scholarly journals RNA Flow Cytometry for the Study of T Cell Metabolism

2021 ◽  
Vol 22 (8) ◽  
pp. 3906
Author(s):  
Alessandra Rossi ◽  
Ilenia Pacella ◽  
Silvia Piconese
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
T Cell ◽  
Cell Metabolism ◽  
Metabolic Reprogramming ◽  
Mitochondrial Activity ◽  
Through Flow ◽  
Invaluable Tool ◽  
Epigenetic Remodeling ◽  
The One

T cells undergo activation and differentiation programs along a continuum of states that can be tracked through flow cytometry using a combination of surface and intracellular markers. Such dynamic behavior is the result of transcriptional and post-transcriptional events, initiated and sustained by the activation of specific transcription factors and by epigenetic remodeling. These signaling pathways are tightly integrated with metabolic routes in a bidirectional manner: on the one hand, T cell receptors and costimulatory molecules activate metabolic reprogramming; on the other hand, metabolites modify T cell transcriptional programs and functions. Flow cytometry represents an invaluable tool to analyze the integration of phenotypical, functional, metabolic and transcriptional features, at the single cell level in heterogeneous T cell populations, and from complex microenvironments, with potential clinical application in monitoring the efficacy of cancer immunotherapy. Here, we review the most recent advances in flow cytometry-based analysis of gene expression, in combination with indicators of mitochondrial activity, with the aim of revealing and characterizing major metabolic pathways in T cells.

scholarly journals Targeting T cell metabolism in the tumor microenvironment: an anti-cancer therapeutic strategy

2019 ◽  
Vol 38 (1) ◽  
Author(s):  
Zhongping Yin ◽  
Ling Bai ◽  
Wei Li ◽  
Tanlun Zeng ◽  
Huimin Tian ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Tumor Microenvironment ◽  
Metabolic Pathways ◽  
Cell Metabolism ◽  
Basic Research ◽  
Metabolic Reprogramming ◽  
Activated T Cells ◽  
Cell Functions ◽  
Anti Cancer

Abstract T cells play important roles in anti-tumor immunity. Emerging evidence has revealed that distinct metabolic changes impact the activation and differentiation of T cells. Tailoring immune responses by manipulating cellular metabolic pathways and the identification of new targets may provide new options for cancer immunotherapy. In this review, we focus on recent advances in the metabolic reprogramming of different subtypes of T cells and T cell functions. We summarize how metabolic pathways accurately regulate T cell development, differentiation, and function in the tumor microenvironment. Because of the similar metabolism in activated T cells and tumor cells, we also describe the effect of the tumor microenvironment on T cell metabolism reprogramming, which may provide strategies for maximal anti-cancer effects and enhancing the immunity of T cells. Thus, studies of T lymphocyte metabolism can not only facilitate the basic research of immune metabolism, but also provide potential targets for drug development and new strategies for clinical treatment of cancer.

scholarly journals 1-Methylnicotinamide is an immune regulatory metabolite in human ovarian cancer

2020 ◽  
Author(s):  
Marisa K. Kilgour ◽  
Sarah MacPherson ◽  
Lauren Zacharias ◽  
Sarah Keyes ◽  
Brenna Pauly ◽  
...  
Keyword(s):  
Ovarian Cancer ◽  
Flow Cytometry ◽  
T Cells ◽  
T Cell ◽  
Tumor Cells ◽  
Immune Modulation ◽  
Human Cancer ◽  
Mitochondrial Activity ◽  
Necrosis Factor Alpha ◽  
Cd8 Cells

