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1.
Biotinylation Assay to Determine LFA-1 Recycling in Motile T-Lymphocytes.
Samuelsson, M, Svensson, LM
Methods in molecular biology (Clifton, N.J.). 2019;:115-122
Abstract
The cycles of internalization of the cell surface β2 integrin receptor lymphocyte function-associated antigen 1 (LFA-1) and its re-exposure on the plasma membrane are important for T-cell trafficking. Biotinylation of cells enables to measure surface expression of receptors, and after reducing surface biotin with reducing buffer, enables to measure the internalization of receptors. Here, by using biotin in combination with reducing buffer and recombinant intercellular adhesion molecule-1 (rICAM-1)-coated dishes and subsequent Western immunoblot analysis, we describe how to measure internalization of the LFA-1 receptor and its re-expression back to the cell surface in motile T-lymphocytes.
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2.
Tracking Dye-Independent Approach to Identify and Isolate In Vitro Expanded T Cells.
Elias, G, Ogunjimi, B, Van Tendeloo, V
Cytometry. Part A : the journal of the International Society for Analytical Cytology. 2019;(10):1096-1107
Abstract
T cell proliferation is routinely identified in vitro using tracking dyes or through detecting intracellular upregulation of the nuclear protein, Ki-67. However, labeling with tracking dyes is cumbersome, associated with cellular toxicity, while Ki-67 cannot be used to identify and isolate viable T cells, and both techniques are incompatible with MACS technology. Here, we introduce a simple tool to identify and isolate in vitro T cell expansion that is tracking dye-independent and allows for sorting of viable T cells. We show that CD71, a transferrin receptor, and CD98, a heterodimer glycoprotein involved in both integrin signaling and amino-acid transport, are both highly upregulated on proliferating T cells upon in vitro stimulation, and that CD71 expression is maximal on the more recent progeny T cells, while CD98 upregulation remains stable across different generations of progeny T cells. Moreover, we demonstrate that the upregulation of CD71 and CD98 identifies CFSElow T cells and provides further proof of the antigen-specificity of T cells identified by CD71 and CD98 dual upregulation based on tetramer staining. We further show that CD71 can be used to enrich for in vitro expanding T cells using MACS technology. In conclusion, we show that CD71 and CD98 can be used to identify and isolate expanded T cells following in vitro stimulation and that CD71 is an MACS-compatible alternative to tracking dyes or Ki-67 detection. © 2019 International Society for Advancement of Cytometry.
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3.
Functionalization Of T Lymphocytes With Citrate-Coated Superparamagnetic Iron Oxide Nanoparticles For Magnetically Controlled Immune Therapy.
Mühlberger, M, Janko, C, Unterweger, H, Friedrich, RP, Friedrich, B, Band, J, Cebulla, N, Alexiou, C, Dudziak, D, Lee, G, et al
International journal of nanomedicine. 2019;:8421-8432
Abstract
PURPOSE Immune activation with T cell tumor infiltration is beneficial for the prognosis of patients suffering from solid cancer. Depending on their immune status, solid tumors can be immunologically classified into three groups: "hot" tumors are infiltrated with T lymphocytes, "cold" tumors are not infiltrated and "immune excluded" tumors are only infiltrated in the peripheral tumor tissue. Checkpoint inhibitors provide new therapeutic options for "hot" tumors by triggering the immune response of T cells. In order to enable this for cold tumors as well, T cells must be enriched in the tumor. Therefore, we use the principle of magnetic targeting to guide T cells loaded with citrate-coated superparamagnetic iron oxide nanoparticles (SPIONCitrate) to the tumor by an externally applied magnetic field. METHODS SPIONCitrate were produced by alkaline coprecipitation of iron(II) and iron(III) chloride and in situ coating with sodium citrate. The concentration-dependent cytocompatibility of the particles was determined by flow cytometry and blood stability assays. Atomic emission spectroscopy was used for the quantification of the particle uptake into T lymphocytes. The attractability of the loaded cells was observed by live-cell imaging in the presence of an externally applied magnetic field. RESULTS SPIONCitrate displayed good cytocompatibility to T cells and did not show any sign of aggregation in blood. Finally, SPIONCitrate-loaded T cells were strongly attracted by a small external magnet. CONCLUSION T cells can be "magnetized" by incorporation of SPIONCitrate for magnetic targeting. The production of the particle-cell hybrid system is straightforward, as the loading process only requires basic laboratory devices and the loading efficiency is sufficient for cells being magnetically controllable. For these reasons, SPIONCitrate are potential suitable candidates for magnetic T cell targeting.
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4.
Optogenetic control shows that kinetic proofreading regulates the activity of the T cell receptor.
