B cell development in GALT: role of bacterial superantigen-like molecules

Abstract

Intestinal bacteria drive the formation of lymphoid tissues, and in rabbit, bacteria also promote development of the preimmune Ab repertoire and positive selection of B cells in GALT. Previous studies indicated that Bacillus subtilis promotes B cell follicle formation in GALT, and we investigated the mechanism by which B. subtilis stimulates B cells. We found that spores of B. subtilis and other Bacillus species, including Bacillus anthracis, bound rabbit IgM through an unconventional, superantigen-like binding site, and in vivo, surface molecules of B. anthracis spores promoted GALT development. Our study provides direct evidence that B cell development in GALT may be driven by superantigen-like molecules, and furthermore, that bacterial spores modulate host immunity.

FIGURE 1

Flow cytometric, morphological, and Western blot analyses of scFv-Ig binding to lumenal bacteria. A, Schematic representation of scFv-Ig with V_H (V_Ha), flexible linker ([Gly₄Ser₁]₃), V_κ, and C_γ2 and Cγ3 domains of Fcγ. B, Flow cytometry of appendix lumenal bacteria stained with scFv-Ig. Left, FSC versus SSC. Gate, population analyzed. Right, Staining with scFv-Ig (unshaded histogram); secondary Abs alone (shaded histogram). Bar and number indicate the percentage of scFv-Ig⁺ events FACS sorted. C, FACS-sorted bacteria were grown in vitro and restained with scFv-Ig; one representative scFv-Ig–binding bacterial isolate (scFv-Ig⁺, left) and scFv-Ig–nonbinding bacterial isolate (scFv-Ig⁻, right) are shown. D, Gram stain (left column) and phase-contrast (right column) images of scFv-Ig–binding bacterial isolates (for phase-contrast microscopy, bacteria were cultured on sporulation media). Intestinal isolates were identified by 16S rRNA gene sequence analysis (Supplemental Table 2). E, Flow cytometry of scFv-Ig binding to purified spores from Bacillus intestinal isolates. Top, FSC versus SSC. Gates indicate population used for scFv-Ig staining below; bottom, SSC versus scFv-Ig staining. The percentage of scFv-Ig⁺ spores is indicated in bottom right quadrants. F, Western blot of spore extracts from the intestinal Bacillus isolates probed with scFv-Ig. See also Supplemental Fig. 2. FSC, forward light scatter; SSC, side light scatter.

FIGURE 2

Microscopic and flow cytometric analyses of B. anthracis spores. A, Transmission electron micrograph of a WT B. anthracis endospore. Exosporium marked with arrow. B, Phase-contrast (top) and fluorescence (bottom) images of WT and bclA mutant B. anthracis spores stained with scFv-Ig (left) or rabbit IgM (right). C, Flow cytometry of WT and bclA mutant B. anthracis spores stained with scFv-Ig (left) or rabbit IgM (right). Top row, FSC versus SSC. Gates, population for staining with scFv-Ig or IgM (unshaded histograms) and indirect reagents alone (shaded histograms, bottom row). See also Supplemental Fig. 3. D, Flow cytometry of bclA mutant B. anthracis vegetative cells (left) and spores (right) stained with scFv-Ig. scFv-Ig staining, unshaded histograms; indirect reagents alone, shaded histograms.

FIGURE 3

Western blot analyses of B. anthracis spore extracts and E. coli lysates probed with scFv-Ig or rabbit IgM. A, WT and cotO mutant spore extracts probed with scFv-Ig (left) or rabbit IgM (hybridoma supernatant, right). B, Coomassie blue-stained proteins from extracts of WT and cotO mutant spores. Arrow, region excised for mass spectrometry analysis. C, Western blot of unreduced E. coli lysates U or I to produce the indicated B. anthracis protein, probed with scFv-Ig. Arrow indicates scFv-Ig binding. See also Supplemental Fig. 4. D, Western blot of WT or exsK mutant spore extracts probed with scFv-Ig. Molecular size markers are shown. I, induced; U, uninduced.

FIGURE 4

Immunofluorescence of mutant scFv-Ig binding to Bacillus spores. A, Schematic diagrams of scFv-Ig proteins used for spore-binding experiments. See also Supplemental Fig. 5. B–I, Phase-contrast (top) and immunofluorescence (bottom) images of B. anthracis bclA mutant spores stained with the indicated Ig. Rabbit IgM, polyclonal Ig from serum; human IgM and human IgG are polyclonal Ig from serum; human IgM (V_κ) and human IgM (V_λ) are myeloma proteins. See also Supplemental Fig. 6.

FIGURE 5

Flow cytometric analyses of spore binding and calcium flux of human (Ramos) B cells incubated with B. anthracis spores. A, Histogram of human Ramos B cells incubated with spores (unshaded histogram) or without spores (shaded histogram; negative control) and then stained with FITC rabbit anti-ExsK. B, Histograms of fluo-3-AM:fura red fluorescence detected in Ramos cells following stimulation with goat F(ab′)₂ anti-human Ig (top line), bclA spores (middle line), or HBSS buffer (bottom line).

FIGURE 6

Immunohistological analyses of appendix sections. A–C, Anti–Ki-67 (green) and anti-IgM (red) staining of appendix sections from rabbits injected with WT B. anthracis cells (A), cotO mutant B. anthracis cells (B), or PBS (C) at 4 wk of age and analyzed 3 wk later (original magnification × 100). D, Quantification of the average number of IgM⁺Ki-67⁺ follicles per appendix section observed in appendices from A–C. A minimum of four nonserial tissue sections was analyzed from each appendix. n is the number of appendices examined. Error bars, SEM.