Engagement of the T cell receptor complex reprograms T cells for proliferation, cytokine production and differentiation towards effector cells. This process depends on activating costimulatory signals and is counteracted by coinhibitory molecules. Three transcription factors, namely NF-κB, NFAT and AP-1, have a major role in inducing the transcriptional program that is required for T cell activation and differentiation. Here we describe the generation of a triple parameter reporter based on the human Jurkat T cell line, where response elements for NF-κB, NFAT and AP-1 drive the expression of the fluorescent proteins CFP, eGFP and mCherry, respectively. The emission spectra of these proteins allow simultaneous assessment of NF-κB, NFAT and AP-1 activity in response to stimulation. Ligation of the TCR complex induced moderate reporter activity, which was strongly enhanced upon coengagement of the costimulatory receptors CD2 or CD28. Moreover, we have generated and tested triple parameter reporter cells that harbor costimulatory and inhibitory receptors not endogenously expressed in the Jurkat cells. In these experiments we could show that engagement of the costimulatory molecule 4-1BB enhances NF-κB and AP-1 activity, whereas coinhibition via PD-1 or BTLA strongly reduced the activation of NF-κB and NFAT. Engagement of BTLA significantly inhibited AP-1, whereas PD-1 had little effect on the activation of this transcription factor. Our triple parameter reporter T cell line is an excellent tool to assess the effect of costimulatory and coinhibitory receptors on NF-κB, NFAT and AP-1 activity and has a wide range of applications beyond the evaluation of costimulatory pathways.

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 PCR 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<0.001). 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=0.004) 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.

Inhibition of Janus-kinase 1/2 (JAK1/2) is a mainstay to treat myeloproliferative neoplasms (MPN). Sporadic observations reported the co-incidence of B-cell non-Hodgkin lymphomas during treatment of MPN with JAK1/2 inhibitors. We assessed 626 patients with MPN, including 69 with myelofibrosis receiving JAK1/2 inhibitors for lymphoma development. B-cell lymphomas evolved in 4 (5.8%) of 69 patients receiving JAK1/2 inhibition compared with 2 (0.36%) of 557 with conventional treatment (16-fold increased risk). A similar 15-fold increase was observed in an independent cohort of 929 patients with MPN. Considering primary myelofibrosis only (N = 216), 3 lymphomas were observed in 31 inhibitor-treated patients (9.7%) vs 1 (0.54%) of 185 control patients. Lymphomas were of aggressive B-cell type, extranodal, or leukemic with high MYC expression in the absence of V617F or other MPN-associated mutations. Median time from initiation of inhibitor therapy to lymphoma diagnosis was 25 months. Clonal immunoglobulin gene rearrangements were already detected in the bone marrow during myelofibrosis in 16.3% of patients. Lymphomas occurring during JAK1/2 inhibitor treatment were preceded by a preexisting B-cell clone in all 3 patients tested. Sequencing verified clonal identity in 2 patients. The effects of JAK1/2 inhibition were mirrored in mice: 16 of 24 mice developed a spontaneous myeloid hyperplasia with the concomitant presence of aberrant B cells. Transplantations of bone marrow from diseased mice unmasked the outgrowth of a malignant B-cell clone evolving into aggressive B-cell leukemia-lymphoma. We conclude that JAK/STAT1 pathway inhibition in myelofibrosis is associated with an elevated frequency of aggressive B-cell lymphomas. Detection of a preexisting B-cell clone may identify individuals at risk.

No abstract

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.

Advanced macrolides, such as azithromycin (AZM) or clarithromycin (CLM), are antibiotics with immunomodulatory properties. Here we have sought to evaluate their in vitro influence on the activation of CD4+ T-cells. Isolated CD4+ T-cells were stimulated with agonistic anti-CD3/anti-CD28 monoclonal antibodies in the presence of 0.6 mg/L, 2.5 mg/L, 10 mg/L or 40 mg/L AZM or CLM. Cell proliferation, cytokine level in supernatants and cell viability was assessed. Intracellular signaling pathways were evaluated using reporter cell lines, FACS analysis, immunoblotting and in vitro kinase assays. AZM inhibited cell proliferation rate and cytokine secretion of CD4+ T-cells in a dose-dependent manner. Similarly, high concentrations of CLM (40 mg/L) also suppressed these T-cell functions. Analysis of molecular signaling pathways revealed that exposure to AZM reduced the phosphorylation of the S6 ribosomal protein, a downstream target of mTOR. This effect was also observed at 40 mg/L CLM. In vitro kinase studies using recombinant mTOR showed that AZM inhibited mTOR activity. In contrast to rapamycin, this inhibition was independent of FKBP12. We show for the first time that AZM and to a lesser extent CLM act as immunosuppressive agents on CD4+ T-cells by inhibiting mTOR activity. Our results might have implications for the clinical use of macrolides.

Background-Increasing evidence suggests that the low-affinity receptor for IgE, CD23, plays an important role in controlling the activity of allergen-specific T cells through IgE-facilitated allergen presentation.

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.