Tumor necrosis factor α promotes clonal dominance of KIT D816V+ cells in mastocytosis: role of survivin and impact on prognosis.

ABSTRACT: Systemic mastocytosis (SM) is defined by the expansion and accumulation of neoplastic mast cells (MCs) in the bone marrow (BM) and extracutaneous organs. Most patients harbor a somatic KIT D816V mutation, which leads to growth factor-independent KIT activation and accumulation of MC. Tumor necrosis factor α (TNF) is a proapoptotic and inflammatory cytokine that has been implicated in the clonal selection of neoplastic cells. We found that KIT D816V increases the expression and secretion of TNF. TNF expression in neoplastic MCs is reduced by KIT-targeting drugs. Similarly, knockdown of KIT or targeting the downstream signaling cascade of MAPK and NF-κB signaling reduced TNF expression levels. TNF reduces colony formation in human BM cells, whereas KIT D816V+ cells are less susceptible to the cytokine, potentially contributing to clonal selection. In line, knockout of TNF in neoplastic MC prolonged survival and reduced myelosuppression in a murine xenotransplantation model. Mechanistic studies revealed that the relative resistance of KIT D816V+ cells to TNF is mediated by the apoptosis-regulator BIRC5 (survivin). Expression of BIRC5 in neoplastic MC was confirmed by immunohistochemistry of samples from patients with SM. TNF serum levels are significantly elevated in patients with SM and high TNF levels were identified as a biomarker associated with inferior survival. We here characterized TNF as a KIT D816V-dependent cytokine that promotes clonal dominance. We propose TNF and apoptosis-associated proteins as potential therapeutic targets in SM.

Hereditary α tryptasemia is a valid genetic biomarker for severe mediator-related symptoms in mastocytosis.

Mastocytosis is a hematopoietic neoplasm characterized by expansion of KIT D816V-mutated clonal mast cells in various organs and severe or even life-threatening anaphylactic reactions. Recently, hereditary α-tryptasemia (HαT) has been described as a common genetic trait with increased copy numbers of the α-tryptase encoding gene, TPSAB1, and associated with an increased basal serum tryptase level and a risk of mast cell activation. The purpose of our study was to elucidate the clinical relevance of HαT in patients with mastocytosis. TPSAB1 germline copy number variants were assessed by digital polymerase chain reaction in 180 mastocytosis patients, 180 sex-matched control subjects, 720 patients with other myeloid neoplasms, and 61 additional mastocytosis patients of an independent validation cohort. α-Tryptase encoding TPSAB1 copy number gains, compatible with HαT, were identified in 17.2% of mastocytosis patients and 4.4% of the control population (P < .001). Patients with HαT exhibited higher tryptase levels than patients without HαT (median tryptase in HαT+ cases: 49.6 ng/mL vs HαT- cases: 34.5 ng/mL, P = .004) independent of the mast cell burden. Hymenoptera venom hypersensitivity reactions and severe cardiovascular mediator-related symptoms/anaphylaxis were by far more frequently observed in mastocytosis patients with HαT than in those without HαT. Results were confirmed in an independent validation cohort. The high prevalence of HαT in mastocytosis hints at a potential pathogenic role of germline α-tryptase encoding TPSAB1 copy number gains in disease evolution. Together, our data suggest that HαT is a novel emerging robust biomarker in mastocytosis that is useful for determining the individual patient´s risk of developing severe anaphylaxis.

Packed red blood cells inhibit T-cell activation via ROS-dependent signaling pathways.

