Charakterisierung von TREM-B1

Charakterisierung von TREM-B1: ein hemmender Rezeptor auf Thrombozyten des Haushuhns TREM-B1 ist ein potentiell immunregulatorischer Oberflächenrezeptor des Haushuhns. Der Rezeptor besitzt zwei Ig-Domänen vom V-Typ, einen ungeladenen Transmembranbereich sowie zwei ITIM und ein ITSM in der zytoplasmatischen Region. Mit diesen strukturellen Merkmalen und den typisch inhibitorischen Signalmotiven besitzt er die charakteristischen Eigenschaften eines hemmenden Ig-ähnlichen Rezeptors. Die TREM Familie umfasst beim Huhn drei Mitglieder und ist auf Chromosom 26 lokalisiert. Neben TREM-B1 konnte der potentiell aktivierende Rezeptor TREM-A1 und der potentiell hemmende Rezeptor TREM-B2 identifiziert werden. Sequenz- und Strukturmerkmale weisen darauf hin, dass TREM B1 ein Homolog des humanen TLT-1 darstellt. Im Rahmen dieser Arbeit wurden das Expressionsmuster, die biochemischen und funktionellen Eigenschaften sowie die intrazelluläre Signaltransduktion von TREM-B1 eingehend untersucht. Zu diesem Zweck wurden TREM-B1-spezifische monoklonale Antikörper hergestellt und anschließend dazu verwendet, die Proteinexpression auf Leukozyten verschiedener Gewebe zu analysieren. Durch die Kombination mit spezifischen Markern für unterschiedliche Zellpopulationen konnte gezeigt werden, dass TREM-B1 ausschließlich auf Thrombozyten exprimiert wird. Darüber hinaus ergab die Untersuchung verschiedener Aktivierungsstadien von Thrombozyten, dass der Rezeptor, im Gegensatz zum TLT-1, konstitutiv auf der Zelloberfläche exprimiert wird. Biochemische Untersuchungen mittels Western Blot ergaben, dass TREM B1 mit einem Molekulargewicht von 50 kDa als glykosiliertes Monomer auf der Thrombozytenoberfläche exprimiert wird. Die Pervanadat-induzierte Phosphorylierung des Rezeptors führte zur Rekrutierung der Protein-Tyrosin-Phosphatase SHP-2. Darüber hinaus konnte gezeigt werden, dass die Kreuzvernetzung von TREM-B1 auf aktivierten Thrombozyten zu einer Abnahme der Oberflächenexpression des Degranulationsmarkers CD107 führt. Ein Ligand für TREM-B1 konnte bisher noch nicht identifiziert werden und auch die Funktion des Rezeptors ist derzeit noch gänzlich ungeklärt. Die drei typisch inhibitorischen Signalmotive sowie die Assoziation des Rezeptors mit SHP-2 und die TREM-B1-induzierte Hemmung der Degranulation von Thrombozyten lassen auf eine inhibitorische Funktion schließen. Eine zu TLT-1 analoge Aufgabe in der Blutgerinnung durch Interaktion mit Fibrinogen ist aufgrund der konstitutiven Oberflächenexpression von TREM-B1 unwahrscheinlich. Zusammengenommen handelt es sich bei TREM-B1 um einen Rezeptor, der konstitutiv auf Thrombozyten des Huhns exprimiert ist und einen hemmenden Effekt auf die Degranulation aktivierter Thrombozyten ausübt.Characterization of TREM-B1: an inhibitory receptor on chicken thrombocytes TREM-B1 in chickens is a potential immunoregulatory cell surface receptor. The receptor possesses two V-set Ig-domains, an uncharged transmembrane region and a cytoplasmic tail bearing two ITIM and one ITSM. These structural features and the typically inhibitory motifs define TREM-B1 as a representative inhibitory Ig-like receptor. The chicken TREM family, located on chromosome 26, comprises three members. In addition to TREM-B1, the potential activating receptor TREM-A1 and the potential inhibitory receptor TREM-B2 were identified. Sequence and structural poperties indicate TREM-B1 might be a homologue to human TLT 1. In this study, TREM-B1 was characterized by detailed analyses of expression pattern, biochemical and functional features and intracellular signaling capabilities. For this purpose, TREM-B1 specific antibodies were generated and used to analyze protein expression on leukocytes obtained from different tissues. Combination with markers specific for different cell populations showed a TREM-B1 cell surface expression restricted to thrombocytes. In contrast to TLT-1, analysis of different activation stages of thrombocytes revealed a constitutive expression of TREM-B1 on the cell surface. Biochemical investigations by western blot demonstrated that TREM-B1 is expressed as a glycosylated monomeric protein of 50 kDa. Pervanadate treatment resulted in phosphorylation of the receptor and subsequent recruitment of the protein tyrosine phosphatase SHP-2. Moreover, TREM B1 crosslinking has been shown to decrease cell surface expression of the degranulation marker CD107 on activated thrombocytes. A ligand of TREM-B1 has not been identified yet and functions of the receptor are still unresolved. Three typically inhibitory motifs as well as association with SHP-2 and TREM-B1 induced reduction of thrombocyte degranulation suggest an inhibitory function of the receptor. Unlike TLT-1, TREM-B1 is constitutively expressed. Therefore, the receptor seems to have a function different from TLT-1, which facilitates hemostasis through interaction with fibrinogen. In summary, these findings show that TREM-B1 is a constitutively expressed receptor on chicken thrombocytes with an inhibitory effect on degranulation of activated thrombocytes.Englische Übersetzung des Titels: Characterization of TREM-B1: an inhibitory receptor on chicken thrombocyte

