XBP-1 is required for the formation of plasma cells as well as several other secretory cell types, including exocrine gland acinar cells and Paneth cells (Lee et al, 2005
; Kaser et al, 2008
). Usually triggered by ER stress-induced IRE-1 activation, XBP-1 drives the expression of numerous genes involved in glycoprotein folding and ER homeostasis, with the overall effect of increasing secretory capacity. Thus, the favoured hypothesis for XBP-1 function in plasma cell development is as follows. Activated B cells rapidly increase production of secreted IgM, some fraction of which is misfolded and accumulates in the ER. Misfolded IgM binds to the chaperone BiP, titrating BiP away from IRE-1. IRE-1 dimerizes, self-activates and initiates the splicing of xbp-1
transcripts. The translation product, XBP-1s, then upregulates transcription of ER chaperones, relieving ER stress and allowing the nascent plasma cell to continue producing IgM. In the absence of XBP-1, misfolded IgM presumably accumulates in the ER and leads to apoptosis, thus explaining the lack of plasma cells in XBP-1-deficient mice (Iwakoshi et al, 2003a
The current model of XBP-1 in plasma cells leads to two testable hypotheses. First, it is misfolded IgM that triggers XBP-1 activation, and second, XBP-1-deficient B cells would be expected to have an increase in misfolded proteins. Neither of these hypotheses is supported by the data shown here. B cells from XBP-1WT
/μS−/− mice can neither produce secreted IgM, nor do they show an increase in membrane-bound IgM during LPS-induced differentiation. Although class-switching rescues serum immunoglobulin levels in vivo
, LPS-stimulated XBP-1WT
/μS−/− LPS plasmablasts show virtually no evidence of switching to other isotypes, as showed by the complete lack of any immunoglobulin heavy chains recovered by immunoprecipitation of kappa chains from culture supernatants (McGehee et al
, manuscript in preparation). Nonetheless, XBP-1WT
/μS−/− plasmablasts activate XBP-1 with similar kinetics as do XBP-1WT
/MD4 plasmablasts (; Supplementary Figure S1
), demonstrating conclusively that XBP-1 activation is a differentiation-dependent event unlinked to accumulation of misfolded IgM. Formally, we cannot exclude the possibility that proteins other than IgM fail to fold properly when B cells initiate their terminal differentiation programme, and so trigger the UPR. However, for the glycoproteins examined, we observed no obvious folding or trafficking defects.
To directly assess the degree of misfolded proteins in XBP-1-deficient B cells, we cross-linked plasmablast lysates and identified misfolded glycoproteins by their aggregation and association with calreticulin and calnexin ( and data not shown). We found no evidence for a global increase in misfolded proteins in XBP-1-deficient plasmablasts. Furthermore, close examination of individual proteins relevant to B-cell function showed no defects in their folding. IgM, both membrane-bound and secreted forms, trafficked through the ER at similar rates () independent of XBP-1 status. The resulting secreted IgM from wild-type and XBP-1-deficient plasmablasts was properly folded, as assessed by its ability to bind antigen, both in an ELISA and by binding to antigen-coated beads (; McGehee et al, manuscript in preparation). Igα and Igβ folding was also normal in the absence of XBP-1 as assessed by rate of synthesis, acquisition of complex glycans, disulfide bond formation and heterodimer stability (; McGehee et al, manuscript in preparation). A variety of B-cell glycoproteins, such as MHC I, MHC II, CD1d, CD40, CD80 and CD86, are expressed normally on the surface of XBP-1-deficient plasmablasts (McGehee et al and data not shown). IL-6, itself a glycoprotein, is secreted normally from XBP-1-deficient plasmablasts on ligation of TLRs (). Signalling through the IL-4 receptor and through TLRs 4 and 9 is uncompromised in XBP-1-deficient B cells, providing further evidence that these receptors are functional and properly folded ().
