13. Role of the erytrocyte protein Band 3 in autoimmune hemolitic anemia associated to CLL

MORANDE PABLO; GALLETTI JEREMÍAS; BORGE MERCEDES; NANNINI PAULA ROMINA; GAMBERALE ROMINA; OPPEZZO PABLO; GIORDANO MIRTA

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Congreso; 1° French-Argentine Immunology Congress,; 2010

Chronic lymphocytic leukemia (CLL) is the most common cause of autoimmune hemolytic anemia (AHA). We have previously shown that leukemic B cells are able to bind the erythrocyte protein Band 3 (B3), through its N-terminal domain (B3N), an ability that could allow the neoplastic clone to trigger the autoimmune process. The aims of the present work were: 1) to identify molecules in the cell membrane of leukemic B cells as candidates for being B3N binding site and 2) to investigate the process of binding and endocytosis of erythrocyte-derived vesicles to leukemic B cells. In order to accomplish our first objective, we removed non-integral membrane proteins from DAUDI cells (a B cell line) by acidic elution using  potasium phosphate 0.1M, sodium citrate 0.05M buffer, pH=2.5. By flow cytometry analysis, we found that this treatment decreased the binding of B3N (50 ug/ml) to cells (MFI control cells: 160±8, MFI treated cells: 70±10, n=5, p<0.05). After neutralizing the acid eluates, they were passed through an affinity column coupled with B3N, and molecules interacting with B3N were recovered and analyzed by mass spectrometry. HMGN2, a nucleosomal protein reported to be secreted by leukocytes, was detected in acid eluates of DAUDI and CLL B cells (n=3).  To evaluate the binding and endocytosis of erythrocyte-derived vesicles to leukemic B cells, we used CFDA staining and flow cytometry. We found that 18% of CLL cells were able to bind inside-out vesicles (with B3N exposed to the outer surface) while only 4% of T lymphocytes did. Images obtained by confocal microscopy suggest that the vesicles are found inside the B cells after 2 hours of culture at 37ºC. We propose that leukemic B cells can specifically bind and uptake erythrocyte-derived vesicles to act as antigen-presenting cells of erythrocyte proteins, and that HMGN2 is a candidate molecule to function as a receptor in this system.