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, 28 (11), 1624-36

XBP-1 Regulates Signal Transduction, Transcription Factors and Bone Marrow Colonization in B Cells

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XBP-1 Regulates Signal Transduction, Transcription Factors and Bone Marrow Colonization in B Cells

Chih-Chi Andrew Hu et al. EMBO J.

Abstract

XBP-1, a transcription factor that drives the unfolded protein response (UPR), is activated in B cells when they differentiate to plasma cells. Here, we show that in the B cells, whose capacity to secrete IgM has been eliminated, XBP-1 is induced normally on induction of differentiation, suggesting that activation of XBP-1 in B cells is a differentiation-dependent event, but not the result of a UPR caused by the abundant synthesis of secreted IgM. Without XBP-1, B cells fail to signal effectively through the B-cell receptor. The signalling defects lead to aberrant expression of the plasma cell transcription factors IRF4 and Blimp-1, and altered levels of activation-induced cytidine deaminase and sphingosine-1-phosphate receptor. Using XBP-1-deficient/Blimp-1-GFP transgenic mice, we find that XBP-1-deficient B cells form antibody-secreting plasmablasts in response to initial immunization; however, these plasmablasts respond ineffectively to CXCL12. They fail to colonize the bone marrow and do not sustain antibody production. These findings define the role of XBP-1 in normal plasma cell development and have implications for management of B-cell malignancies.

