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 Ab response because it diversifies Ab effector functions. Like somatic hypermutation, CSR requires activation-induced cytidine deaminase (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(+/C)γ(1-cre)Rab7(fl/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(+/C)γ(1-cre)Rab7(fl/fl) mice displayed normal B and T cell development and were deficient in Rab7 only in B cells undergoing Igh(C)γ(1-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(+/C)γ(1-cre)Rab7(fl/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 noncanonical 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(+/C)γ(1-cre)Rab7(fl/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.

FIGURE 1. Generation of Igh^+/Cγ^1-creRab7^fl/fl mice

Schematics of expression of the Cre recombinase in “induced” Igh^+/Cγ^1-creRab7^fl/fl B cells (bottom panel) through germline Iγ1-Sγ1-Cγ1-cre transcription in the Igh^Cγ^1-cre allele and IRES-dependent translation (germline Iγ1-Sγ1-Cγ1 transcription in the Igh⁺ allele is also indicated), as well as the consequent Cre-mediated deletion of the first coding exon of the two Rab7 alleles (47) – one Rab7 allele will remain intact in induced Igh^+/Cγ^1-creRab7^+/fl B cells (top panel). This Rab7^fl allele is different from a recently reported Rab7 mutant allele in which the second and third exons are floxed (51). In all of our experiments, Igh^+/Cγ^1-creRab7^fl/fl mice were used as conditional knockout mice and Igh^+/Cγ^1-creRab7^+/fl mice were used as their “wildtype” littermate controls, as these mice did not display hypomorphism in the Rab7 protein expression. Igh^+/+Rab7^fl/fl mice were not chosen as “wildtype” controls because they have two Igh⁺ alleles capable of encoding IgG1, while Igh^+/Cγ^1-creRab7^fl/fl mice have only one.

FIGURE 2. Abrogation of Rab7 protein expression in Igh^+/Cγ^1-creRab7^fl/fl B cells stimulated by a primary stimulus plus IL-4

(A) DNA electrophoresis of PCR products from the genotyping of four littermates born from breeding of an Igh^+/Cγ^1-creRab7^+/fl mouse with an Igh^+/+Rab7^fl/fl mouse. (B) PCR analysis of the kinetics of Cre-mediated genomic DNA deletion in the Rab7^fl allele and appearance of post-deletion Rab7^del allele in Igh^+/Cγ^1-creRab7^fl/fl B cells (top panels) and their Igh^+/Cγ^1-creRab7^+/fl B cell counterparts (bottom panels) after stimulation with CD154 plus IL-4 for 0, 12, 24, 36 and 48 h – in Igh^+/Cγ^1-creRab7^+/fl B cells, the Rab7⁺ allele should remain constant. The proportion of remaining Rab7^fl allele DNA was analyzed by the ratio of band intensity of Rab7^fl allele DNA (quantified by the ImageJ software) to that of Rab7⁺ allele DNA (used as the internal loading control) in Igh^+/Cγ^1-creRab7^+/fl B cells, and was expressed as percentages of the value at 0 h (set as 100%). (C) Immunoblotting analysis of Rab7 expression in Igh^+/Cγ^1-creRab7^fl/fl B cells stimulated with CD154, LPS, or CpG plus anti–δ/dex (as indicated) plus IL-4 for 48 h and their Igh^+/Cγ^1-creRab7^+/fl B cell counterparts. (D) Immunofluorescence staining and confocal microscopy analysis of Rab7 and B220 expression (pseudo-colored) in Igh^+/Cγ^1-creRab7^fl/fl B cells stimulated with CD154 plus IL-4 for 48 h and their Igh^+/Cγ^1-creRab7^+/fl B cell counterparts. (E) Stimulated emission depletion (STED) confocal microscopy analysis of Rab7 and CD40 distribution (pseudo-colored) in Igh^+/Cγ^1-creRab7^+/fl B cells stimulated with CD154 plus IL-4 for 48 h (two different cells are shown). Data are representative of three independent experiments.

