Glenn R. Bantug

Complement Regulates Nutrient Influx and Metabolic Reprogramming during Th1 Cell Responses

Summary Expansion and acquisition of Th1 cell effector function requires metabolic reprogramming; however, the signals instructing these adaptations remain poorly defined. Here we found that in activated human T cells, autocrine stimulation of the complement receptor CD46, and specifically its intracellular domain CYT-1, was required for induction of the amino acid (AA) transporter LAT1 and enhanced expression of the glucose transporter GLUT1. Furthermore, CD46 activation simultaneously drove expression of LAMTOR5, which mediated assembly of the AA-sensing Ragulator-Rag-mTORC1 complex and increased glycolysis and oxidative phosphorylation (OXPHOS), required for cytokine production. T cells from CD46-deficient patients, characterized by defective Th1 cell induction, failed to upregulate the molecular components of this metabolic program as well as glycolysis and OXPHOS, but IFN-γ production could be reinstated by retrovirus-mediated CD46-CYT-1 expression. These data establish a critical link between the complement system and immunometabolic adaptations driving human CD4+ T cell effector function.

CD8+ T lymphocytes control murine cytomegalovirus replication in the central nervous system of newborn animals.

Human CMV infection of the neonatal CNS results in long-term neurologic sequelae. To define the pathogenesis of fetal human CMV CNS infections, we investigated mechanisms of virus clearance from the CNS of neonatal BALB/c mice infected with murine CMV (MCMV). Virus titers peaked in the CNS between postnatal days 10–14 and infectious virus was undetectable by postnatal day 21. Congruent with virus clearance was the recruitment of CD8+ T cells into the CNS. Depletion of CD8+ T cells resulted in death by postnatal day 15 in MCMV-infected animals and increased viral loads in the liver, spleen, and the CNS, suggesting an important role for these cells in the control of MCMV replication in the newborn brain. Examination of brain mononuclear cells revealed that CD8+ T cell infiltrates expressed high levels of CD69, CD44, and CD49d. IE1168-specific CD8+ T cells accumulated in the CNS and produced IFN-γ and TNF-α but not IL-2 following peptide stimulation. Moreover, adoptive transfer of brain mononuclear cells resulted in decreased virus burden in immunodepleted MCMV-infected syngeneic mice. Depletion of the CD8+ cell population following transfer eliminated control of virus replication. In summary, these results show that functionally mature virus-specific CD8+ T cells are recruited to the CNS in mice infected with MCMV as neonates.

The Immune-Metabolic Basis of Effector Memory CD4+ T Cell Function under Hypoxic Conditions.

Effector memory (EM) CD4+ T cells recirculate between normoxic blood and hypoxic tissues to screen for cognate Ag. How mitochondria of these cells, shuttling between normoxia and hypoxia, maintain bioenergetic efficiency and stably uphold antiapoptotic features is unknown. In this study, we found that human EM CD4+ T cells had greater spare respiratory capacity (SRC) than did naive counterparts, which was immediately accessed under hypoxia. Consequently, hypoxic EM cells maintained ATP levels, survived and migrated better than did hypoxic naive cells, and hypoxia did not impair their capacity to produce IFN-γ. EM CD4+ T cells also had more abundant cytosolic GAPDH and increased glycolytic reserve. In contrast to SRC, glycolytic reserve was not tapped under hypoxic conditions, and, under hypoxia, glucose metabolism contributed similarly to ATP production in naive and EM cells. However, both under normoxic and hypoxic conditions, glucose was critical for EM CD4+ T cell survival. Mechanistically, in the absence of glycolysis, mitochondrial membrane potential (ΔΨm) of EM cells declined and intrinsic apoptosis was triggered. Restoring pyruvate levels, the end product of glycolysis, preserved ΔΨm and prevented apoptosis. Furthermore, reconstitution of reactive oxygen species (ROS), whose production depends on ΔΨm, also rescued viability, whereas scavenging mitochondrial ROS exacerbated apoptosis. Rapid access of SRC in hypoxia, linked with built-in, oxygen-resistant glycolytic reserve that functionally insulates ΔΨm and mitochondrial ROS production from oxygen tension changes, provides an immune-metabolic basis supporting survival, migration, and function of EM CD4+ T cells in normoxic and hypoxic conditions.

