Expression of NTB-A in Normal Tissue, Non-Hodgkin Lymphomas and Investigation of Its Potential Therapeutic Impact for Chronic Lymphocytic Leukemia.

Introduction: Expressed on T, B, NK cells and neutrophils, NTB-A belongs to the signaling lymphocytic activation molecule (SLAM) family of immune receptors. It was reported recently that cross-linking of NTB-A induces T cell activation and Ly108 (the presumed mouse orthologue of NTB-A) controls the production of reactive oxygen species in neutrophils. To date, little is known about its expression at the protein level in normal tissues, or in hematologic malignancies.

Methods: We have generated monoclonal antibodies (mAbs) to NTB-A and used tissue microarrays (TMAs) to screen normal tissues and lymphomas. Western blotting and flow cytometry were used for confirmation of selected entities. Complement dependent cytotoxicity (CDC) assays were performed using the CellTiter-Glo Luminescent Cell Viability Assay kit (Promega).

Results: NTB-A was not detected in normal tissues including heart, liver, breast, kidney, brain, lung, uterus, small intestine, skin, prostate, pancreas, ovary, bladder, testis and stomach (5/5 negative cases for all above tissues) by IHC. Expression was observed in lymphocytes of normal tonsil and spleen. In lymphomas, we found NTB-A expression in diffuse large B-cell lymphoma (DLBL), follicular lymphoma, small lymphocytic lymphoma (SLL), mantle cell lymphoma and Burkitt lymphoma. Western blotting and flow cytometry of B-cell lymphoma cell lines DOHH2 (DLBL) and Raji (Burkitt) confirmed NTB-A expression in these lines. Myeloma cell lines were negative for expression. Flow cytometry of normal blood cells showed expression of NTB-A in B-cells, T-cells and NK cells (95.5 ± 6.7%, 62.6 ± 29.5% and 72.7 ± 26.5%, respectively) but not in CD66+ granulocytes. Furthermore, CD34+ progenitor cells from bone marrow were negative. Because of the expression in SLL, we focused on chronic lymphocytic leykemia (CLL) cells. All (15/15) patient samples were confirmed positive by flow cytometry. No significant differences in expression levels were observed comparing CLL B-cells vs normal B-cells (n = 10). However, anti-NTB-A antibodies were capable of inducing CDC in white blood cells from CLL patients (n=5) but not in normal white blood cells (n=5), in addition to CDC activity against Raji cells. Preliminary data also suggests that the CDC effect is greater in purified CLL B-cells than purified normal B-cells.

Conclusions: These results show that NTB-A is expressed in normal lymphocytes but not in other solid tissues or in CD34+ progenitor cells. B cell malignancies, including Non-Hodgkin lymphoma cells appear to express NTB-A. In particular CLL patient cells express NTB-A, and an anti-NTB-A mouse mAb is capable of inducing CDC, suggesting NTB-A may be a potential immunotherapeutic target. Further studies examining the expression patterns in hematologic malignancies are ongoing.