Eosinophilic gastrointestinal disease and peanut allergy are alternatively associated with IL-5⁺ and IL-5⁻ T_H2 responses

Article Outline

• Abstract
• Methods
  • Subjects
  • Cell culture, staining, and flow cytometry
  • Data analysis and statistics
• Results
• Discussion
• Acknowledgment
• Methods
  • Cell culture and staining
  • Flow cytometry
  • Subjects
• Fig E1. 
• Fig E2. 
• Fig E3. 
• Fig E4. 
• Table E1. 
• References
• References
• Copyright

Background

Both anaphylactic food allergy and eosinophil-associated gastrointestinal disorders are associated with T_H2 responses and food-specific IgE, yet they have very different clinical presentations.

Objective

To determine whether the clinical differences between anaphylactic food allergy and eosinophil-associated gastrointestinal disorders are reflected in different T_H2 responses to foods.

Methods

Subjects with peanut allergy (PA), subjects with allergic eosinophilic gastroenteritis (AEG), and nonatopic subjects were enrolled. Antigen-specific IL-4, IL-5, IFN-γ, and TNF T-cell responses to peanut, soy, and shrimp were measured by using intracellular cytokine staining and polychromatic flow cytometry.

Results

Two distinct subpopulations of T_H2 cells were found: IL-5⁺ T_H2 (IL-4⁺, IL-5⁺) and IL-5^– T_H2 (IL-4⁺, IL-5^–) cells. Peanut-specific IL-5⁺ T_H2 cells were present at a 20-fold greater frequency in AEG versus PA (81 vs 4 per 10⁶ CD4 cells; P = .05), whereas there were similar frequencies of IL-5^- T_H2 cells (67 vs 41 per 10⁶). For all foods, IL-5⁺ T_H2 cells accounted for a significantly greater fraction of the antigen-specific cells in AEG relative to PA (29% vs 4%; P < .0001). In PA but not AEG, IL-5^– T_H2 responses to peanut were highly correlated with peanut-specific IgE (r = 0.87 vs 0.55, respectively). All subject groups elicited similar very low-magnitude T_H1 responses to food antigens.

Conclusion

T_H2 responses are composed of 2 subpopulations: IL-5⁺ T_H2 and IL-5^– T_H2 cells. IL-5⁺ T_H2 food allergen–specific T cells are singularly associated with AEG, whereas PA is associated with a dominant IL-5^– T_H2 response. These results suggest heterogeneity within the T_H2 cytokine response, with different T_H2 responses alternatively favoring IgE-mediated or eosinophil-dominant immunopathology.

Key words: Food allergy, peanut, T cell, T_H2, eosinophil-associated gastrointestinal disease, IL-4, IL-5, flow cytometry

Abbreviations used: AEG, Allergic eosinophilic gastroenteritis, EG, Eosinophilic gastroenteritis, EGID, Eosinophil-associated gastrointestinal disorder, EoE, Eosinophilic esophagitis, NA-EG, Nonallergic eosinophilic gastroenteritis, NIAID, National Institute of Allergy and Infectious Diseases, PA, Peanut allergy

Food allergies are increasingly common and affect approximately 4% of the population.¹, ² Eosinophil-associated gastrointestinal disorders (EGIDs), including eosinophilic esophagitis (EoE) and eosinophilic gastroenteritis (EG), are a spectrum of increasingly recognized inflammatory diseases that are strongly associated with hypersensitivity to multiple foods.³, ⁴, ⁵ Furthermore, EGIDs, in particular pediatric EoE, are responsive to an elemental diet,⁶ suggesting that food allergen–driven immune responses contribute to disease pathogenesis.

T_H2 cells are critical to both the IgE and eosinophilic responses that underlie allergic diseases.⁷, ⁸ Both peanut allergy⁹, ¹⁰ (PA) and EGIDs¹¹, ¹², ¹³, ¹⁴ are associated with abnormal cytokine (T_H2-like) and IgE responses to food antigens. However, the 2 diseases have markedly dissimilar clinical presentations. The dominant feature of PA is immediate hypersensitivity and anaphylaxis, whereas EGIDs are associated with chronic eosinophilic gut inflammation, but typically not anaphylaxis.³ T_H2 responses play a dominant role in directing both IgE class switching (IL-4) and tissue eosinophila (in part IL-5–mediated). However, it is not clear how a uniform T_H2 response could differentially direct the IgE-mediated or eosinophil dominant pathology characteristic of PA and EGIDs, respectively.

To understand how T_H2 cells may differentially drive classic IgE-mediated food allergy versus the eosinophilic gut inflammation, we used polychromatic flow cytometry to examine antigen-specific T-cell cytokine responses in EG, PA, and healthy nonatopic control subjects. Notably, the IgE versus eosinophil dominance of these diseases was reflected in the food allergen–specific T_H2 cytokine response. These results suggest heterogeneity within T_H2 responses that may alternatively favor IgE production versus eosinophilic inflammation.

