Oncogene induced TIM-3 ligand expression dictates susceptibility to anti-TIM-3 therapy in mice.

Leukemia relapse is a major cause of death after allogeneic hematopoietic cell transplantation (allo-HCT). We tested the potential of targeting TIM-3 for improving graft-versus-leukemia (GVL) effects. We observed differential expression of TIM-3 ligands when hematopoietic stem cells overexpressed certain oncogenic-driver mutations. Anti-TIM-3 Ab-treatment improved survival of mice bearing leukemia with oncogene-induced TIM-3 ligand expression. Conversely, leukemia cells with low ligand expression were anti-TIM-3 treatment-resistant. In vitro, TIM-3 blockade or genetic deletion in CD8+ T cells (Tc) enhanced Tc activation, proliferation and IFN-γ production while enhancing GVL effects, preventing Tc exhaustion and improving Tc cytotoxicity and glycolysis in vivo. Conversely, TIM-3 deletion in myeloid cells did not affect allogeneic Tc proliferation and activation in vitro, suggesting that anti-TIM-3-treatment-mediated GVL effects are Tc-induced. In contrast to anti-PD-1 and anti-CTLA-4-treatment, anti-TIM-3-treatment did not enhance acute graft-versus-host-disease (aGVHD). TIM-3 and its ligands were frequently expressed in acute myeloid leukemia (AML) cells of patients with post-allo-HCT relapse. We deciphered the connection between oncogenic mutations found in AML and TIM-3 ligands expression and identify anti-TIM-3-treatment as a strategy to enhance GVL effects via metabolic and transcriptional Tc-reprogramming, without exacerbation of aGVHD. Our findings support clinical testing of anti-TIM-3 Abs in patients with AML relapse post-allo-HCT.

Targeted Metabolomics on Rare Primary Cells.

Cellular function critically depends on metabolism, and the function of the underlying metabolic networks can be studied by measuring small molecule intermediates. However, obtaining accurate and reliable measurements of cellular metabolism, particularly in rare cell types like hematopoietic stem cells, has traditionally required pooling cells from multiple animals. A protocol now enables researchers to measure metabolites in rare cell types using only one mouse per sample while generating multiple replicates for more abundant cell types. This reduces the number of animals that are required for a given project. The protocol presented here involves several key differences over traditional metabolomics protocols, such as using 5 g/L NaCl as a sheath fluid, sorting directly into acetonitrile, and utilizing targeted quantification with rigorous use of internal standards, allowing for more accurate and comprehensive measurements of cellular metabolism. Despite the time required for the isolation of single cells, fluorescent staining, and sorting, the protocol can preserve differences among cell types and drug treatments to a large extent.

Prostaglandin E2 controls the metabolic adaptation of T cells to the intestinal microenvironment.

Immune cells must adapt to different environments during the course of an immune response. Here we study the adaptation of CD8+ T cells to the intestinal microenvironment and how this process shapes the establishment of the CD8+ T cell pool. CD8+ T cells progressively remodel their transcriptome and surface phenotype as they enter the gut wall, and downregulate expression of mitochondrial genes. Human and mouse intestinal CD8+ T cells have reduced mitochondrial mass, but maintain a viable energy balance to sustain their function. We find that the intestinal microenvironment is rich in prostaglandin E2 (PGE2), which drives mitochondrial depolarization in CD8+ T cells. Consequently, these cells engage autophagy to clear depolarized mitochondria, and enhance glutathione synthesis to scavenge reactive oxygen species (ROS) that result from mitochondrial depolarization. Impairing PGE2 sensing promotes CD8+ T cell accumulation in the gut, while tampering with autophagy and glutathione negatively impacts the T cell pool. Thus, a PGE2-autophagy-glutathione axis defines the metabolic adaptation of CD8+ T cells to the intestinal microenvironment, to ultimately influence the T cell pool.

Metabolic rewiring tunes dermal macrophages in staphylococcal skin infection.