Figures

Figure 1
Figure 1
B cells from XBP-1KO/MD4 mice fail to differentiate into plasma cells. (A, B) XBP-1 deficiency leads to reduced production of plasma cells, whereas all other B-cell compartments of the XBP-1KO mice appear normal. B cells were isolated from the bone marrow, peritoneal cavity and spleen, and stained with indicated markers. Cells shown were gated on B220+ or CD19+ populations. The B-cell populations indicated in each panel are as follows: (i) pro-B cells, (ii) pre-B cells, (iii) immature B cells, (iv) pro-B cells, (v) pre-B cells, (vi) immature B cells, (vii) plasma cells, (viii) B-1 cells, (ix) transitional B cells, (x) follicular B cells, (xi) marginal zone B cells, (xii) T-1 transitional B cells, (xiii) T-2 transitional B cells, (xiv) germinal centre B cells and (xv) plasma cells. The numbers indicate the percentages of cells. (C) XBP-1WT/MD4 and XBP-1KO/MD4 mice were not immunized. XBP-1WT and XBP-1KO mice were repeatedly immunized with HEL. The levels of anti-HEL IgM in the sera were determined by ELISA.
Figure 2
Figure 2
XBP-1 activation is a differentiation-dependent event in B cells, and the lack of XBP-1 leads to IRE-1 upregulation. (A) XBP-1 deficiency does not lead to accumulation of misfolded proteins. Four-day LPS-stimulated XBP-1WT/MD4 and XBP-1KO/MD4 plasmablasts were treated with or without 30 μM thapsigargin for 3 h before lysis. Lysates were treated with the indicated concentrations of the cross-linker bis[sulfosuccinimidyl]suberate (BS3) and immunoblotted for calreticulin. (B) B cells purified from spleens of either XBP-1WT/MD4 and XBP-1KO/MD4 mice (upper three panels) or XBP-1WT/μS−/− and XBP-1KO/μS−/− mice (lower three panels) were cultured in LPS (20 μg/ml) to induce differentiation. Cell lysates were immunoblotted for XBP-1, p97 (AAA-ATPase) and actin. (C) XBP-1WT/μS−/− and XBP-1KO/μS−/− B cells were stimulated by LPS to induce differentiation. Lysates were immunoblotted for IRE-1, p97 and actin.
Figure 3
Figure 3
Transport of membrane-bound IgM and the heterodimeric Igα/Igβ to the cell surface appears to be normal in XBP-1-deficient B cells. (A) Four-day LPS-stimulated XBP-1WT/MD4 and XBP-1KO/MD4 plasmablasts were labelled by 35S-[methionine] and -[cysteine] for 10 min and chased for indicated time. Cells were lysed in Triton X-114 and lysates were subjected to phase separation. The intracellular membrane-bound IgM was immunoprecipitated using the anti-μ antibody from Triton X-114-associated protein fractions, whereas the intracellular secreted IgM was immunoprecipitated from Triton X-114 supernatant fractions. The extracellular secreted IgM was immunoprecipitated from the culture media. The asterisk denotes endo-H-resistant complex glycans. (B) Four-day LPS-stimulated XBP-1WT/μS−/− and XBP-1KO/μS−/− plasmablasts were radiolabelled for 10 min and chased for indicated time. Lysates were immunoprecipitated using the anti-μ antibody. (C) Naive XBP-1WT/MD4 and XBP-1KO/MD4 B cells were stimulated by LPS for 4 days to allow differentiation. Each day cells were surface-stained by an FITC-conjugated anti-μ antibody and analysed by flow cytofluorimetry. (D) Three-day LPS-stimulated XBP-1WT/MD4 and XBP-1KO/MD4 plasmablasts were lysed. Lysates were treated with either endo-H or PNGase F before immunoblotting for Igα or Igβ. CHO, CHO* and NAG represent high mannose-type glycans, complex-type glycans and N-acetylglucosamines, respectively. (E) Four-day LPS-stimulated XBP-1WT/MD4 and XBP-1KO/MD4 plasmablasts were lysed. Lysates were cross-linked with BS3 of indicated concentrations and analysed by immunoblot for Igα or Igβ.
Figure 4
Figure 4
XBP-1 deficiency causes defective phosphorylation of Igα, Igβ and Syk in LPS-induced plasmablasts on antigen engagement. (A) LPS-stimulated XBP-1WT/MD4 and XBP-1KO/MD4 plasmablasts were activated by trimeric HEL for 2 min and lysed immediately. Lysates were immunoprecipitated using anti-Igα and anti-Igβ antibodies. The immunoprecipitates were analysed by SDS–PAGE and immunoblotted using an anti-phospho-Igα antibody or an anti-phosphotyrosine antibody. Total lysates were immunoblotted using an anti–actin antibody as a loading control. (B) XBP-1 plays a role in the maintenance of persistent Syk phosphorylation on BCR activation. Day 4 LPS-stimulated XBP-1WT/MD4 and XBP-1KO/MD4 plasmablasts were stimulated with trimeric HEL for indicated time and lysed immediately. Lysates were immunoblotted for phospho-Syk, Syk, actin, p97 and calreticulin. Quantitation of protein bands was performed, and the numbers represent relative band intensity within each gel. (C) Naive XBP-1WT/MD4 and XBP-1KO/MD4 B cells were induced to differentiate into plasmablasts by LPS. At the end of each indicated LPS stimulation time, cells were exposed to trimeric HEL for 2 min and lysed immediately. Day 0 cells were not exposed to LPS. Lysates were immunoblotted for phospho-Syk, Syk, protein disulfide isomerase (PDI), calnexin, calreticulin, p97 and actin. Note the imbalanced Syk phosphorylation between XBP-1WT/MD4 and XBP-1KO/MD4 plasmablasts from day 1 to day 4. (D) Protein bands in the Syk immunoblot in (C) were quantified and data were plotted as fold changes. (E) Protein bands in the phospho-Syk immunoblot in (C) were quantified.
Figure 5
Figure 5
IgM- or HEL-stimulated XBP-1-deficient B cells produce less IL-6. (A) Naive B cells were cultured in the media with or without IL-4 for 4 h, and some cells were subsequently stimulated by trimeric HEL for 2 min. Besides, naive B cells were cultured in LPS for 3 days and subsequently treated with IL-4 for another 24 h. Lysates were immunoblotted for phospho-Stat6 (upper panels) and actin (lower panels). Naive (B), 2-day LPS stimulated (C) and 4-day LPS stimulated (D) XBP-1WT/MD4 and XBP-1KO/MD4 B cells were cultured with plate-bound LPS, CpG, anti-IgM or HEL. (E) Sorted XBP-1WT/MD4 and XBP-1KO/MD4 follicular B cells (using CD1d and CD23 markers) were stimulated with LPS for 4 days and then cultured with plate-bound HEL. The level of secreted IL-6 in the culture supernatants was measured after 24 h by ELISA. *P<0.005.
Figure 6
Figure 6
XBP-1 deficiency leads to altered expression of IRF4, Blimp-1, activation-induced cytidine deaminase (AID) and sphingosine-1-phosphate receptor (S1P1). (A) XBP-1WT/μS−/− and XBP-1KO/μS−/− B cells were cultured in LPS to induce differentiation for indicated time. Lysates were immunoblotted for IRF4, Blimp-1, Pax5, BCL6, p97 and actin. (B) XBP-1WT and XBP-1KO B cells were stimulated by LPS to induce differentiation. Lysates were immunoblotted for AID, S1P1 and p97. (C) A model that illustrates the inhibitory effect of XBP-1 on IRF4, Blimp-1 and its activating enzyme IRE-1. The red arrows indicate that the function of IRF4 and Blimp-1 to regulate plasma cell differentiation requires XBP-1.
Figure 7
Figure 7
XBP-1-deficient plasma cells mount only a short lived but robust antibody response due to their failure to colonize into the bone marrow. (A) XBP-1WT/MD4/Blimp-1-GFP and XBP-1KO/MD4/Blimp-1-GFP mice were immunized with HEL at the following time points: days −34, −20 and −7 (3 × immunized); days −20 and −7 (2 × immunized); day −7 (1 × immunized); or unimmunized (0 × immunized). Splenocytes and bone marrow cells from all these mice were isolated on the same day (day 0), stained for CD138 and analysed by flow cytometry. The percentage of Blimp-1-GFP-positive/CD138-positive cells is indicated in the upper right quadrants. The results are representative of two independent experiments. Note that very few cells (0.05 and 0.09%) in the bone marrow of XBP-1KO/MD4/Blimp-1-GFP mice are Blimp-1-GFP-positve and CD138-positive after reimmunization. (B) The percentages of CD138+/Blimp-1-GFP+ B cells from the spleens and bone marrow of immunized mice (shown in A) were plotted. (C) HEL-specific antibody titers in the sera from mice described in (A) were measured by ELISA. (D) Day 4 LPS-stimulated XBP-1WT/MD4 and XBP-1KO/MD4 plasmablasts were stimulated with CXCL12 for indicated time and lysed immediately. Lysates were immunoblotted for phospho-ERK1/2, ERK1/2 and actin.

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