Back to Article Outline

Methods 

Subjects 

Subjects were 18 to 60 years of age. Subjects with EG had peak tissue eosinophil counts of ≥44 eosinophils/high power field (hpf) in gastric or duodenal biopsies (see this article's Table E1 in the Online Repository at www.jacionline.org). The diagnosis of EG was based on typical gastrointestinal symptoms, tissue eosinophilia in stomach or duodenal biopsy specimens, and the exclusion of other causes of gut eosinophilia, including helminth infection. Crohn disease was excluded by lack of typical pathologic findings (ulcerations, granulomata, or crypt architectural distortion) and clinical features (fistula, abdominal mass, and surgical obstructive disease). Subjects with immunodeficiency or a positive Fip1-like 1 -platelet-derived growth factor receptor alpha fusion gene PCR were excluded. Subjects with EG were post hoc grouped as having either allergic EG (AEG) or nonallergic EG (NA-EG) as noted in Table I. Subjects AEG 1 to 4 and 6 to 9 are identical to EG 1 to 4 and 6 to 9 reported previously¹⁵; all samples were obtained before the study drug on that study. Four of the subjects with AEG had concurrent EoE (Table E1). Subjects with PA had a history of systemic symptoms including at least 1 extracutaneous site (ie, wheezing, gastrointestinal symptoms, laryngeal edema, angioedema, or hypotension) within 30 minutes of peanut ingestion, and a peanut-specific IgE of ≥4 kIU/L. Subjects with PA did not undergo a peanut challenge as part of their work-up. Sixteen healthy nonatopic subjects had a negative history for allergy, asthma, and food allergy; a negative Phadiatop atopy screen (Phadia, Uppsala, Sweden); and undetectable levels (<0.35 kIU/L) of food allergen–specific IgE to peanut, soy, shrimp, wheat, egg, and milk. The National Institute of Allergy and Infectious Diseases (NIAID) Institutional Review Board approved the clinical protocols used for this study. All subjects signed informed consent.

Table I. Subject characteristics

AEC, Absolute eosinophil count; F, female; M, male; N, no; ND, not detectable, ≤ 0.35 kIU/L; Y, yes.

Therapy: Bud, budesonide; Pred, prednisone; dose noted in mg/d.

Cell culture, staining, and flow cytometry 

T-cell activation and intracellular cytokine staining were performed on cryopreserved samples according to previously published methods¹⁶ that are detailed in this article's Methods in the Online Repository at www.jacionline.org. The following 9-color panel was used: Violet LIVE/DEAD, CD154 fluorescein isothiocyanate (clone TRAP1), IL-4 phycoerythrin (clone 25D2), CD4 phycoerythrin/Cy5 (clone SK3), IFN-γ phycoerythrin/Cy7 (clone B27), IL-5 allophycocyanin (clone TRFK-5), and TNF Alexa 700 (clone MAb11), all BD Biosciences, San Jose, Calif; and CD8 phycoerythrin/TR (clone 3B5) and CD3 allophycocyanin/Alexa 750 (clone UCHT1), both Invitrogen (Carlsbad, Calif).

Data analysis and statistics 

Boolean analysis of cytokine coexpression was performed by using SPICE software (M. Roederer, National Institutes of Health, Bethesda, Md)¹⁷ as detailed in the Methods in the Online Repository. Medians were used as the measure of central tendency. Proportional analyses represent a given subpopulation as a fraction of the total number of antigen-specific cytokine-producing cells. Statistics and linear regression were calculated with Prism software (GraphPad, La Jolla, Calif) by using Mann-Whitney U or Wilcoxon signed-rank tests for comparisons and the Spearman rank test for correlations. Box-and-whisker plots represent median, quartiles, minimum, and maximum.

Back to Article Outline

Results 

Of the 17 subjects with EG enrolled, 13 were designated as having AEG and 4 as having NA-EG on the basis of the presence of multiple food hypersensitivities in the former (Table I). Subjects with PA had significantly higher peanut-specific IgE than did subjects with AEG (16 vs 1.2 kIU/L; P = .028).

After serial gating to identify viable CD4⁺ T cells (Fig 1, A-D), we used the rapid upregulation of CD154 and cytokines to identify food antigen–specific T cells.¹⁸, ¹⁹ CD154⁺, cytokine⁺ cells were readily apparent in the peanut antigen activated samples (Fig 1, E and F) from subjects with PA and AEG, but not in the unstimulated (media control) cultures (Fig 1, G and H). CD8 T cytokine responses to food allergens were not detected (data not shown).

• 
  • View Large Image
  • Download to PowerPoint

• Fig 1. 

  Detection of food allergen–specific T-cell responses. A-D, Gating. CD4⁺ T-cell expression of CD154 and either IL-4 (E and G) or IL-5 (F and H) after incubation with peanut antigen (E and F) or media (G and H). In a separate experiment, peanut antigen–activated cultures were incubated with isotype control (I and J), or anti-MHC class II mAb (K and L), n = 5. SSC, Side scatter; FSC, foward scatter.

