Lack of Gut Secretory Immunoglobulin A in Memory B-Cell Dysfunction-Associated Disorders: A Possible Gut-Spleen Axis

Abstract

Background: B-1a B cells and gut secretory IgA (SIgA) are absent in asplenic mice. Human immunoglobulin M (IgM) memory B cells, which are functionally equivalent to mouse B-1a B cells, are reduced after splenectomy. Objective: To demonstrate whether IgM memory B cells are necessary for generating IgA-secreting plasma cells in the human gut. Methods: We studied intestinal SIgA in two disorders sharing the IgM memory B cell defect, namely asplenia, and common variable immune deficiency (CVID). Results: Splenectomy was associated with reduced circulating IgM memory B cells and disappearance of intestinal IgA-secreting plasma cells. CVID patients with reduced circulating IgM memory B cells had a reduced frequency of gut IgA⁺ plasma cells and a disrupted film of SIgA on epithelial cells. Toll-like receptor 9 (TLR9) and transmembrane activator and calcium-modulator and cyclophilin ligand interactor (TACI) induced IgM memory B cell differentiation into IgA⁺ plasma cells in vitro. In the human gut, TACI-expressing IgM memory B cells were localized under the epithelial cell layer where the TACI ligand a proliferation inducing ligand (APRIL) was extremely abundant. Conclusions: Circulating IgM memory B cell depletion was associated with a defect of intestinal IgA-secreting plasma cells in asplenia and CVID. The observation that IgM memory B cells have a distinctive role in mucosal protection suggests the existence of a functional gut-spleen axis.

Keywords: common variable immune deficiency; gut mucosal immunology; plasma cell; splenectomy; transmembrane activator and calcium-modulator and cyclophilin ligand interactor.

Figure 1

Secretory immunoglobulin A (SIgA) is reduced in splenectomized patients. (A) Cryostat sections of the duodenum were stained in red with phalloidin (a toxin that binds and stains filamentous actin) and in green with antibodies against either IgA (left panels), secretory component (SC, middle panels), or J-chain (right panels). Biopsies of one healthy donor (HD) and two representative splenectomized patients (pt.1 and pt.2) are shown. A reduction of IgA⁺ plasma cells, SIgA, SC, and J chain is observed after removal of the spleen. (B) IgM⁺ plasma cells are shown in blue along the axis of the villi in HD and asplenic patients. IgM is not significantly secreted.

Figure 2

Lamina propria expression of secretory immunoglobulin A (SIgA) in the gut of CVID patients who have reduced (Group 1) or normal (Group 2) number of circulating immunoglobulin M (IgM) memory B cells. Cryostat duodenal sections were stained with phalloidin (red) and immunoglobulin A (IgA) (green) in six patients of Group 1 with reduced numbers of circulating IgM memory B cells (upper panels), and a control subject (HD) together with five patients belonging to Group 2 with normal numbers of circulating IgM memory B cells (lower panels).

Figure 3

Immunoglobulin M (IgM) can substitute immunoglobulin A (IgA) in the gut of common variable immune deficiency (CVID) patients. Cryostat sections were stained with phalloidin (red), IgA (green), and IgM (gray). IgA (left panels) and IgM (right panels) staining in the same sections are shown separately. In CVID patients belonging to Group 1, neither IgA nor IgM is expressed. In most CVID patients belonging to Group 2, IgA is the major isotype expressed and transported (pt.22, as representative example). IgM substitutes IgA in one case (pt.30, lower panel) and is transported through epithelial cells.

Figure 4

Immunoglobulin M (IgM) memory B cells differentiate into IgA⁺ plasma cells in response to transmembrane activator and calcium-modulator and cyclophilin ligand interactor (TACI) and toll-like receptor 9 (TLR9) co-stimulation. Flow-cytometry analysis after 7 days in culture with anti-TACI plus IL-4 and IL-21. The loss of intracellular CMFDA tracks cell division. (A) No proliferation is observed in mature-naive and IgM memory B cells, whereas a modest expansion of transitional B cells is observed (upper panels). The IgM/CD27 staining (lower panels) shows the loss of surface IgM in 17.0% of mature-naïve and 43.6% of IgM memory B cells (bottom panels). (B) The addition of CpG induces proliferation as shown by the loss of CMFDA fluorescence, mainly in transitional and IgM memory B cells (top panels). Differentiation into IgM⁺ CD27⁺ plasma cells is observed in all B cell types. Switched plasma cells are only generated from IgM memory B cells. (C) After 7 days with anti-TACI, a small fraction of mature-naïve B cells had switched to IgA. The addition of CpG further increased survival. Upon TACI cross-linking, almost half of the IgM memory B cells switched to IgA, but when CpG was added to the culture, IgM memory B cells differentiated into IgA⁺ plasma cells. (D) Graphs indicate the concentration of IgM and IgA (ng/ml) detected by ELISA in culture supernatants obtained from B cells stimulated with anti-TACI, CpG, IL-4, and IL-21. IgM can be detected in the supernatants of all B cell types, but only stimulated IgM memory B cells secreted IgA. IgG production was never detectable.

Figure 5

Expression of a proliferation inducing ligand (APRIL) in the adult gut. Cryostat sections from adult intestine were stained for actin (phalloidin, red) and APRIL (green). (A) We show the overlay of phalloidin and APRIL staining (left) and APRIL alone (right). APRIL can be detected in the epithelium, but is not equally expressed by each epithelial cell, probably reflecting the topographic distribution of inductive signals (magnification ×60). (B) Details of cryptae analyzed with ×60 objective amplification are shown. We observe a gradient of APRIL expression starting at the luminal side and progressing toward the basal side of the epithelial cell.

Figure 6

Expression of a proliferation inducing ligand (APRIL), transmembrane activator and calcium-modulator and cyclophilin ligand interactor (TACI), CD27, and immunoglobulin M (IgM) in the gut. Cryostat sections from the intestine were stained for actin (phalloidin, red) and APRIL (green) (upper panel). APRIL is expressed by epithelial cells of the crypts. Intestinal sample is stained for CD27 (green) and IgM (gray) (middle panel). From the same field (×20 magnification) we show in separate panels the overlay of phalloidin, TACI, and IgM. CD27⁺ (left) IgM⁺ (right) B cells were distributed among the crypts (middle panels). IgM⁺ B cells express TACI (lower panel).