Numerous observations indicate that red blood cells (RBCs) affect T-cell activation and proliferation. We have studied effects of packed RBCs (PRBCs) on T-cell receptor (TCR) signaling and the molecular mechanisms whereby (P)RBCs modulate T-cell activation. In line with previous reports, PRBCs attenuated the expression of T-cell activation markers CD25 and CD69 upon costimulation via CD3/CD28. In addition, T-cell proliferation and cytokine expression were markedly reduced when T-cells were stimulated in the presence of PRBCs. Inhibitory activity of PRBCs required direct cell-cell contact and intact PRBCs. The production of activation-induced cellular reactive oxygen species, which act as second messengers in T-cells, was completely abrogated to levels of unstimulated T-cells in the presence of PRBCs. Phosphorylation of the TCR-related zeta chain and thus proximal TCR signal transduction was unaffected by PRBCs, ruling out mechanisms based on secreted factors and steric interaction restrictions. In large part, downstream signaling events requiring reactive oxygen species for full functionality were affected, as confirmed by an untargeted MS-based phosphoproteomics approach. PRBCs inhibited T-cell activation more efficiently than treatment with 1 mM of the antioxidant N-acetyl cysteine. Taken together, our data imply that inflammation-related radical reactions are modulated by PRBCs. These immunomodulating effects may be responsible for clinical observations associated with transfusion of PRBCs.

The energy sensor AMPK orchestrates metabolic and translational adaptation in expanding T helper cells.

The importance of cellular metabolic adaptation in inducing robust T cell responses is well established. However, the mechanism by which T cells link information regarding nutrient supply to clonal expansion and effector function is still enigmatic. Herein, we report that the metabolic sensor adenosine monophosphate-activated protein kinase (AMPK) is a critical link between cellular energy demand and translational activity and, thus, orchestrates optimal expansion of T cells in vivo. AMPK deficiency did not affect T cell fate decision, activation, or T effector cell generation; however, the magnitude of T cell responses in murine in vivo models of T cell activation was markedly reduced. This impairment was global, as all T helper cell subsets were similarly sensitive to loss of AMPK which resulted in reduced T cell accumulation in peripheral organs and reduced disease severity in pathophysiologically as diverse models as T cell transfer colitis and allergic airway inflammation. T cell receptor repertoire analysis confirmed similar clonotype frequencies in different lymphoid organs, thereby supporting the concept of a quantitative impairment in clonal expansion rather than a skewed qualitative immune response. In line with these findings, in-depth metabolic analysis revealed a decrease in T cell oxidative metabolism, and gene set enrichment analysis indicated a major reduction in ribosomal biogenesis and mRNA translation in AMPK-deficient T cells. We, thus, provide evidence that through its interference with these delicate processes, AMPK orchestrates the quantitative, but not the qualitative, manifestation of primary T cell responses in vivo.

Delays during PBMC isolation have a moderate effect on yield, but severly compromise cell viability.

OBJECTIVES: Peripheral blood mononuclear cells (PBMCs) are a versatile material for clinical routine as well as for research projects. However, their isolation via density gradient centrifugation is still time-consuming. When samples are taken beyond usual laboratory handling times, it may sometimes be necessary to pause the isolation process. Our aim was to evaluate the impact of delays up to 48 h after the density gradient centrifugation on PBMC yield, purity and viability. METHODS: PBMCs were isolated from samples of 20 donors, either with BD Vacutainer CPT tubes (CPT) or with the standard Ficoll method. Isolation was paused after initial density gradient centrifugation for 0, 24, or 48 h. PBMC yield (% output/input), purity (% PBMCs/total cells) and viability (% Annexin V-/propidium iodide-) were compared. RESULTS: The yield did not change significantly over time when CPT were used (55%/52%/47%), but did after isolation with the standard method (62%/40%[p<0.0001]/53%[p<0.01]). Purity was marginally affected if CPT were used (95%/93%[p=n.s./92%[p<0.05] vs. 97% for all time points with standard method). Whereas viable PBMCs decreased steadily for CPT isolates (62%/51%[p<0.001]/36%[p<0.0001]), after standard Ficoll gradient isolation, cell apoptosis was more pronounced already after 24 h delay, and viability did not further decrease after 48 h (64%/44%[p<0.0001]/40%[p<0.0001]). CONCLUSIONS: In conclusion, our findings suggest that while post-centrifugation delays ≥24 h might have only a minor effect on cell yield and purity, their impact on cell viability is substantial, even when CPT are used.

