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To the Editor,

Food allergy affects a significant number of children and is the most frequent reason for anaphylactic reactions at this age. Specific oral tolerance induction (SOTI) is an increasingly attractive strategy, and success has been achieved in a majority of patients. However, the immunologic mechanisms underlying SOTI are still unclear and additional advances in such therapies will require a greater understanding of the mechanism of tolerance/desensitization induction. We have recently published that allergic children have increased frequencies and absolute counts of effector-memory CD4+ T cells (TEM) in comparison with non-allergic children [1] and tolerance achievement after SOTI normalized the levels of these TEM [1]. TEM is a subset of cells with immediate effector function that can rapidly produce inflammatory mediators, playing a key role in food allergy [1, 2]. In addition to the deletion of reactive lymphocytes, the generation of regulatory T cells (Treg) has been also postulated as a mechanism capable of preventing inappropriate reactivity against innocuous antigens. Treg cells are a subset of CD4+ T cells with suppressive function [3], which have demonstrated in animal models a crucial role in the oral tolerance to food antigens [4]. However, whether Treg are implicated in the SOTI-mediated desensitization to food allergens has not been elucidated in humans.

Here, we performed an analysis of immune subsets in 18 egg-allergic children [mean (range) age = 9.26 (4–14) years], following a SOTI protocol with egg, comparing immune values before SOTI (T0) and when egg desensitization was achieved (Tend). All subjects had histories of acute clinical reactions to egg, including immediate reactions (urticaria, vomiting, and/or anaphylaxis), a positive egg-white skin prick test (SPT), and a positive serum-specific IgE. Prior to being included to the desensitization protocol, the clinical history records and immunological analysis were reassessed and an oral challenge test with egg was carried out on those who had not had clinical episodes within the last 3 months. A SOTI protocol with powdered pasteurized egg mixed with juice or milkshakes was established enabling patients to continue treatment at home, with periodic weekly visits to the outpatient allergy department. On the first day, fractionated doses were administered until reaching 31 mg of egg. Subsequently, weekly increases were made in the clinic until 10 g of powdered egg, the equivalent of one egg, was reached.

All the children enrolled in this study achieved a complete desensitization to egg. A child was considered desensitized when he was capable of ingesting 10 gr of powdered egg without any adverse reactions. A normal diet including egg was recommended to patients after desensitization achievement. 22 age-matched healthy children were also studied as control group [mean (range) age = 7.20 (3–13) years]. The study was conducted according to the Declaration of Helsinki, approved by the clinical ethics committee of the center, and written informed consent was obtained from all legal guardians. Percentage and absolute counts of Treg, CD4+ T-cell subsets, monocytes, basophils, and granulocytes were determined in fresh peripheral blood samples as previously described [5].

SOTI induces an increase in the frequency and number of Treg cells

The results showed that SOTI did not modify the percentages or absolute counts of monocytes, basophils, neutrophils, or eosinophils (Table 1). Thus, the immune mechanism implicated in the SOTI-mediated desensitization seems not to be associated with significant changes in these populations. We observed that desensitization (Tend) was related to a significant increase in the frequency and absolute counts of Treg (Table 1, Fig. 1a,b). Regarding the phenotype of these Treg, we did not observe significant changes in the proportion of naïve Treg (defined as CD4+CD25+Foxp3+CD45RA+) or effector Treg (defined as CD4+CD25+Foxp3+CD45RAneg) after SOTI. However, the increase in the Treg number was more significant for the effector-Treg subset (Table 1).

Table 1. Percentages and absolute counts of immune subsets in egg-allergic children before SOTI (T0) and after tolerance achievement (Tend). Values are given as median (25–75th percentiles)
 PercentageCells/μL
T0TendpT0Tendp
  1. a

    Percentage of Treg cells in the total of CD4+ T cells.

  2. b

    Percentage of naïve or effector Treg in the total of Treg cells.