Yousefi, OS, Günther, M, Hörner, M, Chalupsky, J, Wess, M, Brandl, SM, Smith, RW, Fleck, C, Kunkel, T, Zurbriggen, MD, et al
eLife. 2019
Abstract
The immune system distinguishes between self and foreign antigens. The kinetic proofreading (KPR) model proposes that T cells discriminate self from foreign ligands by the different ligand binding half-lives to the T cell receptor (TCR). It is challenging to test KPR as the available experimental systems fall short of only altering the binding half-lives and keeping other parameters of the interaction unchanged. We engineered an optogenetic system using the plant photoreceptor phytochrome B (PhyB) as a ligand to selectively control the dynamics of ligand binding to the TCR by light. This opto-ligand-TCR system was combined with the unique property of PhyB to continuously cycle between the binding and non-binding states under red light, with the light intensity determining the cycling rate and thus the binding duration. Mathematical modeling of our experimental datasets showed that indeed the ligand-TCR interaction half-life is the decisive factor for activating downstream TCR signaling, substantiating KPR.
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5.
Calcium signalling in T cells.
Trebak, M, Kinet, JP
Nature reviews. Immunology. 2019;(3):154-169
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Abstract
Calcium (Ca2+) signalling is of paramount importance to immunity. Regulated increases in cytosolic and organellar Ca2+ concentrations in lymphocytes control complex and crucial effector functions such as metabolism, proliferation, differentiation, antibody and cytokine secretion and cytotoxicity. Altered Ca2+ regulation in lymphocytes leads to various autoimmune, inflammatory and immunodeficiency syndromes. Several types of plasma membrane and organellar Ca2+-permeable channels are functional in T cells. They contribute highly localized spatial and temporal Ca2+ microdomains that are required for achieving functional specificity. While the mechanistic details of these Ca2+ microdomains are only beginning to emerge, it is evident that through crosstalk, synergy and feedback mechanisms, they fine-tune T cell signalling to match complex immune responses. In this article, we review the expression and function of various Ca2+-permeable channels in the plasma membrane, endoplasmic reticulum, mitochondria and endolysosomes of T cells and their role in shaping immunity and the pathogenesis of immune-mediated diseases.
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6.
Coeliac Disease and Mast Cells.
Frossi, B, De Carli, M, Calabrò, A
International journal of molecular sciences. 2019;(14)
Abstract
Over the last decades, there has been an impressive progress in our understanding of coeliac disease pathogenesis and it has become clear that the disorder is the final result of complex interactions of environmental, genetic, and immunological factors. Coeliac disease is now considered a prototype of T-cell-mediated disease characterized by loss of tolerance to dietary gluten and the targeted killing of enterocytes by T-cell receptor αβ intraepithelial lymphocytes. Accumulating evidence, however, indicates that the induction of a gluten-specific T helper-1 response must be preceded by the activation of the innate immune system. Mast cells are key players of the innate immune response and contribute to the pathogenesis of a multitude of diseases. Here, we review the results of studies aimed at investigating the role of mast cells in the pathogenesis of coeliac disease, showing that these cells increase in number during the progression of the disease and contribute to define a pro-inflammatory microenvironment.
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7.
Serum very long-chain fatty acid-containing lipids predict response to immune checkpoint inhibitors in urological cancers.
Mock, A, Zschäbitz, S, Kirsten, R, Scheffler, M, Wolf, B, Herold-Mende, C, Kramer, R, Busch, E, Jenzer, M, Jäger, D, et al
Cancer immunology, immunotherapy : CII. 2019;(12):2005-2014
Abstract
Checkpoint inhibitors (CPI) have significantly changed the therapeutic landscape of oncology. We adopted a non-invasive metabolomic approach to understand immunotherapy response and failure in 28 urological cancer patients. In total, 134 metabolites were quantified in patient sera before the first, second, and third CPI doses. Modeling the association between metabolites and CPI response and patient characteristics revealed that one predictive metabolite class (n = 9/10) were very long-chain fatty acid-containing lipids (VLCFA-containing lipids). The best predictive performance was achieved through a multivariate model, including age and a centroid of VLCFA-containing lipids prior to first immunotherapy (sensitivity: 0.850, specificity: 0.825, ROC: 0.935). We hypothesize that the association of VLCFA-containing lipids with CPI response is based on enhanced peroxisome signaling in T cells, which results in a switch to fatty acid catabolism. Beyond use as a novel predictive non-invasive biomarker, we envision that nutritional supplementation with VLCFA-containing lipids might serve as an immuno sensitizer.
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Systemic β-Adrenergic Receptor Activation Augments the ex vivo Expansion and Anti-Tumor Activity of Vγ9Vδ2 T-Cells.