The TGF-b/SOX4 axis and ROS-driven autophagy co-mediate CD39 expression in regulatory T-cells.

The ectonucleotidase CD39 on human regulatory T-cells (Treg) is an important immune regulator which is dysregulated in autoimmune diseases and cancer immunosuppression. We here define that CD39 expression on Treg is independent of the Treg-specific transcription factors FOXP3 and HELIOS and promoted by canonical TGF-b- and mTOR-signaling. Furthermore, the TGF-b mediated upregulation of CD39 is counteracted by reactive oxygen species (ROS)-driven autophagy. In line, CD39+ peripheral blood Treg constitute a distinct lineage with low autophagic flux and absent ROS production. Patients with rare genetic defects in autophagy show supraphysiological levels of CD39+ Treg, validating our observations in vivo. These biological processes rely on a distinct transcriptional program with CD39+ Treg expressing low levels of two genes with putative involvement in autophagy, NEFL and PLAC8. Furthermore, the TGF-b downstream transcription factor SOX4 is selectively upregulated in CD39+ Treg. Overexpression of SOX4 in Treg strongly increases CD39 expression, while Crispr/Cas9-mediated knockout of SOX4 in Treg has the opposing effect. Thus, we identify a crucial role of SOX4 in immune regulation and provide new insights involving the interplay of tolerogenic cues and autophagy in Treg.

Allergen alters IL-2/αIL-2-based Treg expansion but not tolerance induction in an allergen-specific mouse model.

BACKGROUND: Regulatory T lymphocytes (Treg) play an important role in preventing allergic diseases. We characterized Treg expansion kinetics, marker profiles, and recirculation behavior in allergen-challenged mice, which had been pretreated with IL-2/αIL-2 complexes in the presence or absence of allergen. Moreover, the ability of induced Treg to control airway hyperreactivity and effector functions of lung T cells was determined. METHODS: Humanized TCR/HLA-transgenic allergy mice were treated in vivo with recombinant IL-2 complexed to the anti-IL-2 mAb JES6-1 in the presence or absence of mugwort pollen extract (MPE) on days 0-2. Afterward, they were intranasally challenged with MPE (days 13-15) followed by determination of airway hyperreactivity and lung T cell effector functions. Multiparametric flow cytometry on peripheral blood T cells was performed on a daily basis. RESULTS: IL-2/αIL-2 complexes highly efficiently expanded peripheral Treg cells, while concomitant allergen exposure altered the phenotype of expanded Treg cells. Notably, application of allergen together with IL-2/αIL-2 complexes induced expression of Treg marker molecules CTLA4, NRP1, Helios, and GITR on conventional T cells. Apart from CD25, GARP was identified as the most reliable surface-expressed lineage discrimination marker of Treg expanded in the presence of IL-2/αIL-2 complexes and allergen. Finally, IL-2/αIL-2 complex-expanded Treg cells could be recalled upon allergen challenge, which was associated with suppression of lung-specific Th2 responses long after initial treatment. CONCLUSION: The characterization of reliable surface and transcription markers of IL-2/αIL-2 complex-expanded Treg along with their expansion kinetics and function will help to identify protocols for their long-term expansion in vivo.

An Organometallic Gold(I) Bis-N-Heterocyclic Carbene Complex with Multimodal Activity in Ovarian Cancer Cells.

The organometallic AuI bis-N-heterocyclic carbene complex [Au(9-methylcaffeine-8-ylidene)2 ]+ (AuTMX2 ) was previously shown to selectively and potently stabilise telomeric DNA G-quadruplex (G4) structures. This study sheds light on the molecular reactivity and mode of action of AuTMX2 in the cellular context using mass spectrometry-based methods, including shotgun proteomics in A2780 ovarian cancer cells. In contrast to other metal-based anticancer agents, this organogold compound is less prone to form coordinative bonds with biological nucleophiles and is expected to exert its drug effects mainly by non-covalent interactions. Global protein expression changes of treated cancer cells revealed a multimodal mode of action of AuTMX2 by alterations in the nucleolus, telomeres, actin stress-fibres and stress-responses, which were further supported by pharmacological assays, fluorescence microscopy and cellular accumulation experiments. Proteomic data are available via ProteomeXchange with identifier PXD020560.

