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%

TREM-B1 is predominantly expressed on PBMC.

<p>Leukocytes obtained from bursa, thymus, caecal tonsils, spleen, bone marrow and blood (PBMC) were incubated with the 7E8 mab and analyzed by flow cytometry. The markers were set according to an isotype-matched negative control and the percentage of positive cells is indicated as a number. One of three representative experiments is shown.</p

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

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

TREM-B1 recruits SHP-2 upon phosphorylation.

<p>TREM-B1-FLAG transfected 2D8 cells were treated with pervanadate for the times indicated, lysed and immunoprecipitated using an anti-FLAG mab. Indicated antibodies were used for detection of the proteins.</p

Oligonucleotides used for cloning.

<p>Oligonucleotides used for cloning.</p

TREM-B1 is expressed as a glycosylated monomer.

<p>2D8 cells stably transfected with a TREM-B1-FLAG construct (A) and PBMC (B) were surface biotinylated, lysed and immunoprecipitated with the 1E9 mab. Deglycosylation was performed with PNGaseF (+) and probes were analyzed under nonreducing (NR) and reducing (R) conditions. Following PAGE, size-fractioned proteins were blotted and detected with a streptavidin-HRP conjugate.</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 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