Although similar intracellular cytokine staining methods have been validated for pathogen-associated T_H1 immune responses,¹⁷, ²⁰ they have not been previously used to analyze T_H2-dominant allergen specific responses. To demonstrate that these food allergen–specific responses are a result of T-cell recognition of MHC-bound antigen, we used an anti-MHC class II mAb to block antigen presentation. Addition of anti-MHC class II decreased peanut antigen–induced cytokine responses (Fig 1, I-L; 80%, 91%, 94%, and 86% inhibition of IL-4, IL-5, IFN-γ, and TNF, respectively). For all subsequent figures, the CD4⁺, CD154⁺, cytokine⁺ 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 (see this article's Fig E1 in the Online Repository at www.jacionline.org; data not shown). On activation with peanut antigen, cytokine expression was highly induced in the subjects with AEG and PA (Fig E1).

Both EGIDs and PA are associated with T_H2 responses and food allergen–specific IgE, yet the 2 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 subjects with AEG, the frequency of peanut antigen–specific IL-4–producing CD4 T cells was not significantly different (Fig 2, A; P = .32). In contrast, subjects with AEG had >10 times more IL-5–producing peanut-specific T cells relative to the PA group (Fig 2, B; P = .038). IFN-γ and TNF expression was not significantly different between the 2 groups (Fig 2, C and D).

• 
  • View Large Image
  • Download to PowerPoint

• Fig 2. 

  AEG is associated with a greater frequency of peanut-specific IL-5–producing T cells. The frequency of (A) IL-4, (B) IL-5, (C) IFN-γ, and (D) TNF-expressing peanut antigen–specific CD4 cells was determined for each subject group. Each symbol represents an individual subject. The median value is denoted by a horizontal bar. Intergroup statistics are shown over the brackets. NA, Nonatopic.

None of the nonatopic subjects had detectable peanut-specific IL-5 responses, and most had undetectable IL-4 expression (Fig 2, A and B). In contrast, small but measurable IFN-γ and TNF responses were detected in about half of nonatopic subjects.

Because previous studies have used ratios of T_H1/T_H2 frequencies to measure relative T_H2 skewing,²¹ we next analyzed the ratio of peanut antigen–specific cytokine-producing cells. Both the AEG and PA responses were highly T_H2-skewed, with IL-4:IFN ratios of 8.4 and 9.4, respectively (see this article's Fig E2, A, in the Online Repository at www.jacionline.org). The corresponding IL-5:IFN ratios were 4.2 and 0.67, respectively (Fig E2, B). In summary, these results demonstrate that both PA and AEG are associated with increased peanut antigen–specific IL-4 responses, whereas IL-5 responses are largely limited to AEG.

We next used polychromatic flow cytometry to analyze IL-4, IL-5, IFN, and TNF simultaneously at the single-cell level. Two major T_H2 subpopulations were discernable: IL-5⁺ T_H2 (IL-4⁺, IL-5⁺) and IL-5^– T_H2 (IL-4⁺, IL-5^–) cells (Fig 3, A and B, right and left upper quadrants, respectively). TNF was highly coexpressed with both IL-4 and IL-5 (Fig 3, D; data not shown), but IFN-γ was not coexpressed with either of these cytokines (Fig 3, C and E).

• 
  • View Large Image
  • Download to PowerPoint

• Fig 3. 

  Food-specific T cells exhibit complex cytokine coexpression patterns. Cytokine coexpression by peanut-specific T cells from subjects with AEG (A, C-E) or PA (B). F, Each of the 15 possible cytokine combinations is shown as a proportion of the total peanut response. Individual subjects and medians are denoted by dots and horizontal bars, respectively. All subjects from the 3 groups were studied. ^∗P ≤ .05; ^∗∗P ≤ .01; ^∗∗∗P ≤ .001. NA, Nonatopic.

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 making up every potential combination of the 4 individual cytokines (Fig 3, F, bottom grid).¹⁷ The frequency of each individual cytokine combination was then assessed as a proportion of the total antigen-specific cytokine response (Fig 3, F).

Both AEG and PA were notable for a dominant T_H2 response to peanut antigen. T_H0 cells, defined as cells coexpressing IFN-γ and either T_H2 cytokine, contributed minimally to the total cytokine-expressing cells. TNF was highly coexpressed with both T_H1 and T_H2 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⁺ T_H2, IL-5^– T_H2, T_H0, T_H1, and TNF alone) as defined in Fig 3, F.

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 antigen. Notably, peanut antigen–specific IL-5⁺ T_H2 cells were 20-fold more frequent in AEG relative to PA (81 vs 4 cells per 10⁶ CD4 cells; P = .05; Fig 4, A), whereas IL-5^– T_H2 cells were present in similar numbers (67 vs 41 per 10⁶; P = .89; Fig 4, B). For all food antigens, AEG was associated with significantly greater frequencies of IL-5⁺ T_H2 cells than the PA population (P = .05, .001, and .01 for peanut, soy, and shrimp, respectively).

• 
  • View Large Image
  • Download to PowerPoint

• Fig 4. 

  AEG is singularly associated with food allergen–specific IL-5⁺ T_H2 cells. A-D, The frequency of food antigen (Ag)–specific CD4 cells for each cytokine subpopulation and subject group. E-H, Stack graphs depict the median values for each cytokine subpopulation summed to represent the total frequency of Ag-specific cytokine-producing cells. NA, Nonatopic; SEB, staphylococcal enterotoxin B.

