The IgD-binding domain of the Moraxella IgD-binding protein MID (MID962-1200) activates human B cells in the presence of T cell cytokines

Abstract

Moraxella catarrhalis immunoglobulin D (IgD)‐binding protein (MID) is an outer membrane protein with specific affinity for soluble and cell‐bound human IgD. Here, we demonstrate that mutated M. catarrhalis strains devoid of MID show a 75% decreased activation of human B cells as compared with wild‐type bacteria. In contrast to MID‐expressing Moraxella, the MID‐deficient Moraxella mutants did not bind to human CD19+ IgD+ B cells. The smallest MID fragment with preserved IgD‐binding capacity comprises 238 amino acids (MID962‐1200). To prove the specificity of MID962‐1200 for IgD, a Chinese hamster ovary (CHO) cell line expressing membrane‐anchored human IgD was manufactured. MID962‐1200 bound strongly to the recombinant IgD on CHO cells. Moreover, MID962‐1200 stimulated peripheral blood lymphocyte (PBL) proliferation 5‐ and 15‐fold at 0.1 and 1.0 μg/ml, respectively. This activation could be blocked completely by antibodies directed against the CD40 ligand (CD154). MID962‐1200 also activated purified B cells in the presence of interleukin (IL)‐2 or IL‐4. An increased IL‐6 production was seen after stimulation with MID962‐1200, as revealed by a human cytokine protein array. MID962‐1200 fused to green fluorescent protein (GFP) bound to human B cells and activated PBL to the same degree as MID962‐1200. Taken together, MID is the only IgD‐binding protein in Moraxella. Furthermore, the novel T cell‐independent antigen MID962‐1200 may, together with MID962‐1200–GFP, be considered as promising reagents in the study of IgD‐dependent B cell activation.

Superantigen- and TLR-Dependent Activation of Tonsillar B Cells after Receptor-Mediated Endocytosis

“…catarrhalis is an important human respiratory pathogen with a high affinity for human IgD (31). Binding of surface-expressed IgD by MID explains the strong mitogenic effect of M. catarrhalis on human lymphocytes (37)(38)(39)43). In this study, we demonstrate the importance of IgD cross-linking in M. catarrhalis-induced Tindependent B cell activation by blocking the IgD and MID interactions with Abs against IgD or the IgD-binding part of MID.…”

“…Moreover, our experiments showed that the B cell activation induced by M. catarrhalis seemed to be independent of the heterogeneity in maturation stages of the tonsillar B cell population because MID-deficient M. catarrhalis would have activated the same memory B cell population as the Moraxella wt. However, MID stimulation alone is not enough to drive B cells into IgM secretion (37,52). Data from Bekeredjian-Ding et al (53) have suggested a synergy in B cell activation between the Staphylococcus aureus B cell superantigen protein A (SpA) and TLR2 signaling.…”

“…We have demonstrated previously that MID is a powerful B cell superantigen by its unique ability to target human IgD (37,38,43). The nonimmune IgD cross-linking by MID-expressing M. catarrhalis leads to B cell proliferation and IgM production (39).…”

“…In all experiments with lymphocytes, 1 ϫ 10 6 cells/ml were cultured in RPMI 1640 medium supplemented with 10% FCS, 2 mM glutamine, 50 g/ml gentamicin, and 100 U/ml penicillin in culture plates (Nunc). rMID fragments were coated on flat-bottom plates, as previously described (38). TLR ligands were added in the concentration range 1-10 g/ml, except for CpG ODN 2006, which was in the range of 0.1-1 M. Proliferation was measured by [methyl-3 H]thymidine incorporation (5 Ci/well; Amersham Biosciences) using an 18-h pulse period.…”

“…An anti-human TLR9 mAb was purchased from InvivoGen. The truncated MID 962-1200 and MID 1000 -1200 fragments and rabbit anti-MID 962-1200 antiserum were prepared, as described earlier (35,38). Chloroquine was obtained from Sigma-Aldrich and dissolved in water, according to the manufacturer's recommendations.…”