Immune Cell Metabolic Fitness for Life.

Adoptive cell therapy holds great promise for treating a myriad of diseases, especially cancer. Within the last decade, immunotherapy has provided a significant leap in the successful treatment of leukemia. The research conducted throughout this period to understand the interrelationships between cancer cells and infiltrating immune cells winds up having one very common feature, bioenergetics. Cancer cells and immune cells both need ATP to perform their individual functions and cancer cells have adopted means to limit immune cell activity via changes in immune cell bioenergetics that redirect immune cell behavior to encourage tumor growth. Current leading strategies for cancer treatment super-charge an individual's own immune cells against cancer. Successful Chimeric Antigen Receptor T Cells (CAR T) target pathways that ultimately influence bioenergetics. In the last decade, scientists identified that mitochondria play a crucial role in T cell physiology. When modifying T cells to create chimeras, a unique mitochondrial fitness emerges that establishes stemness and persistence. This review highlights many of the key findings leading to this generation's CAR T treatments and the work currently being done to advance immunotherapy, to empower not just T cells but other immune cells as well against a variety of cancers.

Adaptation of microplate-based respirometry for hippocampal slices and analysis of respiratory capacity.

Multiple neurodegenerative disorders are associated with altered mitochondrial bioenergetics. Although mitochondrial O(2) consumption is frequently measured in isolated mitochondria, isolated synaptic nerve terminals (synaptosomes), or cultured cells, the absence of mature brain circuitry is a remaining limitation. Here we describe the development of a method that adapts the Seahorse Extracellular Flux Analyzer (XF24) for the microplate-based measurement of hippocampal slice O(2) consumption. As a first evaluation of the technique, we compared whole-slice bioenergetics with previous measurements made with synaptosomes or cultured neurons. We found that mitochondrial respiratory capacity and O(2) consumption coupled to ATP synthesis could be estimated in cultured or acute hippocampal slices with preserved neural architecture. Mouse organotypic hippocampal slices oxidizing glucose displayed mitochondrial O(2) consumption that was well coupled, as determined by the sensitivity to the ATP synthase inhibitor oligomycin. However, stimulation of respiration by uncoupler was modest (<120% of basal respiration) compared with previous measurements in cells or synaptosomes, though enhanced slightly (to ∼150% of basal respiration) by acute addition of the mitochondrial complex I-linked substrate pyruvate. These findings suggest a high basal utilization of respiratory capacity in slices and a limitation of glucose-derived substrate for maximal respiration. The improved throughput of microplate-based hippocampal respirometry over traditional O(2) electrode-based methods is conducive to neuroprotective drug screening. When coupled with cell type-specific pharmacology or genetic manipulations, the ability to measure O(2) consumption efficiently from whole slices should advance our understanding of mitochondrial roles in physiology and neuropathology.

The LRRC8 volume-regulated anion channel inhibitor, DCPIB, inhibits mitochondrial respiration independently of the channel.

There has been a resurgence of interest in the volume-regulated anion channel (VRAC) since the recent cloning of the LRRC8A-E gene family that encodes VRAC. The channel is a heteromer comprised of LRRC8A and at least one other family member; disruption of LRRC8A expression abolishes VRAC activity. The best-in-class VRAC inhibitor, DCPIB, suffers from off-target activity toward several different channels and transporters. Considering that some anion channel inhibitors also suppress mitochondrial respiration, we systematically explored whether DCPIB inhibits respiration in wild type (WT) and LRRC8A-knockout HAP-1 and HEK-293 cells. Knockout of LRRC8A had no apparent effects on cell morphology, proliferation rate, mitochondrial content, or expression of several mitochondrial genes in HAP-1 cells. Addition of 10 µM DCPIB, a concentration typically used to inhibit VRAC, suppressed basal and ATP-linked respiration in part through uncoupling the inner mitochondrial membrane (IMM) proton gradient and membrane potential. Additionally, DCPIB inhibits the activity of complex I, II, and III of the electron transport chain (ETC). Surprisingly, the effects of DCPIB on mitochondrial function are also observed in HAP-1 and HEK-293 cells which lack LRRC8A expression. Finally, we demonstrate that DCPIB activates ATP-inhibitable potassium channels comprised of heterologously expressed Kir6.2 and SUR1 subunits. These data indicate that DCPIB suppresses mitochondrial respiration and ATP production by dissipating the mitochondrial membrane potential and inhibiting complexes I-III of the ETC. They further justify the need for the development of sharper pharmacological tools for evaluating the integrative physiology and therapeutic potential of VRAC in human diseases.

