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LILRB3 supports acute myeloid leukemia development and regulates T-cell antitumor immune responses through the TRAF2–cFLIP–NF-κB signaling axis

Abstract

Leukocyte immunoglobulin-like receptor B (LILRB), a family of immune checkpoint receptors, contributes to acute myeloid leukemia (AML) development, but the specific mechanisms triggered by activation or inhibition of these immune checkpoints in cancer is largely unknown. Here we demonstrate that the intracellular domain of LILRB3 is constitutively associated with the adaptor protein TRAF2. Activated LILRB3 in AML cells leads to recruitment of cFLIP and subsequent NF-κB upregulation, resulting in enhanced leukemic cell survival and inhibition of T-cell-mediated anti-tumor activity. Hyperactivation of NF-κB induces a negative regulatory feedback loop mediated by A20, which disrupts the interaction of LILRB3 and TRAF2; consequently the SHP-1/2-mediated inhibitory activity of LILRB3 becomes dominant. Finally, we show that blockade of LILRB3 signaling with antagonizing antibodies hampers AML progression. LILRB3 thus exerts context-dependent activating and inhibitory functions, and targeting LILRB3 may become a potential therapeutic strategy for AML treatment.

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Fig. 1: LILRB3 promotes monocytic AML progression.
Fig. 2: LILRB3 increases the sensitivity of monocytic AML cells to cytotoxic T cells.
Fig. 3: LILRB3 enhances NF-κB signaling and monocytic AML survival via TRAF2.
Fig. 4: LILRB3 interacted with TRAF2.
Fig. 5: LILRB3 enhancement of NF-κB signaling depends on cFLIP.
Fig. 6: LILRB3 negatively regulates NF-κB signaling with strong LPS stimulation.
Fig. 7: Blocking anti-LILRB3 antibody prevents monocytic AML development.
Fig. 8: Anti-LILRB3-blocking antibody prevents development of patient-derived AML.

Data availability

The RNA-seq datasets generated in the present study have been deposited in the National Center for Biotechnology Information’s Sequence Read Archive (SRA) database with the SRA accession no. SRP292554. Source data for Figs.18 and Extended Data Figs. 1, 2 and 47 have been provided. All other data supporting the findings of the present study are available from the corresponding author on reasonable request.

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Acknowledgements

We thank W. Xiong and H. Deng for their technical support, and G. Salazar for editing the manuscript. This work was supported by the National Cancer Institute (grant no. 1R01 CA248736), Leukemia and Lymphoma Society (grant no. TRP 6629-21), US Army Medical Research and Materiel Command (grant no. W81XWH2010793), the Cancer Prevention and Research Institute of Texas (grant nos. RP180435, RP150551 and RP190561), the Welch Foundation (grant no. AU-0042-20030616) and Immune-Onc Therapeutics, Inc. (Sponsored Research grant no. 111077).

Author information

Authors and Affiliations

Authors

Contributions

G.W. developed the experimental protocol, designed, performed and analyzed experiments, and wrote the manuscript. C.C.Z. directed the project, interpreted the results and wrote the manuscript. Y.X. prepared the antibodies, evaluated the properties of antibodies in vitro, prepared RNA-seq samples, provided extensive discussion and wrote the manuscript. R.S screened and sequenced the antibody from scFv library. H.C., J.X. and X.L. conducted part of the animal experiments. M.D. and H.A. provided technical support. X.G. purified the antibodies. S.J. prepared clinical samples. Z.L. analyzed TCGA clinical data. N.Z. and Z.A. directed antibody production and evaluation of antibody properties, and contributed extensive discussions.

Corresponding author

Correspondence to Cheng Cheng Zhang.

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Competing interests

The Board of Regents of the University of Texas System has filed a patent application covering ‘Methods for identifying LILRB-blocking antibodies’. C.C.Z., G.W., H.C., M.D., Z.A. and N.Z. are listed as inventors. The patent application has been exclusively licensed to Immune-Onc Therapeutic, Inc. by the Board of Regents of the University of Texas System. Z.A., N.Z. and C.C.Z. hold equity in and have Sponsored Research Agreements with Immune-Onc Therapeutics, Inc. Z.A. is a Scientific Advisory Board member with Immune-Onc Therapeutics, Inc. The remaining authors declare no competing interests.

