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. Author manuscript; available in PMC: 2019 May 1.
Published in final edited form as: J Allergy Clin Immunol. 2018 Feb 15;141(5):1699–1710.e7. doi: 10.1016/j.jaci.2018.01.035

Identification and analysis of peanut-specific T effector and T regulatory cells in children allergic and tolerant to peanut

Katherine A Weissler a, Marjohn Rasooly a, Tom DiMaggio a, Hyejeong Bolan a, Daly Cantave a, David Martino b,c, Melanie R Neeland b, Mimi LK Tang b,c,d, Thanh D Dang b,c, Katrina J Allen b,c,d, Pamela A Frischmeyer-Guerrerio a
PMCID: PMC5938104  NIHMSID: NIHMS943319  PMID: 29454004

Abstract

Background

Peanut allergy is potentially life-threatening and generally persists lifelong. Recent data suggests the skin may be an important route of initial sensitization to peanut, while early oral exposure to peanut is protective. In mice, T regulatory cells (Tregs) are central to development of tolerance to food, but their contribution to the pathogenesis of food allergy in humans is less clear.

Objective

We sought to quantify and phenotype peanut-specific CD4+ T effector (ps-Teff) and ps-Tregs in children with and without peanut allergy or sensitization.

Methods

Ps-Teffs and ps-Tregs were identified from peripheral blood of peanut allergic, peanut sensitized, and non-sensitized/non-allergic school-aged children and one year old infants based on upregulation of CD154 or CD137, respectively, following stimulation with peanut extract. Expression of cytokines and homing receptors were evaluated using flow cytometry. Methylation at the FOXP3 locus was measured as a marker of Treg stability.

Results

Differential upregulation of CD154 and CD137 efficiently distinguished ps-Teffs and ps-Tregs. A greater percentage of ps-Teffs from peanut allergic and sensitized infants expressed the skin homing molecule CLA, suggesting activation following exposure through the skin, compared to non-allergic infants. While ps-Teffs in both school-aged and infant peanut-allergic children produced primarily Th2 cytokines, a Th1-skewed anti-peanut response was only seen in non-allergic school-aged children. The frequency, homing receptor expression, and stability of ps-Tregs in infant and school-aged children were similar regardless of allergic status.

Conclusions

Exposure to peanut through the skin may prime the development of Th2 ps-Teffs that promote sensitization to peanut, despite the presence of normal numbers of ps-Tregs.

Keywords: peanut allergy, Treg, antigen specificity, CD154, CD137, CLA, α4β7, route of sensitization, food allergy

Graphical Abstract

graphic file with name nihms943319u1.jpg

Introduction

Food allergy is a growing problem around the world, and the associated anaphylactic responses can have fatal consequences.1 Peanut allergy is the most common cause of severe reactions in the United States2 and is usually lifelong.2–4 A better understanding of the underlying immunologic events that lead to this disease may facilitate efforts to prevent and treat peanut allergy.

Food allergy is postulated to result from a defect in either the establishment or maintenance of oral tolerance, defined as a state of unresponsiveness upon reencounter with an antigen following oral exposure. T regulatory cells (Tregs) are an important component of oral tolerance, and several studies have suggested that deficits in the development, function, and/or stability of Tregs predispose to food allergy. Adoptive transfer of Tregs can suppress anaphylactic responses to food antigens in murine models,5 and mice that cannot produce Tregs outside the thymus spontaneously develop type 2 inflammation at mucosal sites and generate an antibody response against antigens found in mouse chow.6 Loss-of-function mutations in FOXP3, which encodes the main transcription factor essential for Treg development, cause immunodysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX) syndrome that is associated with profound autoinflammatory disease, as well as food allergy and eczema in some patients.7, 8 Additionally, an increase in the frequency and stability of Tregs was associated with more favorable clinical outcomes in children who underwent oral immunotherapy for peanut allergy, and increased numbers of Treg were reported in children who naturally outgrew their milk allergy.9–11

The predominant immune response to peanut in allergic patients is characterized by the release of Th2 effector cytokines (IL-4, IL-5, IL-13) that induce B cells to produce peanut-specific IgE. Crosslinking of IgE bound to the surface of mast cells and basophils results in degranulation and release of inflammatory mediators following peanut exposure, resulting in an allergic reaction.12 Growing evidence points to the skin as the main site of sensitization to food antigens, particularly peanut. Genetic variants in FLG, SPINK5, and DSG1, which encode epithelial barrier proteins, have all been associated with an increased risk of food allergy.13–15 Infants with eczema are eleven times more likely to develop peanut allergy than those without,16 and high household consumption of peanut is associated with increased levels of biologically active peanut allergen in household dust and a greater risk of developing peanut sensitization, particularly if the child has eczema.17–19

While recent studies have provided clues regarding the role of T cells in the pathogenesis of peanut allergy, these efforts have been hindered by technical difficulties in identifying peanut-specific T cells. Here, we have adapted a method previously used to detect T cells that recognize fungal antigens20, 21 to simultaneously identify ps-Teffs and ps-Tregs cells directly ex vivo based on rapid upregulation of CD154 (CD40L) or CD137 (4-1BB), respectively.22 CD154 was originally identified on activated T cells and is crucial for T cell-dependent activation of B cell responses23, 24, while CD137 is a known immunoregulatory molecule and a direct target of Foxp3.25 Using this methodology, we sought to explore the frequency and phenotype of ps-Teffs and ps-Tregs in children who are allergic versus tolerant to peanut, and thereby illuminate the changes in CD4+ T cell responses that lead to the development of peanut allergy.