ABSTRACTImmune regulatory metabolites are key features of the tumor microenvironment (TME), yet with a few notable exceptions, their identities remain largely unknown. We uncovered the immune regulatory metabolic states and metabolomes of sorted tumor and stromal, CD4+, and CD8+ cells from the tumor and ascites of patients with high-grade serous ovarian cancer (HGSC) using high-dimensional flow cytometry and metabolomics supplemented with single cell RNA sequencing. Flow cytometry revealed that tumor cells show a consistently greater uptake of glucose than T cells, but similar mitochondrial activity. Cells within the ascites and tumor had pervasive metabolite differences, with a striking enrichment in 1-methylnicotinamide (MNA) in T cells infiltrating the tumor compared to ascites. Despite the elevated levels of MNA in T cells, the expression of nicotinamide N-methyltransferase, the gene encoding the enzyme that catalyses the transfer of a methyl group from S-adenosylmethionine to nicotinamide, was restricted to fibroblasts and tumor cells. Treatment of T cells with MNA resulted in an increase in T cell-mediated secretion of the tumor promoting cytokine tumor necrosis factor alpha. Thus, the TME-derived metabolite MNA contributes to an alternative and non-cell autonomous mechanism of immune modulation of T cells in HGSC. Collectively, uncovering the tumor-T cell metabolome may reveal metabolic vulnerabilities that can be exploited using T cell-based immunotherapies to treat human cancer.

scholarly journals Amino Acid Metabolism in Lupus

2021 ◽  
Vol 12 ◽  
Author(s):  
Michihito Kono ◽  
Nobuya Yoshida ◽  
George C. Tsokos
Keyword(s):  
T Cells ◽  
Amino Acid ◽  
T Cell ◽  
T Cell Activation ◽  
Amino Acid Metabolism ◽  
Acid Metabolism ◽  
Cell Metabolism ◽  
Cell Subset ◽  
Metabolic Reprogramming ◽  
Acid Oxidation

T cell metabolism is central to cell proliferation, survival, differentiation, and aberrations have been linked to the pathophysiology of systemic autoimmune diseases. Besides glycolysis and fatty acid oxidation/synthesis, amino acid metabolism is also crucial in T cell metabolism. It appears that each T cell subset favors a unique metabolic process and that metabolic reprogramming changes cell fate. Here, we review the mechanisms whereby amino acid transport and metabolism affects T cell activation, differentiation and function in T cells in the prototype systemic autoimmune disease systemic lupus erythematosus. New insights in amino acid handling by T cells should guide approaches to correct T cell abnormalities and disease pathology.

scholarly journals Metabolic and functional impairment of CD8+ T cells from the lungs of influenza-infected obese mice

2020 ◽  
Author(s):  
William D. Green ◽  
Abrar E. Al-Shaer ◽  
Qing Shi ◽  
Nancie J MacIver ◽  
Melinda A. Beck ◽  
...  
Keyword(s):  
T Cells ◽  
Influenza Virus ◽  
T Cell ◽  
Post Infection ◽  
Cytokine Production ◽  
Cell Metabolism ◽  
Metabolic Reprogramming ◽  
Obese Mice ◽  
Diet Induced Obesity ◽  
And Function

ABSTRACTBackgroundObesity increases influenza disease risk in millions of adults worldwide. In this study, we investigated the effect of diet-induced obesity on pulmonary CD8+ T cell metabolism and function as a mechanism of impairment.MethodsMale C57BL/6J mice were fed either control (10% kcal/g) or high-fat (60% kcal/g) diet. Sub-lethal A/PR/8/34 influenza virus infection generated a robust pulmonary immune response. T cell metabolism and function were assessed at day 10 and day 24 post infection.ResultsAt day 10 post infection, CD8+ T cells from obese mice had impaired oxidative and glycolytic metabolism, greater fatty acid uptake, and decreased effector populations and cytokine production. At infection resolution, obese mice had lower numbers of naïve and central memory CD8+ T cell populations in the lungs.ConclusionDiet-induced obesity increases influenza virus pathogenesis through CD8+ T cell mediated metabolic reprogramming resulting in suppressed effector CD8+ T cell function.SummaryDiet-induced obesity impairs the metabolism of pulmonary CD8+ T cells resulting in reduced effector CD8+ T cells and cytokine production following primary influenza infection.

scholarly journals OP0312 METABOLIC REPROGRAMMING IN MEMORY CD4+ T CELLS IS ASSOCIATED WITH REACTIVE OXYGEN INDUCED IMMUNE CELL DYSFUNCTION DURING AGING