Human regulatory T cells lack the cyclophosphamide‐extruding transporter ABCB1 and are more susceptible to cyclophosphamide‐induced apoptosis

ATP‐binding cassette (ABC) transporters, including ABC‐transporter B1 (ABCB1), extrude drugs, metabolites, and other compounds (such as mitotracker green (MTG)) from cells. Susceptibility of CD4+ regulatory T (Treg) cells to the ABCB1‐substrate cyclophosphamide (CPA) has been reported. Here, we characterized ABCB1 expression and function in human CD4+ T‐cell subsets. Naïve, central memory, and effector‐memory CD4+ T cells, but not Treg cells, effluxed MTG in an ABCB1‐dependent manner. In line with this, ABCB1 mRNA and protein was expressed by nonregulatory CD4+ T‐cell subsets, but not Treg cells. In vitro, the ABCB1‐substrate CPA was cytotoxic for Treg cells at a 100‐fold lower dose than for nonregulatory counterparts, and, inversely, verapamil, an inhibitor of ABC transporters, increased CPA‐toxicity in nonregulatory CD4+ T cells but not Treg cells. Thus, Treg cells lack expression of ABCB1, rendering them selectively susceptible to CPA. Our findings provide mechanistic support for therapeutic strategies using CPA to boost anti‐tumor immunity by selectively depleting Treg cells.

The spectrum of T cell metabolism in health and disease

In healthy individuals, metabolically quiescent T cells survey lymph nodes and peripheral tissues in search of cognate antigens. During infection, T cells that encounter cognate antigens are activated and — in a context-specific manner — proliferate and/or differentiate to become effector T cells. This process is accompanied by important changes in cellular metabolism (known as metabolic reprogramming). The magnitude and spectrum of metabolic reprogramming as it occurs in T cells in the context of acute infection ensure host survival. By contrast, altered T cell metabolism, and hence function, is also observed in various disease states, in which T cells actively contribute to pathology. In this Review, we introduce the idea that the spectrum of immune cell metabolic states can provide a basis for categorizing human diseases. Specifically, we first summarize the metabolic and interlinked signalling requirements of T cells responding to acute infection. We then discuss how metabolic reprogramming of T cells is linked to disease.

Mitochondria-Endoplasmic Reticulum Contact Sites Function as Immunometabolic Hubs that Orchestrate the Rapid Recall Response of Memory CD8+ T Cells

&NA; Glycolysis is linked to the rapid response of memory CD8+ T cells, but the molecular and subcellular structural elements enabling enhanced glucose metabolism in nascent activated memory CD8+ T cells are unknown. We found that rapid activation of protein kinase B (PKB or AKT) by mammalian target of rapamycin complex 2 (mTORC2) led to inhibition of glycogen synthase kinase 3&bgr; (GSK3&bgr;) at mitochondria‐endoplasmic reticulum (ER) junctions. This enabled recruitment of hexokinase I (HK‐I) to the voltage‐dependent anion channel (VDAC) on mitochondria. Binding of HK‐I to VDAC promoted respiration by facilitating metabolite flux into mitochondria. Glucose tracing pinpointed pyruvate oxidation in mitochondria, which was the metabolic requirement for rapid generation of interferon‐&ggr; (IFN‐&ggr;) in memory T cells. Subcellular organization of mTORC2‐AKT‐GSK3&bgr; at mitochondria‐ER contact sites, promoting HK‐I recruitment to VDAC, thus underpins the metabolic reprogramming needed for memory CD8+ T cells to rapidly acquire effector function. Graphical Abstract Figure. No caption available. HighlightsmTORC2, AKT, and GSK3&bgr; are present at mitochondria‐ER contact sites of CD8+ T cellsmTORC2‐activated AKT inhibits GSK3&bgr; in nascent activated memory CD8+ T cellsGSK3&bgr; inhibition enables binding of HK‐I to VDAC, promoting pyruvate oxidationPyruvate oxidation is required for rapid generation of IFN‐&ggr; in memory T cells &NA; How glucose metabolism enables rapid acquisition of effector function in memory CD8+ T cells remains poorly understood. Bantug et al. demonstrate that mitochondria‐endoplasmic reticulum contact sites are signaling hubs that enable the metabolic reprogramming required for rapid CD8+ T cell recall responses.

Tissue Distribution Dynamics of Human NK Cells Inferred from Peripheral Blood Depletion Kinetics after Sphingosine‐1‐Phosphate Receptor Blockade

Human natural killer (NK) cell subsets differentially distribute throughout the organism. While CD56dim and CD56bright NK cell subsets similarly reside in the bone marrow (BM), the CD56dim population predominantly accumulates in non‐lymphoid tissues and the CD56bright counterpart in lymphoid tissue (LT). The dynamics with which these NK cell subsets redistribute to tissues remains unexplored. Here, we studied individuals newly exposed to fingolimod, a drug that efficiently blocks sphingosine‐1‐phosphate (S1P)‐directed lymphocyte – including NK cell – egress from tissue to blood. During an observation period of 6h peripheral blood depletion of CD56bright NK cells was observed 3 h after first dose of fingolimod, with 40–50% depletion after 6 h, while a decrease of the numbers of CD56dim NK cells did not reach the level of statistical significance. In vitro, CD56bright and CD56dim NK cells responded comparably to the BM‐homing chemokine CXCL12, while CD56bright NK cells migrated more efficiently in gradients of the LT‐homing chemokines CCL19 and CCL21. In conjuncture with these in vitro studies, the indirectly observed subset‐specific depletion kinetics from blood are compatible with preferential and more rapid redistribution of CD56bright NK cells from blood to peripheral tissue such as LT and possibly also the inflamed central nervous system. These data shed light on an unexplored level at which access of NK cells to LT, and thus, for example antigen‐presenting cells, is regulated.

