This study showed that an in vivo milk challenge induced Treg cells preferentially in previously cow's milk–allergic children who had become tolerant, that is, those who clinically had outgrown their allergy. It is intriguing how the intestinal immune system can discern between pathogens and innocuous antigens such as dietary proteins and respond by eliciting productive immunity or oral tolerance, respectively. The understanding of the latter phenomenon has been hampered by intricate mechanistic complexity involving T cell anergy, clonal deletion, skewing of the cytokine profile (immune deviation), and the action of different Treg cells. We established here for the first time that human CD4+CD25+ Treg cells induced via the gut are involved in the control of food allergy, and that this T cell subset after activation exerts its suppressive function at least partially in a cell contact-dependent manner.
Our study compared phenotypic and functional characteristics of PBMCs from children with clinically active or outgrown cow's milk allergy collected before and 1wk after an in vivo challenge with cow's milk. PBMCs from the tolerant group showed reduced proliferation induced in vitro by the major milk allergen β-LG. Furthermore, the same children had a CD25+
T cell subset within their PBMCs that was able to suppress such proliferation. In this group, 30% of CD4+
T cells expressed the maturation marker CD45RO both before and after challenge with cow's milk, whereas the corresponding figure was only some 5% in the group with active allergy. All children included in our study had negative skin prick test against cow's milk proteins and no elevated specific IgE antibody levels in serum. This suggested that the observed symptoms were most probably produced via cell-mediated immunity. Approximately 50% of children suffering from food allergy appear by standard diagnostic tests to have a non-IgE–mediated allergic reaction (43
), presumably delayed-type hypersensitivity, although the pathological process in the gut may be indistinguishable from an atopic disorder (45
). We selected our patients to be homogeneously nonatopic, but it would certainly be of interest in the future to also study children with elevated IgE antibody levels against food allergens in a similar experimental set-up.
Such studies in young children (with repeated sampling) have profound ethical restrictions. Therefore, we had to design our in vitro experiments on the basis of the limited blood volumes available. We also had to take into account that with the large age range of the patients (6–56 mo), they had a highly variable immunological history in terms of exposure to antigens from vaccines and the environment. This related not only to their systemic but also to their mucosal immune system. Importantly, we investigated tolerance induction via the gut, and recent animal experiments have demonstrated that the systemic and local immunization routes provide different results when it comes to dampening of mucosal allergy (46
). In view of this complex scenario, we decided that PHA stimulation, rather than an arbitrarily selected T cell–dependent antigen, would best ensure a consistent base-line response as a reference for the mucosal induction of Treg cells by cow's milk proteins.
The in vitro proliferative activity of PBMCs against β-LG was presumably a T cell recall response. Notably, however, there was no significant difference when we compared PBMCs collected from the two groups of children before the in vivo cow's milk challenge with regard to proliferation elicited by β-LG, frequency of CD4+CD25+ T cells, and a suppressive effect of CD25+ cells revealed by their depletion. This result probably reflected that the patients with active allergy at this time point did not have a substantially larger number of circulating memory T cells specific for β-LG than tolerant children. The avoidance of cow's milk proteins for at least 2 mo before the challenge might have reduced this number, and specific T cells activated in GALT and mesenteric LNs would presumably start homing back to the intestinal lamina propria during milk challenge. Thus, there was a reduced relative frequency of circulating CD4+CD25+ T cells in most allergic children after the challenge ( B).
It is of note that the β-LG preparations contained LPS comparable to an endotoxin activity of 5000 EU/ml at 5 mg/ml (Limulus Amebocyte Lysate test); but the polyclonal immunostimulation exerted by this contamination did not mask the antigen-specific T cell response elicited by β-LG in vitro (unpublished data). Interestingly, the proliferative response of PBMCs against β-LG was significantly decreased in milk-tolerant compared with allergic children after the in vivo milk challenge. Our findings therefore suggested that in the tolerant children this challenge had resulted in GALT activation and expansion not only of specific effector T cells but also of CD4+CD25+ Treg cells, which suppressed the in vitro proliferative response elicited by β-LG. The observed relative increase of this circulating subset in half of the tolerant children 1 wk after challenge ( B) was probably an underestimate due to homing of allergen-activated T cells to the milk protein–exposed gut lamina propria as alluded to above.
