SIGLEC-G deficiency increases susceptibility to develop B-cell lymphoproliferative disorders

Abstract

The sialic-acid-binding immunoglobulin-like lectin SIGLEC-G is a negative regulator of B-cell receptor-mediated calcium signaling. Its deficiency leads to reduced turnover and increased proliferation and survival of murine B-1a cells. Siglecg(-/-) mice show a premature expansion of polyclonal CD5(+) B cells in the spleen and the peritoneal cavity. Here we studied the fate of B lymphocytes in Siglecg(-/-) mice over time. We demonstrate that in aging animals SIGLEC-G deficiency promotes progressive accumulation of monoclonal B lymphocytes and increases the susceptibility to develop B-cell lymphoproliferative disorders. Lymphoid tumors arising in aged Siglecg(-/-) mice are monoclonal and histologically heterogeneous as they include diffuse large B-cell lymphoma, follicular lymphoma, and medium-to-large B-cell monomorphic lymphoma but surprisingly not chronic lymphocytic leukemia. The tumors express high levels of BCL-2 and are transplantable. In keeping with these findings we have also observed a remarkable down-regulation of the human ortholog SIGLEC10 in human B-cell lymphoma and leukemia cell lines. Taken together, these observations indicate that the down-regulation of negative B-cell receptor regulators such as SIGLEC-G/SIGLEC10 may represent another mechanism relevant to the pathogenesis of B-cell lymphomas.

Figure 1.

Evolution of B-1a, total B cells and T lineage cells in Siglecg^−/− mice. (A–B) Flow cytometric analysis of peripheral blood and splenic B cells of 5-, 10- and 16-month old Siglecg^−/− mice and age-matched control mice (n=6–10). Accumulation of (A) B-1a cells (CD19⁺CD5⁺) in the peripheral blood and of (B) total B cells (CD19⁺) within the spleen of Siglecg^−/− mice over time. (C–D) Cells from 5-, 10- and 16-month old Siglecg^−/− mice and age-matched control mice (n=3–10) were analyzed by flow cytometry to define the percentage of (C) CD3⁺CD4⁺ and (D) CD3⁺CD8⁺ T-cell populations. Each symbol in the dot plots represents one mouse and the horizontal line indicates the mean value (+/+: wild-type, −/−: Siglecg^−/− mice). Statistical significance was analyzed by the t-test.

Figure 2.

Development of B-cell lymphoproliferative disorders in aged Siglecg^−/− mice. (A) Kaplan-Meier survival curves of wild-type (n=9) and Siglecg^−/− (n=8) mice. Statistical analysis between groups was performed using the log-rank test (median survival: 26 and 20.5 for wild-type and Siglecg^−/− mice, respectively). Mice were included in the analysis after spontaneous death. (B) Histopathological examination of spleen of aged Siglecg^−/− mice: representative cases of atypical lymphoproliferative process (ALP), DLBCL, follicular lymphoma (FL) and medium-to-large B-cell monomorphic lymphoma (ML-BCML) compared to a healthy (negative) age-matched wild-type mouse. Spleen was analyzed by hematoxylin and eosin (H&E) staining or immunohistochemical stains for B220, BCL-2, BCL-6, MUM1 and Ki-67. Microscopic evaluation: 10× or 40×, as indicated.

Figure 3.

Enhanced calcium signaling in B lymphocytes from aged Siglecg^−/− mice that developed B-cell lymphoproliferations. (A) Basal levels of intracellular calcium in B cells (gated as CD19⁺) from aged Siglecg^−/− (n=6) and wild-type (n=5) mice. Data are expressed as a whisker plot. Statistical significance was analyzed by the t-test. (B) Extracts from B cells of aged Siglecg^−/− and wild-type mice were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis followed by immunoblotting with NFATc1-specific antibody or β-actin-specific control antibody (three representative samples for each genotype are shown).

Figure 4.

Engraftment of Siglecg^−/− B-cell lymphoproliferative disorders. (A) Kaplan-Meier survival curves of mice that received Siglecg^−/− B-cell lymphoproliferation and lymphoma transplants. Mice were included in the analysis after spontaneous death or when killed because of signs/symptoms of illness. Squares and triangles indicate the primary (I) and secondary (II) transplants, respectively. Statistical analysis between groups was performed using the log-rank test (median survival: 15 months for primary transplant, 7 months for secondary transplant). (B) Histopathological examination of spleen of BALB/c mice injected with Siglecg^−/− B cells (primary transplant, one representative animal): hematoxylin and eosin (H&E) staining and immunohistochemical stain for B220; microscopic evaluation 40×.

Figure 5.

Downregulation of SIGLEC10 expression in human B-cell lymphoma and leukemia cell lines. (A–B) Human B-cell lymphoma/leukemia cell lines, primary B cells and GC B cells purified from tonsils (T) and primary B cells isolated from peripheral blood (PB) of healthy donors were analyzed for the expression of SIGLEC10. (A) Relative mRNA expression of SIGLEC10. The expression of SIGLEC10 was normalized to GAPDH and the results presented as a ratio between normalized SIGLEC10 expression in the target samples and in Raji cells (calibrator sample). Data shown are the average of two independent experiments ± standard deviation. (B) Western blot analysis of SIGLEC10 and β-actin expression on extracts from cell lines and primary cells.