2021 ◽  
Vol 80 (Suppl 1) ◽  
pp. 191.2-191
Author(s):  
Y. Chen ◽  
Y. Ye ◽  
H. Wu ◽  
P. L. Krauß ◽  
P. Löwe ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
T Cell ◽  
Cd4 T Cells ◽  
Immune Cell ◽  
Metabolic Reprogramming ◽  
Ki 67 ◽  
Cell Dysfunction ◽  
Age Related ◽  
Memory Cd4 T Cells

Background:Inflamm-aging is a chronic, sterile, low-grade inflammatory status, characterized by an increase of proinflammatory cytokines which participate in the development of most age-related diseases such as cancer, Alzheimer’s disease, type 2 diabetes mellitus, stroke, cardiovascular diseases, and rheumatoid arthritis (RA). As cellular metabolism modulates T cell function, it can be assumed that metabolic changes accompany the differentiation of memory CD4+ T cells into senescent CD4+ T cell and contribute to memory CD4+ T cells dysfunction during aging.Objectives:Therefore, we hypothesized that metabolic reprograming in memory CD4+ T cells might represent an essential factor promoting immune cell dysfunction during aging, thereby fuelling to the pathogenesis of age-related diseases including RAMethods:To this end, we analysed memory CD4+ T cells isolated from PBMCs of young donors (20-32 years) and old donors (52-67 years) by using MACSTM technology. Ex vivo memory CD4+ T cells were analysed by SeahorseTM Technology to determine proton efflux rate (PER) as a measure of glycolysis (glycPER) and oxygen consumption rate (OCR) as a measure of mitochondrial respiration (mitoOCR). Cytokine expression and secretion was measured by flow cytometry and multiplex assay with and without Mitotempo an inhibitor of reactive oxygen species (ROS). Finally, TCR-stimulated memory CD4+ T cell proliferation was determined using CSFE and Ki-67 after 3 days and 4 days by flow cytometry. ROS and mitochondrial activity were analysed after 24 h using DCF-DA and CellROX Deep Red and Mitotracker by flow cytometry.Results:In a quiescent state, memory CD4+ T cells from elderly individuals demonstrated a decrease in basal glycolysis and compensatory glycolysis, and an increase in the ratio of basal mitochondrial oxygen consumption rate (mitoOCR) to glycolytic proton efflux rate (glycoPER) while their mitochondrial profile was equivalent to that of young donors while the amount of mitochondria was higher with no increase in steady-state ATP level. In this line and in comparison to the younger reference group, memory CD4+ T cells from aged donors presented a greater spare respiratory capacity after TCR-activation and a marked increase in intracellular ROS production. Interestingly, we did not observe an impact of aging on memory CD4+ T cell proliferation as determined by CFSE and Ki-67. Although the capacity of intracellular cytokine expression did not differ between the compared groups, the levels of secreted IFN-γ, IP-10, IL-6, IL-9, and MCAF were significantly higher in the supernatants of memory CD4+ T cells taken from aged donors but were sensitive to ROS inhibition. .Conclusion:These findings suggest that metabolic reprogramming in human memory CD4+ T cells during aging results in an increased expression of proinflammatory cytokines as a result of ROS production and mitochondrial dysfunction. This process may culminate in T cell dysfunction and thus contribute to the pathogenesis of inflamm-aging and the development of age-related diseases such as rheumatoid arthritis (RA).Disclosure of Interests:None declared.

scholarly journals The Role of Non-essential Amino Acids in T Cell Function and Anti-tumour Immunity

2021 ◽  
Vol 69 (1) ◽  
Author(s):  
Helen Carrasco Hope ◽  
Robert J. Salmond
Keyword(s):  
Amino Acids ◽  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Metabolic Pathways ◽  
Cell Metabolism ◽  
Building Blocks ◽  
Essential Amino Acids ◽  
Metabolic Reprogramming ◽  
Tumour Immunity