  3. c

    Percentage of cells in the total of leukocytes.

  4. d

    Percentage of cells in the total of granulocytes.

  5. e

    p < 0.05 Wilcoxon test for paired samples.

Treg Cells7.89 (7.11–9.99)a8.42 (7.41–9.81)0.010e73 (49–93)84 (61–105)0.037e
Naïve Treg48.35 (43.7–55.2)b51.88 (44.5–55.6)0.94834 (24–49)39 (32–59)0.064
Effector Treg52.21 (45.4–56.5)b48.64 (45.0–56.1)0.87935 (26–48)43 (31–55)0.031e
Monocytes5.63 (4.91–7.38)c6.54 (4.95–7.93)0.723327 (263–475)383 (286–506)0.717
Basophils1.26 (0.91–1.87)c1.18 (0.74–1.57)0.98571 (52–86)75 (52–87)0.655
Granulocytes49.40 (42.8–55.4)c48.31 (40.8–52.1)0.1792624 (1772–3911)2499 (2096–3207)0.334
Neutrophils84.17 (74.3–87.7)d84.06 (76.4–90.2)0.6422084 (1530–3337)1982 (1744–2654)0.281
Eosinophils14.36 (10.9–23.6)d14.25 (8.9–20.0)0.796339 (231–660)460 (274–631)0.427
image

Figure 1. Treg values and ratio between Treg and TEM. Percentage (a) and number of Treg cells per μL of peripheral blood (b) in egg-allergic children (n = 18) before SOTI (T0) and after tolerance achievement (Tend). Horizontal lines represent the mean±S.E.M. Doted lines represents the variation from T0 to Tend in 3 representative children. (c) Mean±S.E.M ratio between the number (per μL of peripheral blood) of Treg and effector-memory CD4+ T cell (TEM) at T0, Tend and in a control group of age-matched healthy children (n = 22). *p < 0.05; **p < 0.01 using the Wilcoxon test for paired samples.

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Findings from animal models and human studies show that allergic diseases are due to an aberrant immune response mediated through the T-helper type-2 cells (Th2), and Treg cells have proved to be crucial in the control of allergic responses by suppressing allergen-specific effector T cells [6]. Here, we show that desensitization achieved after SOTI was accompanied by an increase in the frequency and number of Treg. In agreement with these findings, successful venom and aeroallergen immunotherapy have shown to be associated with the induction of peripheral tolerance in T cells by generation of Treg that secrete the suppressive cytokines IL-10 and TGF-β [6]. Little is known about how these Treg are exactly generated. Treg can be generated in the thymus, but they can also be induced in the periphery (named iTreg) by differentiation from conventional T cells [7]. We observed a slight increase in the number of naïve Treg, and thus, an increased thymic output cannot be excluded as the origin of the generated Treg. However, the Treg increase was more marked in the subset of effector Treg, pointing to a peripheral induction of Treg from naïve CD4+ T cells under subimmunogenic antigen presentation [7]. In fact, studies in mice showed that administration of antigen chicken ovalbumin (OVA) by oral route induced the generation of OVA-specific iTreg cells, but concomitant effector T-cell generation was inefficient [8]. Thus, SOTI could be favoring the generation of antigen-specific iTreg without increasing the differentiation or activation of effector CD4 T cells.

Treg increase is associated with a decrease in effector immune cells implicated in the allergic process

Immune response to allergens in health and disease is the result of a balance between Treg cells and effector T cells, and a change in the dominant subset may lead to allergy development or tolerance achievement [6]. We observed that the ratio between the number of Treg and TEM in periphery experimented a 2.3-fold increase after SOTI (p = 0.010), reaching a value comparable with the Treg/TEM ratio observed in healthy controls (p = 0.281) (Fig. 1c). As mentioned above, SOTI could induce an increase in the number of Treg without a concomitant increase in effector cells. This fact joined to the probed effect of Treg suppressing [3] and inducing the senescence of effector T cells would explain the profound change in the Treg/TEM ratio.

