Abstract
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%.
Highlights
A balanced immune reaction is important to avoid either insufficient or exaggerated immune responses
We showed the inhibitory effect of Triggering receptors expressed on myeloid cells (TREM)-B1 in chicken thrombocytes using a CD107 degranulation assay
The mab 1E9 and 7E8 are specific for TREM-B1 and do not cross-react with other TREM members
Summary
A balanced immune reaction is important to avoid either insufficient or exaggerated immune responses. Chicken TREM-B1 which fine-tune the outcome of the immune response Many of these cell-cell interactions are still poorly understood, but a group of so called immunoregulatory receptors are very likely to be involved in this process [1,2,3]. Activating receptors have a short cytoplasmic tail without any signaling capabilities, but display a positively charged amino acid in the transmembrane region, which can be either arginine or lysine. This is associated with the negatively charged residue of an ITAM containing adaptor molecule, which is DAP12 or common γ chain. Interaction with the specific ligand leads to phosphorylation of these tyrosines and subsequent recruitment of tyrosine specific protein phosphatases like src-homology phosphatase-1 (SHP-1), SHP-2 or Src homology 2-containing inositol 5'-phosphatase (SHIP) which are dephosphorylating downstream target molecules [8, 9]
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