Dimethyl fumarate influences innate and adaptive immunity in multiple sclerosis

INTRODUCTION The mode of action of dimethyl fumarate (DMF), an immunomodulatory treatment for relapsing-remitting multiple sclerosis (RRMS), has not yet been fully elucidated. While in-vitro experiments and animal studies suggest effects on immune cell survival, proliferation, migration and oxidative stress response, corresponding observations from human studies are lacking. This study aims to characterize ex-vivo and in-vivo effects in a cohort of DMF treated RRMS patients. METHODS Blood samples were collected from twenty well-characterized RRMS patients at baseline and after 3, 6 and 12 months of DMF treatment and an age- and gender-matched cohort of 20 healthy individuals at 0 and 3 months. Leukocyte subpopulations, immunoglobulin levels and cytokine secretion were measured. T cells were assessed for their levels of reactive oxygen species (ROS), metabolic status and their proliferative capacity. Levels of antioxidants were determined in serum by mass spectrometry. Responses of monocyte activation markers as well as NFkB and MAPK pathways to DMF were analysed. RESULTS Upon DMF treatment, all lymphocyte subpopulations dropped significantly over the course of 12 months with cytotoxic and effector T cells being affected most significantly. DMF induced cell death and inhibited proliferation of T cells in-vitro. Interestingly, this anti-proliferative effect decreased under treatment. In-vivo DMF treatment led to decreased T cell glycolysis and higher turn-over of antioxidants. In line with these results a significant increase of cytosolic ROS levels after 3 months treatment was detected in T cells. In-vitro DMF treatment reduced NFkB (p65) translocation to the nucleus and MAPK (p38) levels decreased upon stimulation with monomethyl fumarate (MMF) in-vitro and ex-vivo. Consequently, the expression of co-stimulatory molecules like CD40 and CD150 was decreased in antigen presenting cells both in-vitro and ex-vivo. CONCLUSION This study translates knowledge from in-vitro and animal studies on DMF into the clinical setting. Our data suggest that DMF not only alters lymphocyte composition, but also has profound effects on proliferation and induces oxidative stress in T cells. It also acts on innate immunity by reducing the activation status of antigen presenting cells (APCs) via NFkB and MAPK inactivation.

Glycolysis and EZH2 boost T cell weaponry against tumors

Low availability of glucose in tumors negatively affects the activity of tumor-infiltrating T cells. Loss of T cell function under these conditions is mediated by the microRNAs miR-101 and miR-26a, which target expression of the methytransferase EZH2 and thereby diminish the expression of anti-tumor cytokines.

T cells specific for different latent and lytic viral proteins efficiently control Epstein-Barr virus–transformed B cells

BACKGROUND AIMS Epstein-Barr virus (EBV)-associated post-transplant lymphoproliferative disorders (PTLD) belong to the most dreaded complications of immunosuppression. The efficacy of EBV-specific T-cell transfer for PTLD has been previously shown, yet the optimal choice of EBV-derived antigens inducing polyclonal CD4(+) and CD8(+) T cells that cover a wide range of human leukocyte antigen types and efficiently control PTLD remains unclear. METHODS A pool of 125 T-cell epitopes from seven latent and nine lytic EBV-derived proteins (EBVmix) and peptide pools of EBNA1, EBNA3c, LMP2a and BZLF1 were used to determine T-cell frequencies and to isolate T cells through the use of the interferon (IFN)-γ cytokine capture system. We further evaluated the phenotype and functionality of the generated T-cell lines in vitro. RESULTS EBVmix induced significantly higher T-cell frequencies and allowed selecting more CD4(+)IFN-γ(+) and CD8(+)IFN-γ(+) cells than single peptide pools. T cells of all specificities expanded similarly in vitro, recognized cognate antigen, and, to a lower extent, EBV-infected cells, exerted moderate cytotoxicity and showed reduced alloreactivity. However, EBVmix-specific cells most efficiently controlled EBV-infected lymphoblastoid cell lines (LCLs). This control was mainly mediated by EBV-specific CD8(+) cells with an oligoclonal epitope signature covering both latent and lytic viral proteins. Notably, EBV-specific CD4(+) cells unable to control LCLs produced significantly less perforin and granzyme B, probably because of limited LCL epitope presentation. CONCLUSIONS EBVmix induces a broader T-cell response, probably because of its coverage of latent and lytic EBV-derived proteins that may be important to control EBV-transformed B cells and might offer an improvement of T-cell therapies.