NOVEL CONSTITUTIVELY ACTIVE POINT MUTATIONS IN THE NH2 DOMAIN OF CXCR2 CAPTURE THE RECEPTOR IN DIFFERENT ACTIVATION STATES

Chemokines are structurally and functionally related 8-10 kDa proteins defined by four conserved cysteine residues. They consist of a superfamily of proinflammatory mediators that promote the recruitment of various kinds of leukocytes and other cell types through binding to their respective chemokine receptor, a member of the GPCR family. Abnormal control of this system results in various diseases including tumorigenesis and cancer metastasis. Deregulation can occur when constitutively active mutant (CAM) chemokine receptors are locked in the “on” position. This can lead to cellular transformation/tumorigenesis. A viral CAM receptor, ORF74, that can cause tumors in humans, also has homology to human CXC chemokine receptor 2 (CXCR2), which is a G-protein-coupled receptor (GPCR) expressed on neutrophils, some monocytes, endothelial cells, and some epithelial cells. CXCR2 activation with ELR+ CXC chemokines induces leukocyte migration, trafficking, cellular differentiation, angiogenesis and cellular transformation. Using a high throughput yeast screen we identified a novel point mutation, D9H, in CXCR2, which leads to constitutive activation (CA). Generation of positively charged substitutions, D9K and D9R, and D143V as a positive control resulted in CA CXCR2 with differential levels of cellular transformation. To further investigate how D9 mutations lead to differential CA, we used inhibitors of known signal transduction pathways. Pertusiss toxin (PTX) sensitivity in foci formation assays demonstrated that D9R uses the Gi subunit like WTCXCR2 and D143V, while D9H and D9K do not. All CA receptors use the JAK pathway based on sensitivity to the inhibitor, AG490. Phosphorylation of PLC-beta 3 and sensitivity to the PLC-beta 3 inhibitor, U73122, implicates that mutant receptors such as D143V, D9H, D9K, and D9R utilize the Gq/11 subunit. Interestingly, D9R use both Gi and Gq/11 subunits. All of the CA receptors induced phosphorylation of the epidermal growth factor receptor (EGFR) indicating a transactivation between CXCR2 and EGFR. These data describe two novel and important findings. First, N-terminal CXCR2 controls activation and signaling using multiple G protein subunits to elicit downstream signaling. Second, our work supports the “functional selectivity” model for GPCR activation. That is, mimicking agonist activation, CA CXCR2 receptors have multiple conformational states that lead to differential activation

STAT3 activation in large granular lymphocyte leukemia is associated with cytokine signaling and DNA hypermethylation

Large granular lymphocyte leukemia (LGLL) is characterized by somatic gain-of-function STAT3 mutations. However, the functional effects of STAT3 mutations on primary LGLL cells have not been studied in detail. In this study, we show that CD8+ T cells isolated from STAT3 mutated LGLL patients have high protein levels of epigenetic regulators, such as DNMT1, and are characterized by global hypermethylation. Correspondingly, treatment of healthy CD8+ T cells with IL-6, IL-15, and/or MCP-1 cytokines resulted in STAT3 activation, increased DNMT1, EZH2, c-MYC, l-MYC, MAX, and NF kappa B levels, increased DNA methylation, and increased oxidative stress. Similar results were discovered in KAI3 NK cells overexpressing gain-of-function STAT3(Y640F) and STAT3(G618R) mutants compared to KAI3 NK cells overexpressing STAT3(WT). Our results also confirm that STAT3 forms a direct complex with DNMT1, EZH2, and HDAC1. In STAT3 mutated LGLL cells, DNA methyltransferase (DNMT) inhibitor azacitidine abrogated the activation of STAT3 via restored SHP1 expression. In conclusion, STAT3 mutations cause DNA hypermethylation resulting in sensitivity to DNMT inhibitors, which could be considered as a novel treatment option for LGLL patients with resistance to standard treatments.Peer reviewe

