Characterisation of murine MICL (CLEC12A) and evidence for an endogenous ligand

Abstract

Inhibitory receptors are required for the control of cellular activation and they play essential roles in regulating homeostasis and immunity. We previously identified a human inhibitory C-type lectin-like receptor, MICL (CLEC12A), a heavily glycosylated monomer predominantly expressed on myeloid cells. Here we characterise the murine homolog of MICL (mMICL), and demonstrate that the receptor is structurally and functionally similar to the human orthologue (hMICL), although there are some notable differences. mMICL is expressed as a dimer and is not heavily glycosylated; however, like hMICL, the receptor can recruit inhibitory phosphatases upon activation, and is down-regulated on leukocytes following stimulation with selected TLR agonists. Using novel monoclonal antibodies, we demonstrate that, like the human receptor, mMICL is predominantly expressed by myeloid cells. However, mMICL is also expressed by B cells and CD8 + T cells in peripheral blood, and NK cells in the bone marrow. Finally, we show that mMICL recognises an endogenous ligand in a variety of murine tissues, suggesting that the receptor plays a role in homeostasis.

MICL controls inflammation in rheumatoid arthritis

“…Characterisation of MICL expression in 8–12-week-old wt and Clec12A −/− mice was performed by flow cytometry on cells isolated from the peripheral blood, bone marrow, peritoneal cavity, spleen and lungs, as previously described. 14 Antibodies used in these experiments included biotin anti-MICL monoclonal antibody 309 7 and isotype control, as well as biotin-Gr-1, FITC-7/4, PE-F480, biotin-F480, PerCpCy5.5-CD11b, PE-CCR3, biotin-NK1.1, PE-CD49, PerCpCy5.5-B220, PE-CD19, PerCpCy5.5-CD3, biotin-CD4, FITC-CD8, biotin-CD11c, PerCpCy5.5-Gr-1, APC-Ly6G, PE-CD11b, PE-Ly6G, PECy7-CD11b, Alexa Fluor 488 anti-STAT5, APC-streptavidin (all from BD Biosciences), and Alexa Fluor 700-F4/80 (BioLegend). Flow cytometry was undertaken using a BD LSRFortessa cell analyser and data analysed using FlowJo.…”

“…MICL is highly expressed on myeloid cells, particularly neutrophils, monocytes and macrophages. 6 It possesses a single extracellular C-type lectin-like domain, that recognises endogenous ligand(s), 7 8 and a cytoplasmic tail containing an immunoreceptor tyrosine-based inhibitory motif that can transduce intracellular inhibitory signals. 8 9 MICL-mediated intracellular signalling can modulate several cellular responses, 8–11 and expression levels of this receptor are substantially altered during cellular activation, 6 7 11 suggesting that it has an immune regulatory role.…”

Macrophage heterogeneity in tissues: phenotypic diversity and functions

“…It was then straightforward to isolate monoclonal antibodies to study tissue expression of Dectin-1 and related antigens, their regulation and signaling. In some cases, we failed to identify ligands for cloned ‘orphan’ lectin-like antigens such as MICL 27 , recently achieved by others 28 .…”

Targeting C-type lectin receptors: a high-carbohydrate diet for dendritic cells to improve cancer vaccines

“…CLEC12a (DCAL-2, CLL-1, MCIL, KLRL1) is an inhibitory, ITIM-containing CLR that is predominantly expressed on myeloid cells but can also be found on B cells, CD8 T cells, and NK cells [ 175 – 178 ]. pDCs and mouse cDC1s express CLEC12a to the greatest extent, but it is also expressed on a subset of mouse cDC2s [ 179 , 180 ].…”