The mTORC1/4E-BP/eIF4E Axis Promotes Antibody Class Switching in B Lymphocytes

Abstract

During an adaptive immune response, activated mature B cells give rise to Ab-secreting plasma cells to fight infection. B cells undergo Ab class switching to produce different classes of Abs with varying effector functions. The mammalian/mechanistic target of rapamycin (mTOR) signaling pathway is activated during this process, and disrupting mTOR complex 1 (mTORC1) in B cells impairs class switching by a poorly understood mechanism. In particular, it is unclear which mTORC1 downstream substrates control this process. In this study, we used an in vitro murine model in which the mTORC1 inhibitor rapamycin, when added after a B cell has committed to divide, suppresses class switching while preserving proliferation. Investigation of mTORC1 substrates revealed a role for eukaryotic translation initiation factor 4E (eIF4E) and eIF4E-binding proteins in class switching. Mechanistically, we show that genetic or pharmacological disruption of eIF4E binding to eIF4G reduced cap-dependent translation, which specifically affected the expression of activation-induced cytidine deaminase protein but not Aicda mRNA. This translational impairment decreased Ab class switching independently of proliferation. These results uncover a previously undescribed role for mTORC1 and the eIF4E-binding proteins/eIF4E axis in activation-induced cytidine deaminase protein expression and Ab class switching in mouse B cells, suggesting that cap-dependent translation regulates key steps in B cell differentiation.

Figure 1.. Low concentrations of rapamycin reduce IgG1 production during SRBC immunization

Mice (5 per group) were immunized with a T-dependent antigen, sheep red blood cells (SRBC) and treated for 7 days i.p. with vehicle control PBS, a low Rap dose of 75 μg/kg that has been shown to preserve lymphocyte activation, or an immunosuppressive dose of 1mg/kg. Serum was obtained from mice on day 8 and SRBC-specific IgG1 (A) and IgM (B) production were measured by detection of serum antibodies bound to SRBC using flow cytometry as described in (45). Significance was calculated using two-way ANOVA with Newman Keuls multiple comparison test. (C) Percentage of spleen cells with germinal center (B220+GL7+Fas+) and T follicular helper cell (CXCR5+PD-1+CD4+) phenotype were analyzed by flow cytometry. (D-F) Mice (5 per group) were immunized with 5 μg NP-LPS and treated for 10 days with PBS, 75 μg/kg, 300 μg/kg or 1mg/kg Rapamycin by IP injection daily. (D) Percentage of spleen cells with germinal center (B220+GL7+Fas+) phenotype and (E) %IgG1+B220+ cells were analyzed by flow cytometry. (F) NP-specific serum IgM was measured by ELISA. Significance was calculated using one-way ANOVA with Newman Keuls multiple comparison test. (*P<0.05; **P<0.01; ***P<0.001; ****P<0.0001)

Figure 2.. mTORC1 control of IgG1 switching is not due to inhibition of division

(A) Purified mouse splenic B cells were stimulated with αCD40+IL-4 and treated with rapamycin or the CDK4/6 inhibitor Palbociclib at the indicated concentrations. The percent of live B cells that have divided at least once (based on CFSE dilution; CFSE-low) expressing IgG1 was determined by flow cytometry at 96h. (B) Intracellular staining of p-S6 and p-4E-BP-1 was measured by flow cytometry. (C-D) Cells were stimulated with αCD40+IL-4, LPS+IL-4, LPS alone, or LPS+IL-4+IL-5+TGF-β+RA. (C) Percent of live B cells that have divided at least once expressing IgG1, IgG3, IgA and (D) IgD was determined by flow cytometry at 96h. Data are representative of three or more independent experiments (3–8 mice) and shown as mean ±SD. Significance was calculated using a paired two-tailed student’s t test. (*P<0.05; **P<0.01; ***P<0.001)

Figure 3.. Glutamine deprivation reduces switching to IgG1

Cells were stimulated with αCD40+IL-4 or LPS+IL-4 in media with different glutamine concentrations as indicated. Percent IgG1 of the divided cells (A) and percent divided (B) is graphed. (C) Cells were stimulated with LPS+IL-4 in media with indicated glutamine concentrations and inhibitors for 24 hours before harvesting for western. p-S6 signal was quantified over multiple experiments. Data are representative of three or more independent experiments (3–4 mice). Significance was calculated using a paired one-tailed student’s t test. (*P<0.05; **P<0.01; ***P<0.001). (D) Cell size was determined by flow cytometry at 24, 48 and 72 hours. (E) %IgG1 gating on Peak 3 was determined by flow cytometry at 96h.