Both allergic groups had significantly greater IL-5⁺ and IL-5^– T_H2 responses to foods than did the nonatopic group (Fig 4, A and B; P < .05 for 11 of 12 comparisons). In contrast, food antigen–specific T_H1 responses were not significantly different between the groups (Fig 4, D). The 4 subjects with NA-EG had responses similar to the nonatopic group (Figs 4 and E1; see this article's Fig E3 in the Online Repository at www.jacionline.org).

To examine the relative contribution of the 5 cytokine subpopulations, we next analyzed these food antigen–specific cytokine responses by frequency (Fig 4, E-G), or alternatively by proportion (Fig E3). Notably, in AEG, T_H2 responses and in particular IL-5⁺ T_H2 responses were greater in both magnitude and in proportion relative to either the PA or nonatopic groups. IL-5⁺ T_H2 cells were a significantly larger fraction of the food antigen response in AEG relative to PA (29% vs 4%; P < .0001). In contrast with the large differences in T_H2 responses, all groups had similar magnitude T_H1 responses (Fig 4, E-G). When individual subject's antigen-specific T_H1 and T_H2 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⁺ T_H2 cells.

To determine whether the T_H2 skewing found in AEG was limited to food antigens, Staphylococcal enterotoxin B–specific responses were examined (Fig 4, H). Although IL-5^– T_H2 responses were similar among the groups, IL-5⁺ T_H2 cells were significantly greater in AEG (0.15% vs 0.06% vs 0.03% for AEG, PA, and nonatopic, respectively; P < .05 for AEG vs either group). Both allergic groups had lower frequencies of T_H1 cells (2.3% vs 2.7% vs 5.0% for AEG, PA, and nonatopic, respectively), although this was only significant for AEG versus nonatopic (P = .04).

Because IL-5 has multiple pro-eosinophil actions, we sought to determine whether there was a relationship between IL-5⁺ T_H2 cells and eosinophilia. Accordingly, in AEG we found that the absolute eosinophil count correlated with the overall frequency of IL-5⁺ T_H2 cells (r = 0.6; Fig 5, A), but less so for IL-5^– T_H2 cells (r = 0.44; data not shown). Similarly, food antigen–specific IL-5 + T_H2 responses for soy and peanut, but not shrimp, correlated with absolute eosinophil count (see this article's Fig E4, A-C, in the Online Repository at www.jacionline.org). The frequency of IL-5⁺ T_H2 cells also correlated with tissue eosinophils in the gastric body but not those in the antrum, duodenum, or esophagus (Fig E4, D-G).

• 
  • View Large Image
  • Download to PowerPoint

• Fig 5. 

  Correlation of T_H2 responses with IgE and eosinophilia. A, Correlation of IL-5⁺ T_H2 cells with eosinophil count. Correlation of peanut-specific IgE with IL-5^– T_H2 (B) and IL-5⁺ T_H2 (C) cells. D, Correlation of soy-specific IgE with IL-5^– T_H2 cells. Linear regression curve fit is shown for subjects with AEG (A) and PA (B and C). SEB, Staphylococcal enterotoxin B. ^∗P < .05.

Because T_H2 responses are required for IgE class switching, we next examined the relationship between T_H2 responses and IgE. In PA but not in AEG, peanut-specific IgE was highly correlated to the peanut-specific T_H2 response (r = 0.87 vs 0.55, respectively, for IL-5^– T_H2; Fig 5, B and C). In contrast, there was minimal correlation between the soy T_H2 and IgE responses in either disease (Fig 5, D).

Back to Article Outline

Discussion 

Both classic food allergy and EGIDs are associated with IgE and T_H2 responses to food allergens, yet have distinct clinical manifestations of anaphylaxis and tissue eosinophilia, respectively. In this study we examined whether these clinical differences are associated with different T_H2 cytokine expression patterns. We thus investigated food allergen–specific T-cell cytokine responses at the single-cell level and demonstrate that T_H2 responses can be divided into 2 subpopulations on the basis of IL-5 expression: IL-5⁺ T_H2 (IL-4⁺, IL-5⁺) and IL-5^– T_H2 (IL-4⁺, IL-5^–) cells. We found that IL-5⁺ T_H2 responses are singularly associated with AEG, whereas PA is associated with a dominant IL-5^– T_H2 response (Fig 3, Fig 4). These results provide evidence for heterogeneity within the T_H2 response and demonstrate fundamental differences in the food allergen–specific T-cell responses found in AEG compared with classic anaphylactic food allergy. These results suggest that distinct T_H2 subpopulations may alternatively contribute to IgE-dominant or eosinophil-dominant allergic disease.

It is unclear whether the IL-5⁺ and IL-5^– T_H2 subpopulations found in this study represent different lineages or T_H2 differentiation states, or simply reflect differential expression of the IL-5 gene caused by probabilistic,²² immunologic,²³ or genetic factors.²⁴ Presumably, IL-5⁺ T_H2 cells serve as an abundant source of IL-5 to drive eosinophil differentiation, survival, and activation.⁸ Our finding that the frequency of IL-5⁺ T_H2 cells correlates with peripheral blood eosinophilia (Fig 5) supports this line of reasoning.

