American Society of Hematology

Figure 4

$Figure 4. Growth responses of CLL cultures to BCR engagement correlate with kinetics of TCL1 and AKT colocalization to BCR membrane activation complexes. Confocal microscopy on primary cultures was done using a TCL1 monoclonal antibody (fluorescein isothiocyanate, green) in combination with AKT1/2, LYN, ZAP70, or SYK polyclonal antibodies (Cy3, red). Depicted are images from the 2-hour time point (C-E), which best distinguishes pAKT responders and correlated most strongly with the differential growth response seen at 48 hours, as in Figure 3A. (A) In tonsillar B cells, TCL1 and AKT showed a predominantly cytoplasmic distribution without apparent colocalization at baseline (or with CD40L stimulation), but recruitment of TCL1 and AKT to membrane complexes after continuous BCR engagement. AKT/TCL1 colocalization had a punctate pattern at 2 hours, with uniform membrane staining at 24 hours. (B) A representative CLL case with a hyperresponsive growth pattern showed a similar rapid localization of TCL1 and AKT to the membrane as early as 15 minutes after BCR stimulation, with persistence of TCL1-AKT complexes for up to 4 days, including nuclear complexes at these later time points. Apoptotic cells were apparent by 24 hours in those cultures from hyperresponsive cases. (C) BCR-induced TCL1 membrane complexes (left column, single cells) colocalize with the BCR-associated kinases LYN, SYK, and ZAP70 within 2 hours of BCR stimulation (control condition shown in inset with +, #, and *, respectively, marking cells shown in enlarged images). (D) A representative CLL case with dose-dependent growth response to BCR engagement showed delayed but strong recruitment of TCL1 (Cy3, red in top panel) and AKT (Cy3, red) to discrete BCR-containing (CD79a in fluorescein isothiocyanate, green in top panel) membrane activation foci along with TCL1/AKT colocalization (middle row), as well as LYN, ZAP70, and pPKC corecruitment (bottom rows). (E) An absence of TCL1-AKT membrane complexes is noted in CLL with no growth response to BCR engagement. (F) Immunoblots of subcellular fractions (left panel) and subsequent spin column eluates (right panel, molecular weight cutoff > 30 kDa) from the same BCR responder as depicted in Figure 4B confirm the enrichment of phospho-activated AKT and to a lesser degree ZAP70 along with complexed TCL1 (14 kDa for monomeric TCL1) in the membrane compartment at 2 hours of successful BCR stimulation. The altered migration properties of CCR4's membrane associated form served as separation control. (G) Western blot analysis of activation complexes detected by AKT immunoprecipitation was performed after 48 hours of BCR stimulation in a BCR-responsive CLL (left) and a nonresponsive case (right). AKT kinase activity was tested on the immunoprecipitated complexes by assaying phosphorylation of an exogenous GSK3α/β substrate. An α-IgM dose-dependent increase in levels of coimmunoprecipitated pAKT, AKT kinase activity, and complexed ZAP70 was detected in the BCR-responsive case, but not in the nonresponder. Coimmunoprecipitated TCL1 was only detected in the BCR responder. Total TCL1 levels in the lysates from both cultures were similar (bottom panel).$

Growth responses of CLL cultures to BCR engagement correlate with kinetics of TCL1 and AKT colocalization to BCR membrane activation complexes. Confocal microscopy on primary cultures was done using a TCL1 monoclonal antibody (fluorescein isothiocyanate, green) in combination with AKT1/2, LYN, ZAP70, or SYK polyclonal antibodies (Cy3, red). Depicted are images from the 2-hour time point (C-E), which best distinguishes pAKT responders and correlated most strongly with the differential growth response seen at 48 hours, as in Figure 3A. (A) In tonsillar B cells, TCL1 and AKT showed a predominantly cytoplasmic distribution without apparent colocalization at baseline (or with CD40L stimulation), but recruitment of TCL1 and AKT to membrane complexes after continuous BCR engagement. AKT/TCL1 colocalization had a punctate pattern at 2 hours, with uniform membrane staining at 24 hours. (B) A representative CLL case with a hyperresponsive growth pattern showed a similar rapid localization of TCL1 and AKT to the membrane as early as 15 minutes after BCR stimulation, with persistence of TCL1-AKT complexes for up to 4 days, including nuclear complexes at these later time points. Apoptotic cells were apparent by 24 hours in those cultures from hyperresponsive cases. (C) BCR-induced TCL1 membrane complexes (left column, single cells) colocalize with the BCR-associated kinases LYN, SYK, and ZAP70 within 2 hours of BCR stimulation (control condition shown in inset with +, #, and *, respectively, marking cells shown in enlarged images). (D) A representative CLL case with dose-dependent growth response to BCR engagement showed delayed but strong recruitment of TCL1 (Cy3, red in top panel) and AKT (Cy3, red) to discrete BCR-containing (CD79a in fluorescein isothiocyanate, green in top panel) membrane activation foci along with TCL1/AKT colocalization (middle row), as well as LYN, ZAP70, and pPKC corecruitment (bottom rows). (E) An absence of TCL1-AKT membrane complexes is noted in CLL with no growth response to BCR engagement. (F) Immunoblots of subcellular fractions (left panel) and subsequent spin column eluates (right panel, molecular weight cutoff > 30 kDa) from the same BCR responder as depicted in Figure 4B confirm the enrichment of phospho-activated AKT and to a lesser degree ZAP70 along with complexed TCL1 (14 kDa for monomeric TCL1) in the membrane compartment at 2 hours of successful BCR stimulation. The altered migration properties of CCR4's membrane associated form served as separation control. (G) Western blot analysis of activation complexes detected by AKT immunoprecipitation was performed after 48 hours of BCR stimulation in a BCR-responsive CLL (left) and a nonresponsive case (right). AKT kinase activity was tested on the immunoprecipitated complexes by assaying phosphorylation of an exogenous GSK3α/β substrate. An α-IgM dose-dependent increase in levels of coimmunoprecipitated pAKT, AKT kinase activity, and complexed ZAP70 was detected in the BCR-responsive case, but not in the nonresponder. Coimmunoprecipitated TCL1 was only detected in the BCR responder. Total TCL1 levels in the lysates from both cultures were similar (bottom panel).

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