Proteomics and metabolomics identify molecular mechanisms of aging potentially predisposing for chronic lymphocytic leukemia.

B cell chronic lymphocytic leukemia (B-CLL), the most common type of leukemia in adults, is still essentially incurable despite the development of novel therapeutic strategies. This reflects the incomplete understanding of the pathophysiology of this disease. A comprehensive proteome analysis of primary human B-CLL cells and B cells from younger as well as elderly healthy donors was performed. For comparison, the chronic B cell leukemia cell line JVM-13 was also included. A principal component analysis comprising 6,945 proteins separated these four groups, placing B cells of aged-matched controls between those of young donors and B-CLL patients, while identifying JVM-13 as poorly related cells. Mass spectrometric proteomics data have been made fully accessible via ProteomeXchange with identifier PXD006570-PXD006572, PXD006576, PXD006578, and PXD006589-PXD006591. Remarkably, B cells from aged controls displayed significant regulation of proteins related to stress management in mitochondria and ROS stress such as DLAT, FIS1, and NDUFAB1, and DNA repair, including RAD9A, MGMT, and XPA. ROS levels were indeed found significantly increased in B cells but not in T cells or monocytes from aged individuals. These alterations may be relevant for tumorigenesis and were observed similarly in B-CLL cells. In B-CLL cells, some remarkable unique features like the loss of tumor suppressor molecules PNN and JARID2, the stress-related serotonin transporter SLC6A4, and high expression of ZNF207, CCDC88A, PIGR and ID3, otherwise associated with stem cell phenotype, were determined. Alterations of metabolic enzymes were another outstanding feature in comparison to normal B cells, indicating increased beta-oxidation of fatty acids and increased consumption of glutamine. Targeted metabolomics assays corroborated these results. The present findings identify a potential proteome signature for immune senescence in addition to previously unrecognized features of B-CLL cells and suggest that aging may be accompanied by cellular reprogramming functionally relevant for predisposing B cells to transform to B-CLL cells.

The IL-10/STAT3 axis: Contributions to immune tolerance by thymus and peripherally derived regulatory T-cells.

The signal transducer and activator of transcription (STAT) proteins are important mediators for the integration of extrinsic signals provided by cytokines and hormones and thereby adapt cellular processes to their surroundings. In the past decade, the involvement of STAT3 in the regulation of T-cell responses has become a topic of increasing interest. STAT3 is activated in response to multiple cytokines, many of which have been shown to influence T-cell responses. Interestingly, many of these factors have been described with apparent opposing roles, such as the highly pro-inflammatory potency of IL-6 and the anti-inflammatory properties of IL-10, thus raising the possibility that STAT3 signaling may fulfill diverse roles in CD4+ T-cells. Here, we review the contribution of STAT3 to the induction and function of both peripherally induced as well as thymus-derived regulatory T-cells. Indeed, experimental approaches as well as studies of human patients suffering from e.g. Job's (hyper IgE) syndrome or inflammatory bowel disease (IBD) have now established a clear-cut role for the IL-10/STAT3 axis in immune tolerance; further understanding of these processes could lead to novel therapeutic approaches for autoimmune diseases.

STAT3 governs hyporesponsiveness and granzyme B-dependent suppressive capacity in human CD4+ T cells.

