Comparative analysis of whole plant, flower and root extracts of Chamomilla recutita L. and characteristic pure compounds reveals differential anti-inflammatory effects on human T cells.

Introduction: Chronic inflammation is a hallmark of chronic wounds and inflammatory skin diseases. Due to a hyperactive and prolonged inflammation triggered by proinflammatory immune cells, transitioning to the repair and healing phase is halted. T cells may exacerbate the proinflammatory milieu by secreting proinflammatory cytokines. Chamomilla recutita L. (chamomile) has been suggested for use in several inflammatory diseases, implying a capability to modulate T cells. Here, we have characterized and compared the effects of differently prepared chamomile extracts and characteristic pure compounds on the T cell redox milieu as well as on the migration, activation, proliferation, and cytokine production of primary human T cells. Methods: Phytochemical analysis of the extracts was carried out by LC-MS/MS. Primary human T cells from peripheral blood (PBTs) were pretreated with aqueous or hydroethanolic chamomile extracts or pure compounds. Subsequently, the effects on intracellular ROS levels, SDF-1α induced T cell migration, T cell activation, proliferation, and cytokine production after TCR/CD3 and CD28 costimulation were determined. Gene expression profiling was performed using nCounter analysis, followed by ingenuity pathway analysis, and validation at protein levels. Results: The tested chamomile extracts and pure compounds differentially affected intracellular ROS levels, migration, and activation of T cells. Three out of five differently prepared extracts and two out of three pure compounds diminished T cell proliferation. In line with these findings, LC-MS/MS analysis revealed high heterogeneity of phytochemicals among the different extracts. nCounter based gene expression profiling identified several genes related to T cell functions associated with activation and differentiation to be downregulated. Most prominently, apigenin significantly reduced granzyme B induction and cytotoxic T cell activity. Conclusion: Our results demonstrate an anti-inflammatory effect of chamomile- derived products on primary human T cells. These findings provide molecular explanations for the observed anti-inflammatory action of chamomile and imply a broader use of chamomile extracts in T cell driven chronic inflammatory diseases such as chronic wounds and inflammatory skin diseases. Importantly, the mode of extract preparation needs to be considered as the resulting different phytochemicals can result in differential effects on T cells.

Keratinocyte-induced costimulation of human T cells through CD6 - but not CD2 - activates mTOR and prevents oxidative stress.

In psoriasis and other inflammatory skin diseases, keratinocytes (KCs) secrete chemokines that attract T cells, which, in turn, cause epidermal hyperplasia by secreting proinflammatory cytokines. To date, it remains unclear whether skin-homing T cells, particularly memory T cells, can also be activated by direct cell contact with KCs. In this study, we demonstrated the ability of primary human KCs to activate human memory T cells directly by transmitting costimulatory signals through the CD6/CD166/CD318 axis. Interestingly, despite being negative for CD80/CD86, KCs initiate a metabolic shift within T cells. Blockade of the CD6/CD166/CD318 axis prevents mammalian target of rapamycin activation and T cell proliferation but promotes oxidative stress and aerobic glycolysis. In addition, it diminishes formation of central memory T cells. Importantly, although KC-mediated costimulation by CD2/CD58 also activates T cells, it cannot compensate for the lack of CD6 costimulation. Therefore, KCs likely differentially regulate T cell functions in the skin through two distinct costimulatory receptors: CD6 and CD2. This may at least in part explain the divergent effects observed when treating inflammatory skin diseases with antibodies to CD6 versus CD2. Moreover, our findings may provide a molecular basis for selective interference with either CD6/CD166/CD318, or CD2/CD58, or both to specifically treat different types of inflammatory skin diseases.

Expression of TRX1 optimizes the antitumor functions of human CAR T cells and confers resistance to a pro-oxidative tumor microenvironment.

Use of chimeric antigen receptor (CAR) T cells to treat B cell lymphoma and leukemia has been remarkably successful. Unfortunately, the therapeutic efficacy of CAR T cells against solid tumors is very limited, with immunosuppression by the pro-oxidative tumor microenvironment (TME) a major contributing factor. High levels of reactive oxygen species are well-tolerated by tumor cells due to their elevated expression of antioxidant proteins; however, this is not the case for T cells, which consequently become hypo-responsive. The aim of this study was to improve CAR T cell efficacy in solid tumors by empowering the antioxidant capacity of CAR T cells against the pro-oxidative TME. To this end, HER2-specific human CAR T cells stably expressing two antioxidant systems: thioredoxin-1 (TRX1), and glutaredoxin-1 (GRX1) were generated and characterized. Thereafter, antitumor functions of CAR T cells were evaluated under control or pro-oxidative conditions. To provide insights into the role of antioxidant systems, gene expression profiles as well as global protein oxidation were analyzed. Our results highlight that TRX1 is pivotal for T cell redox homeostasis. TRX1 expression allows CAR T cells to retain their cytolytic immune synapse formation, cytokine release, proliferation, and tumor cell-killing properties under pro-oxidative conditions. Evaluation of differentially expressed genes and the first comprehensive redoxosome analysis of T cells by mass spectrometry further clarified the underlying mechanisms. Taken together, enhancement of the key antioxidant TRX1 in human T cells opens possibilities to increase the efficacy of CAR T cell treatment against solid tumors.

