Prolonged Antigen Persistence Within Nonterminal Late Endocytic Compartments of Antigen-Specific B Lymphocytes

Abstract

Although Ag-specific B lymphocytes can process Ag and express peptide-class II complexes as little as 1 h after Ag exposure, it requires 3–5 days for the immune system to develop a population of Ag-specific effector CD4 T lymphocytes to interact with these complexes. Presently, it is unclear how B cells maintain the expression of cell surface antigenic peptide-class II complexes until effector CD4 T lymphocytes become available. Therefore, we investigated B cell receptor (BCR)-mediated Ag processing and presentation by normal B lymphocytes to determine whether these cells have a mechanism to prolong the cell surface expression of peptide-class II complexes derived from the processing of cognate Ag. Interestingly, after transit of early endocytic compartments, internalized Ag-BCR complexes are delivered to nonterminal late endosomes where they persist for a prolonged period of time. In contrast, Ags internalized via fluid phase endocytosis are rapidly delivered to terminal lysosomes and degraded. Moreover, persisting Ag-BCR complexes within nonterminal late endosomes exhibit a higher degree of colocalization with the class II chaperone HLA-DM/H2-M than with the HLA-DM/H2-M regulator HLA-DO/H2-O. Finally, B cells harboring persistent Ag-BCR complexes exhibit prolonged cell surface expression of antigenic peptide-class II complexes. These results demonstrate that B lymphocytes possess a mechanism for prolonging the intracellular persistence of Ag-BCR complexes within nonterminal late endosomes and suggest that this intracellular Ag persistence allows for the prolonged cell surface expression of peptide-class II complexes derived from the processing of specific Ag.

Functional and structural requirements for the internalization of distinct BCR‐ligand complexes

“…4 M, there was no significant depletion of b-hex activity during the first 15 min of the assay in untreated cells, consistent with a lack of Ag trafficking in b-hex-positive compartments early after internalization [30]. In agreement with our previous report [29], in signaling-competent cells Ag causes depletion of b-hex activity more prominently after 30 min. However, treatment of cells with Src kinase trafficking of Ag-BCR complexes to processing compartments as suggested by earlier studies.…”

“…Due to the altered distribution of internalized Ag observed in cells pretreated with PP1 and PP2 (Fig. 4), we determined the effect of inhibition of BCR signaling on the trafficking of HRP-labeled Ag to b-hexosaminidase (b-hex)-positive late endocytic compartments using an HRP-catalyzed 3,3 0 -diaminobenzidine (DAB)-mediated protein cross-linking approach [29]. As a control, the depletion of a-mannosidase, a marker enzyme present in the Golgi complex which is not accessed by the BCR, was also measured.…”

“…Ag-BCR complexes were then allowed to internalize at 37 C for 15 or 30 min (in the continued presence of vehicle or inhibitors). Cells were then allowed to bind to alcian blue-treated coverslips, fixed and permeabilized (or not) as previously described [29]. Subsequently, cells were stained with SA-Alexa 594 (Molecular Probes) followed by mouse anti-B220-FITC (Southern Biotechnology).…”

The Syk-binding Ubiquitin Ligase c-Cbl Mediates Signaling-dependent B Cell Receptor Ubiquitination and B Cell Receptor-mediated Antigen Processing and Presentation

“…Flow Cytometric Antigen Processing and Presentation AssayFlow cytometric analysis of HEL 46 -61 -I-A k expression was done as reported previously (16,18,(27)(28)(29), except for the following modifications. Splenocytes from either MD4.B10.Br or B10.Br mice were pretreated with inhibitors (4-amino-5-(4-methylphenyl)-7-(t-butyl) pyrazolo [3,4- Cells were infected with lentiviral particles at 37°C with constant inversion for 30 min and returned to culture in complete media to allow viral integration for 48 h. After 48 h, flow cytometric analysis confirmed a transfection efficiency of 8 -15% on the basis of GFP expression.…”

“…Ag⅐BCR complexes were then allowed to internalize at 37°C for 0 -60 min. Cells were attached to Alcian blue-treated coverslips, fixed, and permeabilized (or not) as described previously (29). For staining of the LAMP-2 positive compartment, cells were permeabilized in 0.01% saponin and stained with anti-LAMP-2 (1:100) and anti-rat IgG (HϩL) Alexa Fluor 647.…”