The skin needs to balance tolerance of colonizing microflora with rapid detection of potential pathogens. Flexible response mechanisms would seem most suitable to accommodate the dynamic challenges of effective antimicrobial defense and restoration of tissue homeostasis. Here, we dissected macrophage-intrinsic mechanisms and microenvironmental cues that tune macrophage signaling in localized skin infection with the colonizing and opportunistic pathogen Staphylococcus aureus. Early in skin infection, the cytokine granulocyte-macrophage colony-stimulating factor (GM-CSF) produced by γδ T cells and hypoxic conditions within the dermal microenvironment diverted macrophages away from a homeostatic M-CSF- and hypoxia-inducible factor 1α (HIF-1α)-dependent program. This allowed macrophages to be metabolically rewired for maximal inflammatory activity, which requires expression of Irg1 and generation of itaconate, but not HIF-1α. This multifactorial macrophage rewiring program was required for both the timely clearance of bacteria and for the provision of local immune memory. These findings indicate that immunometabolic conditioning allows dermal macrophages to cycle between antimicrobial activity and protection against secondary infections.

Phosphoinositide acyl chain saturation drives CD8+ effector T cell signaling and function.

How lipidome changes support CD8+ effector T (Teff) cell differentiation is not well understood. Here we show that, although naive T cells are rich in polyunsaturated phosphoinositides (PIPn with 3-4 double bonds), Teff cells have unique PIPn marked by saturated fatty acyl chains (0-2 double bonds). PIPn are precursors for second messengers. Polyunsaturated phosphatidylinositol bisphosphate (PIP2) exclusively supported signaling immediately upon T cell antigen receptor activation. In late Teff cells, activity of phospholipase C-γ1, the enzyme that cleaves PIP2 into downstream mediators, waned, and saturated PIPn became essential for sustained signaling. Saturated PIP was more rapidly converted to PIP2 with subsequent recruitment of phospholipase C-γ1, and loss of saturated PIPn impaired Teff cell fitness and function, even in cells with abundant polyunsaturated PIPn. Glucose was the substrate for de novo PIPn synthesis, and was rapidly utilized for saturated PIP2 generation. Thus, separate PIPn pools with distinct acyl chain compositions and metabolic dependencies drive important signaling events to initiate and then sustain effector function during CD8+ T cell differentiation.

LC-MS-Based Targeted Metabolomics for FACS-Purified Rare Cells.

Metabolism plays a fundamental role in regulating cellular functions and fate decisions. Liquid chromatography-mass spectrometry (LC-MS)-based targeted metabolomic approaches provide high-resolution insights into the metabolic state of a cell. However, the typical sample size is in the order of 105-107 cells and thus not compatible with rare cell populations, especially in the case of a prior flow cytometry-based purification step. Here, we present a comprehensively optimized protocol for targeted metabolomics on rare cell types, such as hematopoietic stem cells and mast cells. Only 5000 cells per sample are required to detect up to 80 metabolites above background. The use of regular-flow liquid chromatography allows for robust data acquisition, and the omission of drying or chemical derivatization avoids potential sources of error. Cell-type-specific differences are preserved while the addition of internal standards, generation of relevant background control samples, and targeted metabolite with quantifiers and qualifiers ensure high data quality. This protocol could help numerous studies to gain thorough insights into cellular metabolic profiles and simultaneously reduce the number of laboratory animals and the time-consuming and costly experiments associated with rare cell-type purification.

TFEB induces mitochondrial itaconate synthesis to suppress bacterial growth in macrophages.

Successful elimination of bacteria in phagocytes occurs in the phago-lysosomal system, but also depends on mitochondrial pathways. Yet, how these two organelle systems communicate is largely unknown. Here we identify the lysosomal biogenesis factor transcription factor EB (TFEB) as regulator for phago-lysosome-mitochondria crosstalk in macrophages. By combining cellular imaging and metabolic profiling, we find that TFEB activation, in response to bacterial stimuli, promotes the transcription of aconitate decarboxylase (Acod1, Irg1) and synthesis of its product itaconate, a mitochondrial metabolite with antimicrobial activity. Activation of the TFEB-Irg1-itaconate signalling axis reduces the survival of the intravacuolar pathogen Salmonella enterica serovar Typhimurium. TFEB-driven itaconate is subsequently transferred via the Irg1-Rab32-BLOC3 system into the Salmonella-containing vacuole, thereby exposing the pathogen to elevated itaconate levels. By activating itaconate production, TFEB selectively restricts proliferating Salmonella, a bacterial subpopulation that normally escapes macrophage control, which contrasts TFEB's role in autophagy-mediated pathogen degradation. Together, our data define a TFEB-driven metabolic pathway between phago-lysosomes and mitochondria that restrains Salmonella Typhimurium burden in macrophages in vitro and in vivo.