Baker, FL, Bigley, AB, Agha, NH, Pedlar, CR, O'Connor, DP, Bond, RA, Bollard, CM, Katsanis, E, Simpson, RJ
Frontiers in immunology. 2019;:3082
Abstract
TCR-gamma delta (γδ) T-cells are considered important players in the graft-vs.-tumor effect following allogeneic hematopoietic cell transplantation (alloHCT) and have emerged as candidates for adoptive transfer immunotherapy in the treatment of both solid and hematological tumors. Systemic β-adrenergic receptor (β-AR) activation has been shown to mobilize TCR-γδ T-cells to the blood, potentially serving as an adjuvant for alloHCT and TCR-γδ T-cell therapy. We investigated if systemic β-AR activation, using acute dynamic exercise as an experimental model, can increase the mobilization, ex vivo expansion, and anti-tumor activity of TCR-γδ T-cells isolated from the blood of healthy humans. We also sought to investigate the β-AR subtypes involved, by administering a preferential β1-AR antagonist (bisoprolol) and a non-preferential β1 + β2-AR antagonist (nadolol) prior to exercise as part of a randomized placebo controlled cross-over experiment. We found that exercise mobilized TCR-γδ cells to blood and augmented their ex vivo expansion by ~182% compared to resting blood when stimulated with IL-2 and ZOL for 14-days. Exercise also increased the proportion of CD56+, NKG2D+/CD62L-, CD158a/b/e+ and NKG2A- cells among the expanded TCR-γδ cells, and increased their cytotoxic activity against several tumor target cells (K562, U266, 221.AEH) in vitro by 40-60%. Blocking NKG2D on TCR-γδ cells in vitro eliminated the augmented cytotoxic effects of exercise against U266 target cells. Furthermore, administering a β1 + β2-AR (nadolol), but not a β1-AR (bisoprolol) antagonist prior to exercise abrogated the exercise-induced enhancement in TCR-γδ T-cell mobilization and ex vivo expansion. Furthermore, nadolol completely abrogated while bisoprolol partially inhibited the exercise-induced increase in the cytotoxic activity of the expanded TCR-γδ T-cells. We conclude that acute systemic β-AR activation in healthy donors markedly augments the mobilization, ex vivo expansion, and anti-tumor activity of TCR-γδ T-cells and that some of these effects are due to β2-AR signaling and phenotypic shifts that promote a dominant activating signal via NKG2D. These findings highlight β-ARs as potential targets to favorably alter the composition of allogeneic peripheral blood stem cell grafts and improve the potency of TCR-γδ T-cell immune cell therapeutics.
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9.
Effects of probiotics supplementation and circuit training on immune responses among sedentary young males.
Ibrahim, NS, Ooi, FK, Chen, CK, Muhamad, AS
The Journal of sports medicine and physical fitness. 2018;(7-8):1102-1109
Abstract
BACKGROUND Growing evidence suggests that probiotics may have positive benefits on immune responses following endurance exercise. However, little attention has been given to its possible beneficial effects on immune responses following resistance exercise. METHODS Forty-one healthy sedentary males were recruited and randomised into four groups: sedentary control with placebo (C), probiotics (P), circuit training with placebo (Ex), and circuit training with probiotics (PEx) groups. Participants in the Ex and PEx groups performed a progressive load of circuit training at 3 times/week for 12 weeks. Each circuit comprised 10 exercises with work to rest ratio of 1:2. Participants consumed either multi-strain probiotics or placebo twice daily for 12 weeks. Body height and weight, blood pressure, resting heart rate, saliva and blood samples were collected at pre- and post-tests. RESULTS Saliva flow rate and salivary IgA, α-amylase, lactoferrin and lysozyme responses were not significantly different (P>0.05) between groups and also between pre- and post-test within each group. Similarly, total leukocytes, total lymphocytes, T lymphocytes, T-helper, T-cytotoxic, B lymphocytes, and natural killer cells counts were not significantly affected (P>0.05) by the probiotics and/or circuit training. However, circuit training significantly increased (P<0.05) immune cells count at post-test as compared to pre-test. Yet, a combination of circuit training and probiotics showed no significant (P>0.05) effects on immune cells count. CONCLUSIONS This study did not provide enough support for the positive effects of probiotics on immune responses among sedentary young males following resistance exercise. However, 12 weeks of circuit training enhanced immune cells count.
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10.
Dynamics of virus-specific T cell immunity in pediatric liver transplant recipients.
Arasaratnam, RJ, Tzannou, I, Gray, T, Aguayo-Hiraldo, PI, Kuvalekar, M, Naik, S, Gaikwad, A, Liu, H, Miloh, T, Vera, JF, et al
American journal of transplantation : official journal of the American Society of Transplantation and the American Society of Transplant Surgeons. 2018;(9):2238-2249
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Abstract
Immunosuppression following solid organ transplantation (SOT) has a deleterious effect on cellular immunity leading to frequent and prolonged viral infections. To better understand the relationship between posttransplant immunosuppression and circulating virus-specific T cells, we prospectively monitored the frequency and function of T cells directed to a range of latent (CMV, EBV, HHV6, BK) and lytic (AdV) viruses in 16 children undergoing liver transplantation for up to 1 year posttransplant. Following transplant, there was an immediate decline in circulating virus-specific T cells, which recovered posttransplant, coincident with the introduction and subsequent routine tapering of immunosuppression. Furthermore, 12 of 14 infections/reactivations that occurred posttransplant were successfully controlled with immunosuppression reduction (and/or antiviral use) and in all cases we detected a temporal increase in the circulating frequency of virus-specific T cells directed against the infecting virus, which was absent in 2 cases where infections remained uncontrolled by the end of follow-up. Our study illustrates the dynamic changes in virus-specific T cells that occur in children following liver transplantation, driven both by active viral replication and modulation of immunosuppression.