The Orphan Immune Receptor LILRB3 Modulates Fc Receptor–Mediated Functions of Neutrophils

Abstract

Neutrophils are critical to the generation of effective immune responses and for killing invading microbes. Paired immune receptors provide important mechanisms to modulate neutrophil activation thresholds and effector functions. Expression of the leukocyte Iglike receptor (LILR)A6 (ILT8/CD85b) and LILRB3 (ILT5/CD85a) paired-receptor system on human neutrophils has remained unclear because of the lack of specific molecular tools. Additionally, there is little known of their possible functions in neutrophil biology. The objective of this study was to characterize expression of LILRA6/LILRB3 receptors during human neutrophil differentiation and activation, and to assess their roles in modulating Fc receptor-mediated effector functions. LILRB3, but not LILRA6, was detected in human neutrophil lysates following immunoprecipitation by mass spectrometry. We demonstrate high LILRB3 expression on the surface of resting neutrophils and release from the surface following neutrophil activation. Surface expression was recapitulated in a human PLB-985 cell model of neutrophil-like differentiation. Continuous ligation of LILRB3 inhibited key IgA-mediated effector functions, including production of reactive oxygen species, phagocytic uptake, and microbial killing. This suggests that LILRB3 provides an important checkpoint to control human neutrophil activation and their antimicrobial effector functions during resting and early-activation stages of the neutrophil life cycle.

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

“…However, as LILRB3 shares high sequence homology (>95%) in its extracellular domain with LILRA6, there is a possibility that some LILRB3 mAb may also recognize shared epitopes on LILRA6, if co-expressed (9). These initial data might receive further evidence from other reagents as well as investigation as to whether LILRA6 protein is detectable in leukocyte subsets, e.g., using proteomics approaches similar to a recent study with neutrophils (41). Epitope mapping experiments revealed that the specific LILRB3 mAb reported here were generated against two specific ectodomains, either Ig-like domain two or four.…”

“…In this study, we investigated another inhibitory LILR family member, LILRB3, whose function, largely due to lack of suitable reagents and experimental systems, is not yet fully determined. Limited previous studies investigated the consequences of LILRB3 activation on granulocytes and have demonstrated its inhibitory function on neutrophils (41) and basophils (42) in culture. Here, we largely concentrated on myelomonocytic cells and the subsequent regulation of adaptive immune responses.…”

Leukocyte Immunoglobulin-Like Receptors (LILRs) on Human Neutrophils: Modulators of Infection and Immunity

“…In summary, activating receptors LILRA2, LILRA3, and LILRA5 are expressed on neutrophils. Recent immunoprecipitation and mass spectrometry analysis was unable to confirm the presence of LILRA6 in neutrophil lysates (17). Inhibitory receptors LILRB1, LILRB2, and LILRB3 are expressed on neutrophils, but there is little support for expression of LILRB4 and LILRB5.…”

“…This permits docking of SH2 domain containing tyrosine phosphatases and suppression of downstream signaling cascades. Inhibitory receptors on neutrophils include leukocyte-associated Ig-like receptor 1 (LAIR-1) (14), signal inhibitory receptor on leukocytes 1 (SIRL-1) (15), Siglec-5 (16), leukocyte immunoglobulin-like receptor (LILR)B2 (13), LILRB3 (17), and CEACAM1 (18). ITIM signaling can regulate activation of ITAM signaling receptors, or receptors that couple with ITAM-containing co-receptors including FcRγ or DAP12.…”

Bacterial protein domains with a novel Ig-like fold target human CEACAM receptors

“…In brief, gBlocks containing open reading frames (ORFs) encoding the extracellular domain of CEACAMs, and a C terminal LPETGGSHHHHHH tag, were cloned into the pcDNA3.4 expression vector (Invitrogen) (Supplementary Table 2). Recombinant CEACAM-His proteins were expressed in Expi293F cells (Life Technologies) and purified by affinity chromatography (ÄKTA Pure, GE Healthcare Life Sciences) using a Nickel column (GE Healthcare Life Sciences) as previously described (Zhao Y et al, 2020). Eluate was dialysed against 300 mM NaCl 50 mM Tris pH 7.8 at 4 o C. Unglycosylated CEACAMs were expressed and purified from E. coli cultures.…”