Targeting MDM2 enhances antileukemia immunity after allogeneic transplantation via MHC-II and TRAIL-R1/2 upregulation.

Patients with acute myeloid leukemia (AML) often achieve remission after allogeneic hematopoietic cell transplantation (allo-HCT) but subsequently die of relapse driven by leukemia cells resistant to elimination by allogeneic T cells based on decreased major histocompatibility complex II (MHC-II) expression and apoptosis resistance. Here we demonstrate that mouse-double-minute-2 (MDM2) inhibition can counteract immune evasion of AML. MDM2 inhibition induced MHC class I and II expression in murine and human AML cells. Using xenografts of human AML and syngeneic mouse models of leukemia, we show that MDM2 inhibition enhanced cytotoxicity against leukemia cells and improved survival. MDM2 inhibition also led to increases in tumor necrosis factor-related apoptosis-inducing ligand receptor-1 and -2 (TRAIL-R1/2) on leukemia cells and higher frequencies of CD8+CD27lowPD-1lowTIM-3low T cells, with features of cytotoxicity (perforin+CD107a+TRAIL+) and longevity (bcl-2+IL-7R+). CD8+ T cells isolated from leukemia-bearing MDM2 inhibitor-treated allo-HCT recipients exhibited higher glycolytic activity and enrichment for nucleotides and their precursors compared with vehicle control subjects. T cells isolated from MDM2 inhibitor-treated AML-bearing mice eradicated leukemia in secondary AML-bearing recipients. Mechanistically, the MDM2 inhibitor-mediated effects were p53-dependent because p53 knockdown abolished TRAIL-R1/2 and MHC-II upregulation, whereas p53 binding to TRAILR1/2 promotors increased upon MDM2 inhibition. The observations in the mouse models were complemented by data from human individuals. Patient-derived AML cells exhibited increased TRAIL-R1/2 and MHC-II expression on MDM2 inhibition. In summary, we identified a targetable vulnerability of AML cells to allogeneic T-cell-mediated cytotoxicity through the restoration of p53-dependent TRAIL-R1/2 and MHC-II production via MDM2 inhibition.

Targeted LC-MS/MS-based metabolomics and lipidomics on limited hematopoietic stem cell numbers.

Metabolism is important for the regulation of hematopoietic stem cells (HSCs) and drives cellular fate. Due to the scarcity of HSCs, it has been technically challenging to perform metabolome analyses gaining insight into HSC metabolic regulatory networks. Here, we present two targeted liquid chromatography-mass spectrometry approaches that enable the detection of metabolites after fluorescence-activated cell sorting when sample amounts are limited. One protocol covers signaling lipids and retinoids, while the second detects tricarboxylic acid cycle metabolites and amino acids. For complete details on the use and execution of this protocol, please refer to Schönberger et al. (2022).

Multilayer omics analysis reveals a non-classical retinoic acid signaling axis that regulates hematopoietic stem cell identity.

Hematopoietic stem cells (HSCs) rely on complex regulatory networks to preserve stemness. Due to the scarcity of HSCs, technical challenges have limited our insights into the interplay between metabolites, transcription, and the epigenome. In this study, we generated low-input metabolomics, transcriptomics, chromatin accessibility, and chromatin immunoprecipitation data, revealing distinct metabolic hubs that are enriched in HSCs and their downstream multipotent progenitors. Mechanistically, we uncover a non-classical retinoic acid (RA) signaling axis that regulates HSC function. We show that HSCs rely on Cyp26b1, an enzyme conventionally considered to limit RA effects in the cell. In contrast to the traditional view, we demonstrate that Cyp26b1 is indispensable for production of the active metabolite 4-oxo-RA. Further, RA receptor beta (Rarb) is required for complete transmission of 4-oxo-RA-mediated signaling to maintain stem cells. Our findings emphasize that a single metabolite controls stem cell fate by instructing epigenetic and transcriptional attributes.