Figure 4.. Genetic targeting of eIF4E activity reduces antibody class switching

(A) (1–2) Model of 4E-BP and eIF4E signaling. (3) Genetic and (4) pharmacological approaches to inhibit eIF4E activity. (B, C) Purified mouse splenic B cells from control mice or DOX-inducible 4E-BP mutant mice were stimulated with αCD40+IL-4 for 48 hours then treated with DOX and harvested at 54 hours to measure (B) transgene expression by western and (C) 96 hours for IgG1 switching among divided B cells. (D) B cells stimulated under the conditions indicated for treated at 48hr with titrations of DOX. The percent of live B cells that have divided at least once (measured by CFSE or eFluor670) or (E) in Peak 3 expressing IgG1, IgG3, or IgA was determined at 96 hours. (F) The histograms on the right show cell division tracking by CFSE in cells treated with different concentrations of DOX and proliferation index, which is the average number of divisions among all divided cells, was also measured. Data are representative of three or more independent experiments (3–4 mice) and shown as mean ±SD. Significance was calculated using a paired one-tailed student’s t test. (*P<0.05; **P<0.01; ***P<0.001)

Figure 5.. Pharmacological targeting of eIF4E activity with SBI-756 reduces antibody class switching

Cells were stimulated then treated with various concentrations of SBI-756 at 0 hrs (A), or at 48 hrs after activation (B). Percent of live B cells that have (C) divided at least once or (D) in Peak 3 expressing IgG1, IgG3, IgA and (E) proliferation index by CFSE or eFluor670 were measured at 96 hours by flow cytometry. Data are representative of three or more independent experiments (4–7 mice) and shown as mean ±SD. Significance was calculated using a paired one tailed student’s t test. (*P<0.05; **P<0.01; ***P<0.001)

Figure 6.. Inhibitors that reduce switching disrupt cap-complex formation

(A) eIF4E-eIF4G interactions detected by proximity ligation assay (PLA) in purified mouse splenic B cells that were treated with inhibitors as indicated and stimulated with LPS+IL-4 for 48 hours before fixing and permeabilizing. Interactions are visualized as red signal. Nuclei are stained with DAPI (blue). 5 images in separate fields were taken for each condition. All data are shown as binary area analysis of PLA/DAPI ±SD. Fold change was calculated by normalizing to vehicle. Each point represents one independent experiment. Significance was calculated using a paired one tailed student’s t test (*P<0.05; **P<0.01; ***P<0.001). (B) We stimulated B cells for 48 hours with LPS+IL-4 and used a bicistronic dual Renilla-Firefly luciferase reporter construct to measure cap-dependent translation (Renilla luciferase) relative to cap-independent, polio IRES mediated translation (firefly luciferase) as an internal control. Each point represents one independent experiment (8 mice). Fold change was calculated using vehicle condition. Significance was calculated using a paired one tailed student’s t test. (*P<0.05; **P<0.01; ***P<0.001)

Figure 7.. 48 hour addition of Rap or SBI-756 reduces AID protein but not Aicda mRNA

(A) B cells were purified and stimulated with LPS+IL-4 for 48 hours then treated with inhibitors as indicated. MK-2206 is an AKT inhibitor. In lanes 2–4 and 6, cells were harvested at 72 hours. Lane 5 cells were activated for 48 hours then treated with Rapamycin for an additional 48 hours before harvesting. AID amounts and 4E-BP phosphorylation were quantitated by western. (B,C) B cells were purified from AID-GFP mice and stimulated with LPS+IL-4, αCD40+IL-4, LPS, or LPS+IL-4+IL-5+TGF-β+RA and treated with inhibitors as indicated. Divided cells were gated on eFluor670-lo cells and AID-GFP MFI among divided cells was analyzed at 96 hrs using flow cytometry. Each point represents one independent experiment (4–5 mice). (D) Purified B cells were stimulated with left to right: LPS+IL-4, αCD40+IL-4, or LPS and treated with inhibitors as indicated. Cells were harvested at 72 hours to measure Aicda mRNA by RT-qPCR. Each point represents one individual experiment (3–6 mice). Fold change was calculated in comparison to vehicle treated. Significance was calculated using a paired one tailed student’s t test. (*P<0.05; **P<0.01; ***P<0.001)