Methods

Patient populations

School-aged subjects were recruited to the NIH under clinical protocol 15-I-0162 (n=17 peanut allergic; n=19 non-allergic). Additional demographic and clinical characteristics of this study group are listed in Table E1 and the Online Repository.

Cryopreserved peripheral blood mononuclear cells (PBMCs) were also obtained from a subset of 1 year old infants enrolled in HealthNuts, a population-based cohort from Melbourne, Australia (n=14 peanut allergic; n=15 peanut sensitized; n=14 non-allergic). Infants were classified as 1) peanut allergic (PA), defined by a positive oral food challenge (OFC) to peanut along with a positive SPT (2 mm or greater ) or IgE test (0.35 kU/L or greater) for peanut; 2) non-allergic (NA), defined as passing an OFC to peanut and a negative SPT and/or IgE test for peanut; and 3) peanut sensitized (PS), defined as having a SPT wheal response to peanut of 2 mm or greater and/or a peanut-IgE level of 0.35 kU/L or greater and a negative OFC to peanut. Additional details can be found in Table E2, references 26 and 27, and the Online Repository.

The study was approved by local institutional review boards, and informed consent/assent was obtained for all subjects. Approval to conduct the HealthNuts study was obtained from the Victorian State Government Office for Children (reference no. CDF/07/492), the Victorian State Government Department of Human Services (reference no. 10/07), and the Royal Children’s Hospital Human Research Ethics Committee (reference no. 27047).

End point titration skin prick tests (SPTs) to measure mast cell reactivity

End point titration SPTs were performed on school-aged PA children using serial 10-fold dilutions of peanut extract (Greer Laboratories, Lenoir NC) with the GREER pick system. The starting concentration was the standard peanut extract (1:20 wt/vol) with serial 10-fold dilutions (1:200, 1:2000, 1:20,000, and 1:200,000 wt/vol). Peanut SPT was not performed on 3 PA subjects since they were unable to discontinue their antihistamines due to eczema.

Crude peanut extract for use in T cell studies

Peeled fresh peanuts were blended at 2 g per 10 mL of saline, rocked overnight at 4 °C, centrifuged at 5000 × g for 30 minutes, and the protein layer was collected. Centrifugation and collection of protein layer was repeated twice more, and the resulting solution was passed through a 0.2 µm filter. Endotoxin was reduced using an endotoxin removal kit from Pierce. The final concentration of endotoxin was approximately 80 EU/mL.

Basophil Activation Test

Basophil activation was measured by upregulation of CD63 as previously described.28 Further details are provided in the Online Repository.

Analysis of peanut-specific T cells

For school-aged subjects recruited at the NIH, blood was collected in sodium heparin and assays were performed using whole blood. For 1 year old infants, PBMCs were thawed and rested overnight prior to being resuspended at 8 × 106 cells/mL in AIM-V medium supplemented with 2.5% human serum, and approximately 5 × 106 cells per well were added to a 48 well plate. Anti-CD40 (clone HB14; Miltenyi Biotec) and anti-CD28 (clone 15E8; Miltenyi Biotec) were added at a final concentration of 1 µg/mL to whole blood cultures (6–7 ml) for school-aged children or PBMCS for 1 year old infants. Crude peanut extract was added where indicated at a final concentration of 300 µg/mL, and cultures were incubated at 37 °C. Brefeldin A (5 µg/mL) was added after 4.75 hours if an intracellular cytokine stain was being performed, and cells were harvested after an additional 2 hours. CD4+ T cells were enriched from whole blood only using a magnetic depletion method that resulted in approximately 85% purity (EasySep Direct Human CD4+ T Cell Isolation Kit; StemCell Technologies). Live/dead stain (ebioscience) was used to discriminate between live and dead cells. Human Ig was used as a blocking agent prior to staining. Cells were surface stained in PBS supplemented with 1% BSA for 25 minutes. When needed, intracellular staining was performed using the ebioscience Foxp3 staining kit according to manufacturer’s instructions. A BD LSRII was used for flow cytometry and between 8×105 and 2×106 events were collected. Analysis was performed using FlowJo software (Supplemental methods). Peanut-specific effector T cells and Tregs were identified by upregulation of CD154 or CD137, respectively. The number of cells expressing a given marker were calculated by subtracting the number of cells in that gate per million CD4+ T cells in the unstimulated control sample from the number in the peanut stimulated sample.

