Bioenergetic analysis of suspension cells: hematopoietic stem cells and lymphocytes

A real time assay that quantifies the ATP and biosynthetic demands of immune cell proliferation, differentiation and effector function.

Immobilization of suspension cells makes it possible to monitor mitochondrial respiration and glycolysis of leukocytes in the XF Analyzer and to connect cellular energy metabolism to cellular function such as immune responses or dysfunction such as leukemia.

Normal suspension cells, such as hematopoietic cells and lymphocytes are responsible for supplying oxygen to the body and protecting the host. Cellular metabolism is markedly dynamic in immune responses during proliferation and immune effector functions. For example, antigen stimulated T cells display a metabolic switch to aerobic glycolysis accompanied by cell growth and proliferation, similar to the Warburg effect observed in cancer cells. Manipulation of the lymphocyte-specific metabolic control pathways may prove to be useful in treating diseases characterized by immune hyperactivation, including leukemia and autoimmune disorders.

Immobilization of non-adherent cells in XF cell culture microplates enables bioenergetic analysis of lymphocytes and hematopoietic cells with the XF Extracellular Flux Analyzer. Immobilization can be achieved by using microplates coated with Cell-Tak™ Adhesive, a formulation of non-immunogenic, polyphenolic proteins extracted from the marine mussel.

Figure 1

Metabolic rates in wild-type and HVCN1-deficient B cells before (−) and after (+) stimulation for 24 h with F(ab')2 anti-IgM (20 μg/ml), presented as the oxygen consumption rate (OCR) and extracellular acidification rate (ECAR), measures of mitochondrial respiration and glycolysis, respectively. Each symbol represents an individual mouse; longer horizontal lines indicate the mean and small horizontal lines indicate the standard error. P values, Student's T-test. Data are representative of three experiments with four mice (time 0) or five mice (24 hours).

In a recent publication, Cappasso, et al. investigated the role of the voltage-gated proton channel HVCN1 in cellular metabolism of B lymphocytes activation using B cells immobilized with Cell-Tak. Bioenergetic analyses of the B cells were generated by measuring the oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) of B cells as indications of mitochondrial respiration and glycolysis, respectively. The authors assessed mitochondrial respiration and glycolysis in B cells activated for 24 hours with F(ab')2 anti-IgM. The B cell receptor (BCR) activated B cells experience an increase in both mitochondrial respiration and glycolysis when compared to non-stimulated B cells. (Figure 1)

The authors then showed that genetic deletion of HVCN1 significantly impaired the increase in metabolism of activated B cells, but not that of resting B cells. These impairments result from disrupted signal transduction of the BCR complex and downstream signaling of Akt activation. They found that after BCR stimulation, cells lacking HVCN1 showed lower oxygen consumption and acidification than did wild-type cells (Figure 1), which indicated impaired energy production via mitochondrial oxidative phosphorylation and glycolysis in the absence of HVCN1 in the activated B cells.

Consistent with a defect in cellular metabolism, HVCN1-deficient B cells showed less proliferation in vitro after stimulation with F(ab')2 anti-IgM and defective antibody response in vivo.

Discussion

Immobilization of suspension cells makes it possible to monitor mitochondrial respiration and glycolysis of leukocytes in the XF Analyzer and to connect cellular energy metabolism to cellular function such as immune responses or dysfunction such as leukemia.

Liu et al4 showed that polycomb repressor Bmi1 has an important role in maintaining mitochondrial function and redox homeostasis of thymocytes and hematopoietic stem cells. The authors analyzed the mitochondrial function of freshly isolated Bmi1-/- murine thymocytes. They showed that intact Bmi1-/- thymocytes had both reduced basal mitochondrial oxygen consumption and reduced mitochondrial oxidative capacity. Consistent with these results, the authors found significant impairment in the function of isolated Bmi1-/- mitochondria including reduced electron flow, increased NAD(P)H levels and increased MitoSox staining supporting their hypothesis that mitochondria are the major source of increased ROS level in Bmi1-/- thymocytes.

Abnormal metabolism is a hallmark of many types of cancers including leukemia cells. In another study, Wu et al.5 investigated the effect of anticancer drug Imatinib, which target Bcr-Abl oncogene, on Bcr-abl-expressing K562 leukemia cells. They found K562 cells treated with Imatinib for 48 hours reduced mitochondrial respiration and glycolysis, which is accompanied by a decreased cell proliferation rate. These results suggest that Imanitib's anticancer effect is mediated, at least in part, through altering cellular energy metabolism.

Most recently, Gurumurthy et al.6 demonstrated that Lkb1 tumor suppressor is critical for the maintenance of energy homeostasis in haematopoietic cells, independent of AMP-activated protein kinase (AMPK) and mammalian target of rapamycin (mTOR) signaling. They found that Lkb1-deficient bone marrow cells exhibit mitochondrial defects with markedly reduced mitochondrial respiratory capacity, among the other metabolic defects. These results define a central role for Lkb1 in broadly maintaining energy homeostasis in haematopoietic cells through a novel metabolic checkpoint.

A concern about assaying immune cells such as B and T lymphocytes immobilized on culture surface is the potential cell activation by the crosslinking of the cells during immobilization. This is unlikely for at least two reasons. First of all, the immobilized cells are analyzed within a very short time period, typically two hours, which precedes the surface expression of early activation marker CD266. Secondly, as shown in Figure 1, it is evident that stimulated and un-stimulated B cells showed a clear bioenergetic difference suggestion the naïve cells are not activated by the immunization. Poly-D-lysine, another nonspecific attachment factor, treated plates, has also been used successfully to immobilize lymphocytes for XF bioenergetic assay.

Materials & Methods

Cells and Compounds: XF V7 cell culture microplates were coated with Cell-Tak™ (BD Bioscience) prior to plating B cells as described in the Seahorse protocol.

B cells were purified from spleens of 8- to 12-week-old mice either by negative selection with anti-CD43 magnetic beads, with a purity of ~95%, or by positive selection with anti-B220 and anti-CD19 magnetic beads (Miltenyi Biotech), with a purity of ~98%. Cells were cultured in RPMI complete medium containing 10% (vol/vol) FCS, penicillin and streptomycin, l-Glutamax and 50 μM 2-mercaptoethanol. Splenic B cells were stimulated for various times at 37 °C with goat anti–mouse IgM F(ab')2 fragment (20 μg/ml). They were stimulated for 24 hours with F(ab')2 anti-IgM (20 μg/ml). Stimulated or control B cells were resuspanded in bicarbonate-free and low buffered DMEM (pH 7.4) containing 11 mM glucose, 2 mM glutamine and 1 mM pyruvate.

XF Bioenergetic Analysis

Bioenergetic analyses of lymphocytes were performed in the XF Analyzer (Seahorse Bioscience). The XF Analyzer creates a transient micro-chamber of only a few microliters in specialized cell culture micro¬plates. This enables OCR (oxygen consumption rate) and ECAR (extracellular acidification rate) to be monitored in real time.

Wild type and HVCN1 deficient B cells stimulated for 24 hours were plated in Cell-Tak™ coated 24 well XF V7 cell culture microplate at 1×106 cells per well in 100 μL assay medium. They were allowed to become attached for 30 minutes in a 37°C non-CO2 incubator. 500 μL assay medium was added to each well after the cells were stably attached to the bottom of the wells and incubated for one additional hour prior to XF bioenergetic assay. Low buffered bicarbonate-free DMEM assay medium contains 11 mM glucose, 2 mM glutamine and 1 mM pyruvate. Four basal OCR and ECAR of the control and stimulated cells were measured, and the average of two basal rates of each sample were plotted (Figure 1).