Organogenic Role of B Lymphocytes in Mucosal Immunity

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Science  03 Dec 1999:
Vol. 286, Issue 5446, pp. 1965-1968
DOI: 10.1126/science.286.5446.1965


Follicle-associated epithelium (FAE) in the intestinal Peyer's patches contains M cells that deliver pathogens to organized lymphoid tissue. Development of Peyer's patches, FAE, and M cells was found to be impaired in mice that had no B cells. Transgenic expression of membrane-bound immunoglobulin M restored B cells and FAE development. The lack of M cells abrogated infection with a milk-borne retrovirus. Thus, in addition to secretion of antibodies and presentation of antigens, B cells are important for organogenesis of the mucosal immune barriers.

The gut-associated lymphoid tissue (GALT) consists of highly organized Peyer's patches (PPs) in the small intestine and intraepithelial lymphocytes (IELs) found throughout the length of the gastrointestinal tract. The intestinal surface of PPs is characterized by the presence of FAE-covering “domes,” regions free of intestinal villi (1). M cells are found in these domes, scattered among enterocytes (2). M cells lack microvilli on their apical surface (hence the term M, denoting microfold or membranous cells) and are able to tunnel pathogens through the cytoplasm to the basal surface, where deep invaginations of their membrane allow close contact with lymphocytes and macrophages (3). In respiratory epithelium, where M cells are also found, they serve as the entrance gates for pathogens such as mycobacteria (4). In vitro, M cells were shown to develop from a human intestinal epithelial cell line under the influence of lymphocytes, and a B cell lymphoma was a stronger converter of epithelial cells into M cells than was a T cell lymphoma (5). Thus, we investigated whether B cells are responsible for the generation of FAE and M cells in vivo and whether B cell deficiency could affect transepithelial transport of an enterally transmitted retroviral pathogen.

We examined knockout (KO) mice with targeted mutations that lack specific lymphocyte subsets (6) for abnormalities in PP and FAE generation. The absence of B cells, due to KO of either the μ membrane segment (Igh-6) or JH segments of immunoglobulin (Ig) genes (JHD), caused a diminution of numbers of detectable PPs (Table 1). In contrast, mice deficient in either αβ T cells [T cell receptor (TCR)β KO] or γδ T cells (TCRδ KO) had normal numbers of PPs, as did mice deficient in both αβ and γδ T cells (TCRβ, TCRδ KO). The strongest PP deficiency has been observed in mice lacking both B and T cells as a result of the absence of RAG1 recombinase (RAG1 KO). In B cell–negative mice, the size of PPs was much smaller and FAE was abnormal, as revealed in unfixed preparations of PPs seen in transmitted light (Fig. 1, C and D) or in a cryostat section (Fig. 1G). The transgenic expression of a membrane-bound immunoglobulin M (mIgM) on the JHD background, allowing generation of B cells with surface but not secreted IgM, completely restored the development of PPs (Table 1) and FAE (Fig. 1, E, F, and H).

Figure 1

Full development of PPs and FAE is affected in B cell–negative mice and can be restored by transgenic expression of mIgM. Relative to normal B6 mice (A and B), two B cell–deficient mouse stocks analyzed—Igh-6 (C andD) and JHD (G)—showed reductions in PP size, dome numbers, and dome size. Transgenic expression of mIgM led to development of B cells (H), PPs, and domes (Eand F). The inner surface of PPs was photographed in transmitted light using a Wild M10 Stereoscope (Leica) and SPOT charge-coupled device camera (Diagnostic Instruments, Sterling Heights, Michigan). Black rectangle, PP in Igh-6 mouse; black and white arrows, FAE; white rectangle, FAE in Igh-6 mouse. Scale bar for (A), (C), and (E) is shown in (A); bar for (B), (D), and (F) is shown in (B). Cryostat sections of PPs from JHD (G) and transgenic mIgM (H) mice were stained with fluorescein isothiocyanate–coupled polyclonal antibodies to Ig (Sigma).

Table 1

Development of Peyer's patches is affected in mice lacking B lymphocytes.

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Scanning electron microscopy (SEM) was used for further analysis of FAE in mutant mice (Fig. 2). SEM is the most adequate approach for M cell detection, as the luminal surface of M cells has very distinct microfold morphology (Fig. 2C) (7). SEM allows analysis of the large FAE surfaces, an advantage over transmission electron microscopy or histochemical detection of M cells (8). M cells could be found in abundance in the large domes in the PPs of normal mice (Fig. 2B) (9). In contrast, only a few cells with a characteristic microfold surface could be detected in the smaller domes of B cell–deficient animals (Fig. 2, E to G) (9). Cells with an unusual brush border (Fig. 2, F and G) were also found in mice without B cells. These cells are most likely M cell maturation intermediates (9).

