Assessing Changes in Human Natural Killer Cell Metabolism Using the Seahorse Extracellular Flux Analyzer.

Natural killer (NK) cells are cytotoxic cells that mediate anti-tumor and anti-viral immunity. The response of NK cells to different cytokines and stimuli may involve cell survival, proliferation, and changes in their cytotoxic function. These responses will be supported by changes in cellular metabolism. Therefore, changes in NK metabolic parameters could somehow predict changes in NK cell function and cytotoxicity. In this chapter, we describe a protocol to measure NK cell metabolism in primary human NK cells by using an extracellular flux analyzer. This machine measures pH and oxygen changes in the medium and allows the study of NK cell glycolysis and mitochondrial respiration in real time with a small number of cells.

Measurement of Cytosolic Mitochondrial DNA After NLRP3 Inflammasome Activation.

The NLRP3 inflammasome, a key component of the innate immune system that mediates caspase-1 activation, which in turn induces cleavage of the pyroptosis executioner gasdermin D and the proinflammatory cytokines IL-1ß and IL-18, requires two signals to be activated. First, inflammasome priming is achieved after activation of Toll-like receptors, which leads to NF-κB signaling and transcriptional activation of the genes for NLRP3 and IL-1ß. Next, the inflammasome complex is activated by a second signal that induces extrusion of mitochondrial DNA to the cytosol of the cell, which leads to its oligomerization by a not fully understood mechanism. Here we describe a simple method that employs quantitative polymerase chain reaction (qPCR) using SYBR green to measure the presence of mitochondrial DNA (mtDNA) in the cytosol, which can be used to measure cytosolic mtDNA levels after inflammasome activation.

Immunometabolism at the Nexus of Cancer Therapeutic Efficacy and Resistance.

Constitutive activity of the immune surveillance system detects and kills cancerous cells, although many cancers have developed strategies to avoid detection and to resist their destruction. Cancer immunotherapy entails the manipulation of components of the endogenous immune system as targeted approaches to control and destroy cancer cells. Since one of the major limitations for the antitumor activity of immune cells is the immunosuppressive tumor microenvironment (TME), boosting the immune system to overcome the inhibition provided by the TME is a critical component of oncotherapeutics. In this article, we discuss the main effects of the TME on the metabolism and function of immune cells, and review emerging strategies to potentiate immune cell metabolism to promote antitumor effects either as monotherapeutics or in combination with conventional chemotherapy to optimize cancer management.

Analysis of Human Natural Killer Cell Metabolism.

Natural Killer (NK) cells mediate mainly innate anti-tumor and anti-viral immune responses and respond to a variety of cytokines and other stimuli to promote survival, cellular proliferation, production of cytokines such as interferon gamma (IFNγ) and/or cytotoxicity programs. NK cell activation by cytokine stimulation requires a substantial remodeling of metabolic pathways to support their bioenergetic and biosynthetic requirements. There is a large body of evidence that suggests that impaired NK cell metabolism is associated with a number of chronic diseases including obesity and cancer, which highlights the clinical importance of the availability of a method to determine NK cell metabolism. Here we describe the use of an extracellular flux analyzer, a platform that allows real-time measurements of glycolysis and mitochondrial oxygen consumption, as a tool to monitor changes in the energy metabolism of human NK cells. The method described here also allows for the monitoring of metabolic changes after stimulation of NK cells with cytokines such as IL-15, a system that is currently being investigated in a wide range of clinical trials.

Trans-endocytosis of intact IL-15Rα-IL-15 complex from presenting cells into NK cells favors signaling for proliferation.

Interleukin 15 (IL-15) is an essential cytokine for the survival and proliferation of natural killer (NK) cells. IL-15 activates signaling by the ß and common γ (γc) chain heterodimer of the IL-2 receptor through trans-presentation by cells expressing IL-15 bound to the α chain of the IL-15 receptor (IL-15Rα). We show here that membrane-associated IL-15Rα-IL-15 complexes are transferred from presenting cells to NK cells through trans-endocytosis and contribute to the phosphorylation of ribosomal protein S6 and NK cell proliferation. NK cell interaction with soluble or surface-bound IL-15Rα-IL-15 complex resulted in Stat5 phosphorylation and NK cell survival at a concentration or density of the complex much lower than required to stimulate S6 phosphorylation. Despite this efficient response, Stat5 phosphorylation was reduced after inhibition of metalloprotease-induced IL-15Rα-IL-15 shedding from trans-presenting cells, whereas S6 phosphorylation was unaffected. Conversely, inhibition of trans-endocytosis by silencing of the small GTPase TC21 or expression of a dominant-negative TC21 reduced S6 phosphorylation but not Stat5 phosphorylation. Thus, trans-endocytosis of membrane-associated IL-15Rα-IL-15 provides a mode of regulating NK cells that is not afforded to IL-2 and is distinct from activation by soluble IL-15. These results may explain the strict IL-15 dependence of NK cells and illustrate how the cellular compartment in which receptor-ligand interaction occurs can influence functional outcome.

