B lymphocytes are an important cell population of the immune regulation; their role in the regulation of food allergy has not been fully understood yet.
B lymphocytes are an important cell population of the immune regulation; their role in the regulation of food allergy has not been fully understood yet.
This study aims to investigate the role of a subpopulation of tolerogenic B cells (TolBC) in the generation of regulatory T cells (Treg) and in the suppression of food allergy-induced intestinal inflammation in mice.
The intestinal mucosa-derived CD5+ CD19+ CX3CR1+ TolBCs were characterized by flow cytometry; a mouse model of intestinal T helper (Th)2 inflammation was established to assess the immune regulatory role of this subpopulation of TolBCs.
A subpopulation of CD5+ CD19+ CX3CR1+ B cells was detected in the mouse intestinal mucosa. The cells also expressed transforming growth factor (TGF)-β and carried integrin alpha v beta 6 (αvβ6). Exposure to recombinant αvβ6 and anti-IgM antibody induced naive B cells to differentiate into the TGF-β-producing TolBCs. Coculturing this subpopulation of TolBCs with Th0 cells generated CD4+ CD25+ Foxp3+ Tregs. Adoptive transfer with the TolBCs markedly suppressed the food allergy-induced intestinal Th2 pattern inflammation in mice.
CD5+ CD19+ CX3CR1+ TolBCs are capable of inducing Tregs in the intestine and suppress food allergy-related Th2 pattern inflammation in mice.
As one of the antigen-presenting cell types, B cells also capture, process, and present antigens to T cells to modulate the T cells' activities . Similar to a subset of regulatory T cells (Treg), a subpopulation of regulatory B cells (Breg) also expresses TGF-β, which is featured as CD19+ CD5+ and produces large amounts of transforming growth factor-β (TGF-β) in response to specific antigen stimulation [2-4]. Functional impairment of the TGF-β-producing B cells is associated with the pathogenesis of food allergy [2-4]; the fact implicates that this subpopulation of B cells may play an important role in the immune regulation of the intestine. Thus, it is necessary to further elucidate its sources, regulatory mechanism, and its association with Treg-generation in the intestine.
TGF-β is one of the major immune regulatory molecules; it plays multiple roles in the immune regulation  and is also involved in a number of inflammatory processes [6, 7]. After synthesis, TGF-β exists as a precursor, the latent TGF-β (LTGF-β). LTGF-β needs to be activated by removing the latency associated peptide from the complex before possessing its immune regulatory function. A number of approaches have been employed to activate LTGF-β, such as via heating, exposing to acidic pH, or matrix-metalloproteinases. The integrin alpha v beta 6 (αvβ6) can directly bind to the arginine-glycine-aspartic acid binding site to activate LTGF-β . Our previous work indicates that intestinal epithelial cell-derived αvβ6 can activate the LTGF-β in dendritic cells (DC) . Whether the intestinal epithelium-derived αvβ6 is also involved in the conversion of LTGF-β in B cells is unclear.
The significance of TGF-β in the immune tolerance is defined by its effect on the generation of tolerogenic DCs and Tregs . Exposure to TGF-β can induce naive T helper (Th) cells to differentiate into Tregs  and induce immature DCs to tolerogenic DCs . The effect of tolerogenic DC-derived TGF-β on the generation of Tregs has been recognized . B cells can capture and present antigens to T cells. Some B cell subpopulations are involved in the immune regulation. The concept of tolerogenic B cells (TolBC) was proposed early  and further studied recently . However, the TolBCs in the intestine have not been characterized yet. In this study, we observed a subpopulation of TolBC expressed the chemokine receptor, CX3CR1, that was capable of inducing Tregs as well as to suppress the food allergy-induced inflammation in the intestine.
Experimental procedures are presented in the Supporting Information.
We isolated cells from the Peyer's patch (PP), lamina propria mononuclear cells (LPMC), the mesentery lymph nodes (MLN), and the spleen. As assessed by flow cytometry, β6+ B cells were detected in PP cells (7.1%) and LPMC (6.7%), but were scarce in either MLN cells (0.7%) or spleen cells (0.8%) (Fig. 1A). The results indicate that a subpopulation of B cells in the intestine carries αvβ6. To confirm the results, we isolated the CD19+ B cells by magnetic cell sorting (MACS) and extracted the total RNA and proteins to be analyzed by qRT-PCR and Western blotting. The results (Fig. 1B) from Western blotting were in line with the data of flow cytometry; however, β6 mRNA in the cells was below the detectable levels (Fig. 1C). The results indicate that the intestinal B cells do not express αvβ6. The detected αvβ6 in the B cells (Fig. 1A) may be captured from exogenous sources.