Absence of surrogate light chain results in spontaneous autoreactive germinal centres expanding VH81X-expressing B cells

Random recombination of antibody heavy- and light-chain genes results in a diverse B-cell receptor (BCR) repertoire including self-reactive BCRs. However, tolerance mechanisms that prevent the development of self-reactive B cells remain incompletely understood. The absence of the surrogate light chain, which assembles with antibody heavy chain forming a pre-BCR, leads to production of antinuclear antibodies (ANAs). Here we show that the naive follicular B-cell pool is enriched for cells expressing prototypic ANA heavy chains in these mice in a non-autoimmune background with a broad antibody repertoire. This results in the spontaneous formation of T-cell-dependent germinal centres that are enriched with B cells expressing prototypic ANA heavy chains. However, peripheral tolerance appears maintained by selection thresholds on cells entering the memory B-cell and plasma cell pools, as exemplified by the exclusion of cells expressing the intrinsically self-reactive VH81X from both pool

Somatic mTOR mutation in clonally expanded T lymphocytes associated with chronic graft versus host disease

Graft versus host disease (GvHD) is the main complication of allogeneic hematopoietic stem cell transplantation (HSCT). Here we report studies of a patient with chronic GvHD (cGvHD) carrying persistent CD4(+) T cell clonal expansion harboring somatic mTOR, NFKB2, and TLR2 mutations. In the screening cohort (n=134), we detect the mTOR P2229R kinase domain mutation in two additional cGvHD patients, but not in healthy or HSCT patients without cGvHD. Functional analyses of the mTOR mutation indicate a gain-of-function alteration and activation of both mTORC1 and mTORC2 signaling pathways, leading to increased cell proliferation and decreased apoptosis. Single-cell RNA sequencing and real-time impedance measurements support increased cytotoxicity of mutated CD4(+) T cells. High throughput drug-sensitivity testing suggests that mutations induce resistance to mTOR inhibitors, but increase sensitivity for HSP90 inhibitors. Our findings imply that somatic mutations may contribute to aberrant T cell proliferations and persistent immune activation in cGvHD, thereby paving the way for targeted therapies. Chronic graft versus host disease (cGvHD) is a major cause of morbidity and mortality in allogeneic bone marrow transplantation. Here the authors identify a recurrent activating mTOR mutation in expanded donor T-cell clones of 3 cGvHD patients, which suggests somatic mutations may contribute to GvHD pathogenesis and opens avenues to targeted therapies.Peer reviewe

Somatic mTOR mutation in clonally expanded T lymphocytes associated with chronic graft versus host disease

Graft versus host disease (GvHD) is the main complication of allogeneic hematopoietic stem cell transplantation (HSCT). Here we report studies of a patient with chronic GvHD (cGvHD) carrying persistent CD4+ T cell clonal expansion harboring somatic mTOR, NFKB2, and TLR2 mutations. In the screening cohort (n = 134), we detect the mTOR P2229R kinase domain mutation in two additional cGvHD patients, but not in healthy or HSCT patients without cGvHD. Functional analyses of the mTOR mutation indicate a gain-of-function alteration and activation of both mTORC1 and mTORC2 signaling pathways, leading to increased cell proliferation and decreased apoptosis. Single-cell RNA sequencing and real-time impedance measurements support increased cytotoxicity of mutated CD4+ T cells. High throughput drug-sensitivity testing suggests that mutations induce resistance to mTOR inhibitors, but increase sensitivity for HSP90 inhibitors. Our findings imply that somatic mutations may contribute to aberrant T cell proliferations and persistent immune activation in cGvHD, thereby paving the way for targeted therapies.</p

Interleukin 6 Accelerates Mortality by Promoting the Progression of the Systemic Lupus Erythematosus-Like Disease of BXSB. Yaa Mice