Additional information

Peer review information Nature Cancer thanks Iannis Aifantis, Ross Levine and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Extended Data Fig. 1 LILRB3 enhances AML cell survival and promotes monocytic AML progression.

a, Analysis of LILRB3 and LILRB4 expression in patient AML samples (n = 35) as determined by flow cytometry. b, The cell death of THP-1 cells cultured with coated anti-LILRB3 or IgG in presence of DMSO, ABT199 (1 μM) or AZA (10 μM). (n = 3 independent cell cultures). c, Knockdown of LILRB3 in AML cell lines does not affect cell growth in culture (n = 3 independent cell cultures). d, THP-1 cells expressing Tet-on Cre and loxp U6 driven shRNAs were treated with Dox (1 μg/ml) for one day, and surface LILRB3 expression was analyzed by flow cytometry one week later. e, Percentages of dead cells in AML cultures treated with anti-LILRB3 antibody or IgG in the presence of different concentrations of TNFα (n = 3 independent cell cultures). f, Percentages of dead cells in THP-1 cells treated with anti-LILRB3 antibody or IgG in the presence of anti-TNFα (5 μg/ml) or control IgG (n = 3 independent cell cultures). g, Percentages of GFP+ AML cells in peripheral blood (PB), bone marrow (BM), spleen (SPL), and liver of mice transplanted with C1498 AML cells expressing B3-FL or B3del ICD (n = 4 mice). h, Survival curve of the mice treated as in panel e. i, THP-1 cell growth in plates coated anti-LILRB3 or IgG (n = 3 independent cell cultures). j, Serial colony-forming unit (CFU) replating with MLL-AF9 mouse AML cells (n = 3 independent cell cultures). k, Percentages of dead cells in U937 cells overexpressing LILRB3 or a control vector (n = 3 wells). The data are presented as mean ± s.e.m, and p values were calculated by two-tailed t-test except for h by log-rank test.

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Extended Data Fig. 2 LILRB3 increases the survival of monocytic AML cells against cytotoxic T cells.

a, Percentages of CD4 and CD8 T cells in spleens of mice injected with mouse IgG or anti-mCD8 (10 mg/kg). b, CFU assays (MethoCult™ GF M3434) of regular BM cells mixed with mouse T cells specific to MLL-AF9 AML cells (T-AF9) or non-specific T cells (T-LPS) (n = 3 independent cell cultures). c, Expression of INFγ,TNFα, and PD-1 on CD4 and CD8 T cells from spleens of mice engrafted with MLL-AF9 AML cells expressing LILRB3 FL or LILRB3 with intracellular domain truncation.

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Extended Data Fig. 3 LILRB3 enhances NF-κB signaling but not JNK signaling.

a, KEGG analysis of the top 20 processes affected by LILRB3 in mouse MLL-AF9 AML cells with whole-genome RNA-seq analysis. RNA was isolated from mouse MLL-AF9 AML cells expressing B3-FL or B3del ICD. “Down” and “Up” indicate genes expressed at lower or higher levels in AML cells that express B3del ICD versus those that express B3-FL. b, GSEA of the correlation between NF-κB signaling and LILRB3 in mouse MLL-AF9 AML cells (p values were calculated by Kolmogorov Smirnov (K-S) test in GSEA analysis). c, LILRB3 does not enhance the JNK signaling. GSEA of gene expression in THP-1 cells cultured in plates coated with anti-LILRB3 antibody or IgG.

Extended Data Fig. 4 TRAF2 and cFLIP interact, stimulate NF-κB signaling, and increase resistance of AML cells to the killing of cytotoxic T cells.

a, Relative NF-κB activities in 293 T cells co-transfected with NF-κB reporter plus empty vector, p22-FLIP, p43-CFLIP, or full-length cFLIP (n = 3 independent experiments). b, Relative NF-κB activities in 293 T cells co-transfected with NF-κB reporter plus empty vector or tet-on cFLIP in the presence of dox (n = 3 independent experiments). c, Co-immunoprecipitation assay of exogenous expressed FLAG-cFLIP and HA-TRAF2 in 293 T cells. d, e, Overexpression of TRAF2 and cFLIP increase the resistance of monocytic AML cells to cytotoxic T cells. CFSE-stained THP-1 cells that overexpress TRAF2 or empty vector (EV) (d) or cFLIP or empty vector (e) were co-cultured with activated T cells at the different ratios for 12 h and cell death was quantified. Left: Plots of percentage of dead cells versus E:T ration. Right: FACS analyses with E:T ratio of 2 (n = 3 independent experiments). f, West blotting of pMLKL (pS358) and MLKL in THP-1 cells treated with coated IgG or anti-LILRB3 for 12 h. g, Percentages of dead cells in THP-1 cells treated with anti-LILRB3 antibody or IgG in the presence of DMSO or NF-κB inhibitor QNZ (10 μM) (n = 3 independent cell cultures). The data are presented as mean ± s.e.m, and p values were calculated by two-tailed t-test.