Our previous reports  indicate that intestinal epithelial cells produce αvβ6. Thus, the detected αvβ6 in the intestinal B cells (Fig. 1A) may be derived from the epithelial source. As intestinal epithelial cells produce fractalkine, a ligand of CX3CR1 , we postulated that some of the intestinal B cells might express the receptor of fractalkine, the CX3CR1. We then analyzed the expression of CX3CR1 in the cells of Fig. 1A. The results showed that more than 90% of the αvβ6-carrying B cells expressed CX3CR1, while only 6.14% of the non-αvβ6-carrying B cells expressed CX3CR1 (Fig. 1D). To elucidate whether B cells can capture exogenous αvβ6, we isolated spleen B cells and cultured in the presence of αvβ6. The cells were collected at several time points from 0 to 30 min and analyzed by Western blotting. The results showed that the B cells did capture exogenous αvβ6 in the exposure time-dependent manner (Fig. 1E). Further analysis showed that this subpopulation of B cells also expressed CD5 (92.6%), CD45R (90.1%), CD35 (79.4%), and CD80 (10.6%); none of the cells expressed CD138. It was noteworthy that 91.5% of the cells also expressed TGF-β (Fig. 1F).
We next observed whether the endocytic αvβ6 enhanced the TGF-β levels in the αvβ6-carrying B cells. We isolated the IL-7R+ CD19+ CD45R+ naïve B cells from the BALB/c mouse bone marrow and analyzed by Western blotting. TGF-β was barely detected in the naive B cells (Fig. 2A). However, mild levels of LTGF-β were detected in naïve B cells, which were markedly increased after the activation by anti-IgM stimulation (Fig. 2B). The results imply that the LTGF-β may be converted to TGF-β in B cells. To test the hypothesis, we treated the isolated naive B cells with anti-IgM or/and αvβ6 in the culture for 3 days. The results showed that moderate levels of TGF-β were detected in the B cells after treating with both anti-IgM and αvβ6, but not with either anti-IgM or αvβ6 alone (Fig. 2C–D). The TGF-β was also released to the culture supernatants (Fig. 2E). The results imply that these B cells may fulfill the task of TolBCs with the TGF-β as immune regulatory mediator. Therefore, we designated this subpopulation of B cells as TolBCs.
We next tested the immune suppressor function of the generated TolBCs. The TolBCs were co-cultured with Th0 cells in the presence of activators for 3 days. As shown by Fig. 3, about 27% Th0 cells proliferated after stimulating with anti-CD3/CD28, which was inhibited by the presence of activated TolBCs in a TolBC number-dependent manner, which was abolished by adding neutralizing anti-TGF-β antibody to the culture; while the addition of neutralizing anti-IL-10 antibody, or an irrelevant protein, bovine serum albumin (BSA), did not show any detectable inhibitory effect. To elucidate whether the cell–cell contact was required in the suppressor action, in separate experiments, the TolBCs and Th0 cells were physically separately cocultured in Transwells. The activation of T cell was still suppressed by the activated TolBCs (data not shown). Collectively, the results indicate that the activating TolBCs have an immune suppressor effect on T-cell activation via releasing TGF-β; the cell–cell contact is not required in the process.
It is reported that TGF-β can induce the inducible Tregs ; thus, we postulated that the αvβ6-carrying TGF-β-producing TolBCs might be able to convert naïve T cells to Tregs. To test the hypothesis, we repeated the experimental procedures of Fig. 3 to detect the possibly generated Foxp3+ Tregs. Indeed, coculturing with TolBCs, a portion of naïve CD4+ T cells expressed Foxp3 in a coculturing TolBC number-dependent manner (Fig. 4). The induced Tregs had the immune suppressor function on other T-cell activation (data not shown).
To further test the immune regulatory capability of the TolBCs, we developed an animal model of Th2 inflammation of the intestine. The mice were adoptively transferred with TolBCs or naïve B cells or saline on day 0 and day 15, respectively, in the course of the 35-day sensitization. The mice were challenged with 5 mg specific antigen via gavage-feeding on day 35 and killed on day 36; the mice were subjected to analysis of food allergy symptoms and the Th2 inflammation of the intestine. As compared with the group received TolBCs, the group of food allergy mice treated with saline or received naïve B cells showed (1) higher serum levels of IL-4, IL-5, IL-13, OVA-specific IgE, mouse mast cell protease-1 (mMCP-1) (Fig. 5A–E); (2) higher frequency of mast cells and eosinophils in the intestine (Fig. S1); (3) higher frequency of intestinal OVA-specific Th2 cells (Fig. S2); and (4) drop of core temperature and had diarrhea (Fig. S3). (5) In addition, higher frequency of Tregs was detected in the intestine of mice received TolBCs (Fig. S4). The results indicate that the TolBCs can inhibit food allergy symptoms and the antigen-specific Th2 inflammation in the intestine.
Currently, the specific immunotherapy is regarded as the only specific remedy for the treatment of allergic diseases currently [16-18]. The present study provides a novel specific method to suppress the allergic inflammation. A subpopulation of B cells carries αvβ6 in the mouse intestine. This subpopulation of B cells expresses CD19 and does not express CD138, indicating the cells are not plasma cells. Analysis by qRT-PCR revealed that this subpopulation of B cells did not express αvβ6 mRNA, indicating the αvβ6 detected inside the cells was captured from exogenous sources. This subpopulation of B cells has high levels of TGF-β, can convert Th0 cells to Tregs, suppress T-cell activation, and inhibit food allergy symptoms and the Th2 inflammation in the intestine. On the basis of these features, we designate this subpopulation of B cells the TolBCs.