Methylation analysis

Peanut specific Teffs and ps-Tregs were sorted from peripheral blood of school-aged children using a BD FACSAria and snap frozen; methylation status was assessed by bisulfite modification and direct pyrosequencing, and was performed by EpigenDX (Hopkinton, MA). Percentage indicated is the average of percent methylation at 11 different CpG sites in intron 1 (-2236 to -2376 from ATG) of the FOXP3 locus.

In vitro expansion of ps-Teffs and ps-Tregs, restimulation, and suppression assay

PBMCs were resuspended at 10 × 106 cells/mL and stimulated for 6 h with 300 µg/mL CPE, 1 µg/mL anti-CD40 (clone HB14; Miltenyi Biotec) and 1 µg/mL anti-CD28 (clone 15E8; Miltenyi Biotec). CD154+ and CD137+ cells were isolated using CD154 and CD137 Microbead kits (Miltenyi Biotec) and sequential magnetic columns. CD154+ cells were cultured at 5 × 105 cells/mL with CPE-loaded irradiated autologous feeder PBMCs at 5 × 106 cells/mL in XVIVO-15 media (Lonza) supplemented with 2.5% (v/v) human AB serum and 4 ng/mL recombinant human IL-2 (Peprotech). CD137+ cells were cultured at 2.5 × 104 cells per well in a 96 well U-bottom plate in XVIVO-15 media (Lonza) supplemented with 2.5% (v/v) human AB serum, 30 nM rapamycin, and 10 ng/mL recombinant human IL-2 (Peprotech). Media was replaced and cultures were split as necessary. After 17–21 days of culture, restimulation and suppression assays were set up using 2 × 105 irradiated autologous CD3-depleted PBMCs alone or loaded with CPE or wheat extract, 1 × 105 expanded CD154+ cells labeled with CellTrace Violet, and/or expanded CD137+ cells labeled with CFSE at a 1:1, 1:2 and 1:4 ratio with expanded CD154+ cells in a 96 well U-bottom plate in XVIVO-15 media supplemented with 2.5% (v/v) human AB serum. Cells were harvested after 6 days and dilution of CFSE and CellTrace Violet was assessed by flow cytometry and division index was identified using FlowJo software. The percent suppression was calculated as (100-((division index of CD154+ cells in the presence of CD137+ cells division index of CD154+ cells alone / division index of CD154+ cells alone)*100).

Statistical Analysis

Statistical analysis was performed using Prism (Graphpad Software). A Students t test with Welch’s correction for unequal variance or a Kruskal-Wallis test were used to determine statistical significance where appropriate. Comparisons between pairs following a significant result by a Kruskal-Wallis test were performed using Dunn’s multiple comparison test. A P value of less than 0.05 was considered significant.

Results

Identification and quantification of peanut-specific CD4+ T cells in school-aged children with or without peanut allergy

17 subjects with peanut allergy (PA; median age 8 years; IQR 8.5; 71% male) and 19 non-allergic subjects (NA; median age 12 years; IQR 5; 53% male) were recruited. Subjects with PA had a median total IgE of 1148 kU/L (IQR 7617 kU/L), peanut (PN)-specific IgE of 36.1 kUA/L (IQR 88.5 kU/L), and wheal size of 5.5 mm (IQR 4.6 mm) on peanut SPT. All of the PA subjects were allergic to other foods, 16/17 had atopic dermatitis, 12/17 had asthma, and 15/17 had allergic rhinitis (Table E1). All were strictly avoiding peanut in their diet. NA subjects had a median total IgE of 31.9 kU/L (IQR 87.5 kU/L), were not sensitized to peanut (peanut-specific IgE was below the level of detection in either a peanut-specific IgE or FX5 food panel, which includes peanut), and were consuming peanut in their diet. 1/19 had atopic dermatitis, 1/19 had asthma, and 2/19 had allergic rhinitis (Table E1).

To detect peanut-specific CD4+ T cells, whole blood was cultured with crude peanut extract (CPE), and upregulation of CD154 and CD137 was used to identify ps-Teffs (CD154+CD137-) and ps-Tregs (CD154-CD137+), respectively, using the gating strategy depicted in Fig. 1A,B and quantified in Fig. 1C. The absolute numbers of ps-Teffs and ps-Tregs per 1X106 total CD4+ T cells, which were calculated by subtracting the number of cells in each gate per 1X106 total CD4+ T cells in the unstimulated condition from the number in the CPE stimulated condition, were not different between PA and NA children, nor was there a significant difference in the ratio of ps-Tregs/ps-Teffs between groups (Fig. 1D,F). Importantly, the majority of CD154+ ps-Teffs in both groups did not express FOXP3, while approximately 90% of CD137+ ps-Tregs were FOXP3+ (Fig. 1E).