In agreement with the landmark findings of Turcanu et al,²¹ we found that peanut antigen responses were highly T_H2-skewed in subjects with PA relative to the nonatopic controls (Figs 4, E, E2, and E3). The relative T_H1 skewing in nonatopic subjects in this previous report led the authors to conclude that T_H1-skewed responses underlie oral tolerance. Our results indicate that this apparent T_H1 skewing of allergen-specific responses in nonatopic controls is an artifact of the experimental system used. The study by Turcanu et al²¹ analyzed cytokine responses as ratios of T_H1:T_H2 cytokines. When our data were analyzed as ratios (Fig E2) or proportions (Fig E3), similar T_H1 skewing was found. A deficiency of these ratio and proportional analyses is that they do not examine the magnitude of the T-cell response. When our data were analyzed by magnitude by measuring the frequency of food antigen–specific T cells, all subject groups had similar T_H1 responses (Figs 2, C, and 4, C, E-G). In the allergic groups, this small T_H1 response is dwarfed by the T_H2 responses. In contrast, in nonatopic subjects, there is no T_H2 response, resulting in a seemingly dominant T_H1 response. Similar findings of T_H1-dominated aeroallergen-specific responses in nonatopic subjects should be reassessed in this light.

In agreement with a recent report,⁹ we found that in PA, peanut-specific T_H2 responses were highly correlated with peanut-specific IgE (Fig 5). This relationship was not found in either the AEG group or with other food antigens. This tight association indicates that the peanut-specific T_H2 cells in PA may provide better help for IgE production than T_H2 cells found in EGIDs.

Interestingly, subjects with EG neatly dichotomized into those with multiple food hypersensitivities (AEG) or those with none (NA-EG; Table I). Unlike the AEG group, the subjects with NA-EG did not have T_H2 responses to foods, but had responses similar to the nonatopic group (Figs 4, E1, and E3). This suggests that the pathogenesis of NA-EG is distinct from AEG and may be T-cell–independent. Although there are no consensus diagnostic guidelines for EG, the AEG group demonstrated relatively homogeneous clinical and immunologic features, supporting the validity of the diagnosis.

In both subject groups with allergy, the frequency of peanut antigen–specific T cells was 100 to 200 per 10⁶ CD4 T cells. In contrast, HIV and cytomegalovirus–specific responses are approximately 50-fold to 100-fold higher.²⁰, ²⁵ This greater magnitude of viral-specific T-cell responses may be a result of the greater pathogen-associated molecular pattern danger signals, which more effectively prime virus specific immune responses relative to those associated with allergens. This low magnitude appears to be an intrinsic feature of allergen-specific T-cell responses,²⁶ making their detection in translational research a technical challenge.

Notably, TNF was highly coexpressed with both IL-4 and IL-5 (Fig 3, F). Although TNF is often considered a T_H1 cytokine, Liu and colleagues²⁷ have described in vitro differentiated “inflammatory T_H2” cells that coexpress TNF and T_H2 cytokines. However, we have not examined the function of these TNF⁺ cells to determine whether they have inflammatory, or for that matter, regulatory function. To our knowledge, this work is the first demonstration of TNF coexpression by T_H2 cells in an allergen-specific manner and without extensive in vitro culture.

This work is also notable for several limitations. Although 4 of the subjects with AEG had coexisting EoE, AEG differs from the most common EGID clinical entity of solitary EoE. As such, our findings of IL-5⁺ T_H2 cells may not be generalizable to EoE. IL-13 is a major effector cytokine in EGIDs; however, technical limitations did not allow us to examine IL-4, IL-5, and IL-13 concurrently. Pilot experiments indicate that IL-4 and IL-13 are concordantly expressed (data not shown). A greater frequency of food antigen–specific T cells was found in AEG relative to nonatopic subjects. However, this may be a result of the panel of cytokine antibodies we used that may not detect other subpopulations of T cells, such as food allergen–specific regulatory T cells, for which specific markers are less well defined. Although pediatric EoE responds to dietary management, similar studies have not been performed for adult AEG, making it difficult to gauge the pathogenic relevance of the food antigen–specific T_H2 cells in this study. Presumably, if IL-5⁺ T_H2 cells play a pathogenic role in EGIDs, they home to the affected gut. A limitation of this work is that the observations of IL-5⁺ T_H2 cells are limited to the blood and have not been verified in the pathologic gut tissue itself.

In conclusion, we have shown that T_H2 responses are composed of 2 subpopulations, IL-5⁺ T_H2 and IL-5^– T_H2 cells, and that these T_H2 subpopulations are respectively associated with eosinophilic inflammatory versus IgE-mediated food allergy. These findings suggest that different subpopulations of T_H2 cells may alternatively contribute to the immediate hypersensitivity versus the eosinophilic inflammatory components of allergic disease.