Sulfur sequestration promotes multicellularity during nutrient limitation.

The behaviour of Dictyostelium discoideum depends on nutrients1. When sufficient food is present these amoebae exist in a unicellular state, but upon starvation they aggregate into a multicellular organism2,3. This biology makes D. discoideum an ideal model for investigating how fundamental metabolism commands cell differentiation and function. Here we show that reactive oxygen species-generated as a consequence of nutrient limitation-lead to the sequestration of cysteine in the antioxidant glutathione. This sequestration limits the use of the sulfur atom of cysteine in processes that contribute to mitochondrial metabolism and cellular proliferation, such as protein translation and the activity of enzymes that contain an iron-sulfur cluster. The regulated sequestration of sulfur maintains D. discoideum in a nonproliferating state that paves the way for multicellular development. This mechanism of signalling through reactive oxygen species highlights oxygen and sulfur as simple signalling molecules that dictate cell fate in an early eukaryote, with implications for responses to nutrient fluctuations in multicellular eukaryotes.

Metabolic Dynamics of In Vitro CD8+ T Cell Activation.

CD8+ T cells detect and kill infected or cancerous cells. When activated from their naïve state, T cells undergo a complex transition, including major metabolic reprogramming. Detailed resolution of metabolic dynamics is needed to advance the field of immunometabolism. Here, we outline methodologies that when utilized in parallel achieve broad coverage of the metabolome. Specifically, we used a combination of 2 flow injection analysis (FIA) and 3 liquid chromatography (LC) methods in combination with positive and negative mode high-resolution mass spectrometry (MS) to study the transition from naïve to effector T cells with fine-grained time resolution. Depending on the method, between 54% and 98% of measured metabolic features change in a time-dependent manner, with the major changes in both polar metabolites and lipids occurring in the first 48 h. The statistical analysis highlighted the remodeling of the polyamine biosynthesis pathway, with marked differences in the dynamics of precursors, intermediates, and cofactors. Moreover, phosphatidylcholines, the major class of membrane lipids, underwent a drastic shift in acyl chain composition with polyunsaturated species decreasing from 60% to 25% of the total pool and specifically depleting species containing a 20:4 fatty acid. We hope that this data set with a total of over 11,000 features recorded with multiple MS methodologies for 9 time points will be a useful resource for future work.

Metabolic conditioning of CD8+ effector T cells for adoptive cell therapy.

CD8+ effector T (TE) cell proliferation and cytokine production depends on enhanced glucose metabolism. However, circulating T cells continuously adapt to glucose fluctuations caused by diet and inter-organ metabolite exchange. Here we show that transient glucose restriction (TGR) in activated CD8+ TE cells metabolically primes effector functions and enhances tumour clearance in mice. Tumour-specific TGR CD8+ TE cells co-cultured with tumour spheroids in replete conditions display enhanced effector molecule expression, and adoptive transfer of these cells in a murine lymphoma model leads to greater numbers of immunologically functional circulating donor cells and complete tumour clearance. Mechanistically, TE cells treated with TGR undergo metabolic remodelling that, after glucose re-exposure, supports enhanced glucose uptake, increased carbon allocation to the pentose phosphate pathway (PPP) and a cellular redox shift towards a more reduced state-all indicators of a more anabolic programme to support their enhanced functionality. Thus, metabolic conditioning could be used to promote efficiency of T-cell products for adoptive cellular therapy.

Polyamines and eIF5A Hypusination Modulate Mitochondrial Respiration and Macrophage Activation.

How cells adapt metabolism to meet demands is an active area of interest across biology. Among a broad range of functions, the polyamine spermidine is needed to hypusinate the translation factor eukaryotic initiation factor 5A (eIF5A). We show here that hypusinated eIF5A (eIF5AH) promotes the efficient expression of a subset of mitochondrial proteins involved in the TCA cycle and oxidative phosphorylation (OXPHOS). Several of these proteins have mitochondrial targeting sequences (MTSs) that in part confer an increased dependency on eIF5AH. In macrophages, metabolic switching between OXPHOS and glycolysis supports divergent functional fates stimulated by activation signals. In these cells, hypusination of eIF5A appears to be dynamically regulated after activation. Using in vivo and in vitro models, we show that acute inhibition of this pathway blunts OXPHOS-dependent alternative activation, while leaving aerobic glycolysis-dependent classical activation intact. These results might have implications for therapeutically controlling macrophage activation by targeting the polyamine-eIF5A-hypusine axis.