Figure 1. Quantification of peanut-specific Teffs and Tregs by rapid upregulation of CD154 and CD137, respectively, in blood from school-aged children.

Figure 1

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A, Gating strategy used to identify CD3+CD4+ T cells following stimulation of whole blood with CPE for 6.75 hours and subsequent CD4+ T cell enrichment by negative selection. B and C, Percent of CD3+CD4+ Tcells that expressed CD154 or CD137 following culture with media alone (No Peanut) or CPE (Peanut) in nonallergic (NA) or peanut allergic (PA) children. Each point in C represents one subject. Statistics were performed using a paired Student t test. ** P < 0.01; *** P ≤ 0.001 D, The number of CD154+ ps-Teffs and CD137+ ps-Tregs per 1×106 CD3+CD4+ cells was calculated by subtracting the number of CD154+ or CD137+ cells in the unstimulated condition from the CPE stimulated condition for each subject. E, The percentage of CD154+ and CD137+ T cells expressing FOXP3 after CPE stimulation. F, The ratio of ps-Treg:ps-Teff in each individual. G, Percentage of total CD4+ T cells expressing FOXP3 in NA and PA subjects. Statistics were performed using an unpaired t test with Welch’s correction for unequal variation.

We next measured the percentage of CD4+ T cells expressing FOXP3 in the PA and NA subjects, and found no evidence for a global deficiency in Tregs in PA children (Fig. 1G).

Examination of ps-Tregs

Although the expression of FOXP3 by the CD137+ population supports the identification of these cells as Tregs, human effector T cells can also upregulate FOXP3 after stimulation.29 To confirm that the CD137+ cells were bona fide Tregs, we sorted both CD154+ (ps-Teffs) and CD137+ (ps-Tregs) cells following stimulation with CPE and assessed the methylation status across 11 CpG sites in the Treg specific demethylated region (TSDR) of intron 1 of the FOXP3 locus. Demethylation of this region is important in maintaining a suppressive Treg phenotype in both mice and humans.30–34 We found that the CD137+ cell population averaged 0–40% methylation across this region of FOXP3, which is consistent with what has been reported previously in human Tregs.34 In contrast, the sorted CD154+ cells consistently displayed a methylation rate close to 90% (Fig. 2A). No differences in FOXP3 methylation in either ps-Tregs or ps-Teffs were observed between NA and PA subjects (Fig. 2A).

Figure 2. Analysis of Tregs in peanut allergic and non-allergic school-aged subjects.

Figure 2

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A, Average percent methylation across 11 CpG locations in the TSDR of intron 1 of FOXP3 in FACS-isolated ps-Teffs (CD154+) and ps-Tregs (CD137+) from PA and NA school-aged subjects. B, Representative CellTrace Violet dilution by expanded CD154+ T cells isolated from NA and PA donors after restimulation with autologous feeders left unloaded or loaded with peanut extract. Autologous CD137+ T cells were added to cultures at ratios of 1:1, 1:2, and 1:4 to CD154+ cells. Shaded grey histograms show division in response to peanut extract in the absence of CD137+ cells. C, Percent suppression of the proliferation of CD154+ cells stimulated with CPE in the presence of the indicated ratio of CD137+ cells relative to cultures that did not contain CD137+ cells. Statistics were performed using an unpaired t test with Welch’s correction for unequal variation.

To assess the suppressive capacity of the CD137+ cells, CD154+ ps-Teffs and CD137+ ps-Tregs from NA and PA subjects were isolated by magnetic column after stimulation with CPE, and expanded in vitro as previously described20,46 to produce sufficient cell numbers for a suppression assay. CD137+ cells from NA and PA subjects were equally capable of suppressing the proliferation of autologous CD154+ cells in response to CPE when added at ratios of 1:1, 1:2 and 1:4 to cultures (Fig. 2B,C). We set up parallel cultures of CD154+ and CD137+ cells alone with autologous PBMCs that were left unloaded or were loaded with wheat or CPE. Both CD154+ and CD137+ cells divided more in response to CPE than to autologous feeders alone or loaded with wheat, thus confirming their reactivity to peanut antigen (Fig. E1 in Online Repository).

Cytokine production and expression of homing receptors by peanut-specific T cells

Peanut specific-Teffs from PA subjects upregulated IL-13 following stimulation with CPE, while IL-13 was not induced by CPE in ps-Teffs from NA children (Fig. 3A,B). In contrast, a similar percentage of ps-Teffs from PA and NA subjects expressed IFN-γ in response to CPE, although the number of IFN-γ+ ps-Teffs was significantly higher in the NA group compared to those with PA (Fig. 3A,B). Peanut-induced IL-17 was not detectable above background in ps-Teffs from either population. IL-10 was significantly induced by CPE stimulation only in PA children. The total number of IL-10+ ps-Teffs also trended higher in those with PA, but did not reach statistical significance (Fig. 3A,B). For PA children, the percentage of ps-Teffs producing IL-13 correlated positively with both peanut-specific IgE and with peanut-induced basophil activation as quantified by the area under the curve (AUC) of CD63 expression over a range of peanut concentrations (Fig. 3C). Additionally, stimulation of CD137+ ps-Tregs with CPE did not results in a significant increase in IL-13, IFN-γ, IL-10 or IL-17 production above the baseline established by unstimulated cells in either PA or NA subjects (Fig. E2 in Online Repository).

