The Surprising Discovery That TGFβ Specifically Induces the IgA Class Switch

The two Pillars of Immunology articles in this issue (1, 2) were the first to demonstrate that IgA switching is induced by TGFβ. In 1989, when these two important papers were published, it was known that IL-4 directs LPS-activated B cells to switch to IgG1 and IgE (3, 4, 5). It was also known that, in the absence of added cytokine, B cells switch to IgG3 and IgG2b and that the addition of IFN-γ induces IgG2a switching (6). However, how IgA, the most abundant Ig in the body, was induced was unknown. As IgA⁺ cells are most prevalent at mucosal sites, many expected that this induction would involve a factor(s) specific for the mucosa, but no such factor had been identified. TGFβ had been shown to inhibit B cell maturation, proliferation, and IgM and IgG production (7, 8), and therefore seemed an unlikely candidate for a factor that would stimulate class switch recombination (CSR).² Furthermore, although some evidence had suggested that IL-5 increased IgA expression, in these same Pillars articles it was demonstrated that IL-5 acted on postswitch IgA⁺ B cells to increase Ab production and also increased production of other isotypes besides IgA.

Coffman et al. (1) and Sonoda et al. (2) clearly showed that when TGFβ was added to LPS-activated IgM⁺ B cells, purified from either the spleen or Peyer’s patch (PP), they would switch to expressing IgA. They found that the frequency of cells expressing IgA and the amount of IgA secreted into the culture were increased, whereas IgM and IgG isotype secretion (except for IgG2b) were inhibited. They also demonstrated that TGFβ does not stimulate IgA production by postswitch IgA⁺ PP cells, thus supporting the hypothesis that TGFβ (in the presence of LPS) induces IgA switching rather than selectively stimulating postswitch cells. Subsequent limiting dilution experiments performed using IgA⁻ splenic B cells strengthened this conclusion by demonstrating that TGFβ increased the frequency of IgA-producing clones by 20-fold (9).

It had just been discovered that IL-4 treatment of activated B cells induces transcripts of the unrearranged (germline) Ig Cγ1 and Cε genes that initiate upstream of the switch region sequences, where CSR occurs (10, 11). Furthermore, IL-4 was shown to reduce transcripts of the unrearranged Cγ2b gene and to reduce IgG2b CSR (12). The germline transcripts (GLTs) were hypothesized to either create or to be a marker for switch region accessibility, allowing attack by the then unknown enzyme that initiates CSR. Subsequent experiments demonstrated that, in an analogous mechanism for how IL-4 directs CSR, TGFβ signaling induces transcription of α GLTs (13, 14) by activating SMADs 2 and 3, which then associate with SMAD4 and Runx3 that together bind to a tandem repeat element in the promoter for the α GLTs (15, 16, 17, 18, 19). Both mouse and human α GLT promoters are regulated similarly (20). These results led to the conclusion that TGFβ directs uncommitted IgM⁺ B cells to switch to IgA by inducing α GLTs.

IgA is well adapted for functioning in the gut due to its ability to be transported across epithelial cells and its relative stability to proteases. IgA controls the endogenous bacterial flora, preventing access to the gut epithelium and thereby reducing inflammation in the gut; without IgA, pathogenic bacteria expand and the resulting breach of the intestinal tissue barrier can lead to systemic and fatal spread of commensal bacteria (21, 22).

IgA CSR primarily occurs in PP (23, 24), which are specialized clusters of lymphoid cells located along the small intestine. IgA CSR also occurs in isolated lymphoid follicles (ILFs) scattered throughout the intestine (25, 26) and in nonlymphoid sites in lamina propria (LP) (27, 28, 29). IgA CSR in germinal centers within PP is dependent on T cells whereas T-independent responses induce IgA in the ILFs.

To determine how important TGFβ signaling in B cells is for IgA expression in vivo, Cazac and Roes (30) deleted the ligand-binding chain of the TGFβ receptor (TβRII) specifically in CD19⁺ B cells. Very few IgA-expressing cells were detected in the PP or LP, and very low levels of serum IgA were detected in unimmunized mice. Upon i.p. or intranasal immunization, no Ag-specific IgA was detectable in serum nor in nasal and bronchoalveolar lavages (30, 31). The few IgA⁻expressing cells detected (∼3% of normal) might be due to leaky expression of the TβRII receptor by B cells or, alternatively, to inefficient alternative mechanisms for inducing IgA CSR (32, 33, 34). Levels of IgM and IgG isotypes were increased in the TβRII-deficient mice, except for IgG2b, consistent with the inhibitory activity of TGFβ on Ab production and the fact that TGFβ stimulates CSR to IgG2b, in addition to IgA (35).

TGFβ is produced by hematopoietic and parenchymal cells located throughout the body, so how can IgA CSR be nearly entirely restricted to the mucosal region? TGFβ appears to function near where it is produced and processed. It is synthesized in a precursor form that is generally activated by proteolysis at the site of function (reviewed in Ref. 36), and expression of TGFβ in the liver and secretion into the blood does not prevent the fatal autoimmunity that develops in Tgfb^−/− mice (37).