Hijacked Immune Cells in the Tumor Microenvironment: Molecular Mechanisms of Immunosuppression and Cues to Improve T Cell-Based Immunotherapy of Solid Tumors.

The understanding of the tumor microenvironment (TME) has been expanding in recent years in the context of interactions among different cell types, through direct cell-cell communication as well as through soluble factors. It has become evident that the development of a successful antitumor response depends on several TME factors. In this context, the number, type, and subsets of immune cells, as well as the functionality, memory, and exhaustion state of leukocytes are key factors of the TME. Both the presence and functionality of immune cells, in particular T cells, are regulated by cellular and soluble factors of the TME. In this regard, one fundamental reason for failure of antitumor responses is hijacked immune cells, which contribute to the immunosuppressive TME in multiple ways. Specifically, reactive oxygen species (ROS), metabolites, and anti-inflammatory cytokines have central roles in generating an immunosuppressive TME. In this review, we focused on recent developments in the immune cell constituents of the TME, and the micromilieu control of antitumor responses. Furthermore, we highlighted the current challenges of T cell-based immunotherapies and potential future strategies to consider for strengthening their effectiveness.

Piperlongumine Acts as an Immunosuppressant by Exerting Prooxidative Effects in Human T Cells Resulting in Diminished TH17 but Enhanced Treg Differentiation.

Piperlongumine (PL), a natural small molecule derived from the Piper longum Linn plant, has received growing interest as a prooxidative drug with promising anticancer properties. Yet, the influence of PL on primary human T cells remained elusive. Knowledge of this is of crucial importance, however, since T cells in particular play a critical role in tumor control. Therefore, we investigated the effects of PL on the survival and function of primary human peripheral blood T cells (PBTs). While PL was not cytotoxic to PBTs, it interfered with several stages of T cell activation as it inhibited T cell/APC immune synapse formation, co-stimulation-induced upregulation of CD69 and CD25, T cell proliferation and the secretion of proinflammatory cytokines. PL-induced immune suppression was prevented in the presence of thiol-containing antioxidants. In line with this finding, PL increased the levels of intracellular reactive oxygen species and decreased glutathione in PBTs. Diminished intracellular glutathione was accompanied by a decrease in S-glutathionylation on actin suggesting a global alteration of the antioxidant response. Gene expression analysis demonstrated that TH17-related genes were predominantly inhibited by PL. Consistently, the polarization of primary human naïve CD4+ T cells into TH17 subsets was significantly diminished while differentiation into Treg cells was substantially increased upon PL treatment. This opposed consequence for TH17 and Treg cells was again abolished by thiol-containing antioxidants. Taken together, PL may act as a promising agent for therapeutic immunosuppression by exerting prooxidative effects in human T cells resulting in a diminished TH17 but enhanced Treg cell differentiation.

Redox Regulation of the Actin Cytoskeleton in Cell Migration and Adhesion: On the Way to a Spatiotemporal View.

The actin cytoskeleton of eukaryotic cells is a dynamic, fibrous network that is regulated by the concerted action of actin-binding proteins (ABPs). In particular, rapid polarization of cells in response to internal and external stimuli is fundamental to cell migration and invasion. Various isoforms of ABPs in different tissues equip cells with variable degrees of migratory and adhesive capacities. In addition, regulation of ABPs by posttranslational modifications (PTM) is pivotal to the rapid responsiveness of cells. In this context, phosphorylation of ABPs and its functional consequences have been studied extensively. However, the study of reduction/oxidation (redox) modifications of oxidation-sensitive cysteine and methionine residues of actin, ABPs, adhesion molecules, and signaling proteins regulating actin cytoskeletal dynamics has only recently emerged as a field. The relevance of such protein oxidations to cellular physiology and pathophysiology has remained largely elusive. Importantly, studying protein oxidation spatiotemporally can provide novel insights into localized redox regulation of cellular functions. In this review, we focus on the redox regulation of the actin cytoskeleton, its challenges, and recently developed tools to study its physiological and pathophysiological consequences.