Figure 3. Cytokine production by CD154+ ps-Teffs from NA and PA school-aged subjects.

Figure 3

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A, The percentage of CD154+ T cells producing each indicated cytokine was measured by flow cytometry when whole blood from children with (PA) or without peanut allergy (NA) was cultured in media alone or with CPE. Statistics were calculated using a paired t test between unstimulated and CPE stimulated samples from the same individual, and an unpaired t test with Welch’s correction between the NA and PA groups. *** P ≤ 0.001 B, The number of CD154+ T cells per 1×106 CD3+CD4+ T cells expressing the indicated cytokine was calculated by subtracting the number of CD154+cytokine+ cells in the unstimulated condition from the number in the CPE stimulated condition. Statistics were calculated using an unpaired t test with Welch’s correction. *P<0.05; *** P ≤ 0.001 C, Correlation of percentage of ps-Teffs producing IL-13 and peanut-specific IgE (top) or area under the curve of basophil CD63 expression over a range of peanut concentrations (bottom) for children with peanut allergy. Linear regression analysis performed.

We next assessed ps-T cells for expression of the skin-homing molecule cutaneous lymphocyte antigen (CLA) and the gut-homing molecule α4β7, which are strongly correlated with the site of initial antigen encounter for a naïve T cell.35, 36 No differences in expression of CLA or α4β7 on ps-Teffs, ps-Tregs, or total CD4+ T cells were observed between PA and NA children (Fig. 4). Consistent with prior reports of total CD4+ Teffs and Tregs, ps-Tregs expressed relatively more CLA and less α4β7 than ps-Teffs.37

Figure 4. Expression of homing molecules CLA and α4β7on ps-Teff and ps-Tregs from school-aged children.

Figure 4

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Following stimulation of whole blood from NA or PA school-aged children with CPE, the percentage and number of CD154+, CD137+, and total CD3+CD4+ T cells expressing CLA (A,C) and α4β7 (B,D) was determined by flow cytometry. Statistics were calculated using an unpaired t test with Welch’s correction.

Although Tregs have been shown to potently suppress mast cell degranulation and allergic responses via direct interactions between the two cell types,38 we found no significant correlation between the number of total ps-Tregs or CLA+ ps-Tregs and mast cell reactivity to peanut as measured by end point titration SPT to peanut (Fig. E3 in Online Repository).

Characterization of peanut-specific T cells at one year of age

The immune system undergoes rapid development early in life as many antigens are encountered for the first time. Since many children develop peanut allergy during the first year of life, we sought to evaluate how the T cell response to peanut develops during this critical window. PBMCs were obtained from a cohort of infants at 1 year of age enrolled in the HealthNuts study.37 Three groups of children were studied: 1) non-allergic (NA; n=14) defined as negative peanut SPT and negative OFC to peanut; 2) peanut sensitized (PS; n=15) defined as a positive peanut SPT , positive peanut-specific IgE, but negative OFC to peanut; 3) peanut allergic (PA; n=14) defined as positive SPT, positive peanut-specific IgE and positive OFC to peanut (additional details in Table E2). Eczema was common in all 3 groups (57% of NA, 73% of PS, and 79% of PA). All of the allergic/sensitized infants were single sensitized, and none of the children had introduced peanut into their diet, although three of the PS children and one of the PA children had had documented minimal exposures to peanut at some point during the first year of life (Table E2).

As shown in Fig. 5, no significant differences were identified in the number of ps-Teffs or ps-Tregs among NA, PS, and PA children. As expected, the majority of CD137+ ps-Tregs, and the minority of CD154+ ps-Teffs, expressed FOXP3 in all three groups (Fig. 5C). Additionally, the level of expression of CD154, as determined by MFI, was not different among ps-Teffs from the three groups (data not shown). The ratio of ps-Treg/ps-Teffs was also not different among the three groups (Fig. 5D).

Figure 5. Quantification of peanut-specific Teffs and Tregs by rapid upregulation of CD154 and CD137, respectively, in PBMCs from one-year old infants.