Effector CD4+ T cells are differentiated or expanded from naïve CD4+ T cells in the presence of a stimulus. Therefore, the proportion of TEM in periphery could be a good marker of the ‘reactivity’ of T cells in the presence of the allergen. In allergic children, increased number of circulating Treg was correlated with lower frequencies of TEM (Fig. 2a). However, this correlation was not observed in the control group (Fig. 2b). Therefore, we observed that the normalization of the TEM frequency previously described after SOTI [1] would be associated with the increase in the number of circulating Treg. This fact is supported by studies demonstrating that depletion of the Treg cells before in vitro culture significantly enhanced the expansion of antigen-specific effector T cells from allergic children [9].

image

Figure 2. Effect of Treg/TEM balance in effector cells. Correlation between the number of circulating Treg and the percentage of TEM in allergic (a) and healthy donors (b). Correlation between the percentage of TEM and the number of basophils per μL of peripheral blood in allergic (c) and healthy donors (d). Correlation was determined using the Spearman's correlation test.

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Basophils produce cytokines such as Interleukin-4 (IL-4) and IL-13, which are central for the manifestations of allergic disease, and have showed to participate in the tolerance to food allergens [10]. Inflammatory cytokines produced by effector CD4 T cells are essential in the expansion and activation of basophils and mast cells. Therefore, we analyzed how the balance between Treg and effector T cells could affect to basophils and granulocytes. We did not find any significant correlation between TEM and eosinophils or neutrophils (p > 0.05). However, in allergic children, we observed a direct correlation between the frequency of TEM and the number of circulating basophils (p = 0.039; Fig. 2c). Interestingly, this correlation between TEM and basophils was not observed in the control group (p = 0.462; Fig. 2d). Comparing values at T0 and Tend, we did not observed significant changes in the proportion or in the number of basophils after SOTI (Table 1). However, our results suggest that in allergic children, basophils and TEM are correlated, and the recovery of the Treg/TEM balance after SOTI could contribute to the normalization of the basophils values and the disappearance of symptoms.

Summing up, Treg cells are responsible for the control of effector immune responses against harmless environmental antigens, and their ratio determines the development of a healthy or an allergic immune response [6]. Here, we show that desensitization induction after SOTI was associated with an increase in the frequency and number of Treg that participate in the control of the effectors cells (notably TEM) implicated in the allergic process. We have not determined whether the Treg generated are allergen specific, due to the very low proportion of these cells in peripheral blood. However, changes in immune homeostasis after SOTI, which probably are more pronounced in the allergen-specific subsets [8], are also reflected in the peripheral values of total Treg. Due to the high rate of success with SOTI protocols, we have no data about children who do not acquire tolerance, and we cannot determine whether Treg values could constitute a reliable predictive marker of tolerance. However, our results provide an interesting basis for the use of this subset as a predictive marker of clinical outcome and as a tool to follow the efficacy of immunotherapy easily measurable in peripheral blood.

Acknowledgments

We thank all participants in the study and their legal tutors for providing blood samples. Also, we thank the Spanish HIV HGM BioBank and we thank Dr. Laura Díaz for her technical assistance of the Flow Cytometry Unit (CA11/00290) and Dr Maribel Clemente Mayoral for her technical assistance and advice as cell culture technician (CA10/01274). We thank Dr. Alberto Álvarez-Perea for critical reading and comments for this manuscript.

Sources of funding

This work was supported by grants from Fondo de Investigación Sanitaria (FIS PS09/02618; PI12/00934). Victoria Fuentes-Aparicio is supported by a grant from Sociedad Española de Inmunología Clínica y Alergia Pediátrica (SEICAP). Rafael Correa-Rocha is supported by the Fondo de Investigación Sanitaria through the ‘Miguel Servet’ program (CP07/00117).

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