mTOR activation promotes plasma cell differentiation and bypasses XBP-1 for immunoglobulin secretion

Abstract

Plasma cells (PCs) are responsible for the secretion of antibodies. The development of fully functional PCs relies on the activation of the inositol-requiring enzyme 1/X-box binding protein 1 (IRE1/XBP-1) arm of the unfolded protein response (UPR). XBP-1-deficient PCs secrete antibodies poorly and exhibit distensions of the endoplasmic reticulum (ER). The kinase mammalian target of rapamycin (mTOR) promotes anabolic activities and is negatively regulated by the tuberous sclerosis complex (TSC). Deletion of TSC1 renders mTOR hyperactive. To explore the relationship between mTOR and the UPR in PC development and function, mice with conditional deletions of XBP-1 and/or TSC1 in their B cell lineage were generated. Deletion of TSC1 enhanced Ig synthesis and promoted differentiation into PCs independently of XBP-1, as evidenced by comparison of TSC1/XBP-1 double-knockout (DKO) PCs to XBP-1 knockout (KO) PCs. The typical morphological abnormalities of the ER in XBP-1 KO PCs were alleviated in the DKO PCs. Expression profiling identified the glycoprotein Ly6C as an mTOR target. Ly6C expression contributed to the enhanced Ig secretion from DKO PCs. Our data reveal a functional overlap between mTOR and the UPR in promoting PC development. In addition to the classical mTOR role in promoting protein synthesis, the mechanism entails transcription regulation of accessory molecules, such as Ly6C.

FIG 1

TSC1 KO and DKO PCs display elevated mTOR activity and IRE1 expression levels. (A) B cells were isolated from the spleens of wt/YFP, XBP-1 KO/YFP, TSC1 KO/YFP, and DKO/YFP mice and subjected to stimulation with LPS for 3 days. At day 3, live cells were separated over Lympholyte M and total cell extracts were made in RIPA buffer. Shown is a typical immunoblot from three independent analyses with the indicated antibodies. (B) MLN cells were isolated from DKO/YFP mice and subjected to stimulation with APRIL for 6 days. Cells were stained for B220 and YFP⁺ B220⁺, and YFP⁻ B220⁺ cells were sorted. Immunoblotting for TSC1 confirmed the reduction in its expression in the YFP⁺ cells. PCR for the floxed XBP-1 allele confirmed the recombination of the locus in the YFP⁺ cells. Shown are the results of two independent experiments.

FIG 2

Deletion of TSC1 promotes PC differentiation. (A) Sera of wt, XBP-1 KO, TSC1 KO, and DKO mice were analyzed for antibody titers by ELISA (n = 10). (B) Single-cell PCR analysis for XBP-1 recombination of YFP/CD138 double-positive PCs derived from bone marrow of wt, XBP-1 KO, and DKO mice. (C and D) Cells derived from pLN (C) and BM (D) of the various CD19-Cre/YFP strains were stained for the CD138 plasma cell marker and analyzed by flow cytometry according to the markers shown. The graphs show the average results of three independent analyses. Statistical significance was calculated by Student's t test, comparing results for TSC1 KO mice and wt mice and results for DKO mice and XBP-1 KO mice (*, P < 0.05). Error bars indicate standard errors (SE).

FIG 3

ER distensions are reduced in DKO PCs relative to the ER morphology in XBP-1 KO PCs. (A) B cells of XBP-1 KO and DKO mice were isolated and stimulated for 3 days with LPS. At day 3, CD138-positive cells were separated by magnetic sorting and subjected to TEM. (B) Bone marrow of the various CD19-Cre/YFP strain mice was sorted for YFP and CD138 markers and visualized by TEM. More than 50 cells for each genotype were inspected. Shown are typical cells from 2 independent separations. Bars, 1 μm.

FIG 4

mTOR activation promotes IgA secretion in the absence of XBP-1. (A) MLN cells of wt/YFP, XBP-1 KO/YFP, and DKO/YFP mice were extracted, stimulated for 6 days with APRIL, and analyzed by flow cytometry analysis for the CD138 marker and their intracellular content of the Ig light chain as indicators of PCs. (B) Supernatants of APRIL-stimulated MLN cells of wt, XBP-1 KO, and DKO were analyzed for Ig, IgM, and IgA secretion by ELISA (n = 6). O.D., optical density. (C) At day 6 of stimulation, YFP-positive MLN cells of wt/YFP, XBP-1 KO/YFP, TSC-1 KO/YFP, and DKO/YFP mice were sorted and enumerated for antibody-secreting cells (ASC) by IgA ELISpot (n = 3). (D) Equal numbers of YFP-sorted wt, XBP-1 KO, and DKO MLN cells were metabolically labeled for 20 min with [³⁵S]methionine and chased up to 4 h. Anti-Ig immunoprecipitation was performed. Synthesized Ig at the end of the pulse and secreted Ig after 4 h were quantified by using a phosphorimager (n = 3). Error bars indicate standard errors of the means (SEM).