IL-15 enhanced antibody-dependent cellular cytotoxicity mediated by NK cells and macrophages.

The goal of cancer immunotherapy is to stimulate the host immune system to attack malignant cells. Antibody-dependent cellular cytotoxicity (ADCC) is a pivotal mechanism of antitumor action of clinically employed antitumor antibodies. IL-15 administered to patients with metastatic malignancy by continuous i.v. infusion at 2 µg/kg/d for 10 days was associated with a 38-fold increase in the number and activation status of circulating natural killer (NK) cells and activation of macrophages which together are ADCC effectors. We investigated combination therapy of IL-15 with rituximab in a syngeneic mouse model of lymphoma transfected with human CD20 and with alemtuzumab (Campath-1H) in a xenograft model of human adult T cell leukemia (ATL). IL-15 greatly enhanced the therapeutic efficacy of both rituximab and alemtuzumab in tumor models. The additivity/synergy was shown to be associated with augmented ADCC. Both NK cells and macrophages were critical elements in the chain of interacting effectors involved in optimal therapeutic responses mediated by rituximab with IL-15. We provide evidence supporting the hypothesis that NK cells interact with macrophages to augment the NK-cell activation and expression of FcγRIV and the capacity of these cells to become effectors of ADCC. The present study supports clinical trials of IL-15 combined with tumor-directed monoclonal antibodies.

NK Cell Proliferation Induced by IL-15 Transpresentation Is Negatively Regulated by Inhibitory Receptors.

IL-15 bound to the IL-15Rα-chain (IL-15Rα) is presented in trans to cells bearing the IL-2Rß-chain and common γ-chain. As IL-15 transpresentation occurs in the context of cell-to-cell contacts, it has the potential for regulation by and of other receptor-ligand interactions. In this study, human NK cells were tested for the sensitivity of IL-15 transpresentation to inhibitory receptors. Human cells expressing HLA class I ligands for inhibitory receptors KIR2DL1, KIR2DL2/3, or CD94-NKG2A were transfected with IL-15Rα. Proliferation of primary NK cells in response to transpresented IL-15 was reduced by engagement of either KIR2DL1 or KIR2DL2/3 by cognate HLA-C ligands. Inhibitory KIR-HLA-C interactions did not reduce the proliferation induced by soluble IL-15. Therefore, transpresentation of IL-15 is subject to downregulation by MHC class I-specific inhibitory receptors. Similarly, proliferation of the NKG2A(+) cell line NKL induced by IL-15 transpresentation was inhibited by HLA-E. Coengagement of inhibitory receptors, either KIR2DL1 or CD94-NKG2A, did not inhibit phosphorylation of Stat5 but inhibited selectively phosphorylation of Akt and S6 ribosomal protein. IL-15Rα was not excluded from, but was evenly distributed across, inhibitory synapses. These findings demonstrate a novel mechanism to attenuate IL-15-dependent NK cell proliferation and suggest that inhibitory NK cell receptors contribute to NK cell homeostasis.

Cutting Edge: Regulation of Exosome Secretion by the Integral MAL Protein in T Cells.

Exosomes secreted by T cells play an important role in coordinating the immune response. HIV-1 Nef hijacks the route of exosome secretion of T cells to modulate the functioning of uninfected cells. Despite the importance of the process, the protein machinery involved in exosome biogenesis is yet to be identified. In this study, we show that MAL, a tetraspanning membrane protein expressed in human T cells, is present in endosomes that travel toward the plasma membrane for exosome secretion. In the absence of MAL, the release of exosome particles and markers was greatly impaired. This effect was accompanied by protein sorting defects at multivesicular endosomes that divert the exosomal marker CD63 to autophagic vacuoles. Exosome release induced by HIV-1 Nef was also dependent on MAL expression. Therefore, MAL is a critical element of the machinery for exosome secretion and may constitute a target for modulating exosome secretion by human T cells.

Centrosome polarization in T cells: a task for formins.

