The bioenergetic response of B lymphocytes is subject to rapid changes following antigen encounter in order to provide ATP and anabolic precursors necessary to support growth. However, the pathways involved in glucose acquisition and metabolism are unknown. We find that B lymphocytes rapidly increase glucose uptake and glycolysis following B-cell antigen receptor (BCR) crosslinking. Inhibition of glycolysis blocks BCR-mediated growth. Prior to S-phase entry, glucose metabolism shifts from primarily glycolytic to include the pentose phosphate pathway. BCR-induced glucose utilization is dependent upon phosphatidylinositol 3-kinase (PI-3K) activity as evidenced by inhibition of glucose uptake and glycolysis with LY294002 treatment of normal B cells and impaired glucose utilization in B cells deficient in the PI-3K regulatory subunit p85␣. Activation of Akt is sufficient to increase glucose utilization in B cells. We find that glucose utilization is inhibited by coengagement of the BCR and Fc␥RIIB, suggesting that limiting glucose metabolism may represent an important mechanism underlying Fc␥RIIB-mediated growth arrest. Taken together, these findings demonstrate that both growth-promoting BCR signaling and growth-inhibitory Fc␥RIIB signaling modulate glucose energy metabolism. Manipulation of these pathways may prove to be useful in the treatment of lymphoproliferative disorders, wherein clonal expansion of B lymphocytes plays a role. IntroductionIn response to antigen challenge, resting B lymphocytes exit the G 0 phase of the cell cycle and undergo a period of growth before committing to genome replication. 1,2 Growth corresponds to an accumulation of cell mass that is accompanied by increased size and is linked to increased de novo macromolecular synthesis. [3][4][5] That mammalian cell growth may be necessary for genome replication underscores its importance in adaptive immunity in that the clonal expansion of antigen-specific B lymphocytes is a prerequisite for humoral immune responses. Most investigations in B cells have focused on the role of genes whose function are important for B-cell antigen receptor (BCR)-induced protein synthesis and increased cell size. 4,5 It is recognized, however, that antigen receptor-triggered macromolecular synthesis and gene expression places enormous bioenergetic demands on lymphocytes. 5,6 Therefore, one of the fundamental aspects of B-cell responses to antigen challenge that may be critical in vivo is the provision of metabolic substrates to provide ATP and anabolic precursors for cellular growth.Early studies in lectin-stimulated thymocytes highlighted the importance of glucose uptake and catabolism in providing energy and carbon for macromolecular synthesis. 7,8 Further, proliferating thymocytes meet their ATP demand mainly by glycolytic catabolism when sufficient glucose is available. 9 It is widely viewed that glucose metabolism is regulated by homeostatic mechanisms wherein mammalian cells respond to a decreased ATP/ADP ratio by adjusting nutrient uptake and catabolism t...

IL-4 prevents the death of naive B lymphocytes through the up-regulation of antiapoptotic proteins such as Bcl-xL. Despite studies implicating glucose utilization in growth factor-dependent survival of hemopoietic cells, the role of glucose energy metabolism in maintaining B cell viability by IL-4 is unknown. We show that IL-4 triggers glucose uptake, Glut1 expression, and glycolysis in splenic B cells; this is accompanied by increased cellular ATP. Glycolysis inhibition results in apoptosis, even in the presence of IL-4. IL-4-induced glycolysis occurs normally in B cells deficient in insulin receptor substrate-2 or the p85α subunit of PI3K and is not affected by pretreatment with PI3K or MAPK pathway inhibitors. Stat6-deficient B cells exhibit impaired IL-4-induced glycolysis. Cell-permeable, constitutively active Stat6 is effective in restoring IL-4-induced glycolysis in Stat6-deficient B cells. Therefore, besides controlling antiapoptotic proteins, IL-4 mediates B cell survival by regulating glucose energy metabolism via a Stat6-dependent pathway.

We show herein that CNT-cell complexes are formed in the presence of a magnetic field. The complexes were analyzed by flow cytometry as a quantitative method for monitoring the physical interactions between CNTs and cells. We observed an increase in side scattering signals, where the amplitude was proportional to the amount of CNTs that are associated with cells. Even after the formation of CNT-cell complexes, cell viability was not significantly decreased. The association between CNTs and cells was strong enough to be used for manipulating the complexes and thereby conducting cell separation with magnetic force. In addition, the CNT-cell complexes were also utilized to facilitate electroporation. We observed a time constant from CNT-cell complexes but not from cells alone, indicating a high level of pore formation in cell membranes. Experimentally, we achieved the expression of enhanced green fluorescence protein by using a low electroporation voltage after the formation of CNT-cell complexes. These results suggest that higher transfection efficiency, lower electroporation voltage, and miniaturized setup dimension of electroporation may be accomplished through the CNT strategy outlined herein.