Naturally occurring tolerance acquisition to foods in previously allergic children is characterized by antigen specificity and associated with increased subsets of regulatory T cells

Abstract

Background: Food allergy affects approximately 6-8% of children, and increasing in prevalence. Some children naturally outgrow their food allergy without intervention, but the mechanisms by which this occurs remain poorly understood. We sought to investigate the role of regulatory T cells in the development of naturally acquired tolerance.

Methods: Fifty-eight children (1-18 years) with either egg or peanut allergy, recent acquisition of natural tolerance to egg or peanut, or no food allergy were studied. Peripheral blood mononuclear cells (PBMC) from these groups were stimulated with relevant antigen for 48 h and flow cytometry performed to characterize both surface (CD3, CD4, CD25, CD14, CD19, and CD127) and intracellular markers (IL-10, Foxp3, and IL-5).

Results: Resting PBMC from naturally tolerant patients had significantly increased CD3+CD4+CD25+CD127loFoxp3+ cells, when compared to allergic or control patients (mean 6.36 vs. 2.37 vs. 2.62%, respectively, P < 0.05). Upon stimulation with relevant antigen, naturally tolerant patients also had increased IL-10-expressing CD25+CD127lo cells (6.33 vs. 1.65 vs. 0.7, P < 0.01), Foxp3+ cells (mean 12.6 vs. 5.42 vs. 3%, P < 0.01), and CD4+ cells (mean 4.48 vs. 1.59 vs. 0.87%, P < 0.01); the increase was not observed in PBMCs from allergic or control patients. Additionally, this upregulation was only seen with relevant antigen stimulation and not upon stimulation with unrelated antigen.

Conclusion: The increased CD3+CD4+CD25+CD127lo cells at baseline and upon stimulation and increased induction of IL-10-producing cells of several types, including Tr1 cells, from naturally tolerant patients suggests an important role for regulatory T cell subsets in the acquisition of natural tolerance.

Keywords: IL-10; T regulatory cells; antigen specificity; food allergy; natural tolerance.

Fig. 1. Naturally Tolerant Patients had Increased CD4+CD25+CD127loFoxp3+ T Regulatory Cells at Baseline

A. Tolerant patients had increased thymically derived T regulatory cells as compared to allergic and control patients. B. There was no difference in Foxp3 MFI in these cells among tolerant patients as compared to allergic and control patients. C. At baseline, there was no difference in CD25+CD127lo cells among control, allergic, and tolerant patients. Data are expressed as the mean percentage cells within gated CD3+CD4+ T cells. Box plots with median as dark horizontal line in wide bars representing lower and upper quartiles. Whiskers are minimum and maximum. * represents p-value <0.05 calculated with Kruskal-Wallis test and Mann Whitney for pairwise comparisons.

Fig. 2. Naturally Tolerant Patients had Increased IL-10 Producing T Regulatory Cell Subsets and Decreased IL-5+Foxp3+ Cells After Stimulation with Relevant Antigen Compared to Control and Allergic Patients

Upon stimulation with relevant antigen: A. tolerant patients had increased IL-10 producing CD25+CD127lo cells when compared to control and allergic patients. B. Tolerant patients had increased IL-10 producing Foxp3+ cells compared to control and allergic patients. C. Tolerant patients had increased IL-10 producing CD4+ cells compared to control and allergic patients. D. Tolerant patients had decreased IL-5 producing Foxp3+ cells compared to control and allergic patients. Scatterplots with dark horizontal line representing mean and whiskers representing standard deviation. * represents p-value <0.05 calculated with Kruskal-Wallis test and Mann Whitney for pairwise comparisons.

Fig. 3. Tolerant Patients Increased IL-10 Producing CD4+ Cells and CD25+CD127lo Cells Upon Stimulation with Relevant Antigen

A. The percentage of IL-10 producing CD4+ cells increased in tolerant patients upon stimulation with relevant antigen. B. The percentage of CD25+CD127loFoxp3+ cells decreased in tolerant patients and did not change in allergic or control patients. C. Percent change in IL-10 producing CD4+ cells in tolerant patients increased and not in allergic and control patients. D. Percent change in IL-10 producing CD25+CD127lo cells increased in tolerant patients compared to allergic and control patients. Data expressed in A, B represent the mean percentage cells within gated CD3+CD4+ T cells. Data in C, D represent the absolute difference between unstimulated and stimulated conditions. Bar graph with whiskers representing standard deviation. Box plots with median as dark horizontal line in wide bars representing lower and upper quartiles. Whiskers are minimum and maximum. * represents p-value <0.05 calculated with Kruskal-Wallis test and Mann Whitney for pairwise comparisons. ** represents p-value <0.005 calculated with Kruskal-Wallis test and Mann Whitney for pairwise comparisons.

Fig. 4. Tolerant Patients Increased IL-10 Producing Cells in Response to Relevant Antigen Only

Upon stimulation with relevant antigen versus unrelated antigen: A. Tolerant patients increased IL-10 producing CD4+ cells in response to relevant antigen only and not unrelated antigen. B. There was no increase in IL-10+CD4+ cells in allergic patients in response to unrelated or relevant antigen. C. Tolerant patients increased IL-10 producing CD25+CD127lo cells in response to relevant antigen only and not irrelevant antigen. D. There was no increase in IL-10+CD25+CD127lo cells in allergic patients in response to unrelated or relevant antigen. Data are expressed as the mean percentage cells within gated CD3+CD4+ T cells (A,B) and within gated CD25+CD127loCD4+ cells (C,D). Scatter plots with mean as dark horizontal line. * represents p-value <0.05 calculated with Kruskal-Wallis test and Mann Whitney for pairwise comparisons.