Inflammatory macrophage dependence on NAD+ salvage is a consequence of reactive oxygen species-mediated DNA damage.

The adoption of Warburg metabolism is critical for the activation of macrophages in response to lipopolysaccharide. Macrophages stimulated with lipopolysaccharide increase their expression of nicotinamide phosphoribosyltransferase (NAMPT), a key enzyme in NAD+ salvage, and loss of NAMPT activity alters their inflammatory potential. However, the events that lead to the cells' becoming dependent on NAD+ salvage remain poorly defined. We found that depletion of NAD+ and increased expression of NAMPT occurred rapidly after inflammatory activation and coincided with DNA damage caused by reactive oxygen species (ROS). ROS produced by complex III of the mitochondrial electron-transport chain were required for macrophage activation. DNA damage was associated with activation of poly(ADP-ribose) polymerase, which led to consumption of NAD+. In this setting, increased NAMPT expression allowed the maintenance of NAD+ pools sufficient for glyceraldehyde-3-phosphate dehydrogenase activity and Warburg metabolism. Our findings provide an integrated explanation for the dependence of inflammatory macrophages on the NAD+ salvage pathway.

A metabolic interplay coordinated by HLX regulates myeloid differentiation and AML through partly overlapping pathways.

The H2.0-like homeobox transcription factor (HLX) regulates hematopoietic differentiation and is overexpressed in Acute Myeloid Leukemia (AML), but the mechanisms underlying these functions remain unclear. We demonstrate here that HLX overexpression leads to a myeloid differentiation block both in zebrafish and human hematopoietic stem and progenitor cells (HSPCs). We show that HLX overexpression leads to downregulation of genes encoding electron transport chain (ETC) components and upregulation of PPARδ gene expression in zebrafish and human HSPCs. HLX overexpression also results in AMPK activation. Pharmacological modulation of PPARδ signaling relieves the HLX-induced myeloid differentiation block and rescues HSPC loss upon HLX knockdown but it has no effect on AML cell lines. In contrast, AMPK inhibition results in reduced viability of AML cell lines, but minimally affects myeloid progenitors. This newly described role of HLX in regulating the metabolic state of hematopoietic cells may have important therapeutic implications.

Mitochondrial Priming by CD28.

T cell receptor (TCR) signaling without CD28 can elicit primary effector T cells, but memory T cells generated during this process are anergic, failing to respond to secondary antigen exposure. We show that, upon T cell activation, CD28 transiently promotes expression of carnitine palmitoyltransferase 1a (Cpt1a), an enzyme that facilitates mitochondrial fatty acid oxidation (FAO), before the first cell division, coinciding with mitochondrial elongation and enhanced spare respiratory capacity (SRC). microRNA-33 (miR33), a target of thioredoxin-interacting protein (TXNIP), attenuates Cpt1a expression in the absence of CD28, resulting in cells that thereafter are metabolically compromised during reactivation or periods of increased bioenergetic demand. Early CD28-dependent mitochondrial engagement is needed for T cells to remodel cristae, develop SRC, and rapidly produce cytokines upon restimulation-cardinal features of protective memory T cells. Our data show that initial CD28 signals during T cell activation prime mitochondria with latent metabolic capacity that is essential for future T cell responses.

Microbial biosynthesis of polyglutamic acid biopolymer and applications in the biopharmaceutical, biomedical and food industries.

This review article provides an updated critical literature review on the production and applications of Polyglutamic Acid (PGA). alpha-PGA is synthesized chemically, whereas gamma-PGA can be produced by a number of microbial species, most prominently various Bacilli. Great insight into the microbial formation of gamma-PGA has been gained thanks to the development of molecular biological techniques. Moreover, there is a great variety of applications for both isoforms of PGA, many of which have not been discovered until recently. These applications include: wastewater treatment, food products, drug delivery, medical adhesives, vaccines, PGA nanoparticles for on-site drug release in cancer chemotherapy, and tissue engineering.