Back to Article Outline

We thank M. Young, D. Cantave, L. Bernardino, L. Scott, and W. Lu for study support; C. Matthews and D. Gladden of the National Institutes of Health Clinical Center Department of Transfusion Medicine for apheresis support; E. Brittain and M. Nason of the NIAID Division of Clinical Research Biostatistics Research Branch for statistical consultation; and M. Roederer and S. Perfetto of the NIAID Vaccine Research Center for SPICE analysis software and technical consultation. We thank T. Nutman, R. Rabin, K. Stone, and J. Milner for critical review of the manuscript.

Back to Article Outline

Methods 

Cell culture and staining 

Subjects underwent lymphapheresis (NIH Clinical Center Department of Transfusion Medicine), and PBMCs were isolated by using density gradient separation (Lymphocyte Separation Media-1077 (MO Biomedicals, LLC, Aurora, Ohio). PBMCs were washed twice in HBSS (Invitrogen) and cryopreserved in liquid nitrogen.

T-cell activation and intracellular cytokine staining were performed according to previously published methods.^E1 PBMCs were thawed, washed twice, resuspended in AIM V medium (Invitrogen) with antigen or staphylococcal enterotoxin B (Toxin Technology, Sarasota, Fla), and cultured at 4 × 10⁶ cells in 2 mL in 16-mm × 125-mm culture tubes (Corning, Corning, NY) in a 5% CO₂ incubator. After 2 hours, brefeldin A 10 μg/mL was added. After a total of 6 hours, the samples were washed twice in cold PBS, labeled with LIVE/DEAD Fixable Violet Dead Cell Stain (Invitrogen) according to the manufacturer's instructions, washed twice in cold PBS, and fixed in 4% paraformaldehyde (Sigma, St Louis, Mo). Cells were then resuspended in PBS with 10% dimethyl sulfoxide (Sigma) and cryopreserved at –80°C.

For staining, cells were thawed, washed in PBS with 0.1% BSA, and blocked in PBS with 0.1% saponin and 5% nonfat dried milk for 60 minutes. The following 9-color panel was used: Violet LIVE/DEAD, CD154 fluorescein isothiocyanate (clone TRAP1), IL-4 phycoerythrin (clone 25D2), CD4 phycoerythrin/Cy5 (clone SK3), IFN-γ phycoerythrin/Cy7 (clone B27), IL-5 APC (clone TRFK-5), and TNF Alexa 700 (clone MAb11), all BD Biosciences; and CD8 phycoerythrin/TR (clone 3B5) and CD3 APC/Alexa 750 (clone UCHT1), both Invitrogen. Cells were stained with mAb diluted in PBS/saponin/nonfat dried milk for 30 minutes, washed twice, and resuspended in PBS for analysis. Parallel samples were stained with isotype-matched controls replacing the anticytokine mAbs.

Food antigens were prepared as aqueous extracts of defatted peanuts, soy flour, and shrimp and used at 100 μg/mL protein in culture. Treatment with endotoxin-reducing beads (Associates of Cape Cod, East Falmouth, Mass) yielded antigen preparations with <0.01 endotoxin units/mL in culture. In specified experiments, mAb to MHC class II (clone IVA12; ATCC, Manassas, Va) was added to cultures 30 minutes before antigen. Complete blood counts and total serum IgE were performed by the NIH Clinical Center. Allergen-specific IgE and Phadiatop assays were performed by using an ImmunoCAP 100E instrument (Phadia).

Flow cytometry 

Samples were acquired on an LSR II flow cytometer (BD Biosciences). A total of 500,000 to 2,000,000 events were collected to yield 300,000 to 1,000,000 viable CD4 T cells per sample. Analysis was performed by using FlowJo software (TreeStar, Inc, Ashton, Ore). Cell doublets were excluded by using forward scatter area versus height parameters (Fig 1, A). Viable CD4⁺ T cells were identified by first gating on typical lymphocyte forward versus side scatter (Fig 1, B), then gating on CD3⁺, LIVE/DEAD^neg cells (Fig 1, C), and finally gating on CD4⁺, CD8^- cells (Fig 1, D).

Boolean analysis of cytokine coexpression was performed by using SPICE software (M. Roederer, NIH, Bethesda, Md) to determine the frequency of each cytokine pattern based on all possible combinations of IL-4, IL-5, IFN-γ, and TNF. Responses to antigen are expressed as net values (media [unstimulated] control values being subtracted from antigen values) and negative values set to 0.

Subjects 

As detailed in Table I, 4 subjects with EG were maintained on budesonide, which was used off label as a topical corticosteroid targeted to the stomach and duodenum.^E2, ^E3 Patients opened Entocort budesonide controlled ileal release capsules (AstraZeneca AB, Södertälje, Sweden), crushed the contents in a mortar and pestle, and mixed this with a small glass of water taken as a single daily dose. Contact with gastric and duodenal mucosa was maximized by giving the drug on an empty stomach at bedtime.

Back to Article Outline

Fig E1. 

• 
  • View Large Image
  • Download to PowerPoint

• Peanut antigen–specific CD4 T-cell cytokine responses. PBMCs from all 4 subject groups were incubated with either media or peanut antigen for 6 hours, and the frequency of CD154⁺, cytokine-producing cells was determined by flow cytometry. Each symbol represents a subject with AEG. Red symbols and lines denote the 2 subjects with AEG treated with prednisone. The bracketed numbers at the base of some plots denote the number of overlapping 0 value results. NA, Nonatopic.