AbstractT cell activation, differentiation and proliferation is dependent upon and intrinsically linked to a capacity to modulate and adapt cellular metabolism. Antigen-induced activation stimulates a transcriptional programme that results in metabolic reprogramming, enabling T cells to fuel anabolic metabolic pathways and provide the nutrients to sustain proliferation and effector responses. Amino acids are key nutrients for T cells and have essential roles as building blocks for protein synthesis as well as in numerous metabolic pathways. In this review, we discuss the roles for uptake and biosynthesis of non-essential amino acids in T cell metabolism, activation and effector function. Furthermore, we highlight the effects of amino acid metabolism and depletion by cancer cells on T cell anti-tumour function and discuss approaches to modulate and improve T cell metabolism for improved anti-tumour function in these nutrient-depleted microenvironments.

scholarly journals 4-1BB costimulation induces T cell mitochondrial function and biogenesis enabling cancer immunotherapeutic responses

2018 ◽  
Vol 215 (4) ◽  
pp. 1091-1100 ◽  
Author(s):  
Ashley V. Menk ◽  
Nicole E. Scharping ◽  
Dayana B. Rivadeneira ◽  
Michael J. Calderon ◽  
McLane J. Watson ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Tumor Microenvironment ◽  
Cd8 T Cells ◽  
Cell Function ◽  
Costimulatory Molecule ◽  
Resistance Mechanism ◽  
Metabolic Reprogramming ◽  
Mitochondrial Activity ◽  
Cancer Immunotherapeutic

Despite remarkable responses to cancer immunotherapy in a subset of patients, many patients remain resistant to these therapies. The tumor microenvironment can impose metabolic restrictions on T cell function, creating a resistance mechanism to immunotherapy. We have previously shown tumor-infiltrating T cells succumb to progressive loss of metabolic sufficiency, characterized by repression of mitochondrial activity that cannot be rescued by PD-1 blockade. 4-1BB, a costimulatory molecule highly expressed on exhausted T cells, has been shown to influence metabolic function. We hypothesized that 4-1BB signaling might provide metabolic support to tumor-infiltrating T cells. 4-1BB costimulation of CD8+ T cells results in enhanced mitochondrial capacity (suggestive of fusion) and engages PGC1α-mediated pathways via activation of p38-MAPK. 4-1BB treatment of mice improves metabolic sufficiency in endogenous and adoptive therapeutic CD8+ T cells. 4-1BB stimulation combined with PD-1 blockade results in robust antitumor immunity. Sequenced studies revealed the metabolic support afforded by 4-1BB agonism need not be continuous and that a short course of anti–4-1BB pretreatment was sufficient to provide a synergistic response. Our studies highlight metabolic reprogramming as the dominant effect of 4-1BB therapy and suggest that combinatorial strategies using 4-1BB agonism may help overcome the immunosuppressive metabolic landscape of the tumor microenvironment.

scholarly journals The Impact of Exercise Serum on Selected Parameters of CD4+ T Cell Metabolism

Immuno ◽  
2021 ◽  
Vol 1 (3) ◽  
pp. 119-131
Author(s):  
Jana Palmowski ◽  
Kristina Gebhardt ◽  
Thomas Reichel ◽  
Torsten Frech ◽  
Robert Ringseis ◽  
...  
Keyword(s):  
T Cells ◽  
Oxygen Consumption ◽  
T Cell ◽  
Real Time ◽  
Cd4 T Cells ◽  
Cell Metabolism ◽  
Cd4 T Cell ◽  
Autologous Serum ◽  
Cell Phenotype ◽  
The Impact

CD4+ T cells are sensitive to peripheral changes of cytokine levels and metabolic substrates such as glucose and lactate. This study aimed to analyze whether factors released after exercise alter parameters of human T cell metabolism, specifically glycolysis and oxidative phosphorylation. We used primary human CD4+ T cells activated in the presence of autologous serum, which was collected before (CO) and after a 30-min exercise intervention (EX). In the course of activation, cells and supernatants were analyzed for cell viability and diameter, real-time oxygen consumption by using PreSens Technology, mRNA expression of glycolytic enzymes and complexes of the electron transport chain by real-time PCR, glucose, and lactate levels in supernatants, and in vitro differentiation by flow cytometry. EX did not alter T cell phenotype, viability, or on-blast formation. Similarly, no difference between CO and EX were found for CD4+ T cell activation and cellular oxygen consumption. In contrast, higher levels of glucose were found after 48 h activation in EX conditions. T cells activated in autologous exercise serum expressed lower HK1 mRNA and higher IFN-γ receptor 1. We suggest that the exercise protocol used was not sufficient to destabilize the immune metabolism of T cells. Therefore, more intense and prolonged exercise should be used in future studies.