IL6 is a multifunctional cytokine that drives terminal B cell differentiation and secretion of immunoglobulins. IL6 also cooperates with IL21 to promote differentiation of CD4(+) T follicular helper cells (TFH). Elevated serum levels of IL6 correlate with disease flares in patients with systemic lupus erythematosus (SLE). We previously reported that IL21 produced by T-FH plays a critical role in the development of the SLE-like disease of BXSB. Yaa mice. To examine the possible contributions of IL6 to disease, we compared disease parameters in IL6-deficient and IL6-competent BXSB. Yaa mice. We report that survival of IL6-deficient BXSB. Yaa mice was significantly prolonged in association with significant reductions in a variety of autoimmune manifestations. Moreover, B cells stimulated by co-engagement of TLR7 and B cell receptor (BCR) produced high levels of IL6 that was further augmented by stimulation with Type I interferon (IFN1). Importantly, the frequencies of T-FH and serum levels of IL21 were significantly reduced in IL6-deficient mice. These findings suggest that high-level production of IL6 by B cells induced by integrated signaling from the IFN1 receptor, TLR7 and BCR promotes the differentiation of IL21-secreting T-FH in a signaling sequence that drives the lethal autoimmune disease of BXSB. Yaa mice.Peer reviewe

Electrical impedance measurements predict cellular transformation

Cellular transformation is the first step in cancer development. Two features of cellular transformation are proliferation in reduced serum and loss of contact inhibition. Electronic Cell-Substrate Impedance Sensing (ECIS) measurements have been used to measure cellular proliferation, cytotoxicity, apoptosis, and attachment. We have used impedance measurements to distinguish normal cells from cells transformed with a constitutively active chemokine receptor, CXCR2. CXCR2, a member of the G-protein coupled receptor (GPCR) family, is normally involved in cellular activation and migration, but a single amino acid substitution leads to constitutive activity. NIH3T3 cells were transformed with a constitutively active CXCR2 (D143V_CXCR2) and growth in reduced serum and foci formation were measured using established biological assays and compared to data from ECIS. The results of this study show that impedance measurements provide a quick and reliable way of measuring cellular transformation and provide real time assessment of transformed cellular parameters. Use of the ECIS system could allow a rapid screening of anti-cancer drugs that alter cellular transformation

Fault Detection of PMSM under Non-Stationary Conditions Based on Wavelet Transformation Combined with Distance Approach

This paper proposes a new method to detect mechanical faults of permanent magnet synchronous motors (PMSMs) under variable speed conditions. Several prior studies have proposed motor current signature analysis (MCSA) based methods for transient conditions; however, these methods have limitations because they require the characteristic frequency of the motor or they only verify the performance of the methods for a restricted time-varying region. Thus, the research outlined in this paper suggests a method for detecting motor faults using stator currents. The proposed method uses two techniques, continuous wavelet transform (CWT) and distance approach. In this method, after the influence of the non-stationary condition is reduced in the wavelet coefficients, the distance of the residual signal from the distribution of normal state is calculated. The performance of the proposed method is confirmed with the simulation result examining unbalance. From the results, the proposed method demonstrates better performance in small-load under non-stationary conditions.N

Inter-turn Short Circuit Fault Detection in Permanent Magnet Synchronous Motors Based on Reference Voltage

This paper proposes a method for detecting inter-turn short circuit faults of surface-mounted permanent magnet synchronous motors (SPMSMs) based on reference voltage. The proposed method consists of three steps. First, from the mathematical model of faulty PMSM, a reference voltage vector in the dq-frame is separated into a healthy voltage and a faulty voltage. Second, the faulty voltage is further decomposed into a DC component and a 2nd harmonic component. Third, the relationship between these components and the inter-turn fault severity is identified. Based on the relationship, this paper proposes two fault indicators; DC bias and 2nd harmonic amplitude. Simulation results verify the effectiveness of the proposed method. The proposed method can detect inter-turn short circuit faults under low sampling rate and without the need for additional sensors. This strength can facilitate on-line fault detection.N