Back to Article Outline

Fig E2. 

• 
  • View Large Image
  • Download to PowerPoint

• Ratiometric analysis of peanut antigen–specific cells. The frequency of antigen-specific cytokine-producing cells as shown in Fig 2 was used to calculate IL-4:IFN-γ (A) and IL-5:IFN-γ (B) ratios for each subject. Each symbol represents a unique subject. NA, Nonatopic.

Back to Article Outline

Fig E3. 

• 
  • View Large Image
  • Download to PowerPoint

• AEG is singularly associated with food allergen–specific IL-5⁺ T_H2 cells. Pie graphs represent the proportional contribution of each cytokine subpopulation to the total antigen-specific response. Each pie corresponds to the same lettered stack graph in Fig 4, E-H. NA, Nonatopic; SEB, Staphylococcal enterotoxin B.

Back to Article Outline

Fig E4. 

• 
  • View Large Image
  • Download to PowerPoint

• Correlation of IL-5⁺ T_H2 cells to tissue and blood eosinophilia. A-C, The correlation between food antigen (Ag)–specific IL-5⁺ T_H2 cells and blood eosinophil count. D-G, The correlation between staphylococcal enterotoxin B–specific IL-5⁺ T_H2 cells and gut tissue eosinophilia. Gut tissue eosinophilia was measured as the upper quartile count of eosinophils (eos)/high power field.

Back to Article Outline

Table E1. 

Allergic eosinophilic gastroenteritis subject characteristics

eo, Eosinophil; eos, eosinophils; ND, not detectable, ≤0.35 kIU/L.