The tolerogenic DCs has been well described in the immune regulation . Besides, some macrophages also have the ability of inducing immune tolerance . The concept of TolBC was proposed in the middle of 1990s , and the research in this area was strengthened in the recent years . Thus, all the three types of antigen-presenting cells can be the tolerogenic cells. Our data reveal a novel subset of TolBCs. In line with previous reports, which indicate that TolBCs express CD5 and CD19 [2, 4, 20], the present subset of TolBCs also expresses CD5 and CD19. In addition, this subset of TolBCs expresses a chemokine receptor, the CX3CR1; the latter may enable these cells migrate to the ligand sources, the intestinal epithelium, where the epithelial cells express the ligand of CX3CR1, the fractalkine [20, 21]. The expression of CX3CR1 also defines the sources of αvβ6 are the intestinal epithelial cells because the epithelial cells produce αvβ6, the latter can be released out the epithelial cells carrying by exosomes [9, 22].
As TGF-β exists as the precursor form, the LTGF-β, after synthesis, the conversion of LTGF-β to TGF-β is a required procedure. A number of factors can convert the LTGF-β by cleaving the latency association peptide from TGF-β, such as high temperature, acidic pH, reactive oxygen species, integrins, plasmin, and the matrix protein thrombospondin-1 [9, 23]. Similar to our previous study  that shows the intestinal epithelial cell-derived αvβ6 activates the LTGF-β in DCs to promote the DCs' tolerogenic property, the present data show that αvβ6 also converts LTGF-β to be TGF-β in B cells and converts the B cells to be TolBCs. Intestinal epithelial cells are the source of αvβ6 in the intestine. A large number of B cells gather in the PP of the intestinal mucosa; this anatomical feature implies that only those B cells in PP have the potential to become TolBCs. However, the B cells isolated from LPMC also showed similar rates of TolBCs to that of PP as shown by the present data. The results were explained by the expression of CX3CR1 of the TolBCs that facilitates the B cells to migrate to the direction of the epithelium to capture αvβ6. Our ex vivo data support this inference that the isolated naïve B cells do capture exogenous αvβ6.
The major tolerogenic action of the tolerogenic cells is to induce Tregs via producing some cytokines, which include TGF-β, IL-10, indoleamine 2,3-dioxygenase (IDO), and some others [10, 24, 25]. By releasing the tolerogenic molecules, tolerogenic cells induce naïve Th cells to be Tregs. Similar to tolerogenic DCs, the TolBCs can also induce Tregs as shown by the present data. TGF-β, not IL-10, was the tolerogenic molecule in the induction as blocking TGF-β abolished the induction of Tregs, which was not affected by the blocking IL-10. Similar results were reported by other investigators; Zheng et al. reported that CD40-activated B cells are more potent than immature dendritic cells to induce and expand CD4+ Tregs , in which the cell–cell contact was required, while in the induction of Tregs by our generated TolBCs, the cell–cell contact is unnecessary. The differences between our results may be because that Zheng et al. found that the CD40-activated B cells inducing Tregs partially rely on modulating the expression of human leukocyte antigen (HLA)-DR and CD80/86, while our data indicate that the CX3CR1+ TolBC-derived TGF-β is the major functional cytokine in the induction of Tregs, which can be released out of the cells.
Apart from the clone deletion and T-cell anergy, the activities of Tregs play an important role in the immune tolerance . TGF-β is one of the most important mediators released by Tregs to fulfill the immune suppressor actions. In addition to the conversion of naïve T cells into Tregs, TGF-β suppresses the effector Th-cell differentiation, inhibits the proliferation of T cells and B cells, down-regulates the effector cytokine production, and suppresses macrophages, DCs, and natural killer (NK) cells . As the TolBCs express high levels of TGF-β, the cells have the potential to suppress the skewed immune responses by themselves in addition to induction of Tregs. Indeed, the CX3CR1+ TolBCs showed the suppressor effect on T-cell activation in vitro and also showed the suppressive effect on the antigen-specific Th2 inflammation in the intestine.
Taken together, the present study has revealed a subpopulation of B cells in the mouse intestine. The cells express cell markers of CX3CR1, CD5, and CD19, produce TGF-β, induce naïve T cells to Tregs, and suppress food allergy symptoms and Th2 inflammation in the intestine.
This study was supported by grants from 973 Program (#2011CB512006) of China, the Natural Science Foundation of SZU (#000004), the innovation of science and Technology Commission of Shenzhen Municipality (JCYJ20120613172559904), the National Natural Science Foundation of China (No.81271950、31101280; 31070799), and the Key Laboratory Project of Shenzhen (No. SW201110010).
Z.Q.L., Y.W., J.P.S., X.L., S.H., Z.L. and P.Y.Z. were involved in performing experiments, analyzing data, and reviewing the manuscript. P.C.Y. designed the project, supervised the experiments, and wrote the manuscript.
The authors have no conflict of interest to declare.