Although it was originally believed that CD4+
Treg cells are anergic, at least two independent reports recently showed that mouse CD4+
T cells initially multiply in vivo after appropriate antigen stimulation (26
). We cannot exclude that the increased frequency of circulating CD4+
T cells after challenge in tolerant children partly reflected expansion of ordinary memory–effector T cells, but the striking increase (fivefold) of proliferative activity against β-LG exhibited by PBMCs from these children after depletion of CD25+
cells strongly suggested that their CD4+
subset contained a substantial fraction of Treg cells. This homeostatic mechanism most likely explained that they had outgrown their cow's milk allergy. For ethical reasons we could not obtain blood at a later time point after the milk challenge, so we have no information with regard to the persistence of the elevated level of CD4+
T cells in the circulation of the tolerant children.
Expression of integrin α4β7 and CCR9 on T cells determines their homing into the small intestinal lamina propria (48
). We analyzed the frequency of T cells with these markers, but there were no apparent differences between the two groups of children. It has been reported that the α4β7 subset is increased in peripheral blood from children with cow's milk allergy, but that finding was obtained with T cells kept in culture for 1 wk (49
). Also, it might be unrealistic to expect that we should be able to identify a phenotypic change induced by milk proteins in the total circulating pool of T cells with gut-homing properties.
Most functional studies of Treg cells have been performed in animal models for autoimmunity, inflammatory bowel disease, and transplantation. The first human studies of CD4+
Treg cells were in fact published quite recently (39
). Evidence suggests that Treg cells may be antigen nonspecific, at least in the effector phase, and bystander suppression is a well-known phenomenon in oral tolerance (53
). A nonspecific effect of Treg cells could explain why we obtained a moderate enhancement of proliferation against PHA in PBMCs from tolerant children after depletion of CD25+
cells (). Altogether, our results implied that mucosally induced Treg cells responding to milk antigens were involved in the observed tolerance induction or, alternatively, that centrally generated Treg cells had become activated and expanded in the children with outgrown cow's milk allergy.
Expression of CTLA-4 has been associated with human CD4+
Treg cells (55
). This molecule is constitutively expressed in the cytoplasm (27
), but once the cell is activated via the TcR complex it appears on the surface. However, the mechanistic role of surface CTLA-4 in cell contact–dependent suppression in humans is debated. Several studies show that blocking CTLA-4 with antibodies does not abolish the suppressive activity of Treg cells (39
). We did not find any difference in the expression level of this surface marker, either between the two groups of children or the two time points for sampling of PBMCs. Nevertheless, transwell experiments demonstrated that CD4+
Treg cells from milk-tolerant children exert their antiproliferative effect via a contact-dependent mechanism. Regrettably, we had no possibility to study the involvement of other inhibitory molecules such as PD-1 (39
The role in oral tolerance of the immunosuppressive cytokine TGF-β derived from so-called regulatory Th3 cells induced in experimental animals is extensively documented (57
). The reduced production of TGF-β by β-LG–stimulated PBMCs after the in vivo milk challenge in the allergic group ( A) is in line with a recent study by Perez-Machado et al. (58
) which shows that T cells obtained from duodenal mucosa of food-allergic children had a decreased potential for TGF-β production compared with healthy controls. Conversely, we found that PBMCs stimulated by β-LG 1 wk after the in vivo challenge tended to show increased production of TGF-β in children with outgrown allergy, which could have contributed to the observed suppressive effect on proliferation. The major source for IL-10 production in β-LG–stimulated PBMC cultures in our experiments remains unknown, and the results obtained were inconclusive.
It has been shown in mouse models that the thymic epithelium is important in the generation of naturally occurring CD4+
Treg cells involved in the maintenance of tolerance (59
). Because our study strongly suggests that CD4+
Treg cells reactive to dietary antigens can be induced via the gut, it will be of considerable interest to investigate whether exosomes released from the intestinal epithelium play a role in the development of such cells. We have previously obtained results indicative of a tolerance-inductive function of epithelial exosomes produced in the gut of experimental animals and therefore called these structures “tolerosomes” (60
In conclusion, the observations presented here provide new insight into the possible relationship between mucosal induction of CD4+CD25+ Treg cells and protection against food allergy in humans. These results may aid the development of better diagnostic tools for such disorders and perhaps contribute to promote a future goal of generating Treg cells in vitro for immunotherapy of allergic diseases.