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 Our new, unpublished experiments suggest interactions between ANGPTL proteins and LILRA/B may be more widespread than originally thought and provide new avenues for immune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
Human Angiopoietin-1 Human Angiopoietin-2 Human ANGPTL4  Human ANGPTL5
Human ANGPTL6 Human ANGPTL7 Human LILRA1 Human LILRA2
Human LILRA4 Human LILRA5 Human LILRA6 Human LILRB1
Human LILRB2 Human LILRB3 Human LILRB4 Human LILRB5
Mouse Angiopoietin-2 Mouse ANGPTL3  Mouse ANGPTL4 Mouse ANGPTL7
Mouse LILRB4      

New 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 proteins2,3,9

    Major Function
Name Alternate Names Expression Regulate Angiogenesis Regulate Lipid Metabolism Promote the Expansion of Hematopoietic Stem Cells
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 Proteins8,10. 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

References

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