Substantial progress has been made recently in defining the nature of the unique mucosal milieu that promotes IgA CSR. There are four potential functionally relevant sources of TGFβ in the gut: B cells, T cells, APCs, and stromal cells.

Activated B cells (human and mouse) secrete TGFβ, and treatment of cultured B cells with blocking Ab for TGFβ increases IgG and suppresses IgA production, indicating that TGFβ from B cells can act as an autocrine factor (38, 39); but whether B cell-produced TGFβ contributes to IgA induction in vivo has not been tested.

Activated T cells are a major source of TGFβ (40), particularly T cells rendered tolerant to Ags and those capable of imposing lymphocyte self-tolerance in trans. TGFβ-producing CD4⁺ T cells have been cloned from mesenteric lymph nodes of mice that were fed myelin basic protein to induce tolerance to this Ag (41). These and other TGFβ-producing T cells have been shown to suppress inflammatory immune diseases such as experimental autoimmune encephalitis and inflammatory bowel disease (36, 41, 42, 43). Some, but not all, of the CD4⁺ cells that produce TGFβ are FOXP3⁺ regulatory T cells. TGFβ originating from T cells is critical for controlling colitogenic T cells, regulatory T cell function, and effector T cell differentiation, especially the generation of proinflammatory Th17 cells (36), but perhaps not for IgA induction. It is not clear that there are more TGFβ-secreting T cells associated with the mucosal lymphoid tissue than in other parts of the body, and there is no convincing evidence that T cell-produced TGFβ is essential for inducing IgA CSR.

Recent data have solidified the idea that local mucosal dendritic cells (DCs) are the primary regulators of IgA production. In the mesenteric lymph nodes, CD103⁺ DCs produce abundant amounts of TGFβ and the vitamin A metabolite retinoic acid, the latter implicated as a cofactor in IgA CSR (44, 45, 46). Similar DCs are found in PP and LP of the intestine and colon, and at a significantly higher frequency in LP than in other lymphoid tissues (47, 48). Although CD103⁺ DCs are likely to be involved in IgA CSR, their exact role has not been demonstrated, and their lineage relationships to other DC subsets in the intestine implicated in IgA synthesis that are discussed below are also imprecisely defined.

As mentioned above, IgA CSR occurs in the absence of T cells in ILFs that are induced in the small intestine upon bacterial infection and during constant surveillance of commensals (26). The ILFs contain lymphoid tissue inducer cells, gut stromal cells, and DCs, and they recruit B cells that then switch to IgA in the ILF. Stromal cells are most likely the major source of TGFβ in the ILF (26). TGFβ in ILFs is made as an inactive pre-pro form and must be activated by the integrin α_vβ₈ on DCs (49) and by TNF-α-induced metalloproteinases, which are produced by both gut stromal cells and DCs. Lymphoid tissue inducer cells and DCs in ILFs produce TNF-α (26). Interestingly, splenic DCs and stromal cells do not express high levels of these metalloproteinases, pointing to another level of regulation that biases IgA production to the mucosa.

The LP of the mouse small intestine is rich in stromal cells that secrete TGFβ (22). LP DCs take up Ags from the intestinal lumen and are hypothesized to directly activate Ag-specific IgM⁺B220⁺ B cells to undergo IgA class switching in the absence of cognate T cell help (27, 29). One DC subset especially prevalent in LP that might be central to IgA production is the TNF-α-producing, inducible NO synthetase (iNOS)⁺ DCs (Tip-DCs) (45, 50). Tip-DCs are missing in mice with defects in TLR signaling, suggesting that Tip-DCs constitute a central coordinator of bacteria sensing and IgA production in mucosal tissues. Interestingly, the bacterial constituent-sensor iNOS produced by these DCs has been implicated in regulating TβRII expression on B cells, and iNOS^−/− mice exhibit impaired IgA production (45). Hence, distinct microenvironments and cell subsets within the gut display remarkable flexibility and drive toward IgA synthesis that is dependent primarily on local TGFβ acting in synergy with other cytokines.

Although the finding that TGFβ induces IgA CSR was originally surprising, it is now clear that the high levels of active TGFβ present at mucosal tissues drive IgA induction in concert with several cofactors (e.g., retinoic acid, vasoactive intestinal peptide, BAFF, and APRIL) (26, 29, 32, 33, 45, 46) and with DCs that are specialized not only in sampling heavy bacterial burden but are capable of eliciting appropriate, finely tuned immune responses in a spatially restricted fashion. In retrospect, these Pillars articles are a prelude to the emergence and current prominence of TGFβ as the critical cytokine for maintaining a delicate life-and-death balance between inflammation and immune tolerance at the mucosal frontier, which is continually assaulted by commensal bacteria, food-borne Ags, and foreign pathogens.