Back to Article Outline

References 

1. Branum A, Lukacs S. Food allergy among U.S. children: trends in prevalence and hospitalizations. NCHS Data Brief. 2008;1–8
   • View In Article
2. Sicherer SH, Sampson HA. Food allergy: recent advances in pathophysiology and treatment. Annu Rev Med. 2008;60:261–277
   • View In Article
   • CrossRef
3. Rothenberg ME. Eosinophilic gastrointestinal disorders (EGID). J Allergy Clin Immunol. 2004;113:11–28
   • View In Article
   • Abstract
   • Full Text
   • Full-Text PDF (278 KB)
   • CrossRef
4. Liacouras CA, Spergel JM, Ruchelli E, Verma R, Mascarenhas M, Semeao E, et al. Eosinophilic esophagitis: a 10-year experience in 381 children. Clin Gastroenterol Hepatol. 2005;3:1198–1206
   • View In Article
   • Abstract
   • Full Text
   • Full-Text PDF (516 KB)
   • CrossRef
5. Roy-Ghanta S, Larosa DF, Katzka DA. Atopic characteristics of adult patients with eosinophilic esophagitis. Clin Gastroenterol Hepatol. 2008;6:531–535
   • View In Article
   • Abstract
   • Full Text
   • Full-Text PDF (151 KB)
   • CrossRef
6. Spergel JM, Shuker M. Nutritional management of eosinophilic esophagitis. Gastrointest Endosc Clin N Am. 2008;18:179–194xi
   • View In Article
   • Abstract
   • Full Text
   • Full-Text PDF (192 KB)
   • CrossRef
7. Woodfolk JA. T-cell responses to allergens. J Allergy Clin Immunol. 2007;119:280–294
   • View In Article
   • Abstract
   • Full Text
   • Full-Text PDF (1239 KB)
   • CrossRef
8. Blanchard C, Rothenberg ME. Basic pathogenesis of eosinophilic esophagitis. Gastrointest Endosc Clin N Am. 2008;18:133–143x
   • View In Article
   • Abstract
   • Full Text
   • Full-Text PDF (413 KB)
   • CrossRef
9. Turcanu V, Winterbotham M, Kelleher P, Lack G. Peanut-specific B and T cell responses are correlated in peanut-allergic but not in non-allergic individuals. Clin Exp Allergy. 2008;38:1132–1139
   • View In Article
   • CrossRef
10. Thottingal TB, Stefura BP, Simons FE, Bannon GA, Burks W, HayGlass KT. Human subjects without peanut allergy demonstrate T cell-dependent, TH2-biased, peanut-specific cytokine and chemokine responses independent of TH1 expression. J Allergy Clin Immunol. 2006;118:905–914
    • View In Article
    • Abstract
    • Full Text
    • Full-Text PDF (321 KB)
    • CrossRef
11. Jaffe JS, James SP, Mullins GE, Braun-Elwert L, Lubensky I, Metcalfe DD. Evidence for an abnormal profile of interleukin-4 (IL-4), IL-5, and gamma-interferon (gamma-IFN) in peripheral blood T cells from patients with allergic eosinophilic gastroenteritis. J Clin Immunol. 1994;14:299–309
    • View In Article
    • MEDLINE
    • CrossRef
12. Straumann A, Bauer M, Fischer B, Blaser K, Simon HU. Idiopathic eosinophilic esophagitis is associated with a T(H)2-type allergic inflammatory response. J Allergy Clin Immunol. 2001;108:954–961
    • View In Article
    • Abstract
    • Full Text
    • Full-Text PDF (665 KB)
    • CrossRef
13. Yamazaki K, Murray JA, Arora AS, Alexander JA, Smyrk TC, Butterfield JH, et al. Allergen-specific in vitro cytokine production in adult patients with eosinophilic esophagitis. Dig Dis Sci. 2006;51:1934–1941
    • View In Article
    • MEDLINE
    • CrossRef
14. Bullock JZ, Villanueva JM, Blanchard C, Filipovich AH, Putnam PE, Collins MH, et al. Interplay of adaptive th2 immunity with eotaxin-3/c-C chemokine receptor 3 in eosinophilic esophagitis. J Pediatr Gastroenterol Nutr. 2007;45:22–31
    • View In Article
    • CrossRef
15. Foroughi S, Foster B, Kim N, Bernardino LB, Scott LM, Hamilton RG, et al. Anti-IgE treatment of eosinophil-associated gastrointestinal disorders. J Allergy Clin Immunol. 2007;120:594–601
    • View In Article
    • Abstract
    • Full Text
    • Full-Text PDF (860 KB)
    • CrossRef
16. Foster B, Prussin C, Liu F, Whitmire JK, Whitton JL. Detection of intracellular cytokines by flow cytometry. Curr Protoc Immunol. 2007;Unit 6.24, p 6.24.1-6.24.21
    • View In Article
17. Seder RA, Darrah PA, Roederer M. T-cell quality in memory and protection: implications for vaccine design. Nat Rev Immunol. 2008;8:247–258
    • View In Article
18. Meier S, Stark R, Frentsch M, Thiel A. The influence of different stimulation conditions on the assessment of antigen-induced CD154 expression on CD4+ T cells. Cytometry A. 2008;73:1035–1042
    • View In Article
19. Campbell JD, Buchmann P, Kesting S, King B, Coffman RL, Hessel EM. Successful use of CD154 expression and intracellular cytokine staining to monitor changes in allergen-specific Th2 cells in peripheral blood of subjects in a placebo-controlled study of Tolamba (TM), a novel immunotherapeutic vaccine for ragweed allergies. J Allergy Clin Immunol. 2009;123:S144
    • View In Article
20. Betts MR, Nason MC, West SM, De Rosa SC, Migueles SA, Abraham J, et al. HIV nonprogressors preferentially maintain highly functional HIV-specific CD8+ T cells. Blood. 2006;107:4781–4789
    • View In Article
    • MEDLINE
    • CrossRef
21. Turcanu V, Maleki SJ, Lack G. Characterization of lymphocyte responses to peanuts in normal children, peanut-allergic children, and allergic children who acquired tolerance to peanuts. J Clin Invest. 2003;111:1065–1072
    • View In Article
    • MEDLINE
    • CrossRef
22. Guo L, Hu-Li J, Paul WE. Probabilistic regulation in TH2 cells accounts for monoallelic expression of IL-4 and IL-13. Immunity. 2005;23:89–99
    • View In Article
    • MEDLINE
    • CrossRef
23. Zhu J, Paul WE. CD4 T cells: fates, functions, and faults. Blood. 2008;112:1557–1569
    • View In Article
    • CrossRef
24. O'Shea JJ, Hunter CA, Germain RN. T cell heterogeneity: firmly fixed, predominantly plastic or merely malleable?. Nat Immunol. 2008;9:450–453
    • View In Article
    • CrossRef
25. Casazza JP, Betts MR, Price DA, Precopio ML, Ruff LE, Brenchley JM, et al. Acquisition of direct antiviral effector functions by CMV-specific CD4+ T lymphocytes with cellular maturation. J Exp Med. 2006;203:2865–2877
    • View In Article
    • MEDLINE
    • CrossRef
26. Akdis M, Verhagen J, Taylor A, Karamloo F, Karagiannidis C, Crameri R, et al. Immune responses in healthy and allergic individuals are characterized by a fine balance between allergen-specific T regulatory 1 and T helper 2 cells. J Exp Med. 2004;199:1567–1575
    • View In Article
    • MEDLINE
    • CrossRef
27. Ito T, Wang YH, Duramad O, Hori T, Delespesse GJ, Watanabe N, et al. TSLP-activated dendritic cells induce an inflammatory T helper type 2 cell response through OX40 ligand. J Exp Med. 2005;202:1213–1223
    • View In Article
    • MEDLINE
    • CrossRef

Back to Article Outline

References 

1. Foster B, Prussin C, Liu F, Whitmire JK, Whitton JL. Detection of intracellular cytokines by flow cytometry. Curr Protoc Immunol. 2007;Chapter 6:Unit 6 24
2. Siewert E, Lammert F, Koppitz P, Schmidt T, Matern S. Eosinophilic gastroenteritis with severe protein-losing enteropathy: successful treatment with budesonide. Dig Liver Dis. 2006;38:55–59
3. Lombardi C, Salmi A, Savio A, Passalacqua G. Localized eosinophilic ileitis with mastocytosis successfully treated with oral budesonide. Allergy. 2007;62:1343–1345