FIG 5

IgA secretion from DKO cells was reduced by rapamycin treatment. MLN cells from DKO/YFP mice were cultured in the presence of APRIL and 50 ng/ml of rapamycin for last 24 h of a 6-day culture, and the supernatants were analyzed by ELISA for the various Ig isotypes secreted since rapamycin addition (n = 4). Error bars indicate SEM.

FIG 6

Deletion of TSC1 does not affect B cell proliferation but compromises viability following stimulation. (A) Splenic B cells were isolated a week after tamoxifen administration. Cells were labeled with CFSE; a portion were analyzed by flow cytometry, and the rest were treated for 3 days with LPS. Cells were then analyzed by flow cytometry using the same conditions. Cells were gated on live cells based on their side scatter and forward scatter (SSC/FSC) properties. (B) Splenic B cells were isolated from RERT wt, XBP-1 KO, and DKO mice 8 days after injection of tamoxifen. Cells were stimulated with LPS and analyzed on day 3 with propidium iodide (PI) and CD138 marker by flow cytometry. (C) day 2 and day 3 stimulated cells were stained with APC-labeled annexin V and PI. Shown are histograms typical of 3 to 5 independent experiments.

FIG 7

TSC1 deletion promotes Ig secretion and irreversibly corrects ER distensions. (A) IgM synthesis and secretion were determined in LPS-stimulated RERT wt, XBP-1 KO, and DKO cells by pulse-chase analysis. Shown are average densitometry measurements from 3 independent experiments. Error bars indicate SEM. Statistical significance was calculated by Student's t test, comparing results for TSC1 KO and wt cells and results for DKO and XBP-1 cells KO (*, P < 0.05). Error bars indicate SE. HC, heavy chain; LC, light chain. (B) Splenic B cells were isolated from XBP-1 KO and DKO mice. Cells were stimulated with LPS for 3 days. Rapamycin (100 ng/ml) was added between day 2 and day 3 of stimulation, and CD138-positive cells were isolated by magnetically activated cell sorting (MACS). Cells were analyzed by TEM.

FIG 8

Derlin 3 but not ERp72 is induced in DKO plasmablasts. Splenic B cells of wt, RERT/XBP-1^flox/flox, RERT/TSC1^flox/flox, and RERT/DKO mice were isolated a week after tamoxifen administration. Cells were cultured in the presence of LPS for 3 days and then harvested for protein and mRNA analyses. (A) Expression of ATF6 in total cell extracts. The immunoblot shown is typical of three independent extractions. No significant difference in full-length ATF6 expression is seen between the various genotypes. (B) qPCR analysis of mRNA levels of ERp72 and Derlin 3 relative to ubiquitin C (UBC). Shown is the average of three independent extractions. Error bars represent standard deviations. *, P < 0.05 (DKO versus XBP-1 KO).

FIG 9

mTOR promotes Ly6C expression in MLN B cells. (A) MLN cells of wt, XBP-1 KO, TSC-1 KO, and DKO YFP mice, stimulated for 6 days with APRIL, were sorted for YFP, and Ly6C mRNA levels were analyzed by quantitative PCR. Error bars represent standard deviations. (B) MLN cells of wt/YFP, XBP-1 KO/YFP, TSC-1 KO/YFP, and DKO/YFP mice were gated on YFP and analyzed for Ly6C and CD138 markers on day 0 and day 6 after APRIL treatment.

FIG 10

mTOR promotes Ly6C expression in naive and activated splenic B cells in an XBP-1-dependent manner. Splenic B cells were isolated from the YFP strains and stimulated with LPS for up to 3 days. Flow cytometry analysis was performed at day 0 and at day 3 for analysis of Ly6C expression on naive B cells and on PCs. The results shown are typical of three independent experiments.

FIG 11

Block of Ly6C reduces IgA secretion from DKO cells. (A) MLN cells of XBP-1 KO/YFP and DKO/YFP mice were cultivated with APRIL in the presence and absence of a Ly6C blocking antibody. On day 6, cells were assessed for PCs by CD138 staining (histograms show results of gating on YFP⁺ cells). (B) Culture supernatants were analyzed for IgA levels by ELISA (n = 3). Error bars indicate SEM. Statistical significance was calculated by Student's t test, comparing results for DKO cells with antibody to results for the control (*, P < 0.05).