Activation of the endoplasmic reticulum stress-associated transcription factor x box-binding protein-1 occurs in a subset of normal germinal-center B cells and in aggressive B-cell lymphomas with prognostic implications

Abstract

X box-binding protein 1 (Xbp-1) is a transcription factor that is required for the terminal differentiation of B lymphocytes into plasma cells. The Xbp-1 gene is activated in response to endoplasmic reticulum stress signals, which generate a 50-kDa nuclear protein that acts as a potent transactivator and regulates the expression of genes related to the unfolded protein response. Activated Xbp-1 is essential for cell survival in plasma-cell tumors but its role in B-cell lymphomas is unknown. We analyzed the expression of activated Xbp-1 in reactive lymphoid tissues, 411 lymphomas and plasma-cell neoplasms, and 24 B-cell lines. In reactive tissues, Xbp-1 was only found in nuclear extracts. Nuclear expression of Xbp-1 was observed in occasional reactive plasma cells and in a subpopulation of Irf-4(+)/Bcl-6(-)/Pax-5(-) B cells in the light zones of reactive germinal centers, probably representing cells committed to plasma-cell differentiation. None of the low-grade lymphomas showed evidence of Xbp-1 activation; however, Xbp-1 activation was found in 28% of diffuse large B-cell lymphomas, independent of germinal or postgerminal center phenotype, as well as in 48% of plasmablastic lymphomas and 69% of plasma-cell neoplasms. Diffuse large B-cell lymphomas with nuclear Xbp-1 expression had a significantly worse response to therapy and shorter overall survival compared with negative tumors. These findings suggest that Xbp-1 activation may play a role in the pathogenesis of aggressive B-cell lymphomas.

Figure 1

Xbp-1 Expression in reactive lymphoid tissue. Human tonsil. A: In the light zones of secondary lymphoid follicles clusters of nuclear Xbp-1 positive cells can be easily identified (×10). B: The cytoplasmic Xbp-1 is ubiquitously expressed in the cytoplasm of B-cells with a granular and paranuclear pattern (×60 under oil). C: Some cells with plasmacytic features exhibit a strong nuclear positivity of Xbp-1 (×60 under oil). D: Ki-67 positive cells (red) are found in the dark zone of the germinal centers. Ki-67 negative cells in the light zone are positive for nuclear Xbp-1(brown) (×20). E: Double staining for CD20 (purple) and Xbp-1(brown) showed that cells with nuclear Xbp-1 expression are B-cells. F: T cells stained with CD3 (purple) are negative for nuclear Xbp-1(brown). G: Western-blot on compartmental protein extracts showed the unspliced p33 in the cytoplasmic extracts and the p50 protein in the nuclear extracts. A nuclear protein, PAARP was used to assed the quality of the extracts. H: Reverse transcription-PCR flanking the splicing region reveals a two peaks pattern in the Gene Scan. A main 437bp unspliced expression is in agreement with the immunohistochemical results. lz: light zone of the germinal center; dz: dark zone of the germinal center.

Figure 2

Relation of Xbp-1 with other transcription B cell transcription factors. Human tonsil. A: Pax-5 is expressed in virtually all B cells, only in the light zone of the germinal centers a small fraction of cells lack Pax-5 and express nuclear Xbp-1, in a mutually exclusive pattern (×20). B: Bcl-6 expression is restricted to germinal centers and only Bcl-6 negative centrocytes expressed nuclear Xbp-1 (×20). C: The plasma cell associated transcription factor Irf-4 is coexpressed in all nuclear Xbp-1 positive cells (×20).

Figure 3

Xbp-1 Activation in plasma cell and lymphoma cell lines. A: Western blotting. All cell lines have some extent of Xbp-1 activation. Myeloma cell lines had the highest expression of the active form of Xbp-1(p50 Xbp-1) with a very weak band, corresponding to the cytoplasmic form of the protein (p33 Xbp-1) in the immunoglobulin secreting cell lines (KMM-1and OPM2) and in the non-secreting cell lines (KMS-12BM). Ramos 2G6.4C10, Jeko-1 and SUHL-4,6 cell lines show the weakest Xbp-1 activation with a prominent p33Xbp-1 expression. B: Immunocytochemistry. All KMS-12BM cells expressed nuclear Xbp-1 concordantly with the high expression of p50 Xbp-1 in the blots. SUDHL-4 exhibits a homogeneous cytoplasmic localization of the protein, according with the higher expression of p33 Xbp-1 (× 60 under oil).

Figure 4

Xbp-1 activation in B-NHL and plasma cell tumors. A: In chronic lymphocytic leukemia, only scattered positive cells expressed nuclear Xbp-1 in the proliferation centers. The small lymphocytic component was consistently negative (×40). B: Marginal zone lymphoma contains some mature plasma cells that expressed nuclear Xbp-1, while the monocytoid neoplastic cells are negative (×40). C: Nuclear positivity for Xbp-1 in a typical mantle cell lymphoma with a high proliferative index (×40). D: Burkitt lymphoma with plasma cell differentiation has more than 20% of tumor cells with nuclear positivity for Xbp-1. E: Diffuse large B-cell lymphoma, germinal center type with a cytoplasmic expression of Xbp-1 (×40). F: Intense nuclear positivity for Xbp-1 in a diffuse large B-cell lymphoma non-germinal center type (×40).G: Plasmablastic lymphoma (×40) and (H) plasmacytoma exhibit a strong and diffuse nuclear positivity for Xbp-1(×40).

Figure 5

Overall survival of 103 diffuse large B-cell lymphoma patients. Overall survival curves after Kaplan and Meier analysis of 103 diffuse large B-cell lymphoma patients regarding the nuclear expression of active Xbp-1 spliced form by immunohistochemistry.