Recruitment of Cbl-b to B cell antigen receptor couples antigen recognition to Toll-like receptor 9 activation in late endosomes.

Margaret Veselits,Marcus R Clark,Shannon O'Neill,Stanley Lipkowitz,Azusa Tanaka,Alison Finnegan,Jian Zhang,Roger Sciammas

doi:10.1371/journal.pone.0089792

Abstract

Casitas B-lineage lymphoma-b (Cbl-b) is a ubiquitin ligase (E3) that modulates signaling by tagging molecules for degradation. It is a complex protein with multiple domains and binding partners that are not involved in ubiquitinating substrates. Herein, we demonstrate that Cbl-b, but not c-Cbl, is recruited to the clustered B cell antigen receptor (BCR) and that Cbl-b is required for entry of endocytosed BCRs into late endosomes. The E3 activity of Cbl-b is not necessary for BCR endocytic trafficking. Rather, the ubiquitin associated (UBA) domain is required. Furthermore, the Cbl-b UBA domain is sufficient to confer the receptor trafficking functions of Cbl-b on c-Cbl. Cbl-b is also required for entry of the Toll-like receptor 9 (TLR9) into late endosomes and for the in vitro activation of TLR9 by BCR-captured ligands. These data indicate that Cbl-b acts as a scaffolding molecule to coordinate the delivery of the BCR and TLR9 into subcellular compartments required for productively delivering BCR-captured ligands to TLR9.

Highlights

Antigen presentation by B lymphocytes is required to mount high affinity humoral immune responses, for coordinating antigen specific cytotoxicity, and for propagating some T cell responses [1]
To explore if Casitas B-lineage lymphoma-b (Cbl-b) played a role in B cell antigen receptor (BCR) endocytic trafficking, splenic B lymphocytes [10] from wild type (WT) or Cblb2/2 Balb/ c mice were isolated and stimulated in vitro through the BCR with FITC-conjugated anti-Ig F(ab)2 antibodies for 30 minutes at 37uC
Shown are the percentages of each cell population that formed a receptor cap on the cell surface containing more than 50% of visualized BCRs (n = 3 experiments, p = 0.005). (E) Internalization of BCR in Cblb2/2 and WT splenocytes in response to anti-IgM F(ab)2 antibodies

Summary

Introduction

Antigen presentation by B lymphocytes is required to mount high affinity humoral immune responses, for coordinating antigen specific cytotoxicity, and for propagating some T cell responses [1]. B lymphocytes differ from other antigen presenting cells in several fundamental ways. In B cells, most antigens are processed in specialized MHC class II containing late endosomes (MIIC) [3] which are Lamp-1+, acidic and contain cathepsins, thiol reductases, and other molecules required for efficient antigen processing [4]. MIIC vesicles consist of a limiting membrane studded with Lamp-1 and a lumen containing multivesicular bodies [5]. These intraluminal vesicles are derived from BCR-laden transport vesicles that have gained access to the MIIC compartment [6]

Methods

Results

Conclusion