LILRA/B Receptors: Checkpoint Targets with Novel Ligands

Leukocyte immunoglobulin-like receptors A/B (LILRA/B) represent a family of immunoregulatory receptors expressed by a range of hematopoietic cell types¹. The LILRB subset contains immunoreceptor tyrosine based inhibitory motifs (ITIMs) also found on other promising immune checkpoint targets such as PD-1, BTLA, and TIGIT. LILRB ligands include MHC I, S100A8/A9, and some myelin-associated proteins. Some recent evidence has also indicates Angiopoietin-like (ANGPTL) proteins may be soluble ligands for LILRB2. Our new, unpublished experiments suggest interactions between ANGPTL proteins and LILRA/B may be more widespread than originally thought and provide new avenues for immune checkpoint research.

Introduction

Research in understanding the roles of Angiopoietin-like (ANGPTL) proteins in immunology is in its infancy. One reason has been a lack of quality tools to study their activity. R&D Systems scientists have produced a range of purified ANGPTL proteins that are now available (Table 1). Their research has utilized these reagents to demonstrate previously unreported associations between Angiopoietins and ANGPTL proteins with LILRA/B receptors. The results suggest new functions for these proteins and reveal unexplored areas of immunology.

Table 1.

Angiopoietin, ANGPTL and related products

Bioactive Recombinant Proteins

Active Biotinylated Proteins

Figure 1. Recombinant Human ANGPTL5 (Catalog # 6675-AN) Induces Proliferation in Primary Rat Liver Mononuclear Cells. ANGPTL5 enhances proliferation of E16 rat liver mononuclear cells in the presence of Recombinant Mouse SCF/c-kit Ligand (Catalog # 455-MC), Recombinant Mouse Thrombopoietin/TPO (Catalog # 488-TO, and Recombinant Mouse Flt-3 Ligand (Catalog # 427-FL).

Angiopoietins [Angiopoietin-1, Angiopoietin-2, Angiopoietin-3 (mouse), Angiopoietin-4 (human)] and ANGPTL1-8 are secreted glycoproteins with roles in angiogenesis, lipid metabolism, hematopoietic stem cell expansion, and inflammation (Table 2). R&D Systems full length ANGPTL3, ANGPTL4, ANGPTL5, ANGPTL6, and ANGPTL7 recombinant proteins all exhibit bioactivity as expected in accordance with published literature.⁷ Typical data showing ANGPTL5-induced proliferation of primary rat liver mononuclear cells is shown in Figure 1.

Structurally, ANGPTL proteins all contain an N-terminal coiled-coil domain that mediates oligomerization and a C-terminal fibrinogen-like domain. ANGPTL8 is the only exception and is lacking the C-terminal domain. We also have found that truncated versions containing the N-terminal coiled-coil domain of recombinant ANGPTL3 and ANGPTL4 share similar bioactivity to the full length protein (not shown).

Table 2.

Angiopoietin and Angiopoietin-like proteins^2,^3,⁹

Name	Alternate Names	Expression	Regulate Angiogenesis	Regulate Lipid Metabolism	Promote the Expansion of Hematopoietic Stem Cells
		Major Function
Angiopoietin-1	AGP1	Periendothelial Cells	Yes		Yes
Angiopoietin-2		Endothelial Cells	Yes
Angiopoietin-4	ANG3	Lung	Yes
Angiopoietin-like-1	ARP1, ANG Y, Angioarrestin	Liver, muscle	Yes		Yes
Angiopoietin-like-2	ARP2	Heart, adipose tissue	Yes	Yes	Yes
Angiopoietin-like-3	ANG-5	Liver (exclusively)	Yes	Yes	Yes
Angiopoietin-like-4	ARP4	Liver, adipose tissue	Yes	Yes	Yes
Angiopoietin-like-5	ANGL5	Adipose tissue, heart		Yes	Yes
Angiopoietin-like-6	ARP5, AGF	Liver	Yes	Yes	Yes
Angiopoietin-like-7	ANG X	Eye (trabecular meshwork)	Yes		Yes
Angiopoietin-like-8	Betatrophin, Lipasin, RIFL	Liver, adipose tissue		Yes

