Monovalent ligation of the B cell receptor induces receptor activation but fails to promote antigen presentation

Abstract

We explored the role of antigen valency in B cell receptor (BCR) activation and rearrangement of intracellular MHC class II compartments as factors that contribute to the efficacy of antigen presentation. Using primary B cells that express a hen egg lysozyme (HEL)-specific BCR, we found that oligomeric HEL more efficiently promoted both BCR activation and internalization than did monovalent HEL, although monovalent HEL, unlike monovalent Fab fragments of anti-Ig, readily triggered the BCR. Nonetheless, oligovalent ligation positions the BCR in a membrane microdomain that is distinct from one engaged in the course of monovalent ligation, as judged by detergent extraction of the BCR. Furthermore, oligovalent HEL induced more pronounced rearrangement of MHC class II-containing antigen-processing compartments. Using videomicroscopy we observed in real time the rearrangement of MHC class II compartments as well as delivery of antigen in primary B cells. The observed increase in rearrangement of MHC class II-positive compartments and the disposition of antigen-bound BCRs therein correlates with improved presentation of a HEL-derived epitope. Although monomeric HEL efficiently engages the BCR, presentation of HEL-derived epitopes is impaired compared to oligovalent antigens. This trait may help explain the known ability of soluble, disaggregated antigen to induce a state of B cell tolerance.

Fig. 1.

Preparation of HEL conjugates and HEL-induced tyrosine phosphorylation. (a) HEL conjugates crosslinked by glutaraldehyde were purified by gel filtration and resolved on a 12.5% SDS gel. Proteins were stained with Coomassie blue. (b) B cells purified from MD4 transgenic or wild type mice were incubated with 5 μg/ml of monomeric, dimeric, trimeric HEL or 40 μg/ml of F(ab′)₂ fragment of goat anti-mouse IgM for 2 min at 37°C. Tyrosine-phosphorylated polypeptides in cell lysates were detected with anti-phosphotyrosine antibody 4G10. (c) Purified MD4 B cells were incubated with 5 μg/ml of untreated monomeric HEL, glutaraldehyde-treated monomeric, dimeric, trimeric, tetrameric HEL or 20 μg/ml of F(ab) or F(ab′)₂ fragment of goat anti-IgM for 2min at 37°C. Tyrosine phosphorylation in B cell lysates was detected as described above. (d) MD4 B cells were incubated with indicated concentration of monomeric, dimeric, trimeric, or tetrameric forms of HEL for 2 min at 37°C. Tyrosine phosphorylated proteins were detected as described above.

Fig. 2.

Calcium mobilization induced by HEL conjugates in MD4 B cells. Changes in intracellular calcium levels were monitored in purified MD4 B cells loaded with fluorescent calcium indicators Fluo-3 and Fura Red by flow cytometry. After an initial 60 s of baseline recording, indicated concentrations of HEL conjugates or goat anti-IgM antibody fragments were added (indicated by dashed lines). (a) Changes in the mean fluorescent ratio were compared after addition of 1 μg/ml of monomeric, dimeric, or trimeric HEL (Upper) or 10 μg/ml of Fab or F(ab′)₂ fragments of anti-IgM antibodies (Lower). (b) Induction of calcium flux by monomeric, dimeric, or trimeric HEL was compared at two different concentrations (1 μg/ml and 10 μg/ml).

Fig. 3.

Detergent extraction of BCR complex. (a) MD4 B cells activated with 5 μg/ml of monomeric, dimeric, or trimeric HEL at 37°C were lysed in a lysis buffer containing 1% digitonin and the lysate was run on a 10% SDS-PAGE gel. Ig μ and Ig α were then detected by immunoblotting. (b) MD4 B cells were pretreated with 1% DMSO or 10 mM methyl-β-cyclodextrin (MβCD) for 15 min and activated with 5 μg/ml of HEL for 2 min at 37°C. The cells were lysed in a lysis buffer containing either 1% digitonin or 1% Triton X-100 and Ig μ and Ig α were detected by immunoblotting. The open arrow denotes surface Ig μ and the two filled arrows indicate intracellular Ig μ.

Fig. 4.

Redistribution of MHC class II upon activation of B cell receptor. The relative increase in intracellular MHC class II-containing compartments in B cells was measured after stimulation with various forms of HEL. Integrated intensity of intracellular MHC class II-GFP signal above background was measured and the percent increase (mean ± SEM) relative to the media control was plotted for each condition.

Fig. 5.

Time-lapse imaging of HEL uptake and rearrangement of MHC Class II-containing compartments in live B cells. (a) Splenic B cells purified form MD4/MHC class II-GFP were imaged at 37°C. The cells were initially imaged without HEL. After capturing a few frames of images, Alexa Fluor 568-conjugated dimeric HEL (final concentration of 5 μg/ml) was added to cells (indicated by an arrow). Changes in distribution of MHC class II-GFP and Alexa Fluor 568-conjugated HEL were sequentially captured and shown is a gallery view of selected time-lapse images of a cell at one focal plane. Elapsed time is shown at the lower right corner of each frame. (b) Localization of MHC class II-GFP and Alexa Fluor 568-labeled dimeric HEL was examined at 5 s and 40 min after addition of 5 μg/ml of Alexa Fluor 568-labeled dimeric HEL. (c) Localization of MHC class II-GFP and Alexa Fluor 568-labeled monomeric HEL was examined at 5 s, 40 min, and 120 min after addition of 5 μg/ml Alexa Fluor 568-labeled monomeric HEL.

Fig. 6.

HEL-peptide presentation by MD4 B cells. B cells purified from wild-type or MD4 mice were incubated with T cell hybridoma BO4H9 and the indicated concentrations of HEL conjugates for 24 h at 37°C. Secreted IL-2 in the culture supernatants was measured by ELISA.