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

Abstract

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.

Leukocyte Immunoglobulin-Like Receptors A2 and A6 are Expressed in Avian Macrophages and Modulate Cytokine Production by Activating Multiple Signaling Pathways

“…In contrast, there are many differences in the corresponding residues between chicken LILRA2 and group 1 of LILRs in human D1–D2 region, explaining LILRA2 in chicken and human likely do not bind to MHC molecules. On the other hand, human LILRA6 (group 2 of LILRs) amino acids associated with MHC class I and β2m ligand binding (S 17 , G 19 , Q/R 36 , G 41 , L/W 46 , Q/E 67 , Y 77 , T 97 , S 101 , T 117 , R/Q 120 , N/T 160 , M/T 178 and Q/W/R 182 ) in Ig domain D1–D2, in which the Ig D1 region (S 17 , G 19 , Q/R 36 , G 41 , L/W 46 , Q/E 67 , Y 77 and T 97 ) strongly bind to MHC class I and β2m [ 5 , 10 , 24 ] that are highly conserved in chicken and human LILRA6. The results indicated that Ig D1 region of LILRA6 is strongly binds to MHC class I but LILRA2 does not bind to MHC class I and β2m.…”

“…The leukocyte immunoglobulin-like receptor (LILR) family consists of members that play inhibitory or activating roles on the genes of the proteins involved with the immune system and expressed in several primary innate immune cells (monocyte/macrophages, dendritic cells and CD4 + /CD8 + T cells) [ 1 , 2 , 3 ] and cell lines (HEK293 T cells, MDCK cells, HL-60 cells, thymoma BWZ.36 cells, epithelial cells and T cells) [ 1 , 4 , 5 , 6 ]. The LILR genes are highly homologous in the sequence of the extracellular regions and different in their sequences of the intracellular regions [ 7 , 8 ].…”

LILRB3 (ILT5) is a myeloid checkpoint on myeloid cells that elicits profound immununomodulation

“…Subsequently, reporter cells transfected with a chimeric receptor expressing the extracellular domain of LILRB3, fused with the human CD3ζ cytoplasmic domain, were used to investigate whether the generated mAb were able to crosslink the receptor. Cross-linking results in the production of NFAT activation and the subsequent expression of GFP and is indicative of agonistic potential (30). Using these cells, we were able to identify two distinct groups of LILRB3 mAb, those with 'agonistic' activity capable of inducing signaling upon binding to the receptor (e.g., A1) and those which were inert (e.g., A28) ( Fig.…”

“…For staining of 2B4 reporter cells expressing LILR-A1, -A2, -A5, -B1, -B2, -B3, -B4, or -B5 (or non-transfected controls) (30,60) cells were stained with 10 μ g/ml LILRB mAb and incubated at 37°C with 5% CO 2 , overnight. The following day, the cells were washed and stained with a secondary anti-hIgG antibody (Jackson ImmunoResearch, USA) at 4°C, for 45 minutes.…”

LILRA6 copy number variation correlates with susceptibility to atopic dermatitis

“…LILR can regulate the activity of TLR (Brown et al 2004 , Cao et al, 2009 ) and mediate the immunological tolerance (Manavalan et al, 2003 ; Kim-Schulze et al, 2006 ; Anderson and Allen, 2009 ). Currently, there is no data about the role of LILRB3 and LILRA6, although their recently discovered interaction with a cytokeratin 8-associated ligand suggests they could shape local inflammatory responses to epithelial tumours (Jones et al, 2016 ). LILRA3 has been described as a soluble receptor that binds HLA class I molecules and it has been associated with multiple sclerosis (An et al, 2016 ), systemic lupus erythematosus and Sjögren’s syndrome (Du et al, 2014 ).…”