Signal transducer and activator of transcription 3 (STAT3) integrates key signals of cell surface immune receptors, yet its precise role in cluster of differentiation (CD)4(+) T cells is not well-established. Current research has indicated T-helper cell 17-inducing roles but also tolerogenic roles. To address this issue, human T cells were transduced with the constitutively active STAT3 mutant STAT3C. Following stimulation, STAT3C(+) T cells up-regulated IL-10 (4.1 ± 0.5-fold; P < 0.001) and granzyme B (2.5 ± 1.2, P < 0.05) secretion, combined with significantly reduced IFN-γ (35 ± 5%), IL-2 (57 ± 4%), TNF-α (64 ± 8%), and IL-13 (89 ± 3%) secretion (P < 0.001). CD3/CD2- or CD3/CD28-activated STAT3C(+) T cells revealed reduced proliferation (53.4 ± 23.5% and 70.5 ± 10.4%, respectively), which was independent of IL-10 production and significantly suppressed effector T cell proliferation by 68.7 ± 10.6% and 65.9 ± 2.6%, respectively (P < 0.001). Phenotypically, STAT3C-transgenic CD4(+) T cells resembled effector T cells regarding expression of T regulatory cell markers, but up-regulated granzyme B expression levels by 2.4-fold (P < 0.05). Suppression was cell contact dependent and mediated by granzyme B-induced cell death, but was independent of IL-10 and TGF-ß. Notably, peripheral blood CD4(+)CD45RA(-)lymphocyte activation gene-3(+)CD49(+) type 1 regulatory T cells revealed activation-induced hyperphosphorylation of STAT3. In agreement, pharmacological inhibition of STAT3 activation partially reverted hyporesponsiveness of peripheral type 1 regulatory T cells (increasing their division index from 0.46 ± 0.11 to 0.89 ± 0.04; P < 0.01). These observations indicate a clear-cut relation between activation of STAT3 and the acquisition of a tolerogenic program, which is also used by peripheral blood type 1 regulatory T cells.

Sepsis biomarkers in neutropaenic systemic inflammatory response syndrome patients on standard care wards.

BACKGROUND: Neutropaenic patients are at a high risk of contracting severe infections. In particular, in these patients, parameters with a high negative predictive value are desirable for excluding infection or bacteraemia. This study evaluated sepsis biomarkers in neutropaenic patients suffering from systemic inflammatory response syndrome (SIRS). Further, the predictive capacities of evaluated biomarkers in neutropaenic SIRS patients were compared to non-neutropaenic SIRS patients. MATERIAL AND METHODS: In this prospective observational cohort study, patients with clinically suspected sepsis were screened. The predictive capacities of procalcitonin (PCT), C-reactive protein and lipopolysaccharide-binding protein (LBP) in neutropaenic SIRS patients were evaluated in terms of their potential to identify infection or bacteraemia and were compared to results for non-neutropaenic SIRS patients. To select an appropriate control cohort, propensity score matching was applied, balancing confounding factors between neutropaenic and non-neutropaenic SIRS patients. RESULTS: Of 3370 prospectively screened patients with suspected infection, 51 patients suffered from neutropaenic SIRS. For the identification of infection, none of the assessed biomarkers presented a clinically relevant discriminatory potency. Lipopolysaccharide-binding protein and PCT demonstrated discriminatory capacity to discriminate between nonbacteraemic and bacteraemic SIRS in patients with neutropaenia [receiver-operating characteristics-area under the curves (ROC-AUCs): 0.860, 0.818]. In neutropaenic SIRS patients, LBP had a significantly better ROC-AUC than in a comparable non-neutropaenic patient cohort for identifying bacteraemia (P = 0.01). CONCLUSION: In neutropaenic SIRS patients, none of the evaluated biomarkers was able to adequately identify infection. LBP and PCT presented a good performance in identifying bacteraemia. Therefore, these markers could be used for screening purposes to increase the pretest probability of blood culture analysis.

Sepsis in standard care: patients' characteristics, effectiveness of antimicrobial therapy and patient outcome--a cohort study.