scholarly journals 588 Targeting GITR enhances human tumour-infiltrating T cell functionality in mismatch repair proficient primary colorectal carcinoma and liver metastases

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A623-A623
Author(s):  
Yannick Rakké ◽  
Lucia Campos Carrascosa ◽  
Adriaan van Beek ◽  
Valeska de Ruiter ◽  
Michael Doukas ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
T Cell ◽  
Colorectal Carcinoma ◽  
Liver Metastases ◽  
Mismatch Repair ◽  
Immune Checkpoint ◽  
Ex Vivo ◽  
Human Tumour ◽  
Functional Assays

BackgroundImmune checkpoint blockade (ICB; e.g. anti-PD-1/-CTLA-4) has been proven to be clinically effective in mismatch repair deficient (dMMR) colorectal carcinoma (CRC). Yet, the majority of patients carry mismatch repair proficient (pMMR) CRC, especially those with liver metastasis, and do not respond to ICB. Here, we studied the effect of immune checkpoint stimulation via GITR targeting on human tumour-infiltrating lymphocyte (TIL) functionality in pMMR primary CRC and liver metastases (CRLM).MethodsHuman TIL were isolated from freshly resected pMMR tumours of patients with primary CRC (stage 1–3) or liver metastases (table 1). GITR expression on TIL was determined using flow cytometry and compared to leukocytes isolated from blood (PBMC) and tumour-free surrounding tissues (tumour-free colon/liver, resp. TFC and TFL). Ex vivo functional assays were used to assess TIL expansion, activation and cytokine/cytotoxic mediator secretion upon CD3/CD28 bead activation and co-stimulation using an antibody-crosslinked recombinant trimeric GITR ligand (GITRL).ResultsGITR was overexpressed on TIL when compared to other stimulatory immune checkpoints (4-1BB, OX40). GITR expression was enhanced on CD4+ and CD8+ TIL compared to PBMC and TFC or TFL compartments in both primary CRC and CRLM. Among CD4+ TIL, GITR was increasingly expressed on CD45RA± FoxP3- helper T (Th), CD45RA- FoxP3int activated helper T (aTh), and CD45RA- FoxP3hi activated regulatory T cells (aTreg), respectively. Within CD8+ TIL, GITR expression was higher on TOX+ PD1Hi and putatively tumour-reactive CD103+ CD39+ TIL.1 Impaired effector cytokine production upon ex vivo PMA/ionomycin stimulation was observed in CD4+ and CD8+ GITR-expressing TIL, hinting to functional exhaustion of the target population. However, recombinant GITRL reinvigorated ex vivo TIL responses by significantly enhancing CD4+ and CD8+ TIL numbers and proinflammatory cytokine secretion in a dose-dependent manner (figure 1). Treg depletion did not fully abrogate the stimulatory effect of GITR ligation on CD4+ and CD8+ T cell expansion, demonstrating that the stimulatory effect was partly exerted via direct targeting GITR on effector T cells. Importantly, GITR-ligation also enhanced expansion of purified CD8+CD39+ TIL. Dual treatment with GITR ligand and nivolumab (anti-PD-1) further enhanced CD8+ TIL responses compared to GITR ligand monotherapy, whereas nivolumab alone did not show any effect.Abstract 588 Table 1Patient characteristicsPatient characteristics of patients included for FACS analysis and/or functional assays. † Pathologic staging was performed according to the AJCC 8th edition criteriaAbstract 588 Figure 1GITR ligation enhances CD4+ and CD8+ TIL expansionTIL were isolated from CRC or CRLM and cultured upon CD3/CD28 activation with or without GITRL (0.1–1.0 ug/mL) for 8 days. TIL numbers were acquired by flow cytometry and normalized to counting beads. Indicated is fold change relative to ctrl-treated TIL (n=10).ConclusionsAgonistic targeting of GITR enhances ex vivo human TIL functionality in pMMR CRC and might therefore be a promising approach for novel mono- or combinatorial immunotherapies in primary CRC and CRLM.AcknowledgementsN/ATrial RegistrationN/AEthics ApprovalThe study was approved by the medical ethics committee of the Erasmus Medical Center (MEC-2012-331).ConsentN/AReferenceDuhen T, Duhen R, Montler R, et al. Co-expression of CD39 and CD103 identifies tumor-reactive CD8 T cells in human solid tumors. Nat Commun 2018;9(1):2724. doi: 10.1038/s41467-018-05072-0.