Figure 5

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A, Percent of CD3+CD4+ T cells that expressed CD154 (top) and CD137 (bottom) following culture of PBMCs in media alone or with CPE from 1 year old infants who were nonallergic (NA), peanut sensitized (PS), or peanut allergic (PA). Statistics were calculated using a paired student t test. *P<0.05; *** P ≤ 0.001 B, The number of CD154+ ps-Teffs and CD137+ ps-Treg per 1×106 CD3+CD4+ cells was calculated by subtracting the number of CD154+ or CD137+ cells in the unstimulated condition from the CPE stimulated condition for each subject. Statistics were calculated using a Kruskal-Wallis test followed by Dunn’s multiple comparisons test. C, The percentage of CD154+ or CD137+ cells expressing FOXP3 following stimulation with CPE. D, The ratio of the number of CD137+ ps-Tregs to CD154+ ps-Teffs.

We next looked at cytokine production by ps-T cells in response to stimulation with CPE. Both PS and PA groups had a significantly higher percentage of IL13+ ps-Teffs compared to the NA cohort, and there was a trend towards higher numbers of ps-Teffs expressing IL-13 in these groups as well (Fig. 6A,B). As in the older children, IL-13 production by ps-Teff correlated positively with peanut-specific IgE (Fig. 6C). Neither IFN-γ nor IL-17 were significantly induced in ps-Teffs following treatment with CPE in any of the 3 categories of subjects, and the percentage and number of IFN-γ+ or IL-17+ pTeffs were not different across NA, PS, and PA children (Fig. 6 A,B). Both the percentage and the number of IL-10+ ps-Teffs tended to be highest in the PS children, although this did not reach statistical significance (Fig. 6A,B).

Figure 6. Cytokine production by CD154+ ps-Teffs from one-year old infants.

Figure 6

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A, The percentage of CD154+ T cells producing each indicated cytokine was measured by flow cytometry when PBMCs from one-year old infants who were nonallergic (NA), peanut sensitized (PS), or peanut allergic (PA)were left unstimulated or cultured with CPE. Statistics were calculated using a paired t test between unstimulated and CPE stimulated samples from the same subject, and a Kruskal-Wallis test among NA, PS, and PA groups. *** P ≤ 0.001 B, The number of CD154+ cells per 1×106 CD3+CD4+ T cells expressing the indicated cytokine was calculated by subtracting the number of CD154+cytokine+ cells in the unstimulated condition from the number in the CPE stimulated condition. Statistics were calculated using a Kruskal-Wallis test followed by Dunn’s multiple comparisons test. *P<0.05; ** P < 0.01 C, Correlation of percentage of ps-Teffs producing IL-13 and peanut-specific IgE for infants with peanut allergy. Linear regression analysis performed.

As we had seen in the older cohort of children, ps-Tregs in NA, PS, and PA children did not generally produce significant amounts of IL-13, IFN-γ, IL-17, or IL10 following CPE stimulation at 1 year of age (data not shown).

Next, we assessed expression of the homing molecules CLA and α4β7 on ps-T cells from these infants. Expression of the skin-homing molecule CLA was significantly higher on ps-Teffs from both PA and PS infants compared to the NA cohort (Fig. 7A,C). Conversely, α4β7 expression was lower on ps-Teffs from these two groups (Fig. 7 B,C). Importantly, the difference in expression of homing molecules was exclusive to ps-Teffs; neither CLA nor α4β7 expression was different on ps-Tregs or total CD4+ FOXP3- or CD4+ FOXP3+ T cells among NA, PS, and PA children (Fig. 7). Effector cytokines were primarily produced by CLA+ ps-Teffs following both peanut and nonspecific (PMA/ionomycin) stimulation in all infants regardless of allergic status. The percent of IL13+CLA+ ps-Teffs were higher in those sensitized or allergic to peanut compared to nonallergic controls after peanut stimulation (Fig E4).

Figure 7. Expression of CLA and α4β7 by ps-T cells from one year old infants.

Figure 7

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Following CPE stimulation of PBMCs from nonallergic (NA), peanut sensitized (PS), or peanut allergic (PA) infants, the percentage of CD154+, CD137+, and total CD3+CD4+FOXP3- (black) and CD3+CD4+FOXP3+ (green) T cells expressing CLA (A) and α4β7 (B) was determined by flow cytometry, and the numbers of CLA+ (C) and α4β7+ (D) CD154+ and CD137+ cells were calculated. Mean and standard deviation are shown. Statistics were calculated using Kruskal-Wallis test followed by Dunn’s multiple comparison test. *P<0.05; ** P < 0.01

Discussion

Food allergy stems from a failure to develop tolerance to food antigens, resulting in the development of Th2 effector immune responses and generation of allergen-specific IgE. The emergence of pathogenic responses to food allergens is hypothesized to result in part from a deficiency in the development and/or function of Tregs specific for the food. However, technical difficulties in identifying these cells have hampered efforts to investigate a role of Tregs in food allergy pathogenesis in humans. Previous efforts have largely relied on proliferation or tetramer staining, which have inherent limitations. Expansion cultures may select for more proliferative clones and be associated with changes in cellular phenotype as well as specificity due to bystander proliferation.39 Tetramer staining allows characterization of T cells specific for only one epitope at a time and requires knowledge of the patient’s HLA status. Furthermore, neither of these methods has allowed for the profiling of the peanut-specific Treg population. We have adapted a different strategy to simultaneously identify and characterize ps-Teffs and ps-Tregs directly ex vivo that overcomes many of these limitations. Our results suggest that both peanut sensitization and peanut allergy are associated with a Th2-skewed effector T cell response to peanut and increased homing of ps-Teffs to the skin early in life; however, we found no evidence that either peanut sensitization or allergy are associated with a deficiency in the frequency, homing, suppressive function, or stability of ps-Tregs.