Charakterisierung von TREM-B1

Charakterisierung von TREM-B1: ein hemmender Rezeptor auf Thrombozyten des Haushuhns TREM-B1 ist ein potentiell immunregulatorischer Oberflächenrezeptor des Haushuhns. Der Rezeptor besitzt zwei Ig-Domänen vom V-Typ, einen ungeladenen Transmembranbereich sowie zwei ITIM und ein ITSM in der zytoplasmatischen Region. Mit diesen strukturellen Merkmalen und den typisch inhibitorischen Signalmotiven besitzt er die charakteristischen Eigenschaften eines hemmenden Ig-ähnlichen Rezeptors. Die TREM Familie umfasst beim Huhn drei Mitglieder und ist auf Chromosom 26 lokalisiert. Neben TREM-B1 konnte der potentiell aktivierende Rezeptor TREM-A1 und der potentiell hemmende Rezeptor TREM-B2 identifiziert werden. Sequenz- und Strukturmerkmale weisen darauf hin, dass TREM B1 ein Homolog des humanen TLT-1 darstellt. Im Rahmen dieser Arbeit wurden das Expressionsmuster, die biochemischen und funktionellen Eigenschaften sowie die intrazelluläre Signaltransduktion von TREM-B1 eingehend untersucht. Zu diesem Zweck wurden TREM-B1-spezifische monoklonale Antikörper hergestellt und anschließend dazu verwendet, die Proteinexpression auf Leukozyten verschiedener Gewebe zu analysieren. Durch die Kombination mit spezifischen Markern für unterschiedliche Zellpopulationen konnte gezeigt werden, dass TREM-B1 ausschließlich auf Thrombozyten exprimiert wird. Darüber hinaus ergab die Untersuchung verschiedener Aktivierungsstadien von Thrombozyten, dass der Rezeptor, im Gegensatz zum TLT-1, konstitutiv auf der Zelloberfläche exprimiert wird. Biochemische Untersuchungen mittels Western Blot ergaben, dass TREM B1 mit einem Molekulargewicht von 50 kDa als glykosiliertes Monomer auf der Thrombozytenoberfläche exprimiert wird. Die Pervanadat-induzierte Phosphorylierung des Rezeptors führte zur Rekrutierung der Protein-Tyrosin-Phosphatase SHP-2. Darüber hinaus konnte gezeigt werden, dass die Kreuzvernetzung von TREM-B1 auf aktivierten Thrombozyten zu einer Abnahme der Oberflächenexpression des Degranulationsmarkers CD107 führt. Ein Ligand für TREM-B1 konnte bisher noch nicht identifiziert werden und auch die Funktion des Rezeptors ist derzeit noch gänzlich ungeklärt. Die drei typisch inhibitorischen Signalmotive sowie die Assoziation des Rezeptors mit SHP-2 und die TREM-B1-induzierte Hemmung der Degranulation von Thrombozyten lassen auf eine inhibitorische Funktion schließen. Eine zu TLT-1 analoge Aufgabe in der Blutgerinnung durch Interaktion mit Fibrinogen ist aufgrund der konstitutiven Oberflächenexpression von TREM-B1 unwahrscheinlich. Zusammengenommen handelt es sich bei TREM-B1 um einen Rezeptor, der konstitutiv auf Thrombozyten des Huhns exprimiert ist und einen hemmenden Effekt auf die Degranulation aktivierter Thrombozyten ausübt.Characterization of TREM-B1: an inhibitory receptor on chicken thrombocytes TREM-B1 in chickens is a potential immunoregulatory cell surface receptor. The receptor possesses two V-set Ig-domains, an uncharged transmembrane region and a cytoplasmic tail bearing two ITIM and one ITSM. These structural features and the typically inhibitory motifs define TREM-B1 as a representative inhibitory Ig-like receptor. The chicken TREM family, located on chromosome 26, comprises three members. In addition to TREM-B1, the potential activating receptor TREM-A1 and the potential inhibitory receptor TREM-B2 were identified. Sequence and structural poperties indicate TREM-B1 might be a homologue to human TLT 1. In this study, TREM-B1 was characterized by detailed analyses of expression pattern, biochemical and functional features and intracellular signaling capabilities. For this purpose, TREM-B1 specific antibodies were generated and used to analyze protein expression on leukocytes obtained from different tissues. Combination with markers specific for different cell populations showed a TREM-B1 cell surface expression restricted to thrombocytes. In contrast to TLT-1, analysis of different activation stages of thrombocytes revealed a constitutive expression of TREM-B1 on the cell surface. Biochemical investigations by western blot demonstrated that TREM-B1 is expressed as a glycosylated monomeric protein of 50 kDa. Pervanadate treatment resulted in phosphorylation of the receptor and subsequent recruitment of the protein tyrosine phosphatase SHP-2. Moreover, TREM B1 crosslinking has been shown to decrease cell surface expression of the degranulation marker CD107 on activated thrombocytes. A ligand of TREM-B1 has not been identified yet and functions of the receptor are still unresolved. Three typically inhibitory motifs as well as association with SHP-2 and TREM-B1 induced reduction of thrombocyte degranulation suggest an inhibitory function of the receptor. Unlike TLT-1, TREM-B1 is constitutively expressed. Therefore, the receptor seems to have a function different from TLT-1, which facilitates hemostasis through interaction with fibrinogen. In summary, these findings show that TREM-B1 is a constitutively expressed receptor on chicken thrombocytes with an inhibitory effect on degranulation of activated thrombocytes.Englische Übersetzung des Titels: Characterization of TREM-B1: an inhibitory receptor on chicken thrombocyte