FIGURE 3. Igh^+/Cγ^1-creRab7^fl/fl mice show normal B and T lymphocyte development and survival

(A) Flow cytometry analysis of expression of B220 and IgM (B cell markers) in bone marrow, spleen and lymph node cells (left panels), expression of CD5, a B1 cell marker in humans and mice (83), and B220 in peritoneal cavity cells (B2 cells were CD5⁻B220^hi, B1a cells were CD5⁺B220⁺ and B1b cells were CD5⁻ B220⁺) as well as that of CD5 and IgM or IgA (middle panels), and expression of CD4 and CD8 in CD3⁺ T cells from the thymus, spleen and lymph nodes (right panels) in Igh^+/Cγ^1-creRab7^fl/fl and Igh^+/Cγ^1-creRab7^+/fl littermate mice. (B) Flow cytometry analysis of the viability (7–AAD⁻) of IgM⁺ B cells in the bone marrow, spleen and lymph nodes (left panels), as well as that of CD4⁺ (middle panels) and CD8⁺ (right panels) T (CD3⁺) cells in the thymus, spleen and lymph nodes in Igh^+/Cγ^1-creRab7^fl/fl and Igh^+/Cγ^1-creRab7^+/fl mice. Data are representative of three independent experiments.

FIGURE 4. Igh^+/Cγ^1-creRab7^fl/fl mice display reduced titers of overall IgG1 and antigen-specific IgG1

(A) ELISA of serum titers of IgM, IgG1, IgG2a, IgG2b, IgG3 and IgA in Igh^+/Cγ^1-creRab7^fl/fl mice (plum) and their Igh^+/Cγ^1-creRab7^+/fl littermates (teal) (mean and s.e.m. of data from five pairs of mice). (B) ELISA of serum titers of high-affinity NP-specific (NP₃-binding) IgG1, IgG2a, IgG2b, IgG3 and IgA as well as NP₃-binding IgM (due to the high-avidity) in Igh^+/Cγ^1-creRab7^fl/fl (plum) and their Igh^+/Cγ^1-creRab7^+/fl littermate mice (teal) 9 d after injection with NP-CGG (mean and s.e.m. of data from five pairs of mice). (C) ELISA of serum titers of NP₃-binding IgM, IgG1, IgG2a, IgG2b, IgG3 and IgA in Igh^+/Cγ^1-creRab7^fl/fl (plum) and their Igh^+/Cγ^1-creRab7^+/fl littermate mice (teal) 9 d after injection with NP-LPS (mean and s.e.m. of data from three pairs of mice). p values, as calculated by paired student t test, less than 0.05 were considered significant.

FIGURE 5. Igh^+/Cγ^1-creRab7^fl/fl mice show decreased IgG1⁺ B cells and IgG1-secreting AFCs

(A) ELISPOT analysis and quantification ((mean and s.e.m. of data from three pairs of mice) of AFCs that produced NP-specific IgM or IgG1 in spleens of Igh^+/Cγ^1-creRab7^fl/fl mice (plum) and their Igh^+/Cγ^1-creRab7^+/fl littermates (teal) 9 d after injection with NP-CGG. p values, as calculated by paired student t test, less than 0.05 were considered significant. (B) Immunofluorescence staining and confocal microscopy analysis of germinal centers (PNA^hi) within B220⁺ follicles in spleens of Igh^+/Cγ^1-creRab7^+/fl and Igh^+/Cγ^1-creRab7^fl/fl littermate mice 9 d after injection with NP-CGG. (C) Cell cycle analysis (top panels; the quadrant corresponding to the G0/G1, S or G2/M phase of the cell cycle was also depicted) and viability analysis (bottom panels; live cells were Annexin V⁻7–AAD⁻) of spleen B cells isolated ex vivo from Igh^+/Cγ^1-creRab7^fl/fl mice and their Igh^+/Cγ^1-creRab7^+/fl littermates 9 d after injection with NP-CGG. (D) Proportion of germinal centers (PNA^hi) cells among (B220⁺) B cells (top panels) and proportion of (CD138⁺B220^hi) plasmablasts and (CD138⁺B220^lo) plasma cells in spleens of Igh^+/Cγ^1-creRab7^fl/fl mice and their Igh^+/Cγ^1-creRab7^+/fl littermates 9 d after injection with NP-CGG. (E) Proportion of IgG1⁺ (top panels) and IgG3⁺ (bottom panels) cells among (B220⁺PNA^hi) germinal center B cells in spleens of Igh^+/Cγ^1-creRab7^fl/fl mice and their Igh^+/Cγ^1-creRab7^+/fl littermates 9 d after injection with NP-CGG. Data are representative of three independent experiments.