To better understand the role of XBP-1 in plasma cell differentiation and the defects in XBP-1-deficient cells, we analysed the B cell-specific XBP-1 knockout/MD4 transgenic (XBP-1KO
/MD4) mouse, in which B cells express an HEL-specific BCR encoded by a transgene (Goodnow et al, 1988
). As anti-IgM reagents do not interact directly with the antigen-binding site, activation of MD4 B cells by trimeric HEL serves as physiological means to cross-link the BCR and explore signalling alterations in the absence of XBP-1 (Kim et al, 2006
XBP-1-deficient MD4 plasmablasts showed a greatly diminished response to antigen stimulation, producing far less IL-6 than their wild-type counterparts (). Closer examination of proximal signalling in XBP-1-deficient plasmablasts showed impaired phosphorylation of Igα, Igβ and Syk on BCR engagement (; Supplementary Figure S2
). XBP-1-deficient plasmablasts showed a slight increase in Syk phosphorylation when tested after 2 days of LPS culture (), but this apparent increase in proximal signalling did not rescue the diminished IL-6 production on BCR engagement (). Both follicular and marginal zone LPS-stimulated B cells showed impaired signalling (; Supplementary Figure S2
). Heterogeneity of the total LPS-induced plasmablast populations, therefore, cannot explain the differences observed in BCR signalling between wild-type and XBP-1-deficient plasmablasts. Furthermore, XBP-1-deficient plasmablasts responded normally to IL-4, CpG and LPS stimulation (), indicating that signalling downstream of the IL-4 receptor and TLRs 4 and 9 is unaffected by the absence of XBP-1.
How the transcription factor XBP-1 could affect BCR signalling is not immediately obvious. Surface expression of IgM is modestly diminished in XBP-1-deficient plasmablasts, but only after 4 days of culture (), whereas signalling defects are observed earlier (). IgM produced by XBP-1-deficient MD4 B cells is fully capable of antigen binding, and IL-6 production by XBP-1-deficient MD4 plasmablasts cannot be rescued by increasing the concentration of trimeric HEL used for stimulation ( and data not shown). Several mechanisms could contribute to the BCR-specific signalling defect in XBP-1-deficient cells. First, although we found no evidence for protein misfolding in the absence of XBP-1, we cannot exclude the possibility that some proteins necessary for B-cell function are misfolded or abnormally glycosylated. Full engagement of the BCR requires not only the antigen-binding membrane IgM and its signalling accessories, Igα/Igβ, but also co-receptor proteins, such as CD21 and CD81. Any defect in assembly or modification of these proteins as a result of downregulated PDI () altered terminal glycosylation or other as yet unidentified alterations could compromise activation of the BCR. Some differences in glycosylation were observed in XBP-1-deficient cells (McGehee et al
, manuscript in preparation), but the functional importance of these altered terminal glycans in BCR signalling has yet to be explored. Furthermore, the expression of XBP-1 massively induces phosphatidylcholine synthesis and increases transcription of several other genes involved in lipid synthesis and metabolism (Sriburi et al, 2004
). Changes in lipid composition caused by the lack of XBP-1 may lead to failure of assembling functional lipid raft domains, which are critical for BCR clustering on engagement of antigen. Indeed, we have observed a decrease in sphingomyelin and phosphatidylinositol content in membranes of XBP-1-deficient B cells (McGehee et al
, manuscript in preparation). Sphingomyelin is an important component of lipid rafts, and phosphatidylinositol is an essential intermediate in signalling pathways involving phosphatidylinositol-3-phosphate.
Despite the defects in BCR signalling, XBP-1KO
/MD4 mice have normal numbers of B cells and B-cell subsets (). This is not unexpected, given that mice with far more significant alterations in signalling still produce B cells, albeit with slight shifts in the ratios of marginal zone, follicular and B-1 B cells (Pillai et al, 2004
). However, BCR signal strength determines B-cell subset fate in the bone marrow during the immature stage of B-cell development. The large differences in BCR signalling reported here were observed predominately after LPS stimulation () at a time when XBP-1 spliced protein is also present (; Supplementary Figure S1
). Thus, B cells in XBP-1 knockout mice are predicted to be similar to the point when they first contact the antigen. We do observe some baseline differences in S1P1
expression by naive XBP-1-deficient splenic B cells (). Although naive B cells do not express XBP-1, expression of XBP-1 in pro-B cells could affect B-cell development (Brunsing et al, 2008
Blimp-1 is a transcriptional repressor essential for plasma cell development. IRF4 controls transcription of the Blimp-1 gene prdm1
by direct binding to a conserved noncoding sequence between exons 5 and 6 (Sciammas et al, 2006
). Although earlier studies have shown that irf4
are neither direct nor indirect targets of XBP-1 (Acosta-Alvear et al, 2007
), we observed that both IRF4 and Blimp-1 protein levels are upregulated in XBP-1-deficient plasmablasts (; Supplementary Figure S5
). We, therefore, propose that XBP-1 inhibits the expression of IRF4, and consequently Blimp-1, in plasma cells (). Given that IRF4 and Blimp-1 can be expressed in the absence of XBP-1, XBP-1 must be placed downstream of both of these factors, an observation consistent with earlier data showing that IRF4 and Blimp-1 increase xbp-1
transcription (Shaffer et al, 2002
; Klein et al, 2006
). XBP-1 activation is also regulated post-transcriptionally by IRE-1-mediated splicing to remove 26 nucleotides from xbp-1
mRNA. Of note, XBP-1 deficiency greatly enhances IRE-1 protein levels (; Supplementary Figure S1
), demonstrating feedback inhibition of XBP-1 expression on IRE-1, similar to what is seen in hepatocytes (Lee et al, 2008
We propose that XBP-1 activation in B cells is a differentiation-dependent event, and that the failure of XBP-1-deficient B cells to become plasma cells involves misregulation of key transcription factors, possibly due to altered BCR signalling. Paradoxically, loss of XBP-1 leads to increased IRF4 levels, which cause an increase in Blimp-1, both key transcription factors in plasma cell differentiation. However, despite higher levels of these canonical plasma cell proteins, XBP-1-deficient B cells still do not become plasma cells. This block is apparent not only by the lack of antibody secretion, but also by decreased expression of AID (), a key enzyme in class switch recombination and somatic hypermutation. Thus, at least in tissue culture, XBP-1-deficient B cells appear poised to become plasma cells, yet fail to do so.