Chicken TREM-B1, an Inhibitory Ig-Like Receptor Expressed on Chicken Thrombocytes

Triggering receptors expressed on myeloid cells (TREM) form a multigene family of immunoregulatory Ig-like receptors and play important roles in the regulation of innate and adaptive immunity. In chickens, three members of the TREM family have been identified on chromosome 26. One of them is TREM-B1 which possesses two V-set Ig-domains, an uncharged transmembrane region and a long cytoplasmic tail with one ITSM and two ITIMs indicating an inhibitory function. We generated specific monoclonal antibodies by immunizing a Balb/c mouse with a TREM-B1-FLAG transfected BWZ.36 cell line and tested the hybridoma supernatants on TREM-B1-FLAG transfected 2D8 cells. We obtained two different antibodies specific for TREM-B1, mab 7E8 (mouse IgG1) and mab 1E9 (mouse IgG2a) which were used for cell surface staining. Single and double staining of different tissues, including whole blood preparations, revealed expression on thrombocytes. Next we investigated the biochemical properties of TREM-B1 by using the specific mab 1E9 for immunoprecipitation of either lysates of surface biotinylated peripheral blood cells or stably transfected 2D8 cells. Staining with streptavidin coupled horse radish peroxidase revealed a glycosylated monomeric protein of about 50 kDa. Furthermore we used the stably transfected 2D8 cell line for analyzing the cytoplasmic tyrosine based signaling motifs. After pervanadate treatment, we detected phosphorylation of the tyrosine residues and subsequent recruitment of the tyrosine specific protein phosphatase SHP-2, indicating an inhibitory potential for TREM-B1. We also showed the inhibitory effect of TREM-B1 in chicken thrombocytes using a CD107 degranulation assay. Crosslinking of TREM-B1 on activated primary thrombocytes resulted in decreased CD107 surface expression of about 50-70%

The mab 1E9 and 7E8 do not cross-react with other TREM family members.

BWZ.36 cells stably expressing TREM-B1-FLAG-muCD3ζ (A) or TREM-A1-FLAG-muCD3ζ (B) and transiently transfected HEK-293T cells expressing TREM-B2-FLAG-muCD3ζ (C) were stained with an isotype-matched negative control (left panels), an anti-FLAG mab as expression control (middle panels, left) and the mab 1E9 (middle panels, right) and 7E8 (right panels). Numbers indicate the percentage of positive cells.</p

TREM-B1 crosslinking reduces CLEC-2 induced thrombocyte degranulation.