PURPOSE: Fast diagnosis and initiation of appropriate antibiotic therapy is pivotal for the survival of sepsis patients. However, most studies on suspected sepsis patients are conducted in the intensive care unit or in the emergency room setting, neglecting the standard care setting. This study evaluated sepsis risk factors, microbiological accurateness of the initial empiric antimicrobial therapy and its effect on hospital mortality in standard care patients. METHODS: In this prospective observational cohort study, patients with clinically suspected sepsis meeting two or more SIRS criteria were screened on standard care wards. After hospital discharge, occurrence of an infection was assessed according to standardized criteria, and empirical antibiotic therapy was evaluated using antibiograms of recognized pathogens by expert review. RESULTS: Of the 2384 screened patients, 298 fulfilled two or more SIRS criteria. Among these were 28.2 % SIRS patients without infection, 46.3 % non-bacteremic/fungemic sepsis patients and 25.5 % bacteremic/fungemic sepsis patients. Occurrence of a malignant disease and chills were associated with a higher risk of patients having bacteremic/fungemic sepsis, whereas other described risk factors remained insignificant. In total, 91.1 % of suspected sepsis patients received empirical antimicrobial therapy, but 41.1 % of bacteremic sepsis patients received inappropriate therapy. Non-surviving bacteremic sepsis patients received a higher proportion of inappropriate therapy than those who survived (p = 0.022). CONCLUSIONS: A significant proportion of bacteremic sepsis patients receive inappropriate empiric antimicrobial therapy. Our results indicate that rapid availability of microbiological results is vital, since inappropriate antimicrobial therapy tended to increase the hospital mortality of sepsis patients.

Lymphocyte-based model systems for allergy research: a historic overview.

During the last decades, a multitude of studies applying distinct in vitro and in vivo model systems have contributed greatly to our better understanding of the initiation and regulation of inflammatory processes leading to allergic diseases. Over the years, it has become evident that among lymphocytes, not only IgE-producing B cells and allergy-orchestrating CD4(+) helper cells but also cytotoxic CD8(+) T cells, γδ-T cells and innate lymphoid cells, as well as regulatory lymphocytes, might critically shape the immune response towards usually innocuous allergens. In this review, we provide a historic overview of pioneering work leading to the establishment of important lymphocyte-based model systems for allergy research. Moreover, we contrast the original findings with our currently more refined knowledge to appreciate the actual validity of the respective models and to reassess the conclusions obtained from them. Conflicting studies and interpretations are identified and discussed. The tables are intended to provide an easy overview of the field not only for scientists newly entering the field but also for the broader readership interested in updating their knowledge. Along those lines, herein we discuss in vitro and in vivo approaches to the investigation of lymphocyte effector cell activation, polarization and regulation, and describe depletion and adoptive transfer models along with gene knockout and transgenic (tg) methodologies. In addition, novel attempts to establish humanized T cell antigen receptor tg mouse models for allergy research are described and discussed.

Evaluation of the automated coagulation analyzer CS-5100 and its utility in high throughput laboratories.

BACKGROUND: Automated analyzers are an important component of modern laboratories. As a representative of the newest generation of coagulation analyzers, the CS-5100 features several technical refinements including a pre-analytical assessment unit as well as multi-wavelength optical detection units. Therefore, the CS-5100 is supposed to rapidly and accurately perform a broad panel of coagulation tests. In the current study, the CS-5100 was evaluated regarding its precision and practicability in a clinical laboratory setting. METHODS: The CS-5100 was evaluated regarding its intra- and inter-assay precision using commercially available control samples. RESULTS of patient samples, including hemolytic, icteric and lipemic specimens, measured on the CS-5100 were compared to reference analyzers, which are used in our accredited laboratory. RESULTS: The coefficients of variation, assessed in the intra- and inter-assay precision analyses were below 5% representatively for most parameters. RESULTS, obtained by the CS-5100 showed predominantly a high comparability to used reference analyzers, with correlation coefficients ranging from 0.857 to 0.990. Only minor ranged systemic or proportional differences were found in Passing-Bablok regression between the CS-5100 and reference analyzers regarding most of the tested parameters. Lipemic samples had a tendency to deteriorate correlation coefficients, but an overall effect of the sample's triglyceride level could be ruled out. In a routine setting, the analyzer reached a sample throughput rate of 160 tests per hour. CONCLUSIONS: The CS-5100 is able to rapidly and precisely measure patient samples. No considerable influence on test comparability was found for elevated levels of free hemoglobin, bilirubin or triglycerides.

