1. The Soluble Leukocyte-Associated Ig-Like Receptor (LAIR)-2 Antagonizes the Collagen/LAIR-1 Inhibitory Immune The Journal of Immunology, 2008 2011,09,06 Xu Zhenjie

2. 1. Background  Known: LAIR-1 （ leukocyte-associated Ig-like receptor-1 ） is an ITIM- containing inhibitory immune receptor. Collagens are high-affinity ligands for the LAIR-1 molecules.  Unknown: Whether LAIR-2 is produced in vivo is unknown. Whether LAIR-2 is a secreted protein. Whether it may function as a soluble competitor for the interaction between collagens and hLAIR-1.  Findings ： a novel mechanism of immune regulation ： sLAIR-2 regulated the inhibitory potential of the membrane-bound hLAIR-1 via competition for the same ligands.

3. Human monocytic U937 cells; HEK293T human embryonic kidney cells; Human colon carcinomas lines HT29, DLD-1, LS174, SW480, and HCT116; SKBR3 human breast cancer line; YT.2C2 human NK-like cells; 721.221 lymphoblastoid cells; THP-1 human monocyte-like cells; Jurkat human T cells; CEM human leukemia T cells; human erythroleukemia K562 cells. K562 cells and 2B4 NFAT-GFP T cell reporter cells were stably transfected with hLAIR-1 or hLAIR-1a-CD3 respectively. Cells lines 2. Materials and methods

4. Subjects: 10 healthy individuals (5 males and 5 females) and 6 healthy pregnant women (16–37 wk of gestation) volunteers. 14 patients suffering from rheumatoid arthritis (RA) and 16 patients suffering from osteoarthritis (OA). Method: RT-PCR Western blot analysis Surface plasmon resonance ( 表面等离子共振 )? Flow cytometry Reporter cell assay ? Sandwich ELISA

5. contents 1. LAIR-2 is expressed by immune cells. 2. LAIR-2 is a secreted protein. 3. LAIR-2 is a high-affinity collagen receptor. 4. LAIR-2 prevents binding of hLAIR-1 to its ligand. 5. LAIR-2 prevents hLAIR-1 cross-linking by collagens. 6. LAIR-2 is produced by primary cells and in vivo. LAIR-2 is a secreted protein; and it functions as a soluble competitor for the interaction between collagens and hLAIR-1

6. 3.1. LAIR-2 is expressed by immune cells Total RNA RT-PCR LAIR-2 specific primers Cell lines 3. Results

7. His-tagged LAIR-2aWild type LAIR-2b 239T cells supernatant FMU-LAIR2.1 anti-LAIR-2 mAbs peroxidase-conjugated rabbit anti- mouse mAb as secondary Ab Western blot Flow cytometry 3.2. LAIR-2 is a secreted protein anti-LAIR-2 mAbs

8. 3.3. LAIR-2 is a high-affinity collagen receptor LAIR-2-IgG fusion proteins surface plasmon resonance To measured binding and affinity of LAIR-2-IgG fusion proteins to collagen: Collagen Ⅰ and collagen Ⅲ

9. 3.4. LAIR-2 prevents binding of hLAIR-1 to its ligand HT29 cells Incubate with unlabeled LAIR-2-IgG Incubate with biotinylated hLAIR-1-IgG Incubate with hLAIR-1-IgG Incubate with biotinylated LAIR-2-IgG Can express endogenous hLAIR-1 ligands Both human LAIR-1 and LAIR-2 fusion proteins bind the same sites on collagen molecules.

10. Incubate with K562 cells or LAIR-1-transfected K562 cells Oregon Green 488-labeled collagen IV Incubate with LAIR-2-IgG Incubate with control Ig fusion proteins (isotype)

11. As documented before, hLAIR-1-transfected K562 cells bind firmly to plate-bound collagens I and III. plate-bound collagens I and III Incubate with LAIR-2-IgG Incubate with hLAIR-1-transfected K562 cells Incubate with irrelevant fusion protein

12. To assess whether wild-type LAIR-2 protein is capable of interfering with the hLAIR-1/collagen interaction: supernatant from LAIR-2-transfected 293T cells concentrated Incubate with hLAIR-1-transfected K562 cells control supernatant plate-bound collagens I and III LAIR-2 binds the same sites on various collagen species as hLAIR-1, suggesting that LAIR-2 may function as a soluble competitor to hLAIR-1/collagen binding.

13. 3.5. LAIR-2 prevents hLAIR-1 cross-linking by collagens By using NFAT-GFP reporter cells expressing a chimeric protein containing the extracellular domain of hLAIR-1 and the transmembrane and intracellular domain of CD3ζ, we have shown that collagen I and III are capable to functionally cross-link hLAIR-1. plate-bound collagens I and III

14. 3.6. LAIR-2 is produced by primary cells and in vivo PBMC anti-CD4/ CD8/CD3/C D19 Abs anti-CD16/ CD14/CD3/ CD56 Abs CD3 + CD4 + T cells CD3 + CD8 + T cells CD3 - CD19 + B cells CD3 - CD16 + CD56 + NK cells CD3 - CD14 + monocytes To detecte LAIR-2 expression stimulated with PMA and ionomycin Sandwich ELISA day 4

15. 10 healthy individuals and 6 pregnant women Plasma, serum and urine 14 patients with RA and 16patients with OA SF Sandwich ELISA