Characterisation of a novel leukocyte receptor complex-encoded receptor TARM1

Cellular immune responses are orchestrated by an intricate balance of activating and inhibitory signals transmitted by cell surface receptors. Perturbations in this balance by overamplified or dysregulated signalling underlie many severe immunopathologies such as sepsis and cancer. In this work I describe the identification and characterisation of a novel, evolutionarily conserved immunoreceptor encoded within the human leukocyte receptor complex and syntenic region of mouse chromosome 7, named T cell–interacting, activating receptor on myeloid cells-1 (TARM1). The transmembrane region of TARM1 contained a conserved arginine residue, consistent with association with a signalling adaptor. Co-immunoprecipitation experiments confirmed that TARM1 associated with the ITAM adaptor FcR-gamma but not with DAP10 or DAP12. Flow cytometric screening of cells and tissues from pathogen-free mice showed that the TARM1 protein was constitutively expressed on the cell surface of mature and immature CD11b+Gr+ neutrophils isolated from bone marrow but not at peripheral sites. Following ip LPS treatment or systemic bacterial challenge, TARM1 protein expression was upregulated by myelocytes, mature neutrophils and inflammatory monocytes and TARM1+ cells were rapidly recruited to sites of inflammation. TARM1 expression was also upregulated by bone marrow–derived macrophages and dendritic cells following stimulation with TLR agonists in vitro. Ligation of the TARM1 receptor with specific antibody in the presence of TLR ligands, such as LPS, enhanced the secretion of proinflammatory cytokines by bone marrow–derived macrophages and primary mouse neutrophils, whereas TARM1 stimulation alone had no effect. Finally, an immobilised TARM1 Fc fusion protein suppressed CD4+ T cell activation and proliferation in vitro. These results suggest that a putative T cell ligand can interact with TARM1 receptor, resulting in bidirectional signalling and raising the T cell activation threshold while costimulating the release of proinflammatory cytokines by macrophages and neutrophils

Allele-specific recognition by LILRB3 and LILRA6 of a cytokeratin 8-associated ligand on necrotic glandular epithelial cells.

The LILRs are a family of receptors that regulate the activities of myelomonocytic cells. We found that specific allelic variants of two related members of the LILR family, LILRB3 and LILRA6, interact with a ligand exposed on necrotic glandular epithelial cells. The extracellular domains of LILRB3 and LILRA6 are very similar and their genes are highly polymorphic. A commonly occurring allele, LILRB3*12, displayed particularly strong binding of these necrotic cells and further screening of the products of LILRB3 alleles identified motifs that correlated with binding. Immunoprecipitation of the ligand from epithelial cell lysates using recombinant LILRB3*12, identified cytokeratins 8, 18 and 19. Purified proteins obtained from epithelial cell lysates, using anti-cytokeratin 8 antibodies, were able to activate LILRB3*12 reporter cells. Knock-down of cytokeratin 8 in epithelial cells abrogated expression of the LILRB3 ligand, while staining with recombinant LILRB3*12 showed co-localisation with cytokeratin 8 and 18 in permeabilised breast cancer cells. Necrosis is a common feature of tumours. The finding of a necrosis-associated ligand for these two receptors raises the possibility of a novel interaction that alters immune responses within the tumour microenvironment. Since LILRB3 and LILRA6 genes are highly polymorphic the interaction may influence an individual's immune response to tumours.DCJ and JT are funded by Worldwide Cancer Research (formerly the AICR), project grant number 13-0074. Additional funding was provided by grants from the MRC (G0901682) and Wellcome Trust (094207 and 089821). MRL-A was funded by Ministerio de Educación of Spain, under the program “Programa Nacional de Movilidad de Recursos Humanos del Plan Nacional de I-D+i 2008-2011” and Fundación Séneca 04087/GERM/06 Project

TARM1 Is a Novel Leukocyte Receptor Complex-Encoded ITAM Receptor That Costimulates Proinflammatory Cytokine Secretion by Macrophages and Neutrophils.

We identified a novel, evolutionarily conserved receptor encoded within the human leukocyte receptor complex and syntenic region of mouse chromosome 7, named T cell-interacting, activating receptor on myeloid cells-1 (TARM1). The transmembrane region of TARM1 contained a conserved arginine residue, consistent with association with a signaling adaptor. TARM1 associated with the ITAM adaptor FcRγ but not with DAP10 or DAP12. In healthy mice, TARM1 is constitutively expressed on the cell surface of mature and immature CD11b(+)Gr-1(+) neutrophils within the bone marrow. Following i.p. LPS treatment or systemic bacterial challenge, TARM1 expression was upregulated by neutrophils and inflammatory monocytes and TARM1(+) cells were rapidly recruited to sites of inflammation. TARM1 expression was also upregulated by bone marrow-derived macrophages and dendritic cells following stimulation with TLR agonists in vitro. Ligation of TARM1 receptor in the presence of TLR ligands, such as LPS, enhanced the secretion of proinflammatory cytokines by macrophages and primary mouse neutrophils, whereas TARM1 stimulation alone had no effect. Finally, an immobilized TARM1-Fc fusion protein suppressed CD4(+) T cell activation and proliferation in vitro. These results suggest that a putative T cell ligand can interact with TARM1 receptor, resulting in bidirectional signaling and raising the T cell activation threshold while costimulating the release of proinflammatory cytokines by macrophages and neutrophils.This work was supported by grants from the Cancer Research UK, the Wellcome Trust, Medical Research Council, UK, and a Marie Curie International Outgoing Fellowship awarded to A.D.B. with additional support from the Wellcome Trust and the National Institute for Health Research (NIHR) Cambridge Biomedical Research Centre.This is the author accepted manuscript. The final version is available from the American Association of Immunologists via http://dx.doi.org/10.4049/jimmunol.140184