Patterns of cell-cell coupling in embryonic spinal cord studied via ballistic delivery of gap-junction-permeable dyes.

Intercellular communication mediated by gap junctions is developmentally regulated in many tissues, including the nervous system. In rodent lumbar spinal cord, extensive gap junctional coupling among motor neurons innervating the same muscle is present at birth but is no longer present 1 week later. Little is known about how this motor-pool-specific coupling arises during embryonic development. To address this question, we developed a novel method of visualizing patterns of cell-cell coupling that can be applied to a wide range of tissues. Gap-junction-permeable dyes adsorbed to metal beads were delivered into individual cells in embryonic cerebral cortex or spinal cord using pressure. Dye diffused off of the bead surface into the cytoplasm, crossed gap junctions, and labeled clusters of coupled cells. For embryonic cerebral cortex, this method revealed patterns of cell-cell coupling similar to those reported with other techniques. In embryonic lumbar spinal cord, cell-cell coupling is widespread in the ventricular zone at E11, and the extent of coupling decreases until birth. In the ventral horn, motor neurons are coupled into clusters at E14, with little change in the extent of coupling at E16, and a similar extent of coupling is present at birth. The cell types within clusters, identified by using antibodies against homeodomain transcription factors, were surprisingly heterogeneous in both the ventricular zone and the motor columns. Taken together, these data suggest that the spatial and temporal patterns of cell-cell coupling are dynamic and that cell-type-specific gap junctional coupling arises gradually during spinal cord development.

Connexin expression in homotypic and heterotypic cell coupling in the developing cerebral cortex.

Intercellular communication through gap junction channels is a prominent feature of the developing cerebral cortex. In the first 2 weeks after birth, a time critical in the development of the rat neocortex, extensive cell coupling has been documented that diminishes as the cortex matures. Among the family of gap junction proteins, connexins 26, 36, and 43 are differentially expressed during cortical development. We used intracellular dye injections and connexin immunohistochemistry to investigate the coupling patterns and connexin expression between the different neuronal and glial cell types of the developing cortex of the rat. We found that neurons and glia couple homotypically and heterotypically at postnatal days 7 and 14. Although the prevalence of coupling was homotypic, there was considerable heterotypic coupling that involved pyramidal and nonpyramidal neurons, the principal neuronal cell types of the cortex, or neurons and astrocytes. Coupling between different cell types appeared to be mediated by differential expression of connexins 26, 36, and 43. It may be that coupling between cells in the developing neocortex is a function of the spatial and temporal expression of these and other connexin proteins.

Analysis of regional brain mitochondrial bioenergetics and susceptibility to mitochondrial inhibition utilizing a microplate based system.

The analysis of mitochondrial bioenergetic function typically has required 50-100 µg of protein per sample and at least 15 min per run when utilizing a Clark-type oxygen electrode. In the present work we describe a method utilizing the Seahorse Biosciences XF24 Flux Analyzer for measuring mitochondrial oxygen consumption simultaneously from multiple samples and utilizing only 5 µg of protein per sample. Utilizing this method we have investigated whether regionally based differences exist in mitochondria isolated from the cortex, striatum, hippocampus, and cerebellum. Analysis of basal mitochondrial bioenergetics revealed that minimal differences exist between the cortex, striatum, and hippocampus. However, the cerebellum exhibited significantly slower basal rates of Complex I and Complex II dependent oxygen consumption (p<0.05). Mitochondrial inhibitors affected enzyme activity proportionally across all samples tested and only small differences existed in the effect of inhibitors on oxygen consumption. Investigation of the effect of rotenone administration on Complex I dependent oxygen consumption revealed that exposure to 10 pM rotenone led to a clear time dependent decrease in oxygen consumption beginning 12 min after administration (p<0.05). These studies show that the utilization of this microplate based method for analysis of mitochondrial bioenergetics is effective at quantifying oxygen consumption simultaneously from multiple samples. Additionally, these studies indicate that minimal regional differences exist in mitochondria isolated from the cortex, striatum, or hippocampus. Furthermore, utilization of the mitochondrial inhibitors suggests that previous work indicating regionally specific deficits following systemic mitochondrial toxin exposure may not be the result of differences in the individual mitochondria from the affected regions.