Keratinocytes costimulate naive human T cells via CD2: a potential target to prevent the development of proinflammatory Th1 cells in the skin.

The interplay between keratinocytes and immune cells, especially T cells, plays an important role in the pathogenesis of chronic inflammatory skin diseases. During psoriasis, keratinocytes attract T cells by releasing chemokines, while skin-infiltrating self-reactive T cells secrete proinflammatory cytokines, e.g., IFNγ and IL-17A, that cause epidermal hyperplasia. Similarly, in chronic graft-versus-host disease, allogenic IFNγ-producing Th1/Tc1 and IL-17-producing Th17/Tc17 cells are recruited by keratinocyte-derived chemokines and accumulate in the skin. However, whether keratinocytes act as nonprofessional antigen-presenting cells to directly activate naive human T cells in the epidermis remains unknown. Here, we demonstrate that under proinflammatory conditions, primary human keratinocytes indeed activate naive human T cells. This activation required cell contact and costimulatory signaling via CD58/CD2 and CD54/LFA-1. Naive T cells costimulated by keratinocytes selectively differentiated into Th1 and Th17 cells. In particular, keratinocyte-initiated Th1 differentiation was dependent on costimulation through CD58/CD2. The latter molecule initiated STAT1 signaling and IFNγ production in T cells. Costimulation of T cells by keratinocytes resulting in Th1 and Th17 differentiation represents a new explanation for the local enrichment of Th1 and Th17 cells in the skin of patients with a chronic inflammatory skin disease. Consequently, local interference with T cell-keratinocyte interactions may represent a novel strategy for the treatment of Th1 and Th17 cell-driven skin diseases.

3D Cellular Architecture Modulates Tyrosine Kinase Activity, Thereby Switching CD95-Mediated Apoptosis to Survival.

The death receptor CD95 is expressed in every cancer cell, thus providing a promising tool to target cancer. Activation of CD95 can, however, lead to apoptosis or proliferation. Yet the molecular determinants of CD95's mode of action remain unclear. Here, we identify an optimal distance between CD95Ligand molecules that enables specific clustering of receptor-ligand pairs, leading to efficient CD95 activation. Surprisingly, efficient CD95 activation leads to apoptosis in cancer cells in vitro and increased tumor growth in vivo. We show that allowing a 3D aggregation of cancer cells in vitro switches the apoptotic response to proliferation. Indeed, we demonstrate that the absence or presence of cell-cell contacts dictates the cell response to CD95. Cell contacts increase global levels of phosphorylated tyrosines, including CD95's tyrosine. A tyrosine-to-alanine CD95 mutant blocks proliferation in cells in contact. Our study sheds light into the regulatory mechanism of CD95 activation that can be further explored for anti-cancer therapies.

Spatial oxidation of L-plastin downmodulates actin-based functions of tumor cells.

Several antitumor therapies work by increasing reactive oxygen species (ROS) within the tumor micromilieu. Here, we reveal that L-plastin (LPL), an established tumor marker, is reversibly regulated by ROS-induced thiol oxidation on Cys101, which forms a disulfide bridge with Cys42. LPL reduction is mediated by the Thioredoxin1 (TRX1) system, as shown by TRX1 trapping, TRX1 knockdown and blockade of Thioredoxin1 reductase (TRXR1) with auranofin. LPL oxidation diminishes its actin-bundling capacity. Ratiometric imaging using an LPL-roGFP-Orp1 fusion protein and a dimedone-based proximity ligation assay (PLA) reveal that LPL oxidation occurs primarily in actin-based cellular extrusions and strongly inhibits cell spreading and filopodial extension formation in tumor cells. This effect is accompanied by decreased tumor cell migration, invasion and extracellular matrix (ECM) degradation. Since LPL oxidation occurs following treatment of tumors with auranofin or γ-irradiation, it may be a molecular mechanism contributing to the effectiveness of tumor treatment with redox-altering therapies.

Sulforaphane Inhibits Inflammatory Responses of Primary Human T-Cells by Increasing ROS and Depleting Glutathione.

