Characterization of lymphocyte responses to peanuts in normal children, peanut-allergic children, and allergic children who acquired tolerance to peanuts

Abstract

Comparing lymphocyte responses to allergenic and nonallergenic foods could reveal the differences between pathogenic and normal immune responses to foods. Defining the cytokine-producing phenotypes of peanut-specific lymphocytes from peanut-allergic children, children who outgrew peanut allergy, and children who have always tolerated peanuts may be useful for understanding the mechanisms of food tolerance. Investigating immune responses against foods is hindered, however, by the fact that circulating food antigen-specific lymphocytes are very rare. In a novel approach we used carboxyfluorescein succinimidyl ester to detect peanut-specific lymphocytes by flow cytometry. We confirmed that these cells are indeed peanut specific by cloning. Peanut-allergic donors show Th2 polarization of cytokine production by peanut-specific cells (IFN-gamma (low), TNF-alpha (low), IL-4 (high), IL-5 (high), IL-13 (high)). Conversely, nonallergic children and children who have outgrown their allergy show Th1 skewing to peanut antigens (IFN-gamma(high), TNF-alpha (high), IL-4 (low), IL-5 (low), IL-13(low)), similarly to nonallergenic food antigens (beta-lactoglobulin, OVA). This finding suggests that peanut antigens do not intrinsically induce Th2 skewing, but that the type of response depends upon the donor's allergic status. In conclusion, food allergic status is characterized by a Th2 response whereas Th1-skewed responses underlie oral tolerance.

Figure 1

PBMC proliferation monitored using CFSE labeling. PBMCs from a PA donor were isolated, labeled with CFSE, and cultured in the presence of 100 μg/ml peanut extract (a-c), 200 μg/ml OVA (d-f), 50 μg/ml PPD (g-i), or without exogenous antigens (j-l). On days 3, 5, and 7 after setting the cultures, cells were collected, stained with anti-CD4 PE, and analyzed by FACS. Figures represent the percentage of (CFSE^low CD4⁺) cells included in the respective gates. Similar results were obtained in three other donors

Figure 2

Flow-cytometry sorting of CFSE^low and CFSE^high CD4⁺ lymphocytes. PBMCs from a PA donor were labeled with CFSE and cultured with 100 μg/ml peanut extract for 7 days. Cells were then collected, stained with anti-CD4 PE, and sorted by flow cytometry. Figures show the percentage of the cell populations within the sorting gates from dot plots (a) before sorting, (b) CD4⁺ CFSE^low after sorting, and (c) CD4⁺ CFSE^high after sorting.

Figure 3

Th2 skewing of peanut-specific responses from a PA donor contrasts with the Th1-biased response induced by nonallergenic food antigens. PBMCs isolated from a PA donor, labeled with CFSE, and cultured in the presence of antigens for 7 days were restimulated with PMA and ionomycin in the presence of brefeldin A for 6 h, then cytokine production was assessed using intracellular cytokine staining. Plots show IFN-γ/IL-4 production (percentage of cytokine positive cells) by the antigen-specific (CFSE^low) cells specific for PPD (a), TT (b), β-lactoglobulin (c), OVA (d), and peanuts (e).

Figure 4

Th subset polarization of antigen-specific T lymphocytes from PA donors (n = 9). PBMCs were isolated, CFSE labeled, and cultured in the presence of antigens for 7–8 days, then the cytokine-producing phenotype of antigen-specific (CFSE^low) cells was assessed by flow cytometry. Th subset skewing was determined as ratios of CFSE^low cell percentages that produce Th1-type (IFN-γ) or Th2-type (IL-4) cytokines (and also TNF-α or IL-13). Points represent data from different individuals, while bars show median values. Significance was determined using the paired t test for log-transformed data (*P < 0.05; **P < 0.02). β-lg, β-lactoglobulin.

Figure 5

Th subset polarization of antigen-specific T lymphocytes from NA donors (n = 9). PBMCs were isolated, CFSE labeled, and cultured in the presence of antigens for 7 days, then the cytokine-producing phenotype of antigen-specific (CFSE^low) cells was assessed by flow cytometry. Th subset skewing was determined as ratios of CFSE^low cell percentages that produce Th1-type (IFN-γ) or Th2-type (IL-4) cytokines (and also TNF-α and IL-13). Points represent data from different individuals, while bars show median values. Significance was determined using the paired t test for log-transformed data (*P < 0.05; **P < 0.020).

Figure 6

Comparison of the Th subset polarization of antigen-specific T lymphocytes from PA (n = 9), NA (n = 9), and PA-outgrown donors (n = 4). PBMCs were isolated, CFSE labeled, and cultured in the presence of antigens for 7 days, then the cytokine-producing phenotype of antigen-specific (CFSE^low) cells was assessed by flow cytometry. Th subset skewing was determined as ratios of CFSE^low cell percentages that produce Th1-type (IFN-γ) or Th2-type (IL-4) cytokines (and also TNF-α and IL-13). Points represent data from different individuals, while bars show median values. Significance was determined using the Mann-Whitney test (*P < 0.05; **P < 0.02).

Figure 7

Comparison of the responses of Ara h2–specific T lymphocytes from PA (n = 4) and NA donors (n = 4). PBMCs were isolated, CFSE labeled, and cultured in the presence of 20 μg/ml purified Ara h2 for 7 days, then the cytokine-producing phenotype of antigen-specific (CFSE^low) cells was assessed by flow cytometry. Th-subset skewing was determined as ratios of CFSE^low cell percentages that produce Th1-type (IFN-γ) or Th2-type (IL-4) cytokines (and, respectively, TNF-α and IL-13). Points represent data from different individuals, while bars show median values. Significance was determined using the Mann-Whitney test (***P < 0.001; **P < 0.02).