T-cell antigen receptor (TCR) engagement triggers the rapid reorientation of the centrosome, which is associated with the secretory machinery, toward the immunological synapse (IS) for polarized protein trafficking. Recent evidence indicates that upon TCR triggering the INF2 formin, together with the formins DIA1 and FMNL1, promotes the formation of a specialized array of stable detyrosinated MTs that breaks the symmetrical organization of the T-cell microtubule (MT) cytoskeleton. The detyrosinated MT array and TCR-induced tyrosine phosphorylation should coincide for centrosome polarization. We propose that the pushing forces produced by the detyrosinated MT array, which modify the position of the centrosome, in concert with Src kinase dependent TCR signaling, which provide the reference frame with respect to which the centrosome reorients, result in the repositioning of the centrosome to the IS.

INF2 promotes the formation of detyrosinated microtubules necessary for centrosome reorientation in T cells.

T cell antigen receptor-proximal signaling components, Rho-family GTPases, and formin proteins DIA1 and FMNL1 have been implicated in centrosome reorientation to the immunological synapse of T lymphocytes. However, the role of these molecules in the reorientation process is not yet defined. Here we find that a subset of microtubules became rapidly stabilized and that their α-tubulin subunit posttranslationally detyrosinated after engagement of the T cell receptor. Formation of stabilized, detyrosinated microtubules required the formin INF2, which was also found to be essential for centrosome reorientation, but it occurred independently of T cell receptor-induced massive tyrosine phosphorylation. The FH2 domain, which was mapped as the INF2 region involved in centrosome repositioning, was able to mediate the formation of stable, detyrosinated microtubules and to restore centrosome translocation in DIA1-, FMNL1-, Rac1-, and Cdc42-deficient cells. Further experiments indicated that microtubule stabilization was required for centrosome polarization. Our work identifies INF2 and stable, detyrosinated microtubules as central players in centrosome reorientation in T cells.

MAL protein controls protein sorting at the supramolecular activation cluster of human T lymphocytes.

T cell membrane receptors and signaling molecules assemble at the immunological synapse (IS) in a supramolecular activation cluster (SMAC), organized into two differentiated subdomains: the central SMAC (cSMAC), with the TCR, Lck, and linker for activation of T cells (LAT), and the peripheral SMAC (pSMAC), with adhesion molecules. The mechanism of protein sorting to the SMAC subdomains is still unknown. MAL forms part of the machinery for protein targeting to the plasma membrane by specialized mechanisms involving condensed membranes or rafts. In this article, we report our investigation of the dynamics of MAL during the formation of the IS and its role in SMAC assembly in the Jurkat T cell line and human primary T cells. We observed that under normal conditions, a pool of MAL rapidly accumulates at the cSMAC, where it colocalized with condensed membranes, as visualized with the membrane fluorescent probe Laurdan. Mislocalization of MAL to the pSMAC greatly reduced membrane condensation at the cSMAC and redistributed machinery involved in docking microtubules or transport vesicles from the cSMAC to the pSMAC. As a consequence of these alterations, the raft-associated molecules Lck and LAT, but not the TCR, were missorted to the pSMAC. MAL, therefore, regulates membrane order and the distribution of microtubule and transport vesicle docking machinery at the IS and, by doing so, ensures correct protein sorting of Lck and LAT to the cSMAC.

Formin INF2 regulates MAL-mediated transport of Lck to the plasma membrane of human T lymphocytes.

Expression of the src-family kinase lymphocyte-specific protein tyrosine kinase (Lck) at the plasma membrane is essential for it to fulfill its pivotal role in signal transduction in T lymphocytes. MAL, an integral membrane protein expressed in specific types of lymphoma, has been shown to play an important role in targeting Lck to the plasma membrane. Here we report that MAL interacts with Inverted Formin2 (INF2), a formin with the atypical property of promoting not only actin polymerization but also its depolymerization. In Jurkat T cells, INF2 colocalizes with MAL at the cell periphery and pericentriolar endosomes and along microtubules. Videomicroscopic analysis revealed that the MAL(+) vesicles transporting Lck to the plasma membrane move along microtubule tracks. Knockdown of INF2 greatly reduced the formation of MAL(+) transport vesicles and the levels of Lck at the plasma membrane and impaired formation of a normal immunologic synapse. The actin polymerization and depolymerization activities of INF2 were both required for efficient Lck targeting. Cdc42 and Rac1, which bind to INF2, regulate Lck transport in both Jurkat and primary human T cells. Thus, INF2 collaborates with MAL in the formation of specific carriers for targeting Lck to the plasma membrane in a process regulated by Cdc42 and Rac1.