Increased mitochondrial mass and glucose uptake in LNs and recent emigrants and effects of CD40 and BCR triggering on CLL metabolism. (A) Combined staining of CXCR4 and CD5 within the CD19/CD5⁺ CLL population from PB mononuclear cells (PBMCs) to discriminate LN immigrants (CD5^dim and CXCR4^high) and emigrants (CD5^high and CXCR4^dim). The outer 10% of these populations is indicated by green and red squares, respectively. Glucose uptake and mitochondrial mass were measured with 2-NBDG and MitoTracker Green, respectively, in the upper and lower 10% percentiles of these populations and in paired LN samples (blue lines). Representative histograms are shown. (B) Summary data of glucose uptake and mitochondrial mass (PBMCs, n = 6; LNs, n = 3; paired samples are indicated). Data are represented as individual values and mean. Statistical analyses were performed with paired Student t test. (C) CLL cells were stimulated by coculture on CD40L-expressing 3T3 cells (CD40) or by beads coated with anti–immunoglobulin M (IgM; BCR) for 24 hours. Glucose uptake and mitochondrial mass were measured by fluorescence-activated cell sorting (FACS) with 2-NBDG or MitoTracker Green, respectively, in unstimulated cells (3T3; blue line) or CD40-stimulated cells (red line). Representative histograms are shown. Relative glucose uptake and mitochondrial mass of CD40-stimulated cells compared with 3T3 condition were plotted in bar graph (2-NBDG, n = 16; MitoGreen, n = 7). (D) CD95 was measured after 24 hours of CD40 stimulation by FACS (n = 5), and pS6 was measured after 30 minutes of BCR stimulation by FACS (n = 5). (E) MitoStress test was performed on Seahorse XF analyzer in CLL cells activated by CD40 (n = 17)/BCR (n = 5) for 24 hours and in the corresponding unstimulated control cells (UN). Representative oxygen consumption rate (OCR) curve is shown. Basal OCR, maximal OCR, and spare respiratory capacity were calculated in 5 independent experiments. Extracellular acidification rate (ECAR) was also measured, and basal ECAR is shown. Data arerepresented as mean ± standard error of the mean of the indicated individual values. Statistical analyses were performed with paired Student t test. *P < .05, **P < .01, ***P < .001, ****P < .0001. MFI, mean fluorescence intensity; ns, not significant.