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Extended Data Fig. 5 LILRB3 balances NF-κB signaling with TRAF2 and SHP1/2.

a, Relative luciferase activity from THP-1-Lucia™ cells at different times after activation with anti-LILRB3 antibody or IgG (n = 3 individual samples). b, TRAF2 mRNA levels in AML cell lines and normal monocytes (n = 3 independent experiments) c, The percentage of GFP+ MLL-AF9 AML cells (with PirB knockout) expressing B3-FL or B3del ICD in peripheral blood (PB), bone marrow (BM), spleen (SPL), and liver in mice treated with PBS or LPS (n = 4 independent mice). d, Survival of mice engrafted with AML cells as treated in panel d (n = 4 independent mice). The data are presented as mean ± s.e.m, and p values were calculated by two-tailed t-test except for e by log-rank test. e, Mechanistic scheme of LILRB3 signaling. Without ligand-induced crosslinking of LILRB3, TRAF2 remains associated with LILRB3 but does not stimulate downstream signaling. When NF-κB signaling is at a low level, upon ligand-induced crosslinking of LILRB3, TRAF2 recruits cFLIP, and cFLIP is cleaved to p22-FLIP by caspase 8 (whose activity can be inhibited by zVAD-FMK). p22-FLIP binds to the IKK complex and stimulates NF-κB signaling. Meanwhile, after ligand binding to LILRB3, the ITIMs of LILRB3 are phosphorylated, which recruits SHP-1 and SHP-2. When there is a high level of NF-κB signaling stimulated by other cues (for example, LPS), higher expression of cFLIP and A20 (TNFAIP3) is induced. Increased cFLIP inhibits caspase 8 activity, and A20 disrupts the interaction between TRAF2 and LILRB3. Thus the inhibitory effect of LILRB3 on NF-κB signaling mediated by SHPs becomes dominant.

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Extended Data Fig. 6 Development of anti-LILRB3 blocking antibodies for suppressing AML development.

a, Upper: Flow chart of strategy for development of fully humanized antibodies against LILRB3. Lower: The identified antibodies were tested in the LILRB3 chimeric receptor reporter cell assay. b, ELISA results for LILRB3 binders. c, EC50 values of the anti-LILRB3 antibodies based on ELISA. d, Affinities of antibodies #32, #33, #67, and #45 to LILRB3 as determined by Octet. e, Cross-reactivity of the anti-LILRB3 antibodies with LILRAs evaluated with LILRA binding analyses. f, Cross-reactivity of the anti-LILRB3 antibodies with other LILRBs evaluated with LILRB binding analyses. g, Interaction with TRAF2 contributes to the effect of LILRB3 on AML development. CFU assay of MLL-AF9 AML cells expressing wild-type LILRB3 or mutant LILRB3 with mutations disrupting TRAF2 binding (AAA, QEE509-511AAA) or disrupting SHP-1/2 interactions (Y596/626 F) in the presence of control or anti-LILRB3 antibodies (n = 3 independent cell cultures). h, Evaluation of the effect of Fc-mediated effector functions of anti-LILRB3. Upper: Schematic of treatment. Lower: Percentages of GFP + MLL-AF9 mouse AML cells in PB, BM, SPL and LV of mice transplanted with AML cells expressing B3-FL or B3delICD and injected with IgG or anti-LILRB3 #6 (n = 4 independent mice). i, Survival of mice as treated in panel h. j, NF-κB signaling target gene expression (measured by qPCR) in THP-1 cells from BM of xenografted NSG mice treated with anti-LILRB3 #1NA or IgG (n = 3 independent experiments). The data are presented as mean ± s.e.m, and p values were calculated by two-tailed t-test except for i by log-rank test.

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Extended Data Fig. 7 Anti-LILRB3 #1N297A antibody did not affect normal hematopoiesis and leukocytosis.

a, Schematic of generation of myeloid-specific LILRB3 transgenic mice. b, c, LILRB3 is expressed on myeloid cells in peripheral blood (PB), spleen (SPL) and bone marrow (BM) of LysM-Cre driven LILRB3 transgenic mice, which were treated by anti-LILRB3 #1 antibody (n = 2 mice) or IgG (n = 3 mice). Shown are representative flow cytometry plots (b) and result summary (c).

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Wu, G., Xu, Y., Schultz, R.D. et al. LILRB3 supports acute myeloid leukemia development and regulates T-cell antitumor immune responses through the TRAF2–cFLIP–NF-κB signaling axis. Nat Cancer 2, 1170–1184 (2021). https://doi.org/10.1038/s43018-021-00262-0

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