Naturally occurring regulatory T cells: markers, mechanisms, and manipulation.

Naturally occurring CD4(+)CD25(high) forkhead box protein 3 (FOXP3)(+) regulatory T cells (nTregs) are key mediators of immunity, which orchestrate and maintain tolerance to self and foreign antigens. In the recent 1.5 decades, a multitude of studies have aimed to define the phenotype and function of nTregs and to assess their therapeutic potential for modulating immune mediated disorders such as autoimmunity, allergy, and episodes of transplant rejection. In this review, we summarize the current knowledge on the biology of nTregs. We address the exact definition of nTregs by specific markers and combinations thereof, which is a prerequisite for the state-of-the-art isolation of defined nTreg populations. Furthermore, we discuss the mechanism by which nTregs mediate immunosuppression and how this knowledge might translate into novel therapeutic modalities. With first clinical studies of nTreg-based therapies being finished, questions concerning the reliable sources of nTregs are becoming more and more eminent. Consequently, approaches allowing conversion of CD4(+) T cells into nTregs by coculture with antigen-presenting cells, cytokines, and/or pharmacological agents are discussed. In addition, genetic engineering approaches for the generation of antigen-specific nTregs are described.

Human TCR transgenic Bet v 1-specific Th1 cells suppress the effector function of Bet v 1-specific Th2 cells.

Pollinosis to birch pollen is a common type I allergy in the Northern Hemisphere. Moreover, birch pollen-allergic individuals sensitized to the major birch pollen allergen Bet v 1 frequently develop allergic reactions to stone fruits, hazelnuts, and certain vegetables due to immunological cross-reactivity. The major T cell epitope Bet v 1(142-153) plays an important role in cross-reactivity between the respiratory allergen Bet v 1 and its homologous food allergens. In this study, we cloned and functionally analyzed a human αß TCR specific for the immunodominant epitope Bet v 1(142-153). cDNAs encoding TCR α- and ß-chains were amplified from a Bet v 1(142-153)-specific T cell clone, introduced into Jurkat T cells and peripheral blood T lymphocytes of allergic and nonallergic individuals, and evaluated functionally. The resulting TCR transgenic (TCRtg) T cells responded in an allergen-specific and costimulation-dependent manner to APCs either pulsed with Bet v 1(142-153) peptide or coexpressing invariant chain::Bet v 1(142-153) fusion proteins. TCRtg T cells responded to Bet v 1-related food and tree pollen allergens that were processed and presented by monocyte-derived dendritic cells. Bet v 1(142-153)-presenting but not Bet v 1(4-15)-presenting artificial APCs coexpressing membrane-bound IL-12 polarized allergen-specific TCRtg T cells toward a Th1 phenotype, producing high levels of IFN-γ. Coculture of such Th1-polarized T cells with allergen-specific Th2-differentiated T cells significantly suppressed Th2 effector cytokine production. These data suggest that human allergen-specific TCR can transfer the fine specificity of the original T cell clone to heterologous T cells, which in turn can be instructed to modulate the effector function of the disease initiating/perpetuating allergen-specific Th2-differentiated T cells.

A multi-omics based anti-inflammatory immune signature characterizes long COVID-19 syndrome.