FIGURE 6. Rab7 deficiency in B cells results in defective CSR to IgG1 in vitro

(A) Flow cytometry analysis of proliferation and CSR to IgG1 in CFSE-labeled Igh^+/Cγ^1-creRab7^fl/fl B cells after stimulation by a primary stimulus (CD154, LPS, a TLR ligand, alone or together with anti–Igδ mAb/dex, as indicated) plus IL-4 for 4 d and their Igh^+/Cγ^1-creRab7^+/fl B cell counterparts. (B) Depiction of the proportion of switched IgG1⁺ cells in CFSE-labeled Igh^+/Cγ^1-creRab7^fl/fl B cells and their Igh^+/Cγ^1-creRab7^+/fl B cell counterparts that had completed each cell division after stimulation by a primary stimulus (CD154, LPS, a TLR ligand or together with anti–Igδ mAb/dex, as indicated) plus IL-4 for 4 d. Data are representative of three independent experiments. (C) ELISA of titers of IgG1 secreted into culture supernatants by Igh^+/Cγ^1-creRab7^fl/fl B cells and their Igh^+/Cγ^1-creRab7^+/fl B cell counterparts after stimulation by the same stimuli as in (B) for 7 d (mean and s.e.m. of data from three independent experiments).

FIGURE 7. Igh^+/Cγ^1-creRab7^fl/fl B cells are normal in IgM expression, class-switching to other IgG isotypes and plasma cell differentiation

(A) Flow cytometry analysis of CSR to IgG1 in B cells from Igh^+/+Rab7^fl/fl and Igh^+/Cγ^1-creRab7^fl/fl littermate mice stimulated by LPS plus IL-4 for 96 h. (B) Flow cytometry analysis of proliferation and CSR to IgG2b, IgG3 or IgG2a in CFSE-labeled Igh^+/Cγ^1-creRab7^fl/fl B cells and their counterparts from Igh^+/Cγ^1-creRab7^+/fl littermates stimulated by a selected primary stimulus, as indicated, plus a cytokine (TGF-β, nil or IFN-γ, as indicated) for 4 d (left panels), and depiction of the proportion of switched (IgG2b⁺, IgG3⁺ or IgG2a⁺) cells in B cells that had completed each cell division (right panels). (C) Flow cytometry analysis of CSR to IgA in Igh^+/Cγ^1-creRab7^fl/fl and Igh^+/Cγ^1-creRab7^+/fl B cells pre-stimulated by CD154 (left panels) or LPS (right panels) plus IL-4 for 48 h and then stimulated by LPS plus TGF-β and anti–Igδ mAb/dex for 72 h to undergo CSR to IgA. (D) Flow cytometry analysis of CSR to IgG1 in Igh^+/Cγ^1-creRab7^fl/fl B cells stimulated by CD154 or LPS plus IL-4 for 5 d and their Igh^+/Cγ^1-creRab7^+/fl B cell counterparts and analysis of CSR to IgG3 after those B cells were stimulated by LPS for 4 d (left panels; cells within red gates were unswitched IgM⁺IgG1⁻ or IgM⁺IgG3⁻ cells, and those within blue gates were switched IgG1⁺IgM⁻ or IgG3⁺IgM⁻ cells), quantification of IgM expression levels in unswitched IgM⁺IgG1⁻ or IgM⁺IgG3⁻ cells (middle panels). (E) Proportion of (CD138⁺B220^lo) plasma cells after Igh^+/Cγ^1-creRab7^fl/fl B cells and their counterparts from Igh^+/Cγ^1-creRab7^+/fl littermates were stimulated by CD154 or LPS plus IL-4 or by LPS alone for 4 d. Data are representative of three independent experiments.

FIGURE 8. Igh^+/Cγ^1-creRab7^fl/fl mice/B cells display impairment in CSR to IgE in vivo and in vitro

(A) Intracellular staining and flow cytometry analysis of CSR to IgE in CFSE-labeled Igh^+/Cγ^1-creRab7^fl/fl B cells and their counterparts from Igh^+/Cγ^1-creRab7^+/fl littermates stimulated by CD154 or LPS plus IL-4 for 5 d (left panels) and depiction of the proportion of switched IgE⁺ cells in B cells that had completed each cell division (right panels). (B) Intracellular staining and flow cytometry analysis of CSR to IgE and IgG1 in Igh^+/Cγ^1-creRab7^fl/fl B cells stimulated by CD154 plus IL-4 for 5 d and their Igh^+/Cγ^1-creRab7^+/fl B cell counterparts (cells within red gates were switched IgE⁺IgG1⁻ cells, and those within blue gates were switched IgG1⁺IgE⁻ cells). Data are representative of three independent experiments. (C) ELISA of titers of total (top panels) and ovalbumin-binding (bottom panels) IgM, IgG1 and IgE in Igh^+/Cγ^1-creRab7^fl/fl mice (plum) and their Igh^+/Cγ^1-creRab7^+/fl littermates (teal) 14 d after injection with ovalbumin (mean and s.e.m. of data from three pairs of mice). p values, as calculated by paired student t test, less than 0.05 were considered significant.

