B Cell Rab7 Mediates Induction of Activation-Induced Cytidine Deaminase Expression and Class-Switching in T-Dependent and T-Independent Antibody Responses

Abstract

Class switch DNA recombination (CSR) is central to the maturation of the antibody response, as it diversifies antibody effector functions. Like somatic hypermutation, CSR requires AID, whose expression is restricted to B cells, as induced by CD40 engagement or dual TLR-BCR engagement (primary CSR-inducing stimuli). By constructing conditional knockout Igh+/Cγ1-creRab7fl/fl mice, we identified a B cell-intrinsic role for Rab7, a small GTPase involved in intracellular membrane functions, in mediating AID induction and CSR. Igh+/Cγ1-creRab7fl/fl mice displayed normal B and T cell development, and were deficient in Rab7 only in B cells undergoing IghCγ1-cre Iγ1-Sγ1-Cγ1-cre transcription, as induced – like Igh germline Iγ1-Sγ1-Cγ1 and Iε-Sε-Cε transcription – by IL-4 in conjunction with a primary CSR-inducing stimulus. These mice could not mount T-independent or T-dependent class-switched IgG1 or IgE responses, while maintaining normal IgM levels. Igh+/Cγ1-creRab7fl/fl B cells showed, in vivo and in vitro, normal proliferation and survival, normal Blimp-1 expression and plasma cell differentiation, as well as intact activation of the non-canonical NF-κB, p38 kinase and ERK1/2 kinase pathways. They, however, were defective in AID expression and CSR in vivo and in vitro, as induced by CD40 engagement or dual TLR1/2-, TLR4-, TLR7- or TLR9-BCR engagement. In Igh+/Cγ1-creRab7fl/fl B cells, CSR was rescued by enforced AID expression. These findings, together with our demonstration that Rab7 mediated canonical NF-κB activation, as critical to AID induction, outline a novel role of Rab7 in signaling pathways that lead to AID expression and CSR, likely by promoting assembly of signaling complexes along intracellular membranes.

Small Molecule Inhibition of Rab7 Impairs B Cell Class Switching and Plasma Cell Survival To Dampen the Autoantibody Response in Murine Lupus

“…As we have shown (10), Rab7 is induced in activated B cells in vivo (i.e., in PNA hi germinal center B cells) and in vitro, e.g., by CD154 and TLR ligands, the same stimuli that induce AID expression and CSR. It plays a B cell-intrinsic role in antibody responses, as mice that conditionally knockout Rab7 in activated B cells cannot mount mature antibody responses to T cell-dependent or -independent antigens (10). Rab7 promotes CSR (to IgG, IgE and IgA) and does so by mediating AID induction, as enforced expression of AID rescues CSR in Rab7 knockout B cells.…”

“…C57 (Stock # 000664), MRL/ Fas lpr/lpr (Stock #000485) and C57/ Sle1Sle2Sle3 (Stock #007228) mice were from The Jackson Laboratory and maintained in a pathogen-free vivarium. Conditional Rab7 knockout Igh +/C γ 1-cre Rab7 fl/fl mice and their “wildtype” Igh +/C γ 1-cre Rab7 +/fl littermates on the C57 background were as described (10). Rosa26 +/fl-STOP-fl-Ikk β ca mice on the C57 background (19) were from The Jackson Laboratory (Stock #008242).…”

“…CD154 was prepared as membrane fragments isolated from Sf21 insect cells infected by CD154-expressing baculovirus, as we described (10, 21) – membrane fragments from non-infected Sf21 cells failed to stimulate B cells. For CSR induction in mouse B cells, spleen B cells were cultured (3 × 10 5 cell/ml) in RPMI-FBS were stimulated for 96 h (or 48 h for transcript and protein analyses) by the following primary stimuli: CD154 (3 U/ml), LPS (3 μg/ml, from E. coli , serotype 055:B5, deproteinized by chloroform extraction; Sigma-Aldrich) or R-848 (30 ng/ml; Invivogen), a ligand of TLR7, which promotes lupus autoantibody responses in a B cell-intrinsic manner.…”

“…Dead (7–AAD + ) cells were excluded from analysis. For CSR analysis of B cells stimulated in vitro , B cells were analyzed by flow cytometry for surface expression of IgG and IgA as well as intracellular expression of IgE, as we have described (10, 21). …”

“…Retroviral vectors pMIG and pMIG-AID (encoding AID) were as described (10); pMIG-Cre (encoding the Cre recombinase) was from Addgene; pMIG-Rab7 and pMIG-Rab2a (encoding Rab7 and Rab2a, respectively) were constructed using specific oligonucleotides (Supplemental Table 2). These vectors were co-transfected with the packaging plasmid into 293T cells to produce retroviruses, as described (10, 22).…”