Identification of B Cell Receptor Antigens in the Chronic Lymphocytic Leukemia Microenvironment

Background: B cell receptor (BCR) signaling is a central pathway in Chronic Lymphocytic Leukemia (CLL) pathogenesis that is activated by interactions between CLL cells and the microenvironment in secondary lymphoid organs. Nurselike cells (NLCs) are an important component of this microenvironment, and co-culture of CLL cells with NLCs activates BCR signaling. CLL BCRs are able to recognize vimentin and calreticulin proteins exposed on the surface of NLCs and these interactions are responsible for stromal-mediated anti-apoptotic effects. However, the exact mechanism of BCR activation and the nature of the BCR ligands expressed by NLCs still remain incompletely defined.

Aim: The aim of this project is to identify and validate ligands expressed by NLCs that activate BCRs on CLL cells.

Methods: CLL PBMCs from 3 CLL patients were cultured in vitro for 14 days until outgrowth of NLCs.  Then, NLCs were harvested and lysed, followed by immunoprecipitation with recombinant monoclonal antibodies obtained from 4 different CLL patients carrying unmutated IGHV genes (U-CLL). Immunoprecipitation of human hTERT mesenchymal stromal cells was used as a negative control. Immunoprecipitated proteins were analyzed by label-free quantitative mass spectrometry followed by bioinformatic data analysis using the softwares MaxQuant and Perseus. The quantitative mass spectrometric data enabled us to distinguish between unspecific background proteins and putative BCR ligands.

Results: In all samples, around 2600 proteins were identified and around 2000 of them were quantified using mass spectrometry. Unsupervised hierarchical clustering identified the enrichment patterns of NLC-derived BCR ligands. We identified 6 different protein clusters; among them, one cluster included 11 putative CLL BCR antigens with a fold-change cut-off above 10, which were enriched in all 3 NLC samples, but not in hTERT cells. These BCR ligands included cytoskeletal proteins, ER-associated proteins, and membrane-associated proteins, some of them with known auto-antigenic function in other diseases.

Conclusion: Recombinant BCRs from U-CLL patients recognize a large number of proteins expressed by NLCs, identified through immunoprecipitation of NLC lysates with CLL BCRs, followed by label-free mass spectrometry. The identified ligands will be further validated by epitope-mapping and BCR activation functional studies to allow a better characterization of the pathogenic antigens in CLL, and of the mechanisms driving CLL survival in the tissue microenvironment.