<p>(A) Co-crosslinking of CLEC-2 and TREM-B1 with the thrombocyte specific mab 8G8 and 1E9 decreased degranulation (lower panels) compared to co-crosslinking an irrelevant antibody and 8G8 (upper panels). Controls were incubated without crosslinking and subsequent stained with the respective antibodies. Numbers indicate the percentage of cells in the respective quadrants. One of five representative experiments is shown. (B) Percentage of TREM-B1 induced inhibition of CLEC-2 mediated thrombocyte degranulation. Markers represent single values of five different animals. Long bars represent the mean. Short bars represent the standard error of the mean (SEM).</p

Cell preparation has no impact on the expression level of TREM-B1.

<p>PBMC obtained by density centrifugation and whole blood were double stained with the mab 1E9 and K1 (specific for monocytes and thrombocytes), gated on the entire leukocyte population by FSC/SSC and analyzed by flow cytometry. Numbers indicate the percentage of cells in the respective quadrants. The marker quadrants were set according to the unstained cells.</p

TREM-B1 is expressed on thrombocytes.

<p>(A) Viable PBMC were discriminated by 7-AAD staining and gated on lymphocytes/thrombocytes (R1), monocytes/NK cells (R2) or heterophils (R3) in FSC/SSC. Cells were double immunofluorescence stained with either the 1E9 (upper panel) or the 7E8 (lower panel) mab in combination with several cell surface markers as indicated. Numbers indicate the percentage of cells in the respective quadrants. Isotype-matched controls were included in each experiment. One of ten representative experiments is shown. (B) Macrophages were single stained with the TREM-B1 specific mab 7E9 and the macrophage/monocyte specific marker KUL01 (filled histograms). Isotype-matched controls are shown as open histograms and percentages of positive cells are indicated as numbers.</p

Oligonucleotides used for real-time RT-PCR.

<p>Oligonucleotides used for real-time RT-PCR.</p

Real-world treatment utilization and economic implications of lupus nephritis disease activity in the United States.

BACKGROUND: Lupus nephritis (LN) is a common and severe complication of systemic lupus erythematosus (SLE), with approximately 40% of patients with SLE developing LN. Even with treatment, 10%-30% of patients will progress to end-stage renal disease (ESRD). Although many studies have assessed the clinical value of low disease activity in LN, the economic implications are less defined. OBJECTIVE: To evaluate treatment utilization and health care costs associated with active disease, low disease activity, and ESRD in patients with LN. METHODS: A retrospective analysis of Optum pharmacy and medical claims data from 2015 to 2019 was performed and included patients with a diagnosis of SLE (International Classification of Diseases, Ninth Revision or Tenth Revision codes 710.0 or M32, respectively) and additional prespecified criteria for LN. Total health care payer costs for medical and pharmacy services and treatment utilization for commonly prescribed medications were determined for periods of low disease activity, active disease, or ESRD. RESULTS: A total of 21,251 patients (mean age 60.3 years; 87% female; 55% White patients and 18% Black patients) with a mean follow-up period of 30.6 months were included; the majority of patients had active disease (67.3%), followed by low disease activity (51.3%), and ESRD (10.5%). Glucocorticoids were used 2 times more often and mycophenolate mofetil was used 4 times more often in patients with active disease vs low disease activity. Glucocorticoids, mycophenolate mofetil, and tacrolimus were more commonly used in patients with ESRD vs those with low disease activity. Mean medical costs were $4,777 per month in active disease and$18,084 per month in ESRD vs $2,523 per month in low disease activity. CONCLUSIONS: Treatment burden and costs are high for patients with active disease and ESRD in LN. Treatments that allow patients to achieve and maintain low disease activity may help improve patient outcomes and reduce medication use and overall health care costs. DISCLOSURES: Maria DallEra and Kenneth Kalunian are consultants of Aurinia Pharmaceuticals. Eric Turowski, Vanessa Birardi, Neil Solomons, Simrat Randhawa, and Paola Mina-Osorio are employees and stockholders of Aurinia Pharmaceuticals. Michael Eaddy is a former employee of Xcenda, LLC. Augustina Ogbonnaya and Eileen Farrelly are employees of Xcenda, LLC, which was contracted by Aurinia Pharmaceuticals to assist in the conduct of this study and the writing of this manuscript. Aurinia Pharmaceuticals provided funding for this study and the preparation of the manuscript. Aurinia Pharmaceuticals had a role in writing the report and decision to submit for publication

1E9 and 7E8 are specifically recognizing TREM-B1 protein.

<p>TREM-B1-FLAG transfected 2D8 and untransfected 2D8 cells were incubated with either the 1E9 or the 7E8 mab (filled histograms) and analyzed by flow cytometry (upper panels). TREM-B1-FLAG-muCD3ζ transfected BWZ.36 and untransfected BWZ.36 cells were tested in the same way (lower panels). Isotype-matched controls are shown as open histograms and percentages of positive cells are indicated as numbers.</p