FIGURE 9. Igh^+/Cγ^1-creRab7^fl/fl B cells are defective in the induction of AID in vivo and in vitro

(A) qRT-PCR analysis of levels of Aicda, IgH germline Iγ1-Sγ1-Cγ1 transcripts, circle Iγ1-Cμ transcript, post-recombination Iμ-Cγ1 transcripts, 14-3-3γ transcripts and Pdrm1 transcripts in B cells isolated ex vivo from Igh^+/Cγ^1-creRab7^fl/fl mice (plum) and their Igh^+/Cγ^1-creRab7^+/fl littermates (teal) 9 d after injection with NP-CGG. Data were normalized to the level of Cd79b and are expressed as ratios of values in Igh^+/Cγ^1-creRab7^+/fl B cells to those in Igh^+/Cγ^1-creRab7^fl/fl B cells (mean and s.e.m. of data from three pairs of mice). (B) qRT-PCR analysis of levels of Aicda transcripts, IgH germline Iγ1-Cγ1 transcripts, circle Iγ1-Cμ transcripts and post-recombination Iμ-Cγ1 transcripts in Igh^+/Cγ^1-creRab7^fl/fl B cells (plum) stimulated by CD154, LPS or CpG plus anti–δ mAb/dex in the presence of IL-4 for 48 h and in their Igh^+/Cγ^1-creRab7^+/fl B cell counterparts (teal). (C) qRT-PCR analysis of levels of Aicda transcripts, IgH germline Iε-Cε transcripts, circle Iε-Cμ transcripts and post-recombination Iμ-Cε transcripts as well as those of Prdm1 and Xbp1 transcripts in Igh^+/Cγ^1-creRab7^fl/fl B cells (plum) stimulated by CD154 plus IL-4 for 48 h and in their Igh^+/Cγ^1-creRab7^+/fl B cell counterparts (teal). (D) qRT-PCR analysis of transcript levels of genes involved in autophagy, as indicated, in Igh^+/Cγ^1-creRab7^fl/fl B cells (plum) stimulated by LPS plus IL-4 for 48 h and in their Igh^+/Cγ^1-creRab7^+/fl B cell counterparts (teal). Data were normalized to the level of Cd79b transcripts and are expressed as ratios of values in Igh^+/Cγ^1-creRab7^+/fl B cells to those in Igh^+/Cγ^1-creRab7^fl/fl B cells (mean and s.e.m. of data from three independent experiments). ***, p < 0.005; **, p < 0.01; *, p < 0.05 (the p values were calculated by paired student t test). (E) Flow cytometry analysis of (CD19⁺) B cell viability (7-AAD⁻, left panels) and CSR (IgG1^+, right panels) in Igh^+/Cγ^1-creRab7^fl/fl B cells pre-stimulated by CD154 plus IL-4 for 48 h, transduced by pTAC or pTAC-AID retrovirus, and then stimulated by CD154 plus IL-4 for 72 h.

FIGURE 10. Rab7 deficiency in B cells results in defective canonical NF-κB signaling in vitro

(A) Immunoblotting analysis of levels of Rab7, LC3-I (no processing) and LC3-II (processed and conjugated to phosphatidylethanolamine), phosphorylated and total p65 in the canonical NF-κB pathway, p100 and p52 in the non-canonical NF-κB pathway, AID and β-actin in Igh^+/Cγ^1-creRab7^fl/fl B cells and their Igh^+/Cγ^1-creRab7^+/fl control B cells stimulated by CD154, LPS or CpG plus anti–δ mAb/dex, as indicated, with IL-4 for 48 h. (B) Immunoblotting analysis of levels of phosphorylated and total p38 as well as phosphorylated and total ERK1/2 in MAPK pathways, Blimp-1 and β-actin in Igh^+/Cγ^1-creRab7^fl/fl B cells and their Igh^+/Cγ^1-creRab7^+/fl control B cells stimulated by CD154, LPS or CpG plus anti–δ mAb/dex, as indicated, with IL-4 for 48 h.

FIGURE 11. Depiction of defective CSR to IgE in Igh^+/Cγ^1–creRab7^fl/fl B cells

(A) Depiction of direct switching from IgM to IgE (top) and sequential switching from IgM to IgG1 and then IgE in Igh^+/Cγ^1–creRab7^fl/fl B cells. (B) Depiction of defective direct switching from IgM to IgE in Igh^+/Cγ^1–creRab7^fl/fl B cells (top). As the generation of IgG1⁺ B cells is also impaired (bottom) due to defective CSR from IgM to IgG1, sequential switching from IgM to IgG1 and then IgE is blocked in these B cells.