Tregs are essential for the development of oral tolerance, and Treg function has been found to be impaired in a number of monogenic diseases associated with a high risk for food allergy.7, 40–42 These data suggest that disruption in Treg development or function is one mechanism that can predispose to food allergy, but whether defects in Tregs contribute to the epidemic of food allergy in the general population remains a matter of debate. While some groups have found an increase in FOXP3+ Tregs following successful immunotherapy for peanut allergy,11, 43 others have seen no increase44 or only a transient increase.45 These studies all analyzed FOXP3+ cells after several days of culturing PBMCs with CPE or utilized tetramer staining for limited epitopes. Using CD137 upregulation to identify ps-Tregs directly ex vivo, we found these cells were nearly all FOXP3+ and did not produce effector cytokines. Methylation analysis of the FOXP3 locus confirmed Treg identify and stability of the CD137+ cells and revealed no differences between PA and NA subjects. Furthermore, ps-Tregs from NA and PA individuals suppressed the proliferation of autologous ps-Teffs to a similar extent. Collectively, these data suggest that alterations in ps-Tregs may not be able to able to explain the development of food allergy.

Although no discrepancy in the frequency of ps-Tregs was identified in school-aged children with established peanut allergy versus age-matched NA subjects, we considered the possibility that a transient deficiency in ps-Tregs early in life, when peanut is first introduced into the diet, may contribute to the development of peanut allergy. To address this question, we compared the frequency of ps-Tregs in 1 year old infants with OFC-confirmed peanut allergy to age-matched infants sensitized but clinically tolerant to peanut and to non-allergic controls. All three groups of children showed similar numbers of ps-Tregs, arguing against a quantitative defect in Treg development early in life as an explanation for the development of peanut allergy. Our data are consistent with the results of a recent study, which concluded that the development of allergic responses to aeroallergens was also not the result of an impaired Treg response in allergic patients.46 This group found no difference in aeroantigen-specific Treg frequency, phenotype, TCR avidity, suppressive capacity, or TCR-β repertoire diversity between allergic and non-allergic individuals. In both our older cohort of children with peanut allergy and our younger cohorts with peanut allergy or sensitization, we also saw a pronounced Th2-dominanted immune response by ps-Teff cells. Similar to what has been reported for both peanut and cat allergy,47, 48 we found a positive correlation between IL-13 production by ps-Teffs and peanut-specific IgE for both age groups, as well as between IL-13 production and the basophil activation test results for the older cohort. While greater numbers of IFN-γ+ ps-Teffs were evident in the older group of non-allergic children compared to those with peanut allergy, this difference was not seen in the 1 year old infants. In fact, Th1 responses by ps-Teffs were low to peanut in all children at this age, but dominated the effector T cell response to peanut in older non-allergic children who were ingesting peanut in their diet. While these data suggest that an initial deficiency in Th1 responses to peanut may not cause peanut allergy, failure of IFN-γ+ ps-Teffs to expand may contribute to disease persistence. We further found that the total number of ps-Teffs was not higher in PA individuals, and in fact, the NA group tended to have a greater frequency of ps-Teffs among school-aged children. We hypothesize that this is likely a consequence of continued exposure to peanut by the NA individuals resulting in expansion of ps-T cells. In contrast, allergic individuals are instructed to avoid peanut, and subsequently ps-T cell clones may not have the opportunity to expand.

Both Treg and Teff cells acquire expression of homing receptors that reflect the tissue site of their initial activation. T cells activated in the mesenteric lymph node (MLN) or Peyers Patches (PP) express α4β7 that directs their trafficking to the gut, while those activated in skin lymph nodes express CLA that mediates their localization to the skin.35, 36, 49 While expression of CLA and α4β7 were similar on both ps-Tregs and ps-Teffs in school-aged children with and without peanut allergy, a higher percentage of ps-Teffs from peanut sensitized and allergic infants at 1 year of age expressed CLA, and correspondingly fewer ps-Teffs expressed α4β7, compared to their non-allergic counterparts. Importantly, expression of CLA and α4β7 on ps-Tregs was not different among the 3 groups of infants, suggesting that lack of proper ps-Treg homing is not responsible for the development of Th2 effector responses in PS and PA children.

