2 (IL-4+, IL-5+) and IL-5- Th2 (IL-4+, IL-5-) cells (: right and left upper quadrants, respectively). TNF was highly coexpressed with both IL-4 and IL-5 ( and data not shown), but IFN-γ was not coexpressed with either of these cytokines ().
Food specific T cells exhibit complex cytokine coexpression patterns.
To evaluate the complexity of the T cell cytokine response more systematically, we used a Boolean gating analysis to examine the 15 cytokine coexpression patterns comprising every potential combination of the 4 individual cytokines (, bottom grid)17
. The frequency of each individual cytokine combination was then assessed as a proportion of the total Ag specific cytokine response ().
Both AEG and PA were notable for a dominant Th2 response to peanut Ag. Th0 cells, defined as cells coexpressing IFN-γ and either Th2 cytokine, contributed minimally to the total cytokine expressing cells. TNF was highly coexpressed with both Th1 and Th2 cytokines.
Only 7 of the 15 possible cytokine combinations substantially contributed to the response. Because of the complexity of simultaneously analyzing 15 cytokine combinations, for further analysis we grouped these into 5 major cytokine subpopulations (IL-5+ Th2, IL-5- Th2, Th0, Th1, and TNF alone) as defined in .
To address the magnitude and quality of CD4 T cell responses, we next examined the frequency of these cytokine subpopulations responding to peanut, soy and shrimp Ag. Notably, peanut Ag specific IL-5+ Th2 cells were 20-fold more frequent in AEG relative to PA (81 vs. 4 cells per 106 CD4 cells, p=0.05, ), whereas IL-5- Th2 cells were present in similar numbers (67 vs. 41 per 106, p= 0.89, ). For all food Ags, AEG was associated with significantly greater frequencies of IL-5+ Th2 cells than the PA population (p=0.05, 0.001, 0.01 for peanut, soy and shrimp, respectively).
AEG is singularly associated with food allergen specific IL-5+ Th2 cells.
Both allergic groups had significantly greater IL-5+ and IL-5- Th2 responses to foods than did the NA group (; p<0.05 for 11 of 12 comparisons). In contrast, food Ag specific Th1 responses were not significantly different between the groups (). The 4 NA-EG subjects had responses similar to the NA group (, E1, and E3).
To examine the relative contribution of the 5 cytokine subpopulations, we next analyzed these food Ag specific cytokine responses by frequency (), or alternatively by proportion (Fig E3
). Notably, in AEG, Th2 responses and in particular IL-5+
Th2 responses were greater in both magnitude and in proportion, relative to either the PA or NA groups. IL-5+
Th2 cells were a significantly larger fraction of the food Ag response in AEG relative to PA (29% vs. 4%, p<0.0001). In contrast to the large differences in Th2 responses, all groups had similar magnitude Th1 responses (). When individual subject's Ag specific Th1 and Th2 responses were plotted against each other, we found no evidence for reciprocal correlation of these cytokine responses (data not shown). In sum, these findings indicate that AEG is singularly associated with an expansion of food allergen specific IL-5+
To determine if the Th2 skewing found in AEG was limited to food Ags, SEB specific responses were examined (). Although IL-5- Th2 responses were similar among the groups, IL-5+ Th2 cells were significantly greater in AEG (0.15% vs. 0.06% vs. 0.03% for AEG, PA and NA, respectively; p<0.05 for AEG vs. either group). Both allergic groups had lower frequencies of Th1 cells (2.3% vs. 2.7% vs. 5.0 % for AEG, PA and NA, respectively), although this was only significant for AEG vs. NA (p=0.04).
Because IL-5 has multiple pro-eosinophil actions we sought to determine if there was a relationship between IL-5+
Th2 cells and eosinophilia. Accordingly, in AEG we found that the absolute eosinophil count correlated with the overall frequency of IL-5+
Th2 cells (r= 0.6, ), but less so for IL-5-
Th2 cells (r=0.44, data not shown). Similarly, food Ag specific IL-5+ Th2 responses for soy and peanut, but not shrimp, correlated with AEC (Fig E4 A-C
). The frequency of IL-5+
Th2 cells also correlated with tissue eosinophils in the gastric body but not those in the antrum, duodenum, or esophagus (Fig E4 D-G
Correlation of Th2 responses with IgE and eosinophilia.
Because Th2 responses are required for IgE class switching, we next examined the relationship between Th2 responses and IgE. In PA but not in AEG, peanut specific IgE was highly correlated to the peanut specific Th2 response (r=0.87 vs. 0.55, respectively for IL-5- Th2; ). In contrast, there was minimal correlation between the soy Th2 and IgE responses in either disease ().