Figure 2

SEM (29) showed normal FAE and the presence of M cells (marked “M”) in BALB/cJ (Ato D) and transgenic mIgM (H andI) mice. Goblet cells are indicated by “G”; enlarged images of a typical M cell (C) and goblet cell (D) are shown. In JHD mice, FAE was small (E and G) and M cell generation (F and G) (9) was affected by the lack of B cells. Cells with an unusual brush border are marked with an asterisk in (F) and (G). (J) B cells in mice lacking CD40L stimulate normal development of FAE and M cells. (K) The absence of both αβ and γδ T cells did not affect M cell development.

Reconstitution of B cells by mIgM transgene expression resulted in the complete development of FAE and M cells (Fig. 2, H and I). Nonactivated B lymphocytes are likely sufficient for organogenesis, as FAE-containing M cells were normally developed in CD40 ligand–negative (CD40L KO) mice (Fig. 2J), even though B cells in these mice cannot be properly activated (10, 11). The lack of T cells did not have any significant influence on FAE development because TCRβ, TCRδ KO mice had normally developed M cells (Fig. 2K). However, in RAG1 KO mice, domes were even more difficult to find than in mice with no B cells, and there was no evidence of M cells (12); this suggests that the development of residual M cells found in the absence of B cells (Fig. 2, F and G) (9) may be promoted by T lymphocytes, although the input of T cells in M cell development is very limited. A conservative estimate suggests that the overall number of M cells in B cell–deficient mice is smaller by a factor of at least 150 to 840 (one-sixth to one-third the number of PPs × one-quarter to one-half the number of domes × one-fifth the dome surface area × one-seventh to one-fifth the number of M cells in the field). Thus, only 1.2 to 6.6 M cells are present in B cell–deficient mice per every 1000 M cells in normal intestine.

Substantial diminution of M cell numbers should lead to a strong functional impairment. We used mouse mammary tumor virus (MMTV), a classical retrovirus suspected to use M cells to enter GALT (13), to show functional involvement of M cells in retroviral transport from the intestinal lumen. MMTV infects only dividing cells, and it relies on its superantigen (SAg) to induce proliferation of T cells expressing certain Vβ chains (14). T cell activation by SAg depends on direct interaction with B lymphocytes (15). Elimination of either T cells with appropriate Vβ chains (16) or B cells (17) abrogates MMTV infection. Thus, B cells may be needed both for T cell activation and for M cell–dependent translocation of MMTV.

To unmask the possible role of M cells in channeling MMTV through the intestinal wall, we used a bone marrow (BM) chimera approach (18). Newborn Igh-6 or TCRβ KO mice were used as recipients of normal adult B6 BM and were simultaneously briefly exposed to MMTV (19). In all TCRβ KO (having intact B and M cells) recipients of B6 BM, MMTV infection was readily detectable (Fig. 3). In contrast, chimeras with a reconstituted immune system but with underdeveloped FAE were significantly resistant to MMTV infection: Most Igh-6 recipients had severely diminished viral load or had no detectable MMTV proviruses at all (Fig. 3). Only a small fraction of total T cells (T cells expressing cognate Vβ regions) can be used by MMTV (20). Nevertheless, T cell numbers originating from donor BM were sufficient to allow MMTV infection in B6 → TCRβ KO chimeras. Because mouse BM contains 15 to 20% cells of the B cell lineage versus 1 to 2% of the T cell lineage, the number of B cells introduced should be sufficient to support SAg-dependent T cell activation.

Figure 3

Infection with milk-borne retrovirus (MMTV) is controlled by B cell–dependent FAE. (A) Control B6 mice, but not TCRβ KO or Igh-6 mice, fed with MMTV-laden milk became infected. Integrated MMTV proviruses were detected in the spleens at 8 weeks by semiquantitative PCR. Each lane represents a different mouse. (B) When the two immunodeficient strains were reconstituted at birth with B6 BM (18), only B6 → TCRβ KO chimeras demonstrated full-scale infection. Mice in the two experiments shown were fostered by MMTV+ foster mothers for 7 and 3 days, respectively. Densitometric analysis of the PCR bands is shown below.

The B cell–controlled development of FAE (as opposed to mere reduction of PP numbers) is crucial for retroviral infection, as tumor necrosis factor receptor 1 (TNFR1) KO mice with the same reduction in number and size of PPs (21, 22) as B cell–negative mice but with significant numbers of M cells were found to harbor MMTV efficiently (9).

The precise mechanisms of the organogenic function of B cells are unknown. B cells in transgenic mIgM mice do not produce any secreted Ig, excluding involvement of soluble Ig or signaling through Fc receptors in FAE development. The members of the TNFR family have been implicated in organogenesis of lymphoid tissue, including GALT (23), and are likely to be involved in the B cell–dependent development of FAE, similar to B cell–dependent generation of follicular dendritic cells (FDCs) (24).

The organogenic function of B cells in GALT (and possibly in other mucosal barriers, such as respiratory epithelium) is distinct from their immune functions of Ig secretion or antigen presentation. It affects M cell–dependent translocation of pathogens through mucosal barriers, influences an organism's interactions with environmental flora (25), and must be taken into account when interpreting studies that implicate B cells as antigen-presenting cells in immunity and autoimmunity (26, 27).

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