To investigate long COVID-19 syndrome (LCS) pathophysiology, we performed an exploratory study with blood plasma derived from three groups: 1) healthy vaccinated individuals without SARS-CoV-2 exposure; 2) asymptomatic recovered patients at least three months after SARS-CoV-2 infection and; 3) symptomatic patients at least 3 months after SARS-CoV-2 infection with chronic fatigue syndrome or similar symptoms, here designated as patients with long COVID-19 syndrome (LCS). Multiplex cytokine profiling indicated slightly elevated pro-inflammatory cytokine levels in recovered individuals in contrast to patients with LCS. Plasma proteomics demonstrated low levels of acute phase proteins and macrophage-derived secreted proteins in LCS. High levels of anti-inflammatory oxylipins including omega-3 fatty acids in LCS were detected by eicosadomics, whereas targeted metabolic profiling indicated high levels of anti-inflammatory osmolytes taurine and hypaphorine, but low amino acid and triglyceride levels and deregulated acylcarnitines. A model considering alternatively polarized macrophages as a major contributor to these molecular alterations is presented.

Sodium perturbs mitochondrial respiration and induces dysfunctional Tregs.

FOXP3+ regulatory T cells (Tregs) are central for peripheral tolerance, and their deregulation is associated with autoimmunity. Dysfunctional autoimmune Tregs display pro-inflammatory features and altered mitochondrial metabolism, but contributing factors remain elusive. High salt (HS) has been identified to alter immune function and to promote autoimmunity. By investigating longitudinal transcriptional changes of human Tregs, we identified that HS induces metabolic reprogramming, recapitulating features of autoimmune Tregs. Mechanistically, extracellular HS raises intracellular Na+, perturbing mitochondrial respiration by interfering with the electron transport chain (ETC). Metabolic disturbance by a temporary HS encounter or complex III blockade rapidly induces a pro-inflammatory signature and FOXP3 downregulation, leading to long-term dysfunction in vitro and in vivo. The HS-induced effect could be reversed by inhibition of mitochondrial Na+/Ca2+ exchanger (NCLX). Our results indicate that salt could contribute to metabolic reprogramming and that short-term HS encounter perturb metabolic fitness and long-term function of human Tregs with important implications for autoimmunity.

Bet v 1-specific T-cell receptor/forkhead box protein 3 transgenic T cells suppress Bet v 1-specific T-cell effector function in an activation-dependent manner.

BACKGROUND: Regulatory T (Treg) cells establish and maintain tolerance to self-antigens and many foreign antigens, such as allergens, by suppressing effector T-cell proliferation and function. We have previously shown that human T-cell receptor (TCR) αß-chains specific for allergen-derived epitopes confer allergen specificity on peripheral blood T cells of individuals with and without allergy. OBJECTIVE: To study the feasibility of generating allergen-specific human Treg cells by retroviral transduction of a transcription unit encoding forkhead box protein 3 (FOXP3) and allergen-specific TCR αß-chains. METHODS: cDNAs encoding the α and ß-chains of a Bet v 1(142-153)-specific TCR (TCR alpha variable region 6/TCR beta variable region 20) and human FOXP3 were linked via picornaviral 2A sequences and expressed as single translational unit from an internal ribosomal entry site-green fluorescence protein-containing retroviral vector. Retrovirally transduced peripheral blood T cells were tested for expression of transgenes, Treg phenotype, and regulatory capacity toward allergen-specific effector T cells. RESULTS: Transduced T cells displayed a Treg phenotype with clear-cut upregulation of CD25, CD39, and cytotoxic T-lymphocyte antigen 4. The transduced cells were hyporesponsive in cytokine production and secretion and, like naturally occurring Treg cells, did not proliferate after antigen-specific or antigen-mimetic stimulation. However, proliferation was inducible upon exposure to exogenous IL-2. In coculture experiments, TRAV6(+)TRBV20(+)FOXP3(+) transgenic T cells, unlike FOXP3(+) single transgenic T cells or naturally occurring Treg cells, highly significantly suppressed T cell cytokine production and proliferation of corresponding allergen-specific effector T cells in an allergen-specific, dose-dependent manner. CONCLUSION: We demonstrate a transgenic approach to engineer human allergen-specific Treg cells that exert their regulatory function in an activation-dependent manner. Customized Treg cells might become useful for tolerance induction therapies in individuals with allergic and other immune-mediated diseases.