Results

Unlike Angiopoietins that are known to signal through the tyrosine kinases Tie-1 or Tie-2, ANGPTL proteins had long been considered orphan ligands.^{2, 3, 4, 5, 6} Recently, ANGPTL1, 2, 5, and 7 were shown to bind to human leukocyte immunoglobulin like receptor B2 (LILRB2), resulting in enhancement of human hematopoietic stem cell proliferation and expansion.⁷ LILRs (LILRA1-6, LILRB1-5, and LILRP1-2), also named ILTs, LIRs, and CD85s, are a family of immunomodulatory molecules that are expressed on professional APCs and influence immune activation and inhibition.⁸

In order to evaluate new interacting partners for ANGPT and ANGPTL proteins, we tested recombinant Angiopoietins and ANGPTL proteins in binding studies with LILRA and LILRB family members (Figure 2 and Table 3).


Figure 2. Representative Data Showing LILR and ANGPTL Binding. A: ELISA binding assay demonstrates Recombinant Human ANGPLT7 (Catalog # 914-AN) binding to biotinylated Recombinant Human LILRB5 with a Kd=4 nM.B: ELISA binding assay demonstrates biotinylated Recombinant Human ANGPTL3 (Catalog # 3829-AN) binding to Recombinant Human LILRB2/CD85d/ILT4 (Catalog # 2078-T4) with a Kd = 24 nM.

Table 3.

Angiopoietin/ANGPTL Binding Screen with LILRA and LILRB Proteins^8,¹⁰. ELISA screen of binding of LILRA1-6 and LILRB1-5 to Angiopoietins and ANGPTL3-7 proteins. For this screen, recombinant human LILRA/LILRB proteins were coated on an ELISA plate, and biotinylated recombinant human Angiopoietins and ANGPTL proteins were used as ligands. Interactions between ANGPTL and LILRB2 protein were described previously.⁷ ANGPTL1 and ANGPTL2 were not included in that study.⁷ Typical data for such interactions are shown in Figure 2.

Principle Name	CD Name	Other names	Expression	ANGPT/ANGPTL binding


LILRA1	CD85i	LIR-6, LIR6	Macrophages	–	–	–	+	–	–	–	+
LILRA2	CD85h	ILT1, LIR-7, LIR7	Monocytes, macrophages, dendritic cells, NK cells, basophils, eosinophils	–	–	–	+	–	–	+	+
LILRA3	CD85e	ILT6, LIR-4, LIR4, HM43, HM31	Monocytes	+	–	–	+	–	–	–	+
LILRA4	CD85g	ILT7	Plasmacytoid dendritic cells	–	–	–	–	–	–	–	+
LILRA5	CD85f	ILT11, LIR-9, LIR9, LILRB7	CD14⁺ monocytes	–	–	–	–	–	–	–	-
LILRA6	CD85b	ILT8, LILRB6	Unknown	–	–	–	–	–	–	–	-
LILRB1	CD85j	ILT2, LIR-1, LIR1, MIR7	Monocytes, macrophages, dendritic cells, osteoclasts, eosinophils, B cells, T cells, NK cells, placental stromal cells	–	–	–	–	–	–	–	–
LILRB2	CD85d	ILT4, LIR-2, LIR2, MIR10, MIR-10	Monocytes, macrophages, dendritic cells, osteoclasts, basophils, eosinophils, placental vascular smooth muscle	+	–	+⁷	+⁷	+⁷	+⁷	+⁷	+⁷
LILRB3	CD85a	ILT5, LIR-3, LIR3, HL9	Monocytes, macrophages, dendritic cells, osteoclasts, basophils, eosinophils	–	–		–	–	–	–	–
LILRB4	CD85k	ILT3, LIR-5, LIR5, HM18	Monocytes, macrophages, dendritic cells, osteoclasts	+	–	–	+	+	+	–	+
LILRB5	CD85c	LIR-8, LIR8	Unknown	–	–	+⁷	+	–	–	–	+
+ Binding observed in R&D Systems preliminary experiments, +⁷ Binding observed previously, – No binding observed