Our data are consistent with the dual-allergen-exposure hypothesis which theorizes that initial exposure to a food allergen through the skin results in generation of an allergic Th2 response and sensitization, while exposure through consumption induces tolerance.50 A prior study found that sorted CLA+ memory cells from peanut allergic individuals proliferated more in response to peanut than sorted α4β7+ memory cells,51 although our study is the first to show differential expression of these markers on identified ps-T cells. The increase in CLA expression on ps-Teffs from PA and PS infants is unlikely to be explained merely by a higher rate of eczema in these two groups, as the majority of NA infants had been diagnosed with eczema as well, and no difference in CLA expression was evident on total CD4+ T cells (FOXP3+ or FOXP3-) across the 3 groups of subjects. Furthermore, CLA expression was similar on ps-Teffs from PA and NA school-aged children, despite the fact that nearly all of the PA children and only one of the NA children had been diagnosed with eczema in this cohort. The higher frequency of α4β7+ ps-Teffs in the NA infants cannot be explained by greater ingestion of peanut in this group, as none of the 1 year old infants had introduced peanut into their diet. However, we cannot exclude the possibility that NA children had ingested small amounts of peanut antigen either through breast milk52 or as a minor component of other food items. Additionally, the allergic and sensitized groups may have had more contact with peanut through the skin, thus allowing for the development and expansion of CLA+ ps-Teffs. Environmental, non-oral peanut exposure has been found to correlate positively with development of peanut allergy.53 Our observation that CLA+ ps-Teffs were the primary source of effector cytokines in all infants emphasizes the critical role of the skin in determining immune responses to peanut early in life.

Peanut-specific Teffs from both PS and PA infants showed altered homing and Th2-dominated effector responses, suggesting that impairment in the skin barrier early in life, along with environmental peanut exposure, promotes sensitization to peanut while other factors must determine clinical reactivity to peanut. Although not statistically different, we found a trend towards higher numbers of IL10+ ps-Teffs in PS infants relative to their PA and NA counterparts. Prior studies have implicated IL-10 as a key tolerogenic cytokine in food allergy; children who outgrew a food allergy, or who were predisposed to food allergy but remained tolerant to cow’s milk, were more likely to have CD4+ T cells that secreted IL-10 in response to the tolerated food antigen than allergic children or those who had no food allergy.54, 55 Although further validation is needed, these data support a potential tolerogenic role for IL-10 in suppressing clinical allergic responses to peanut in infants who are sensitized to the food.

Our study has several limitations. First, the results from this study did not use paired longitudinal samples but rather samples from allergic children at different ages, and thus we cannot be certain that the changes in T cell responses in our infant and school-aged cohorts were due to age alone. Second, ps-Teffs and ps-Tregs may fail to recirculate after encountering peanut antigen in the gut, and thus our ability to detect them in peripheral blood and draw conclusions may be limited. However, others have found T cells specific for orally-administered antigens in the blood, which suggests recirculation can occur.35, 56 Finally, there may be factors that predispose for food allergy in the innate immune system, which are outside the scope of the present study and require further investigation.57

In summary, our data suggest that peanut allergy does not result from a quantitative deficiency in the development of ps-Tregs or IFN-γ+ ps-Teffs, but rather the emergence of a pathogenic Th2 effector response as a result of impairment in the skin barrier and environmental peanut exposure.

Supplementary Material

Key messages.

  • The frequency, stability (as assessed by methylation status of the FOXP3 locus) and homing receptor expression of peanut specific-Tregs in peripheral blood were similar in peanut allergic and non-allergic children.

  • Peanut-specific Teffs from peanut allergic school-aged children were more likely to produce IL-13 and less likely to express IFN-γ compared to non-allergic controls; however, while peanut-specific Teffs from peanut sensitized or allergic infants were also Th2-skewed, few IFN-γ+ ps-Teffs were detected in one year old infants regardless of allergic status

  • Ps-Teffs from infants sensitized or allergic to peanut at one year of age are more likely to express the skin homing receptor CLA compared to non-allergic controls, suggesting they were likely primed following exposure to peanut through the skin.

Acknowledgments

This research was supported by the Intramural Research Program of the National Institute of Allergy and Infectious Diseases, NIH.

Abbreviations used in this article

Treg

regulatory T cell

ps-Treg

peanut-specific regulatory T cell

ps-Teff

peanut-specific effector T cell

NA

non-allergic

PS

peanut sensitized

PA

peanut allergic

CPE

crude peanut extract

SPT

skin prick test

BAT

basophil activation test

PN

peanut

IPEX

immunodysregulation, polyendocrinopathy, enteropathy, X-linked syndrome

OFC

oral food challenge

FOXP3

Forkhead box protein 3

CLA

cutaneous lymphocyte antigen

TCR

T cell receptor

AUC

area under the curve

FACS

fluorescence activated cell sorting

TSDR

Treg specific demethylated region

PBMC

peripheral blood mononuclear cell

PMA

phorbol myristate acetate

Footnotes

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Conflicts of interest: none.

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NIHMS943319-supplement-supplement_1.pdf (3.1MB, pdf)

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