The activity and function of T-cells are influenced by the intra- and extracellular redox milieu. Oxidative stress induces hypo responsiveness of untransformed T-cells. Vice versa increased glutathione (GSH) levels or decreased levels of reactive oxygen species (ROS) prime T-cell metabolism for inflammation, e.g., in rheumatoid arthritis. Therefore, balancing the T-cell redox milieu may represent a promising new option for therapeutic immune modulation. Here we show that sulforaphane (SFN), a compound derived from plants of the Brassicaceae family, e.g., broccoli, induces a pro-oxidative state in untransformed human T-cells of healthy donors or RA patients. This manifested as an increase of intracellular ROS and a marked decrease of GSH. Consistently, increased global cysteine sulfenylation was detected. Importantly, a major target for SFN-mediated protein oxidation was STAT3, a transcription factor involved in the regulation of TH17-related genes. Accordingly, SFN significantly inhibited the activation of untransformed human T-cells derived from healthy donors or RA patients, and downregulated the expression of the transcription factor RORγt, and the TH17-related cytokines IL-17A, IL-17F, and IL-22, which play a major role within the pathophysiology of many chronic inflammatory/autoimmune diseases. The inhibitory effects of SFN could be abolished by exogenously supplied GSH and by the GSH replenishing antioxidant N-acetylcysteine (NAC). Together, our study provides mechanistic insights into the mode of action of the natural substance SFN. It specifically exerts TH17 prone immunosuppressive effects on untransformed human T-cells by decreasing GSH and accumulation of ROS. Thus, SFN may offer novel clinical options for the treatment of TH17 related chronic inflammatory/autoimmune diseases such as rheumatoid arthritis.

L-plastin regulates the stability of the immune synapse of naive and effector T-cells.

T-cells need to be tightly regulated during their activation and effector phase to assure an appropriate defence against cancer or pathogens and - vice versa - to avoid autoimmune reactions. Regulatory signals are provided via the immune synapse between T-cells and antigen-presenting cells (APCs) or target cells. The stability and kinetics of immune synapse formation is critical for proper T-cell functions. It requires dynamic rearrangements of the actin cytoskeleton necessary for organized spatio-temporal redistribution of receptors and adhesion molecules. We identified glucocorticoid-sensitive phosphorylation of serine 5 on the actin-bundling protein L-plastin as one important signalling event for this regulation. Using imaging flow cytometry as well as confocal and super-resolution microscopy we showed that L-plastin relocalizes to the immune synapse upon antigen encounter, where it associates with the ß2-subunit of LFA-1 (CD11a/CD18). Interfering with L-plastin expression or activation leads to a defective LFA-1 recruitment and unstable T-cell/APC contacts. Consequently, the lack of L-plastin diminishes T-cell activation, proliferation and proximal effector responses such as cytokine production. On the other hand, a pro-oxidative milieu leads to prolonged activation of L-plastin resulting in a stronger enrichment of LFA-1 in the cytolytic immune synapse. Concomitant stabilization of conjugates formed by cytotoxic T-cells (CTLs) and their target cells impairs the ability of CTLs to kill more than one target cells (serial killing), which de facto leads to a downregulation of T-cell cytotoxicity. Together, we demonstrate that activation and spacial distribution of L-plastin regulates the maturation and stability of activating and cytolytic immune synapses important for T-cell activation and effector functions.

Qualitative and quantitative analysis of PMN/T-cell interactions by InFlow and super-resolution microscopy.

Neutrophils or polymorphonuclear cells (PMN) eliminate bacteria via phagocytosis and/or NETosis. Apart from these conventional roles, PMN also have immune-regulatory functions. They can transdifferentiate and upregulate MHCII as well as ligands for costimulatory receptors which enables them to behave as antigen presenting cells (APC). The initial step for activating T-cells is the formation of an immune synapse between T-cells and antigen-presenting cells. However, the immune synapse that develops at the PMN/T-cell contact zone is as yet hardly investigated due to the non-availability of methods for analysis of large number of PMN interactions. In order to overcome these obstacles, we introduce here a workflow to analyse the immune synapse of primary human PMN and T-cells using multispectral imaging flow cytometry (InFlow microscopy) and super-resolution microscopy. For that purpose, we used CD3 and CD66b as the lineage markers for T-cells and PMN, respectively. Thereafter, we applied and critically discussed various "masks" for identification of T-cell PMN interactions. Using this approach, we found that a small fraction of transdifferentiated PMN (CD66b+CD86high) formed stable PMN/T-cell conjugates. Interestingly, while both CD3 and CD66b accumulation in the immune synapse was dependent on the maturation state of the PMN, only CD3 accumulation was greatly enhanced by the presence of superantigen. The actin cytoskeleton was weakly rearranged at the PMN side on the immune synapse upon contact with a T-cell in the presence of superantigen. A more detailed analysis using super-resolution microscopy (structured-illumination microscopy, SIM) confirmed this finding. Together, we present an InFlow microscopy based approach for the large scale analysis of PMN/T-cell interactions and - combined with SIM - a possibility for an in-depth analysis of protein translocation at the site of interactions.