109 Dominant-negative TGFβ receptor 2 enhances GPC3-targeting CAR-T cell efficacy against hepatocellular carcinoma

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A121-A121
Author(s):  
Nina Chu ◽  
Michael Overstreet ◽  
Ryan Gilbreth ◽  
Lori Clarke ◽  
Christina Gesse ◽  
...  
Keyword(s):  
Hepatocellular Carcinoma ◽  
Flow Cytometry ◽  
T Cells ◽  
T Cell ◽  
Dominant Negative ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T

BackgroundChimeric antigen receptors (CARs) are engineered synthetic receptors that reprogram T cell specificity and function against a given antigen. Autologous CAR-T cell therapy has demonstrated potent efficacy against various hematological malignancies, but has yielded limited success against solid cancers. MEDI7028 is a CAR that targets oncofetal antigen glypican-3 (GPC3), which is expressed in 70–90% of hepatocellular carcinoma (HCC), but not in normal liver tissue. Transforming growth factor β (TGFβ) secretion is increased in advanced HCC, which creates an immunosuppressive milieu and facilitates cancer progression and poor prognosis. We tested whether the anti-tumor efficacy of a GPC3 CAR-T can be enhanced with the co-expression of dominant-negative TGFβRII (TGFβRIIDN).MethodsPrimary human T cells were lentivirally transduced to express GPC3 CAR both with and without TGFβRIIDN. Western blot and flow cytometry were performed on purified CAR-T cells to assess modulation of pathways and immune phenotypes driven by TGFβ in vitro. A xenograft model of human HCC cell line overexpressing TGFβ in immunodeficient mice was used to investigate the in vivo efficacy of TGFβRIIDN armored and unarmored CAR-T. Tumor infiltrating lymphocyte populations were analyzed by flow cytometry while serum cytokine levels were quantified with ELISA.ResultsArmoring GPC3 CAR-T with TGFβRIIDN nearly abolished phospho-SMAD2/3 expression upon exposure to recombinant human TGFβ in vitro, indicating that the TGFβ signaling axis was successfully blocked by expression of the dominant-negative receptor. Additionally, expression of TGFβRIIDN suppressed TGFβ-driven CD103 upregulation, further demonstrating attenuation of the pathway by this armoring strategy. In vivo, the TGFβRIIDN armored CAR-T achieved superior tumor regression and delayed tumor regrowth compared to the unarmored CAR-T. The armored CAR-T cells infiltrated HCC tumors more abundantly than their unarmored counterparts, and were phenotypically less exhausted and less differentiated. In line with these observations, we detected significantly more interferon gamma (IFNγ) at peak response and decreased alpha-fetoprotein in the serum of mice treated with armored cells compared to mice receiving unarmored CAR-T, demonstrating in vivo functional superiority of TGFβRIIDN armored CAR-T therapy.ConclusionsArmoring GPC3 CAR-T with TGFβRIIDN abrogates the signaling of TGFβ in vitro and enhances the anti-tumor efficacy of GPC3 CAR-T against TGFβ-expressing HCC tumors in vivo, proving TGFβRIIDN to be an effective armoring strategy against TGFβ-expressing solid malignancies in preclinical models.Ethics ApprovalThe study was approved by AstraZeneca’s Ethics Board and Institutional Animal Care and Use Committee (IACUC).

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