Blocking immunoinhibitory receptor LILRB2 reprograms tumor-associated myeloid cells and promotes antitumor immunity
Hui-Ming Chen, … , Ping-Ying Pan, Shu-Hsia Chen
Hui-Ming Chen, … , Ping-Ying Pan, Shu-Hsia Chen
Published October 22, 2018
Citation Information: J Clin Invest. 2018;128(12):5647-5662. https://doi.org/10.1172/JCI97570.
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Blocking immunoinhibitory receptor LILRB2 reprograms tumor-associated myeloid cells and promotes antitumor immunity

Abstract

Tumor-associated myeloid cells maintain immunosuppressive microenvironments within tumors. Identification of myeloid-specific receptors to modulate tumor-associated macrophage and myeloid-derived suppressor cell (MDSC) functions remains challenging. The leukocyte immunoglobulin-like receptor B (LILRB) family members are negative regulators of myeloid cell activation. We investigated how LILRB targeting could modulate tumor-associated myeloid cell function. LILRB2 antagonism inhibited receptor-mediated activation of SHP1/2 and enhanced proinflammatory responses. LILRB2 antagonism also inhibited AKT and STAT6 activation in the presence of M-CSF and IL-4. Transcriptome analysis revealed that LILRB2 antagonism altered genes involved in cell cytoskeleton remodeling, lipid/cholesterol metabolism, and endosomal sorting pathways, as well as changed differentiation gene networks associated with inflammatory myeloid cells as opposed to their alternatively activated phenotype. LILRB2 blockade effectively suppressed granulocytic MDSC and Treg infiltration and significantly promoted in vivo antitumor effects of T cell immune checkpoint inhibitors. Furthermore, LILRB2 blockade polarized tumor-infiltrating myeloid cells from non–small cell lung carcinoma tumor tissues toward an inflammatory phenotype. Our studies suggest that LILRB2 can potentially act as a myeloid immune checkpoint by reprogramming tumor-associated myeloid cells and provoking antitumor immunity.

Authors

Hui-Ming Chen, William van der Touw, Yuan Shuo Wang, Kyeongah Kang, Sunny Mai, Jilu Zhang, Dayanira Alsina-Beauchamp, James A. Duty, Sathish Kumar Mungamuri, Bin Zhang, Thomas Moran, Richard Flavell, Stuart Aaronson, Hong-Ming Hu, Hisashi Arase, Suresh Ramanathan, Raja Flores, Ping-Ying Pan, Shu-Hsia Chen

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Figure 3

LILRB2 antagonism favors NF-κB/STAT1 inflammatory pathways.

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LILRB2 antagonism favors NF-κB/STAT1 inflammatory pathways. (A–C) THP1 L...
(A–C) THP1 LILRB2+ cells were cultured with IgG or anti-LILRB2 (αLILRB2, 1 μg/ml) for 24 hours followed by acute stimulation with LPS (A), IFN-γ (B), or IL-4 (C) for 5, 10, and 30 minutes. (A) Immunoblot of phosphorylated NF-κB, ERK1/2, and p38 in response to LPS (50 ng/ml) stimulation. (B) Immunoblot of phosphorylated NF-κB, ERK1/2, p38, and STAT1 in response to IFN-γ (20 ng/ml) stimulation. (C) Immunoblot of phosphorylated STAT6 in response to IL-4 (20 ng/ml) stimulation. (D) Immunoblot of SOCS1 and SOCS3 in response to IL-4 (20 ng/ml) stimulation. (E) Immunoblot of phosphorylated AKT from primary M-CSF macrophages matured in the presence of IgG or anti-LILRB2. Representative data from 3 independent PBMC donors. (F) LILRB2, p-SHP1, and total SHP1 immunoblotting from IgG- and anti-LILRB2–treated macrophages. Results from anti-LILRB2 (42D1) immunoprecipitate (top) and total input lysate (bottom). (G) LILRB2 antagonism inhibits monocyte/macrophage-mediated suppression of effector T cell responses. Total CD4+ and CD8+ T cell counts determined by flow cytometry of 72-hour mixed lymphocyte reactions (MLRs) containing mature DCs, sorted allogeneic T cells, and titrated ratios of M-CSF macrophages matured in the presence of IgG (black line) or anti-LILRB2 (red line). (H) Supernatants from MLRs in G were analyzed for secreted IFN-γ by ELISA. (I) Total PBMCs were incubated with anti-LILRB2 (5 μg/ml) or IgG overnight followed by anti–PD-1 treatment (1 μg/ml) in the presence of OKT3 stimulation (0.01 μg/ml) for 3 days. Supernatants were harvested for IFN-γ detection by ELISA. Data are from a representative experiment of 3 independent experiments and are presented as mean ± SEM, and P values were calculated using 1-way ANOVA followed by Tukey’s multiple-comparisons test; *P < 0.05, **P < 0.01.