Background. Proteomics can integrate hybridoma technology in discovering new antigens through the characterization of monoclonal antibodies (mAbs) of unknown specificity, that are generated when using whole cells and even purified known proteins as immunogens.

Aim. To characterize, by means of a new mAb (B28p), a protein sharing an antigenic determinant with IRTA1, i.e. the unrelated immunogen used in the unforeseen generation of B28p.

Methods/Results. Among ~1000 hybridomas from mice immunized with IRTA1, one producing an IgG1 mAb (B28p), cross-reactive with IRTA1, recognized also a 28 kDa cytoplasmic protein expressed strongly by all mature B cell subsets, with the highest levels in plasma cells (PC), and weakly by epithelial cells. This pattern clearly differs from that expected for IRTA1, a 70 kDa cell membrane receptor selectively expressed in MALT marginal zone B cells (

Falini et al, Blood 2003;102:3684
).

To identify the 28 kDa antigen, the proteome of a B28p+ B-NHL was resolved by 2D-PAGE, and tandem mass spectrometry analysis revealed the B28p-immunoreactive spot as human tumor protein D52 (TPD52), previously described to be expressed only in epithelial tissues. The specificity of B28p mAb for TPD52 was confirmed by immunostaining/immunoblotting TPD52-transfected cells and by observing an identical labeling pattern (mature B cells/PC and, less strongly, epithelial cells) upon immunohistology with a specfic anti-TPD52 polyclonal antibody.

In routine tumor biopsies, TPD52 was expressed in all peripheral B-NHL subtypes (except some DLCLs) and most abundantly in multiple myeloma (MM). TPD52-staining proved to be a useful diagnostic tool in the quantification of bone marrow PC infiltration (thus helping in discriminating between MGUS and MM and in assessing minimal residual disease after therapy for MM), as well as in the evaluation of lymphomas associated with PC differentiation (including transplant-associated lymphomas, plasmablastic lymphomas of the oral cavity and primary effusion lymphomas). Notably, TPD52 was also expressed by a proportion of tumor cells in about 40% classical Hodgkin’s lymphomas, including cases misdiagnosed as ALK ALCL with null phenotype.

Unlike other B cell markers (PAX5,CD19,C79a,CD22,CD20), TPD52 represents the first example of a B cell protein whose expression increases during PC differentiation Intriguingly, in the Thiel myeloma cell line (similarly to what previously observed during zymogen secretion in pancreatic acinar cells), we found a strong and Ca+2-dependent co-immunoprecipitation of TPD52 with annexin VI, suggesting TPD52 up-regulation in PC may play a role in immunoglobulin-related secretory processes through interaction with annexin VI (which we also showed to be strongly expressed in PC).

Conclusions. By combining proteomic and hybridoma technologies, we first identified the epithelium-associated TPD52 protein as a new B cell/plasma cell protein showing unique expression pattern, diagnostic value and, likely, biological importance.

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