To analyse plasma cell formation in vivo
, we immunized XBP-1KO
/MD4/Blimp-1-GFP mice, which express GFP under control of the Blimp-1 promoter to allow the unambiguous quantitation of plasma cells by flow cytometry. To our surprise, XBP-1-deficient mice developed a robust plasmablast population in the spleen after a single immunization, which correlated with transiently elevated serum levels of anti-HEL antibodies. On successive immunizations, however, the spleen plasmablast population decreased and no plasma cell population could be found accumulating in the bone marrow (). Specific antibody titers also declined in the XBP-1-deficient animals with each subsequent immunization (). The half-life of serum IgM in the mouse is 2 days (Vieira and Rajewsky, 1988
). Consequently, although XBP-deficient mice can mount a primary antibody response, they do not sustain production of serum antibodies even after reimmunization.
Annexin V staining of spleen plasma cells at each time point showed no differences in apoptosis between wild-type and XBP-1-deficient cells (data not shown). CXCL12, produced by stromal cells, and its receptor CXCR4 are primarily responsible for homing of plasma cell precursors to the bone marrow niches (Muehlinghaus et al, 2005
). CXCR4 is expressed normally on the surface of XBP-1-deficient plasma cells, but signalling through CXCR4 is impaired, as shown by decreased ERK1/2 phosphorylation in XBP-1-deficient plasmablasts exposed to CXCL12 (). The blunted response to CXCL12 could in part account for the failure of XBP-1-deficient plasma cells to colonize the bone marrow. In addition, IL-6 contributes to plasma cell maintenance, and immunoglobulin itself has been proposed to sustain long-lived plasma cells, although the mechanism responsible is not clear (Iwakoshi et al, 2003a
; Kumazaki et al, 2007
). Decreased serum antibody titers and decreased IL-6 produced by B cells could contribute to the lack of long-lived plasma cells in the bone marrow of XBP-1-deficient mice. However, given that XBP-1-deficient MD4 mice in this study have measurable baseline serum antibodies in the same order of magnitude as wild-type MD4 mice ( and ), and given that IL-6 is produced by many cell types other than B cells, we propose that failure of plasma cells to traffick to the bone marrow is the major cause of lack of sustained antibody production in XBP-1-deficient mice.
The short burst of antibody production observed in immunized XBP-1KO
/MD4/Blimp-1-GFP mice is at odds with earlier reports showing that immunized XBP-1-deficient mice have lower serum levels of specific antibodies (Reimold et al, 2001
). This discrepancy could be due to the different time points at which the immunized mice were analysed, given that the antibody production we observed occurred only briefly, and only after a single immunization. Alternatively, the MD4 BCR is expressed from a transgene on >95% of B cells, including B-1 cells in the peritoneal cavity and extrafollicular B cells. Thus, immunization in MD4 mice could induce antibody production primarily from these sources, which would not be a major contributing factor in mice with a polyclonal repertoire.
XBP-1 is upregulated in many human malignancies, particularly multiple myeloma, a cancer for which few treatment options are available. Overexpression of XBP-1 in B cells is sufficient to cause a monoclonal gammopathy of undetermined significance in mice, suggesting that abnormal expression of XBP-1 could be a predisposing factor for the development of myeloma (Carrasco et al, 2007
). Our finding that XBP-1 activation precedes the UPR in normal plasma cell development offers an attractive possibility. If IRE-1-mediated splicing of XBP-1 is triggered by a specific differentiation-dependent event rather than by the accumulation of misfolded aggregates, then XBP-1 activation itself may be an attractive target for drug therapy.